key: cord-0006421-p7feodk1 authors: Shigeta, Shiro title: Recent Progress in Anti-Influenza Chemotherapy date: 2012-10-07 journal: Drugs R D DOI: 10.2165/00126839-199902030-00001 sha: 75db64673bd25ec409a30d07762a31bccbb1a939 doc_id: 6421 cord_uid: p7feodk1 Influenza virus infections in high risk individuals, such as infants, the elderly, and patients with cardiopulmonary disorders or immunocompromised states, cause severe manifestations which often result in fatalities. The emergence of a new antigen type of influenza A virus (H5N1) in Hong Kong during 1997 and 1998 threatened a possible pandemic of a new influenza infection. The investigation for anti-influenza chemotherapies has progressed in the last decade whereas clinical trials of new compounds have been limited to amantadine, rimantadine and ribavirin. Fusion inhibitors which directly inhibit conformational change of haemagglutinin (HA), protease inhibitors which inhibit cleavage of HA to HA1 and HA2, RNA transcription inhibitors which inhibit cap formation of mRNA and antisense oligonucleotides targeted at mRNA of PB2 (a part of viral RNA polymerase) have been reported, in their development phases. Recently, 2 neuraminidase (NA) inhibitors, zanamivir and oseltamivir (GS 4104), were used in clinical trials for the treatment of patients with influenza. Both agents showed promising results. A polyoxometalate, PM-523, inhibits fusion between the virus envelope and cell membrane and inhibits the penetration of the virus into cells. This compound has shown potent anti-influenza activity and synergistic inhibitory activity in combination with ribavirin or zanamivir in vitro and in vivo. Resistant strains for zanamivir, oseltamivir or PM-523 have been isolated. The analysis of mutation points of these strains have contributed to the investigation of the antiviral mechanisms of action of these compounds and the mechanism of resistance of the mutants to these compounds. Influenza is a highly contagious acute respiratory illness. Epidemics of influenza are characterised by a sudden appearance in the winter season, worldwide spread and then a sudden disappearance. Numerous epidemics have been recorded from ancient times; among these, the 1918-1919 pandemic was particularly severe (the Spanish influenza). After the Spanish influenza pandemic, 3 major antigen shifts in the influenza A virus have been encountered, from H1Nl (the Puerto Rico virus) to H2N2 (the agent for the Asian influenza) and then to H3N2 (the Hong Kong virus). Since 1977 we have encountered epidemics by 2 major antigenic subtypes: H3N2 and H1Nl (the Russian virus). Influenza viruses are divided into 3 types (A, B and C) based on the differences in their ribonucleoprotein (RNP) antigenicity. Influenza Avirus is further subdivided into subtypes based on differences in the antigenicity of the haemagglutinin (HA) and neuraminidase (NA) components of the virus. The current nomenclature system for human influenza Table I . Influenza encephalitis/encephalopathy in the Hokkaido area of Japan during epidemic seasons from 1994 to 1998[11 kines could be implicated in the neuropathology in these patients. The reported mortality rate of influenza is not high, on average, throughout all age categories (1.0 per million), as revealed by the annual mortality report of 1997 in Japan. However, the mortality rate increases 7-, 16-and 44-fold among elderly individuals aged over 65 years, over 75 years and over 85 years, respectively.Pl Much time has passed since the emergence of H3N2 in 1968 andHINI in 1977. Anew antigenic shift of influenza A virus may occur in the near future. Influenza B virus also exhibits antigenic drift sequentially every few years. Over time, prophylaxis for both influenza A and B viruses becomes ineffective using current vaccines. In May 1997, a 3-year-old boy in Hong Kong died of respiratory failure caused by an influenza virus infection; an influenza A strain ofH5N1 subtype was isolated from a sample of the bronchial aspirate. Between November 1997 and January 1998, 17 patients infected with the H5Nl subtype of the virus were detected, of whom 5 died. The influenza A H5Nl strain was isolated from chickens in several poultry farms and 1.5 million chickens were sacrificed in order to prevent an epidemic of the new antigenic virus among the human population. Direct person to person spread of this subtype has not yet been reported. [3, 4] In a more recent report from the WHO, another avian influenza A strain (H9N2) was identified in 2 