key: cord-0930896-uzzjvf9q
authors: Shah, Hriday M.; Jain, Ashvi S.; Joshi, Shreerang V.; Kharkar, Prashant S.
title: Crocetin and related oxygen diffusion‐enhancing compounds: Review of chemical synthesis, pharmacology, clinical development, and novel therapeutic applications
date: 2021-04-04
journal: Drug Dev Res
DOI: 10.1002/ddr.21814
sha: 36ec5ef653903df22a309b3923780be45bea41e0
doc_id: 930896
cord_uid: uzzjvf9q

The current pandemic forced us to introspect and revisit our armamentarium of medicinal agents which could be life‐saving in emergency situations. Oxygen diffusion‐enhancing compounds represent one such class of potential therapeutic agents, particularly in ischemic conditions. As rewarding as the name suggests, these agents, represented by the most advanced and first‐in‐class molecule, trans‐sodium crocetinate (TSC), are the subject of intense clinical investigation, including Phase 1b/2b clinical trials for COVID‐19. Being a successor of a natural product, crocetin, TSC is being investigated for various cancers as a radiosensitizer owing to its oxygen diffusion enhancement capability. The unique properties of TSC make it a promising therapeutic agent for various ailments such as hemorrhagic shock, stroke, heart attack, among others. The present review outlines various (bio)synthetic strategies, pharmacological aspects, clinical overview and potential therapeutic benefits of crocetin and related compounds including TSC. The recent literature focusing on the delivery aspects of these compounds is covered as well to paint the complete picture to the curious reader. Given the potential TSC holds as a first‐in‐class agent, small‐ and/or macromolecular therapeutics based on the core concept of improved oxygen diffusion from blood to the surrounding tissues where it is needed the most, will be developed in future and satisfy the unmet medical need for many diseases and disorders.

the severity of the hypoxemia and comparatively milder respiratory discomfort experienced by the patients. Of the several potential therapeutic strategies, supplemental oxygenation is the key to healing of the lungs by improving lung oxygenation mechanics and associated events. Summarily, gas exchange abnormalities coupled with the intravascular thrombi and compromised cortical feedback due to anxiety complicate the clinical presentation and the ensuing treatment such as mechanical ventilation.

Intrigued by the facts and observations related to hypoxemia, the authors reasoned if somehow the gas-exchange abnormalities could be reversed and possibly restored, the multiple organ damage would be prevented or minimized. The literature search on 'oxygen diffusion-enhancing compounds' pointed to an interesting report which described the evaluation of compounds for improved oxygen diffusion in plasma (Kuryel & Akgerman, 1978) . The discussion on the theoretical aspects of one-dimensional mass transport of oxygen from red blood cells (RBCs) to plasma, then through vascular wall followed by interstitial spaces and ultimately to the cells in tissues and organs, highlighted the importance of oxygen diffusion coefficient in plasma. A total of four compoundsprocaine (local anesthetic drug, 1, Figure 1 ), clofibrate (antihyperlipidemic drug, 2), vitamin A (3) and crocetin (natural apocarotinoid acid, 4) were evaluated in vitro using Clark-type polarographic electrode for measuring the oxygen diffusion coefficient in human plasma under unsteady state. The best compound, clofibrate (2), increased the oxygen diffusion coefficient to an extent of 58.5% (0.125 mg/ml of plasma); other tested compounds were effective as well, at the concentrations used.

Interestingly, 1, a carboxylic acid ester, is highly unstable in human plasma (t 1/2 = <5 min), which is hydrolyzed to p-aminobenzoic acid by plasma esterases (Hartman, 2003) Due to its extremely short half-life, it is reasonable to assume that the observed oxygen diffusion-enhancing effect of 1 could arise from its polar acid metabolite. On the similar lines, 2, an ester prodrug, is relatively unstable in plasma (Du et al., 2003) , generating its alkanoic acid metabolite.

