In vitro and in vivo activities of anti-influenza virus compound T-705

T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has been found to have potent and selective inhibitory activity against influenza virus. In an in vitro plaque reduction assay, T-705 showed potent inhibitory activity against influenza A, B, and C viruses, with 50% inhibitory concentrations (IC(50)s) of 0.013 to 0.48 microg/ml, while it showed no cytotoxicity at concentrations up to 1,000 microg/ml in Madin-Darby canine kidney cells. The selectivity index for influenza virus was more than 2,000. It was also active against a neuraminidase inhibitor-resistant virus and some amantadine-resistant viruses. T-705 showed weak activity against non-influenza virus RNA viruses, with the IC(50)s being higher for non-influenza virus RNA viruses than for influenza virus, and it had no activity against DNA viruses. Orally administered T-705 at 100 mg/kg of body weight/day (four times a day) for 5 days significantly reduced the mean pulmonary virus yields and the rate of mortality in mice infected with influenza virus A/PR/8/34 (3 x 10(2) PFU). These results suggest that T-705 may be a compound that is useful and highly selective against influenza virus infections and that has a mode of action different from those of commercially available drugs, such as amantadine, rimantadine, and neuraminidase inhibitors.     

Efficacy of orally administered T-705 on lethal avian influenza A (H5N1) virus infections in mice

T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) was inhibitory to four strains of avian H5N1 influenza virus in MDCK cells, with the 90% effective concentrations ranging from 1.3 to 7.7 microM, as determined by a virus yield reduction assay. The efficacy was less than that exerted by oseltamivir carboxylate or zanamivir but was greater than that exerted by ribavirin. Experiments with mice lethally infected with influenza A/Duck/MN/1525/81 (H5N1) virus showed that T-705 administered per os once, twice, or four times daily for 5 days beginning 1 h after virus exposure was highly inhibitory to the infection. Dosages from 30 to 300 mg/kg of body weight/day were well tolerated; each prevented death, lessened the decline of arterial oxygen saturation (SaO(2)), and inhibited lung consolidation and lung virus titers. Dosages from 30 to 300 mg/kg/day administered once or twice daily also significantly prevented the death of the mice. Oseltamivir (20 mg/kg/day), administered per os twice daily for 5 days, was tested in parallel in two experiments; it was only weakly effective against the infection. The four-times-daily T-705 treatments at 300 mg/kg/day could be delayed until 96 h after virus exposure and still significantly inhibit the infection. Single T-705 treatments administered up to 60 h after virus exposure also prevented death and the decline of SaO(2). Characterization of the pathogenesis of the duck influenza H5N1 virus used in these studies was undertaken; although the virus was highly pathogenic to mice, it was less neurotropic than has been described for clinical isolates of the H5N1 virus. These data indicate that T-705 may be useful for the treatment of avian influenza virus infections.     

Identification of GS 4104 as an Orally Bioavailable Prodrug of the Influenza Virus Neuraminidase Inhibitor GS 4071

GS 4071 is a potent carbocyclic transition-state analog inhibitor of influenza virus neuraminidase with activity against both influenza A and B viruses in vitro. GS 4116, the guanidino analog of GS 4071, is a 10-fold more potent inhibitor of influenza virus replication in tissue culture than GS 4071. In this study we determined the oral bioavailabilities of GS 4071, GS 4116, and their respective ethyl ester prodrugs in rats. Both parent compounds and the prodrug of the guanidino analog exhibited poor oral bioavailability (2 to 4%) and low peak concentrations in plasma (C_maxs; C_max <0.06 μg/ml). In contrast, GS 4104, the ethyl ester prodrug of GS 4071, exhibited good oral bioavailability (35%) as GS 4071 and high C_maxs of GS 4071 (C_max = 0.47 μg/ml) which are 150 times the concentration necessary to inhibit influenza virus neuraminidase activity by 90%. The bioavailability of GS 4104 as GS 4071 was also determined in mice (30%), ferrets (11%), and dogs (73%). The plasma of all four species exhibited high, sustained concentrations of GS 4071 such that at 12 h postdosing the concentrations of GS 4071 in plasma exceeded those necessary to inhibit influenza virus neuraminidase activity by 90%. These results demonstrate that GS 4104 is an orally bioavailable prodrug of GS 4071 in animals and that it has the potential to be an oral agent for the prevention and treatment of influenza A and B virus infections in humans.     