hospitalised virus considers the geographical location of first isolation, strain number, year of isolation and antigenic description of HA and NA, for example AlIshikawa/7/82/(H3N2) or B/Singapore/222/79. The clinical features of influenza in humans range from asymptomatic infection to primary viral pneumonia. The onset of influenza is usually abrupt, with headache, chills and a dry cough, rapidly followed by high fever, myalgia, malaise and anorexia. The fever often peaks to as high as 41°C within 24 hours, but more commonly the temperature ranges between 38°and 40°C. The fever usually begins to decline on the third day of the illness and the patient usually becomes afebrile by the sixth day. When the virus infects children or individuals at high risk for complications (i.e. the elderly, patients with cardiopulmonary disease or immunocompromised patients), the clinical manifestations are more severe and the disease is sometimes fatal. In children, abrupt high fever is sometimes followed by CNS involvement, the manifestations of which can range from drowsiness and confusion to delirium and coma. This condition is termed influenza encephalitis or influenza encephalopathy. Influenza encephalitis [with inflammatory cells in the cerebrospinal fluid (CSF)] and influenza encephalopathy (without inflammatory cells in the CSF) sometimes manifest as convulsions. In Hokkaido (an island located in the northern part of Japan), 53 cases of influenza encephalitis/encephalopathy were reported during epidemics of influenza between 1994 to 1998. Among the 31 cases in whom the diagnosis was confirmed virologically, the average age was 3.7 years and the ratio of males to females was 2 : 1 (table I) . Influenza virus types A(HINl), A (H3N2) and typeB were isolated from the throat swabs of these patients. Mortality was reported to be 51.6%, and 19.4% of the patients exhibited some sequelae after recovery from the disease.I'J The appearance of CNS involvement after infection was rapid (2.8 days after the onset of fever) [table I]. The titres of inflammatory cytokines, such as interleukin-6 and tumour necrosis factor o, were high whereas the virus itself could not be isolated from the CSF. It is possible that these cyto- children in Hong Kong. The illness was not so servere as with the previousl y mentioned strain, and the patients recovered without any medical complications.U From the highly contagious nature of the influenza virus and the relatively high mortality rate in children and the elderl y, the necessity to treat patients with influenza virus infection using antiviral drugs cannot be overemphasised. This article reviews recent progress in anti-influenza chemotherapy. Prophylaxis of influenza virus infection with vaccination is not discussed. to detect influenza A and B viral antigens from patients ' specim ens within 15 minutes. When the extracted specimen is placed on the thin film which contains immobilised enzyme-linked antibodies and the antibodies capture the antigen, the thickn ess of the film changes and the colour of enzyme reacted substrate appears blue to purple (thus called the optical immunoassay). Another diagnostic kit, Directigen FluA (Becton Dickinson Co., Ltd . Cockeysville, Maryland, USA) can detect influenza A antigen but not B antigen. It may be useful to use this kit before prescribing amantadine or rimantadine; these drugs are effective only for influenza A. Amantadine (amantadine hydrochloride) was the first anti-influenza drug appro ved for clinical use.l 8 ] Amantadine is effecti ve for influenza A virus, but not for influenza Band C virus infections. Amantadine has also been used as a drug for the treatm ent of Parkinson 's disease for more than 30 years.Thus, the safety of amantadine for human use has been well established, although some neurological adverse effects, such as depression , difficulty in concentration and sleep disturb ances, have been reported. Rimantadine hydrochloride is an analogue of amantadine hydrochloride (ex-methyl 1adamantanemethylamine hydrochloride) which has been shown to inhibit the replication of influenza Acute respiratory infection (ARI) includes several clini cal diagnostic categories, such as rhinitis, pharyngitis, laryngitis (croup) , tracheitis, bronchitis, bronchiolitis and pneumonia. However, terms such as ' common cold ' or ' influenza-like disease ' are also used to categorise ARI. Influenza virus does not only cause clinical 'influenza' or ' influenza-like disease' but also other ARls such as, 'common