The structural features common to all four molecules, thus, include -

(1) a polar group (either COOH or CH 2 OH) capable of forming stronger H-bond(s) with suitable H-bond acceptor(s) and (2) structural rigidity imparted by the polyene system in 3 and 4. The precise mechanism(s) of oxygen diffusion improvement by the identified leads was missing from the original report. Few follow-up articles by the same group delineated the detailed mechanism of increased O 2 diffusivity from plasma which involved the physical alteration in the plasma water structure upon addition of the oxygen diffusion-enhancing compounds (Gainer, 2008) . The H-bonding groups ( COOH/ CH 2 OH) and the structural rigidity in these molecules have a significant impact on the plasma water structure with direct impact on 1 2 3 4 5 6 F I G U R E 1 Molecular structures of oxygen diffusion-enhancing compounds solute (e.g., O 2 ) diffusivity. Surprisingly, solute diffusivity alterations by physical means has been largely overlooked as seen from dearth of literature reports investigating it.

Further literature search on 4 and its analogs and derivatives as promising oxygen diffusion-enhancing agents led us to a very recent update featuring trans-sodium crocetinate (TSC), a disodium salt of 4 (5, Figure 1 ) as an Investigational New Drug for international phase 1b/2b COVID-19 clinical program based on (Diffusion Pharmaceuticals, 2020).

Additionally, 5 is a clinical candidate for oncology indication (Overview/ Trans Sodium Crocetinate (TSC), 2020) (Glioblastoma multiforme -Phase 3; pancreatic cancer and brain metastasis -Phase 1), stroke (Stroke Program, 2020) and related disorders, owing to its ability to enhance oxygen diffusion in various tissues and organs. It has been shown to improve the outcome of radiotherapy due to increased oxygenation of the otherwise hypoxic cancerous tissue being treated. Gainer (2008) have been investigating naturally-occurring, first-in-class agent 4 since 1970s for its unique ability to enhance oxygen diffusion in plasma across various therapeutic categories and indications such as ischemic stroke and haemorrhagic shock. Several reviews and meta-analyses discussed the pharmacology and therapeutic utility of 4 and related compounds such as 5 and 6 (crocin, crocetin glycoside, Figure 1 ). The recently

proposed indication of 5, that is, COVID-19, can certainly be the game- (Xi & Qian, 2006) . As mentioned earlier, various investigations demonstrated the effectiveness of 4 in increasing oxygen diffusivity and oxygen consumption by the body tissues (Xi & Qian, 2006) . However, saffron is the most expensive spice in the world. The copious amount of water needed, coupled with a difficult irrigation process for saffron cultivation are the key reasons for its abysmally higher cost (Xi & Qian, 2006) . Today, 1 kg of saffron costs~US $200 (estimate). However, 4 has a carotenoid-like structure, a C 20 carbon chain with seven double bonds and a carboxylic acid group at both the termini. This has a direct impact on its aqueous solubility (Gainer, 2000; Lautenschläger et al., 2014;  F I G U R E 2 Retrosynthetic strategy leading to compounds 4 and 5 Mir et al., 2020) . Thus, solving the solubility issue holds the key to fully optimizing the medicinal utility of this product . One feasible solution that has been widely reported is using trans-sodium crocetinate (5). It is a sodium salt of 4. The trans form is supposedly more effective. It is well-known that the cis form counterbalances the increase in oxygen diffusivity shown by the trans form (Gainer, 2000) . Conventionally, 5 is extracted from saffron repeatedly using warm, distilled water.

Interested readers are invited to read up on the extraction methods through the scientific literature (Gainer, 2000; Lautenschläger et al., 2014; Mir et al., 2020) . The main problem with the conventional extraction method is the use of natural saffron which is quite expensive. Thus, the bio/synthetic methods have been pursued over the years to yield the product of interest due to synthetically-challenging chemical strategies.

The literature reports indicated few de novo multi-step approaches to synthetic 4, 5 and/or related compounds. The all-trans C 20 chain was systematically built using repetitive addition of building blocks mimicking the addition of isoprenoid units in biosynthetic pathways.

The retrosynthetic strategy leading to 4 and 5 is shown in Figure 2 .

Compound 5 was derived from its corresponding di-ester 18 by alkaline hydrolysis (yield 58-65%), which in turn, was obtained as the double condensation product of C 10 di-aldehyde (15) and C 5 phosphorane salt (16) (Frederico et al., 2003) . The conversion of C 10 di-ester (13) to 15 involved intermediate reduced product, C 10 di-alkanol (14). The mono condensation of 11 with phosphorane salt (12) yielded 13.