Oral oseltamivir in human experimental influenza B infection

Oseltamivir is the prodrug of Ro64-0802 (GS4071), a potent and selective inhibitor of influenza A and B virus neuraminidases. Three randomized, double-blind, placebo-controlled, parallel-group studies evaluated oral oseltamivir for early treatment (75 or 150 mg twice daily for 5 days) or prevention (75 mg once or twice daily for 7 days) of experimental influenza B virus infection in healthy susceptible adults. Treatment study A (n=60) demonstrated similar trends to treatment study B (n=117), in which 75 mg doses of oseltamivir introduced 24 h after inoculation reduced median area under curve (AUC) virus titre (oseltamivir, 22.7; placebo, 131.1 log10 TCID50 x h/ml; P=0.002) and duration of viral shedding (oseltamivir, 23.9 h; placebo, 95.8 h; P=0.0005). In prevention study C (n=58), oseltamivir did not reduce infection rates (85 versus 84%) but significantly reduced median AUC virus titre (10.0 versus 66.9 log10 TCID50 x h/ml; P=0.03) and duration of viral shedding (36 versus 84 h; P=0.03) compared with placebo. Oseltamivir was well tolerated. No emergence of drug-resistant variants was detected by testing last-day isolates (n=112) in neuraminidase inhibition assays. These results indicate that oseltamivir has significant antiviral activity in experimental human influenza B virus infection when used for prophylaxis or early treatment.     

Comparison of the anti-influenza virus activity of RWJ-270201 with those of oseltamivir and zanamivir

We have recently reported an influenza virus neuraminidase inhibitor, RWJ-270201 (BCX-1812), a novel cyclopentane derivative discovered through structure-based drug design. In this paper, we compare the potency of three compounds, RWJ-270201, oseltamivir, and zanamivir, against neuraminidase enzymes from various subtypes of influenza. RWJ-270201 effectively inhibited all tested influenza A and influenza B neuraminidases in vitro, with 50% inhibitory concentrations of 0.09 to 1.4 nM for influenza A neuraminidases and 0.6 to 11 nM for influenza B neuraminidases. These values were comparable to or lower than those for oseltamivir carboxylate (GS4071) and zanamivir (GG167). RWJ-270201 demonstrated excellent selectivity (>10,000-fold) for influenza virus neuraminidase over mammalian, bacterial, or other viral neuraminidases. Oral administration of a dosage of 1 mg/kg of body weight/day of RWJ-270201 for 5 days (beginning 4 h preinfection) showed efficacy in the murine model of influenza virus infection as determined by lethality and weight loss protection. RWJ-270201 administered intranasally at 0.01 mg/kg/day in the murine influenza model demonstrated complete protection against lethality, whereas oseltamivir carboxylate and zanamivir at the same dose demonstrated only partial protection. In the delayed-treatment murine influenza model, oral administration of a 10-mg/kg/day dose of RWJ-270201 or oseltamivir (GS4104, a prodrug of GS4071) at 24 h postinfection showed significant protection against lethality (P < 0.001 versus control). However, when the treatment was delayed for 48 h, no significant protection was observed in either drug group. No drug-related toxicity was observed in mice receiving 100 mg/kg/day of RWJ-270201 for 5 days. These efficacy and safety profiles justify further consideration of RWJ-270201 for the treatment and prevention of human influenza.     

Neuraminidase inhibitors as anti-influenza virus agents.