cold' or 'catarrhal otitis media' . On the other hand, adenoviruses are commonly thought to be the causative agent of'pharyngo-coniunctival fever ', whereas they can somet imes cause influenza-like disease. Thus , it is rather difficult to determine causative viruses from clinical symptoms.I'vl There are many types of virus that cause ARl, including orthomyxoviruses, paramyxoviruses , picornaviruses, adenoviruses, coronaviruses and herpesviruses. Among these the most import ant viruses are orthomy xoviruses and paramyxoviruses which frequ ently cause acute lower respiratory infections in infants and children (table II) . Recent progress in molecular and immunological techniques has made it possibl e to rapidly detect the genomi c polynucleotides of viruses or virus antig ens using polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELI SA). For rapid diagnosis of influenza virus infection, the influenza virus optical immunoassay (FLU OIA) test (Biota Holding Inc. Melbourne, Australia) is handy and convenient. This compact kit is devised A virus but not that of influenza Band C viruses .Pl This drug has been used in countries such as Russia. Both drugs are absorbed well from the alimentary tract and the usual dosage is 100 to 200 mg/day. Rim antadine was reported to be more effective and safer than amantadine for the treatm ent of patients with influenza ) 10] In a comparative study of amantadine and rimantadine (lOOmg twice daily for 6 weeks ) prophylaxis of influenza A infection, Dolin et a1. [11 J reported that the withdraw al rate frommedication was significantly high er in the amantadine group than in the rimantadine group (13 vs 6%). During the course of chemotherapy for influenza A virus infection with amantadine , the development of resistance to this drug has been reported.l' Fl This disco ve ry shed light on the unique mechanism of action of amantadine as an anti-influ enza A virus drug and also on the function of the matrix protein (M2) of the virus. Amantadine and rimantadine inhibit vi rus replication by blo cking the acidified ion channels formed by virion-associated M2 protein. The M2 ion channel activity is thou ght to facilitate the flow of ions from the lumen of the endosome into the virion interior. This ionic flow brin gs about the dissociation of the RNP and M 1 (inner coat of RNP ). The liberat ed RNP moves easily to the cellular nucleus . The genomic RN A (seg ment 7) ofthe influ enza B vi rus encodes M 1and BM2 (the matrix of influenza B virus) proteins whose functions have not been determined yet. Segment 6 of the influenza B genomic RNA encodes NA and NB (the overlapping protein of neuraminidase gene) protein ; NB protein is speculated to be equivalent to the M2 prot ein of influenza A virus. It is probable that amantadine and rimantadine do not ' fit' properly to the influenza B virus ion chann el; this would explain the lack of channel activity inhibition. Ribavirin is an analogue of guanosine and is well known as an antiviral compound against several RNA viruses, including paramyxoviru ses, bunyaviruses, flaviviruses and arenaviruses. Its anti-influenza v irus activity is reported to be non-specific; it inhibits inosine monophosphate (IMP) dehydro gena se activity in cells , thereb y decreasing the amount of xantho sine mono phosphate and guanosine monophosphate, thus inhibiting viral RNA synthesis.F' l Riba virin is efficacious in infections of influ enza A and B viruses and respiratory syncytial (RS) virus but has 0 nly been approved by tile US Food and Drug Administration (FDA) for use in the treatment of patients with RS virus pneumonia.Uf The usual formulation of ribavirin is an aero sol (20 mg/ml solution) ; [14] howe ver, oral and intravenous infusions have also been reported to be efficacious .Uv ' 1.0 is estimated as antagonistic, 1.0 as additive, <1.0 as synergistic) [ fig. 3 ]. PM-523 was investigated for its anti-influenza A virus activity following administration as an aerosol to mice infected with a lethal dose of the virus (PR8/HlNl). Following exposure to PM-523 4.8 mmol/L for 2 hours twice daily for 4 days, 50% of the infected mice survived, whereas all the infected and untreated mice died by day 9. When we used a mixture ofPM-523 and ribavirinat a ratio of! : 16 (PM-523 2.4 mmol/L and ribavirin 40 nnnol/L) to treat the infected mice, a synergistic and improved therapeutic effect was achieved compared with the individual use of either compound; 80% of the mice survived to day 9 with the combined use of these