Intermediate 16 was derived from tiglic acid (17) in four steps in reasonable yield (32%) (Buchta & Andree, 1960; Duffield & Pettit, 2001; Gainer & Grabiak, 2007; Ingold & Roberts, 1971; Inhoffen et al., 1953; Isler et al., 1957) . The di-ester 18 was purified using silica gel column chromatography to obtain pure all-trans isomer as a brick-red solid.

The contaminating isomers were removed in the process (Gainer & Grabiak, 2007) . The final step, that is, saponification of 18 to generate 5, proved to be cumbersome. Of the several methods tried, MeOH/40% NaOH under reflux conditions gave good results; commercial process may use EtOH/i-PrOH instead of MeOH. The insolubility of 5 in most NMR solvents then presented difficulty in the structural characterization; HPLC, UV, IR and elemental analysis were used for this purpose (Gainer & Grabiak, 2007; Gainer & Lanz, 2013) .

The synthetic scheme leading to major intermediate 11 (Scheme 1) began with the oxidative ring-opening reaction of furan in presence of Br 2 /MeOH leading to (E)-1,1,4,4-tetramethoxybut-2-ene (8), which was further subjected to acidic hydrolysis catalyzed by cation-exchange sulfonic acid resin, Amberlyst-15, yielding monoprotected aldehyde (E)-4,4-dimethoxybut-2-enal (9) (Frederico et al., 2003; Gainer & Grabiak, 2007) . Intermediate 9 was combined with the corresponding ylide (Scheme 1) to give 10 in moderate (45%) yield, which was then deprotected to obtain 11. The ylide was generated by treating ethyl bromoacetate with PPh 3 and MeI in a four-step process with 87% yield (Gainer & Grabiak, 2007; Jansen & Lugtenbura, 1994) . Overall, the discussed retrosynthetic strategy formed the basis of the first synthetic route leading to 5 (reported in 2003) starting from furan (7, Scheme 1) with overall yield of 1.5% (Frederico et al., 2003) . Appreciably large number of synthetic steps, lower yields, higher cost with associated scale-up issues prompted the development of alternate, more efficient, high-yielding and environment-friendly synthetic routes for 5 and its intermediates as well as derivatives. The di-aldehyde 15 (Figure 2 ), being one of strategicallyimportant intermediates and supposedly the bottleneck in the synthetic route to 5, due to 16% yield starting from 7 (Scheme 1) was revisited for optimizing the synthesis. Scheme 2 depicts the alternate pathway to obtain 15 starting from 1,4-dichlorobut-2-ene (19), which in turn was generated following chlorination of 1,3-butadiene (Gainer & Grabiak, 2007) , followed by isomerization using FeCl 3 or a quaternary salt such as trioctylethylammonium bromide (Gordon, 1974) . Condensation of 19 with triethyl phosphite gave tetraethyl but-2-ene-1,4-diyl(E)-bis(phosphonate) (20)

in high yield (94%). Further reaction of 20 with methyl glyoxal generated intermediate (2E,4E,6E)-1,1,8,8-tetramethoxy-2,7-dimethylocta-2,4,6-triene (21) and its isomers in reasonably higher yield (Babler, 1992; Gainer & Grabiak, 2007) . The acidic hydrolysis led to a mixture of desired and undesired isomers of 15, which was reacted with pTSA to convert part of the undesired isomers to the desired ones, thereby modestly improving the yield (59% to 67%) (Gainer & Grabiak, 2007) .

Alternatively, 15 could be assembled starting from acetaldehyde diethyl acetal ( (Richter et al., 2007) . The environment-friendly process avoids use of furan as well as liq. Br 2 and can be carried out at atmospheric pressure in a continuous fashion using an undivided flow-through cell, resulting in better yield (62%) of 8.

Moreover, a suitable ionic liquid could be used as an electrolyte salt in the process.

1.3 | Biological production of Crocetin Dialdehyde (31) As an alternative to synthetic chemistry methods, biological production of crocetin dialdehyde (31, Figure 3 ) using genetic engineering and recombinant DNA technology approaches offers a great avenue.