Influenza virus neuraminidase (NA) catalyses the cleavage of sialic acid residues terminally linked to glycoproteins and glycolipids and plays an important role in the replication of the virus. Recently, several potent NA inhibitors have been synthesized based on the rational design of mimicking the transition state of the sialic acid cleavage. Zanamivir and oseltamivir (GS 4104, the prodrug of GS 4071) have emerged as promising influenza NA inhibitors for the treatment and prophylaxis of human influenza virus infection. This review describes the recent work toward the discovery and development of influenza NA inhibitors.     

Oral Administration of a Prodrug of the Influenza Virus Neuraminidase Inhibitor GS 4071 Protects Mice and Ferrets against Influenza Infection

We have recently described GS 4071, a carbocyclic transition-state analog inhibitor of the influenza virus neuraminidase, which has potent inhibitory activity comparable to that of 4-guanidino-Neu5Ac2en (GG167; zanamivir) when tested against influenza A virus replication and neuraminidase activity in vitro. We now report that GS 4071 is active against several strains of influenza A and B viruses in vitro and that oral GS 4104, an ethyl ester prodrug which is converted to GS 4071 in vivo, is active in the mouse and ferret models of influenza virus infection. Oral administration of 10 mg of GS 4104 per kg of body weight per day caused a 100-fold reduction in lung homogenate viral titers and enhanced survival in mice infected with influenza A or B viruses. In ferrets, a 25-mg/kg dose of GS 4104 given twice daily reduced peak viral titers in nasal washings and eliminated constitutional responses to influenza virus infection including fever, increased nasal signs (sneezing, nasal discharge, mouth breathing), and decreased activity. Consistent with our demonstration that the parent compound is highly specific for influenza virus neuraminidases, no significant drug-related toxicity was observed after the administration of oral dosages of GS 4104 of up to 800 mg/kg/day for 14 days in nonclinical toxicology studies with rats. These results indicate that GS 4104 is a novel, orally active antiviral agent with the potential to be used for the prophylaxis and treatment of influenza A and B virus infections.     

Effects of the Combination of Favipiravir (T-705) and Oseltamivir on Influenza A Virus Infections in Mice