compounds, in contrast to all the mice dying by day 9 with use of the individual compounds.F'l We demonstrated that PM-523 inhibited fusion between the influenza A virus envelope and the cellular membrane, whereas it did not inhibit the adsorption of the virus onto the cell membrane.l-sl Thus, the mechanism of anti-influenza virus activity is quite different from that of ribavirin and zanamivir, which inhibit RNA replication and the NA activity of the influenza virus, respectively. PM-523 and zanamivir also showed a synergistic inhibitory effect against influenza virus in vitro; the synergism was more prominent against influenza B virus than against the A virus (Mori S et al. In order to analyse the antifusionactivity ofPM-523 for the influenza virus, strains of influenza virus A resistant to PM-523 were isolated after sequential passages of the wild strain of AJIshikawa/ H3N2 in PM-523-containing cultures of MDCK cells. The resistant strains were analysed for amino Table III . Antimyxovirus activities of several polyoxotungstates l26] Shigeta acid substitutions of HA after cDNA amplification of the HA genome. All 5 strains which were resistant to PM-523 possessed mutations in HAl at positions Lysl89 and Ile204 compared with the sequence of the wild-type (sensitive) strain. These point mutations were located in the globular head of HAl and the interface edges of the trimer molecules. The trimers open their heads in response to the conformational change ofHA at low pH. Thus, we assumed thatPM-523 binds to the interface edges ofHA trimers and inhibits the opening of HAl trimers, consequently inhibiting fusion of the viral envelope to the cellular membrane by HA2 hydrophobic amino acids at the edge of the cleavage site (unpublished observations) [ fig. 4 ]. In The mechanism of anti-influenza virus activity ofPM-523 is unique and entirely different from that of amantadine, ribavirin or NA inhibitors. The synergistic anti-influenza virus activity of this compound with ribavirin or zamamivir offers promise for the use of PM-523 as a therapeutic drug for influenza virus infection. In addition, PM-523 and its congeners have broad antiviral activities against enveloped RNA viruses and may be developed as broad -spectrum drugs for ARI caused by ortho-and paramyxoviruses. Among the patients who were febrile and began treatment within 30 hours after the onset of symptoms, alleviation of the major symptoms occurred at least 2 days earlier in the treated group compared with the placebo group) 31] A randomised double-blind clinical trial of zanamivir for the treatment of influenza A and B infec-tions was carried out by the Management ofInfluenza in the Southern Hemisphere Trialists (MIST) study group during the winter season of 1997/1998)32] 321 patients were randomly assigned to inhaled zanamivir lOmg or placebo twice daily for 5 days. Zanamivir significantly shortened the time to alleviation of symptoms compared with placebo (5.0 vs 6.5 days; p = 0.004). Patients who were febrile (temperature >37.8°C) and virologically positive for the influenza virus responded (alleviation of clinical symptoms) 2.0 days earlier in the zanamivir group than in the placebo group. High-risk patients (those with respiratory, endocrine, metabolic or cardiovascular disorders, immunocompromised patients or patients aged 65 years or older) responded a median of 2.5 days earlier when treated with zanamivir than when treated with placebo. The most common adverse effects observed with zanamivir inhalation were respiratory effects (bronchitis and cough). However, there was no significant difference in the incidence of adverse effects between the zanamivir inhalation group and the placebo group. The study group stated that zanamivir was well tolerated among high-risk patients.Ivl Drug-resistant viruses were isolated by passaging the virus in MDCK cells in the presence ofzanamivir. Mutations in HA and NA were found in the resistant virus. The HA mutation, 198Thr to Ile, reduced the affinity of the virus for the receptors found on susceptible human cells (Siaa2-6Gal). Weak affinity of HA for the receptor may facilitate the release of the virus from cells, and NAactivity may not contribute significantly to the release of the virus. Thus, zanamivir is thought to inefficiently inhibit the release of the resistant strains with mutations in HA. Two types of NA mutants were also isolated from the culture described above. One had a mutation in the catalytic site ofNA (Arg292 to Lys) and the other a