The (32), which was then cleaved to 31 in E. coli and to 4 (Figure 1) in the corn endosperm (Giuliano et al., 2018) . The E. coli too must be genetically modified to accumulate zeaxanthin (Li et al., 2015) . The gene CCD2 was identified and isolated from the stigmas of saffron and then cloned onto the vector p-ThioDAN1, which allowed its expression in E. coli when induced from arabinose. (Holland et al., 1985) . Overall, 4 causes enhanced O 2 diffusion by ordering the plasma water structure due to its unique set of structural features.

Crocetin and related compounds are known to possess significant free-radical scavenging activity. This is overtly beneficial in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease as long as the molecule is capable of crossing the blood-brain barrier The antioxidant potential of saffron constituents is of particular interest, for example, 6 exerts multivalent effects at several targets and pathways in context with the cellular redox signaling (Bukhari et al., 2018; Korani et al., 2019) . There is a significant overlap in its redox properties and anti-inflammatory effects. This is not surprising,

given the deep involvement of the free-radicals in several inflammatory pathways. Overall, the neuroprotection offered by the saffron constituents due to their antioxidant capacity, is of immense benefit in the neurodegenerative disorders.

Owing to a long history dating back to 1970s and its unique mode of such as intestine, liver, kidneys, heart and brain. The more watersoluble salt of 4, that is, TSC (5) , was reviewed long ago for its potential to treat hypoxia/ischemia and hemorrhagic shock (Gainer, 2008) . 1.8 | Crocetin: Physicochemical, pharmacokinetic, and toxicity aspects

The natural source of 4, that is, saffron, is a coloring agent in food preparations. Daily saffron consumption saffron up to 1.5 g was considered relatively safe. Daily doses beyond 5 g were harmful,

while awfully higher doses of 20 g or so proved lethal (Hashemi & Hosseinzadeh, 2019) . Oral administration of Crocetin glycoside, 6, was advantageous over its metabolite 4 since it led to the higher serum concentrations of 4 in rats compared to 4 itself (Zhang et al., 2017) . Additionally, lower aqueous solubility of 4 (1.238 μg/ mL,~3.7 nM) is a major concern for its utility as a promising Recently granted/published patents/patent applications involving use and clinical trials of 5 emphasize its therapeutic potential and clinical use (Table 1) 

Both 4 and its salt 5 are the subject of intense biological, biochemical, pharmacological, toxicological, biomanufacturing as well as therapeutic investigations lately. The renewed interest in these widely explored and exploited therapeutic entities clearly demonstrate the potential they hold; more so in the current pandemic times. The ultimate use of 5 as a radiosensitizer is particularly more rewarding for inaccessible and inoperable tumors, such as GBM. In addition, the antioxidant potential, ability to cross BBB and accumulate in the brain is so very motivating from the neurodegenerative diseases and disorders perspective, given the lack of promising treatment modalities for such conditions. In future, such an approach based on use of TSC along with radiation therapy, for inoperable and inaccessible solid tumors could bring a radical transformation in oncology. The medical fraternity has waited so long for therapeutic options which could restore the organ functioning post-ischemia by regularizing or reversing the damage caused by lack of oxygen in the damaged tissue. Extension of therapeutic approaches based on enhanced oxygen diffusion by small molecules will certainly contribute to treat such conditions as ischemic stroke, myocardial infarction and several other cardiovascular complications.

The dangerous precipitation of the so-called cytokine storm with major contribution from the immune system and the ensuing inflammatory component in conditions, such as ARDS is therapeutically challenging, wherein the body has to deal with multiple attacks from within and outside; even the multiple administered drugs fail to tame it down.

The pharmacological utility of 4 and 5 in such conditions requiring antioxidant, anti-inflammatory as well as antiapoptotic actions, is particularly valuable. These compounds offer a promise to treat COVID-19 and related diseases including emerging viral infections. The nutraceutical status of 4 is the best part, which in a way, certifies its relatively safer character. The tip of the iceberg is just unraveled. There is much more beneath our current state of knowledge. Further systematic medicinal chemistry and biochemical explorations involving the molecular targets and the associated structural requirements will dictate tailor-made derivatives or analogs with imparted specialized abilities.

The current surge in the biosynthetic and chemical synthetic routes will make it possible, delivering better new chemical entities with defined therapeutic profiles. Crocetin and derivatives are here to stay for a very long time on the horizon of medicinal agents.