Favipiravir (T-705 [6-fluoro-3-hydroxy-2-pyrazinecarboxamide]) and oseltamivir were combined to treat influenza virus A/NWS/33 (H1N1), A/Victoria/3/75 (H3N2), and A/Duck/MN/1525/81 (H5N1) infections. T-705 alone inhibited viruses in cell culture at 1.4 to 4.3 μM. Oseltamivir inhibited these three viruses in cells at 3.7, 0.02, and 0.16 μM and in neuraminidase assays at 0.94, 0.46, and 2.31 nM, respectively. Oral treatments were given twice daily to mice for 5 to 7 days starting, generally, 24 h after infection. Survival resulting from 5 days of oseltamivir treatment (0.1 and 0.3 mg/kg/day) was significantly better in combination with 20 mg/kg of body weight/day of T-705 against the H1N1 infection. Treatment of the H3N2 infection required 50 mg/kg/day of oseltamivir for 7 days to achieve 60% protection; 25 mg/kg/day was ineffective. T-705 was ≥70% protective at 50 to 100 mg/kg/day but inactive at 25 mg/kg/day. The combination of inhibitors (25 mg/kg/day each) increased survival to 90%. The H5N1 infection was not benefited by treatment with oseltamivir (≤100 mg/kg/day for 7 days). T-705 was 30 to 70% protective at 25 to 100 mg/kg/day. Survival improved slightly with combination treatments. Increased activity was seen against H5N1 infection by starting treatments 2 h before infection. Oseltamivir was ineffective at ≤40 mg/kg/day. T-705 was 100% protective at 40 and 80 mg/kg/day and inactive at 20 mg/kg/day. Combining ineffective doses (20 mg/kg/day of T-705 and 10 to 40 mg/kg/day of oseltamivir) afforded 60 to 80% protection and improved body weights during infection. Thus, synergistic responses were achieved with low doses of T-705 combined with oseltamivir. These compounds may be viable candidates for combination treatment of human influenza infections.The emergence of swine influenza H1N1 virus infections in 2009 (2) highlights the need for effective antiviral therapy in a largely immune-naïve population. Treatment options for influenza are becoming more limited because viruses, including the 2009 swine H1N1 virus, are resistant to the antiviral drugs amantadine and rimantadine (3, 4, 11, 13, 20). Oseltamivir-resistant viruses are also becoming more common in the environment, particularly within the last 2 years (1, 5, 19). Thus, more potent and effective treatments are needed to combat these growing threats.More potent antiviral therapy can be achieved by using drugs in combination, as demonstrated in mouse models (10, 14-17, 24, 26, 27). Such treatment can slow down the emergence of drug-resistant viruses (12). The reported animal studies have primarily focused on the known-active antiviral agents amantadine, rimantadine, oseltamivir, peramivir, zanamivir, and ribavirin. The kinds of studies that can be performed have been limited based upon the number of active antiviral compounds that are available.In 2002, Furuta et al. reported a novel pyrazine molecule, T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide, now named favipiravir), as an inhibitor of influenza virus infections in cell culture and in mice (8). T-705 inhibits both influenza A and B viruses (8, 23, 29). The compound converts to nucleoside mono- (T-705 RMP [ribosylated, monophosphorylated]), di-, and triphosphate (T-705 RTP [ribosylated, triphosphorylated]) forms in cells (9). The mode of action of T-705 RTP is similar to that of ribavirin triphosphate as an inhibitor of influenza virus RNA polymerase (6, 9). Unlike ribavirin monophosphate, T-705 RMP is only weakly inhibitory to cellular inosine monophosphate (IMP) dehydrogenase (9, 28), and thus, it is less cytotoxic. These properties make T-705 a viable candidate for the treatment of influenza virus infections in humans. The compound is currently undergoing phase II clinical trials.The use of T-705 in combination with other antiviral substances has not been reported. The purpose of the present work was to evaluate whether the combination of T-705 with the widely used antiviral drug oseltamivir is more beneficial than either substance used alone against influenza virus infections in mice. We chose three mouse-adapted influenza viruses for these comparisons, A/NWS/33 (H1N1), A/Victoria/3/75 (H3N2), and A/Duck/MN/1525/81 (H5N1). The A/NWS and A/Victoria viruses are of seasonal origin and are confined to the respiratory tract following infection. The A/Duck virus is a low-pathogenicity avian virus from the United States that also does not spread beyond the respiratory tract of mice. The experimental influenza A/Duck mouse infection does not fully reflect the type of pathogenesis of the highly pathogenic avian influenza H5N1 viruses from the Old World. This is because the A/Duck virus lacks the multibasic amino acid R-X-R/K-R motif in the hemagglutinin protein, whereas the highly pathogenic avian H5N1 viruses contain it (7). This motif allows for the highly pathogenic viruses to be proteolytically activated by ubiquitous subtilisin-like cellular proteases, allowing the virus to spread in vivo beyond the respiratory tract and to cause multiorgan failure. Nevertheless, the A/Duck virus induces rapid, severe lung infections that are difficult to treat with conventional antiviral therapy. Using these three models, H1N1, H3N2, and H5N1, in mice, we were able to demonstrate the benefits of using oseltamivir and T-705 in combination to treat influenza virus infections.     

In vitro and in vivo activities of T-705 against arenavirus and bunyavirus infections