mutation in the framework residue ofNA (Glu119 to Gly, Asp or Ala) with which the guanidino group of zanamivir interacts.U'l The same research group also isolated zanamivirresistant strains from an immunocompromised child infected with the influenza B virus. The isolates had a Thrl98 to Ile mutation in HA and an Argl52 to © Adis International Limited. All rights reserved. Lys mutation in NA. The HA mutation reduced the affinity of the virus for human cell receptors and the NA mutation reduced the enzymatic NA inhibitory activity of zanamivir.P'l A large scale US clinical trial of zanamivir in the treatment of influenza during 1995 to 1996 was reported by Monto et alV5] In this trial, 1182 patients were randomised to receive double-blind treatment with placebo or zanamivir via inhalation (1Omg, 2 or 4 times daily) and nasal spray (6.4mg, 2 or 4 times daily) for 5 days. Overall, 722 patients had virologically confirmed influenza [including A (H3N2), A (HINl) and B in similar numbers]. Zanamivir reduced the duration of illness by 1 to 1.5 days when given within 30 hours of onset of disease (p = 0.001 when zanamivir was given 4 times daily, IOmg via inhalation or 6.4mg via nasal spray). Possible drug associated adverse events were reported equally in placebo and zanamivir groups. 35 patients were withdrawn from the study because of adverse events; numbers were similar in each group (12 in the placebo group, 13 in the zanamivir 2 times daily group, 10 in the zanamivir 4 times daily group). Thus, this large randomised trial of zanamivir for the treatment of influenza confirms the clinical efficacy of zanamivir when given early in the course of infections and also its tolerability. More recently, a group from Gilead Sciences developed a carbocyclic transition state analogue of sialic acid, oseltamivir (GS 4104) [ fig. 5 ]. Oseltamivir is well absorbed from the alimentary tract (30 to 70%), in contrast to zanamivir which has only a low degree of absorption (3 to 4%) from the alimentary tract. After absorption from the alimentary tract, oseltamivir changes to its nonester active form, GS 4071, and is distributed efficiently to the lungs and other tissuesV6, 37] Therapeutic efficacy of oral oseltamivir in reducing the symptoms of influenza in both experimentally infected volunteers and patients with acute febrile respiratory illness have been reported. 80 infected adults were randomised to receive oral oseltamivir (20 to 200mg twice daily for the volunteers and 75 to 150mg twice daily for the patients) or placebo for 5 days. Treatment was initiated 28 hours after the inoculation of the volunteers and 36 hours after the onset of symptoms in the patients. In both trials, oral oseltamivir significantly reduced the time to alleviation of symptoms compared with placebo) 38, 39] Similar HA and NA mutants were obtained as for the case of zanamivir resistance (see section 6.1) following in vitro culture of influenza A virus in the presence of GS 4071. The mutants were all resistant to the inhibitory action of GS 4071. The HA mutants had substitutions at Ala28 to Thr and Thr Arg124 to Met. The NA had an Arg292 to Lys mutation. The substitution ofNA Glu1l9 to Gly or Asp did not induce resistance for the activity of GS 4071 as an NAinhibitor.l 40 ] From the observations ofthese investigators.l'vl mutant strains resistant to zanamivir or GS 4071 are difficult to generate both in vitro and in vivo. The clinical importance of anti-influenza chemotherapy is evident as antigenic shifts of virus and pandemics of new subtype strains of influenza are predicted in the near future. The prophylactic use of amantadine and rimantadine for influenza A virus infection is recommended, and ribavirin, in the form of an aerosol for patients with severe influenza A and B infections, has been reported to be efficacious. Recent pro gress in anti -influenza chemotherapy has been made with the development of the NA inhibitors, zanamivir and oseltamivir. Clinical trials have shown both drugs to be efficacious in alleviating the main symptoms of influenza. A poly oxometalate, PM-523, was inhibitory for the growth of influenza virus in vitro and in vivo. PM-523 inhibited fusion ofvirus envelope and cell membrane and showed broad spectrum antiviral activity against ortho-and paramyxoviruses. What remains to be done in the future is to examine clinically the combination of 2 or more drugs with different antiviral activity against influenza virus growth. 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