The authors are thankful to Prof. Aniruddha B. Pandit, Vice

Chancellor, Institute of Chemical Technology (ICT), for the literature search facilities and encouragement throughout the preparation of the manuscript.

The authors declare no conflict of interest.

Data sharing is not applicable to this article as no new data were created or analyzed in this study.

https://orcid.org/0000-0003-1955-7223

Comprehensive review on regulatory effects of crocin on ischemia/reperfusion injury in multiple organs

Method of making 2,7-dimethyl-2,4,6-octatrienedial and derivatives thereof

Efficacy and safety of saffron supplementation: Current clinical findings

Eine Totalsynthese des "all"-trans-Crocetin-dimethylesters

A comprehensive review of the pharmacological potential of Crocus sativus and its bioactive apocarotenoids

The Crocus sativus compounds trans-crocin 4 and trans-crocetin modulate the amyloidogenic pathway and tau misprocessing in Alzheimer disease neuronal cell culture models

Trans sodium crocetinate alleviates ischemia/reperfusion-induced myocardial oxidative stress and apoptosis via the SIRT3/FOXO3a/SOD2 signaling pathway

Method for synthesizing 2,7-dimethyl-2,4,6-octatriene-1,8-dialdehyde

Synthesis, characterization and inhibitory effects of crocetin derivative compounds in cancer and inflammation

Crocetin and crocin from saffron in cancer chemotherapy and chemoprevention

Crocetin extracted from saffron shows antitumor effects in models of human glioblastoma

The pathophysiology of 'happy' hypoxemia in COVID-19

Diffusion-Pharma-ceuticals-Files-IND-for-International-Phase-1b2b-COVID-19-Clinical-Program-With-TSC/default.aspx Diffusion Pharmaceuticals TSC MOA

Simultaneous determination of clofibrate and its active metabolite clofibric acid in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet absorbance detection

Synthesis of (7S,15S)-and (7R,15S)-dolatrienoic acid

Beneficial effects of saffron (Crocus sativus L.) in ocular pathologies, particularly neurodegenerative retinal diseases

A short and efficient synthesis of crocetin-dimethyl ester and crocetindial

Bipolar trans carotenoid salts and their uses

Trans-sodium crocetinate, methods of making and methods of use thereof

Trans-sodium crocetinate for treating hypoxia/ischemia

Use of bipolar trans carotenoids as a pretreatment and in the treatment of peripheral vascular disease

Diffusion enhancing compounds and their use alone or with thrombolytics

Use of bipolar trans carotenoids with chemotherapy and radiotherapy for treatment of cancer

Bipolar trans carotenoid salts and their uses

Trans carotenoids, their synthesis, formulation and uses

Trans carotenoids, their synthesis, formulation and uses

Oral formulations of bipolar trans carotenoids

Trans sodium crocetinate with temozolomide and radiation therapy for glioblastoma multiforme

Compositions and methods for treating cancer with atypical Braf mutations

Saffron; an updated review on biological properties with special focus on cardiovascular effects

Carotenoid dioxygenase and methods for the biotechnological production in microorganisms and plants of compounds derived from saffron

Isomerization of 1,2-dichloro-3-butene to 1,4-dichloro-2-butene

Determination of the stability of drugs in plasma

A comprehensive review on biological activities and toxicology of crocetin. Food and Chemical Toxicology

Antioxidant properties of Crocus sativus L. and its constituents and relevance to neurodegenerative diseases; focus on Alzheimer's and Parkinson's disease

Saffron (Crocus sativus L.) in ocular diseases: A narrative review of the existing evidence from clinical studies

Kinetics of O 2 uptake and release by red cells in stopped-flow apparatus: Effects of unstirred layers

The effect of red cell membrane and a diffusion boundary layer on the rate of oxygen uptake by human erythrocytes

Free radical substitution reactions (p. p22)

Synthesen in der Carotinoid-Reihe XXVI. Totalsynthese des Crocetin-dimethylesters

Diet therapy for the treatment of Alzheimer's disease in view of traditional Persian medicine: A review