There is a need for the development of effective antivirals for the treatment of severe viral diseases caused by members of the virus families Bunyaviridae and Arenaviridae. The pyrazine derivative T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) has demonstrated remarkable antiviral activity against influenza virus and, to a lesser degree, against some other RNA viruses (Y. Furuta, K. Takahashi, Y. Fukuda, M. Kuno, T. Kamiyama, K. Kozaki, N. Nomura, H. Egawa, S. Minami, Y. Watanabe, H. Narita, and K. Shiraki, Antimicrob. Agents Chemother., 46:977-981, 2002). Here, we report that T-705 is highly active against a panel of bunyaviruses (La Crosse, Punta Toro, Rift Valley fever, and sandfly fever viruses) and arenaviruses (Junin, Pichinde, and Tacaribe viruses) by cytopathic effect and virus yield reduction cell-based assays. The 50% effective concentrations for T-705 ranged from 5 to 30 microg/ml and 0.7 to 1.2 microg/ml against the bunyaviruses and arenaviruses examined, respectively. We also demonstrate that orally administered T-705 is efficacious in treating Punta Toro virus in the mouse and hamster infection models, as well as Pichinde virus infection in hamsters. When administered twice daily for 5 to 6 days, beginning 4 h pre- or 24 h post-Punta Toro virus challenge, a 30-mg/kg of body weight/day dose provided complete protection from death and limited viral burden and liver disease. A dose of 50 mg/kg/day was found to be optimal for treating Pichinde infection and limiting viral replication and disease severity. In general, T-705 was found to be more active than ribavirin in cell-based assays and in vivo, as reflected by substantially greater therapeutic indexes. Our results suggest that T-705 may be a viable alternative for the treatment of life-threatening bunyaviral and arenaviral infections.     

In vitro characterization of A-315675, a highly potent inhibitor of A and B strain influenza virus neuraminidases and influenza virus replication

A-315675 is a novel, pyrrolidine-based compound that was evaluated in this study for its ability to inhibit A and B strain influenza virus neuraminidases in enzyme assays and influenza virus replication in cell culture. A-315675 effectively inhibited influenza A N1, N2, and N9 and B strain neuraminidases with inhibitor constant (K(i)) values between 0.024 and 0.31 nM. These values were comparable to or lower than the K(i) values measured for oseltamivir carboxylate (GS4071), zanamivir, and BCX-1812, except for the N1 enzymes that were found to be the most sensitive to BCX-1812. The time-dependent inhibition of neuraminidase catalytic activity observed with A-315675 is likely due to its very low rate of dissociation from the active site of neuraminidase. The half times for dissociation of A-315675 from B/Memphis/3/89 and A/Tokyo/3/67 (H3N2) influenza virus neuraminidases of 10 to 12 h are significantly slower than the half times measured for oseltamivir carboxylate (33 to 60 min). A-315675 inhibited the replication of several laboratory strains of influenza virus in cell culture with potencies that were comparable or superior to those for oseltamivir carboxylate and BCX-1812, except for the A/H1N1 viruses that were found to be two- to fourfold more susceptible to BCX-1812. A-315675 and oseltamivir carboxylate exhibited comparable potencies against a panel of A/H1N1 and A/H3N2 influenza virus clinical isolates, but A-315675 was found to be significantly more potent than oseltamivir carboxylate against the B strain isolates. The favorable in vitro results relative to other clinically effective agents provide strong support for the further investigation of A-315675 as a potential therapy for influenza virus infections.     

Mechanism of action of T-705 against influenza virus

T-705, a substituted pyrazine compound, has been found to exhibit potent anti-influenza virus activity in vitro and in vivo. In a time-of-addition study, it was indicated that T-705 targeted an early to middle stage of the viral replication cycle but had no effect on the adsorption or release stage. The anti-influenza virus activity of T-705 was attenuated by addition of purines and purine nucleosides, including adenosine, guanosine, inosine, and hypoxanthine, whereas pyrimidines did not affect its activity. T-705-4-ribofuranosyl-5'-triphosphate (T-705RTP) and T-705-4-ribofuranosyl-5'-monophosphate (T-705RMP) were detected in MDCK cells treated with T-705. T-705RTP inhibited influenza virus RNA polymerase activity in a dose-dependent and a GTP-competitive manner. Unlike ribavirin, T-705 did not have an influence on cellular DNA or RNA synthesis. Inhibition of cellular IMP dehydrogenase by T-705RMP was about 150-fold weaker than that by ribavirin monophosphate, indicating the specificity of the anti-influenza virus activity and lower level of cytotoxicity of T-705. These results suggest that T-705RTP, which is generated in infected cells, may function as a specific inhibitor of influenza virus RNA polymerase and contributes to the selective anti-influenza virus activity of T-705.     