Synthesen in der carotinoid-reihe. 10. Mitteilung. Anwendung der Wittig-reaktion zur synthese von estern des bixins und crocetins. Helvetica

Synthesis of isotopically labelled carotenoids: Investigations on structure and function of carotenoproteins at the atomic level

The efficacy of crocin of saffron (Crocus sativus L.) on the components of metabolic syndrome: A randomized controlled clinical trial

Crocus sativus a natural food coloring and flavoring has potent anti-tumor properties

Therapeutic effects of crocin in autoimmune diseases: A review

Compounds that increase oxygen diffusion in plasma

Effective isolation protocol for secondary metabolites from saffron: Semi-preparative scale preparation of crocin-1 and trans-crocetin

Metabolic engineering of Escherichia coli to produce zeaxanthin

Crocetin attenuates the oxidative stress, inflammation and apoptosis in arsenic trioxide-induced nephrotoxic rats: Implication of PI3K/AKT pathway

Construction of a stable and temperature-responsive yeast cell factory for crocetin biosynthesis using CRISPR-Cas9

The carotenoid compound of saffron crocetin alleviates effects of ischemia reperfusion injury via a mechanism possibly involving MiR-127

Carotenoids: Biochemistry, pharmacology and treatment

Isolation, purification and characterization of naturally derived Crocetin beta-d-glucosyl ester from Crocus sativus L. against breast cancer and its binding chemistry with ERalpha/HDAC2

The antileukemic effects of saffron (Crocus sativus L.) and its related molecular targets: A mini review

Anti-tumor effects of crocetin and related molecular targets

Bioactivity and bioavailability of the major metabolites of Crocus sativus L. flower

Oral crocetin administration suppressed refractive shift and axial elongation in a murine model of lens-induced myopia

Biologically active compounds and pharmacological activities of species of the genus crocus: A review

Saffron (Crocus sativus) in the treatment of gastrointestinal cancers: Current findings and potential mechanisms of action

Herbal nutraceuticals: Safe and potent therapeutics to battle tumor hypoxia

Safety, efficacy, and pharmacokinetics (PK) study of trans sodium crocetinate (TSC) in patients with intermittent claudication

Trans sodium crocetinate (TSC) study of intratumoral oxygen concentration, safety, and pharmacokinetics in patients with high grade glioma

Safety and efficacy of trans sodium crocetinate (TSC) with radiation and temozolomide in newly diagnosed glioblastoma

Safety and efficacy study of trans sodium crocetinate (TSC) in newly diagnosed glioblastoma (GBM) biopsy-only subjects (INTACT) (Diffusion Pharmaceuticals, Inc)

Efficacy and safety of trans sodium crocetinate (TSC) for treatment of suspected stroke (PHAST-TSC) (Diffusion Pharmaceuticals, Inc)

Mechanism behind the antitumour potential of saffron (Crocus sativus L.): The molecular perspective

Combination of oxidizing agents, photosensitizers and wound healing agents for oral disinfection and treatment of oral diseases

Saffron and its derivatives, crocin, crocetin and safranal: A patent review

Saffron: A promising natural medicine in the treatment of metabolic syndrome

Process for preparing 1,1,4,4-tetraalkoxybut-2-ene derivatives. Can

Saffron against components of metabolic syndrome: Current status and prospective

Considerations for target oxygen saturation in COVID-19 patients: Are we under-shooting?

Crocetin overproduction in engineered Saccharomyces cerevisiae via tuning key enzymes coupled with precursor engineering

Synthesis of crocetin derivatives and their potent inhibition in multiple tumor cells proliferation and inflammatory property of macrophage

Preparation of 1,1,4,4-tetramethoxy-2-butene

Delivering crocetin across the blood-brain barrier by using γ-cyclodextrin to treat Alzheimer's disease

Targeting oxidative stress and inflammation to prevent ischemia-reperfusion injury

Pharmacological properties of crocetin and crocin (digentobiosyl ester of crocetin) from saffron

COVID-19 and multiorgan response

Immunoregulatory and anti-inflammatory properties of Crocus sativus (saffron) and its main active constituents: A review

Protective effects of crocetin against radiation-induced injury in intestinal epithelial cells

Sensitive analysis and simultaneous assessment of pharmacokinetic properties of crocin and crocetin after oral administration in rats