Activities of oseltamivir and ribavirin used alone and in combination against infections in mice with recent isolates of influenza A (H1N1) and B viruses

Mouse models have been widely used for evaluating potential influenza virus inhibitors. However, the viral strains traditionally used in these models are fairly old and do not represent currently circulating viruses in nature. We developed two new lethal infection models in mice using mouse-adapted influenza A/New Caledonia/20/99 (H1N1) and influenza B/Sichuan/379/99 viruses. Both virus infections were used to study oral treatment with oseltamivir and ribavirin, both alone and in combination. Oral treatments were given twice daily for 5 days starting 4 h before infection in initial studies. Against influenza A, oseltamivir was active at 10, 20, and 40 mg/kg/day, protected 80-100% of mice from death and reduced lung consolidation - ribavirin was similarly effective at 20, 40, and 80 mg/kg/day. When treatments were initiated after virus challenge, delaying treatment with oseltamivir even 1 day caused it to be ineffective. Ribavirin prevented mortality by 50-80% when treatments were delayed 1-4 days after infection. The combination of the two drugs (oseltamivir at 20 mg/kg/day and ribavirin at 40 mg/kg/day) was no better than ribavirin alone. In contrast to what we observed with influenza A virus infections, oseltamivir and ribavirin showed similar dose-related antiviral activities against influenza B virus infections. The compounds both significantly increased survival when treatments started up to 4 days after infection, but ribavirin was more active than oseltamivir (50-80% survival compared to 30-40% survival, respectively, when starting treatments on days 2-4 after infection). By varying the doses of each drug that were used in combination (oseltamivir at 1.25, 2.5 and 5 mg/kg/day; ribavirin at 5, 10 and 20 mg/kg/day) certain dosage combinations were superior to either compound used alone as assessed by decreased mortality, lung virus titre, lung score and lung weight parameters. These activities differed from published results with older, more established virus strains as oseltamivir was less effective and ribavirin was more active than previously reported.     

Penetration of GS4071, a novel influenza neuraminidase inhibitor, into rat bronchoalveolar lining fluid following oral administration of the prodrug GS4104.

GS4071 is a novel potent inhibitor of influenza neuraminidase (Ki < 1 nM) with low (< 5%) oral bioavailability in animals. An ethyl ester prodrug of GS4071, GS4104, has exhibited good oral bioavailability in rat, mouse, and dog models and is currently being developed for the treatment of influenza A and B virus infections. Since influenza virus replicates primarily in the surface epithelial cells of the respiratory tract, the ability of the prodrug to deliver GS4071 to the bronchoalveolar lining fluid (BALF) following an oral dose of GS4104 should be an important indicator of its potential efficacy. In the present study, we determined the concentration-time profiles of GS4071 in the BALF and plasma of rats following oral administration of GS4104. The BALF was sampled by bronchoalveolar lavage with endogenous urea as a dilution marker. The concentration of GS4071 in BALF reached a peak at 2 h (1 h after the plasma peak) and declined at a slower rate than plasma levels, suggesting slow clearance of drug from the lung acini. The ratios of the area-under-the-curve (AUC) values of GS4071 in BALF to those in plasma were 1.05 for AUC from 0 to 6 h (AUC(0-6)) and 1.51 for AUC(0-infinity), indicating significant penetration of the parent drug into the lower respiratory tracts of rats following oral administration of the prodrug. No unchanged GS4104 was detected in BALF.     

Influence of treatment schedule and viral challenge dose on the in vivo influenza virus-inhibitory effects of the orally administered neuraminidase inhibitor GS 4104.

Experiments were done to determine how an alteration of the treatment schedule of 5 or 32 mg/kg/day per os (p.o.) doses of GS 4104 [the ethyl ester prodrug of the neuraminidase inhibitor (3R, 4R,5S)-4-acetamido-5-amino-3-(1-ethylpropoxy)-1-cylohexene-1 -carboxylic acid (GS 4071)] would affect influenza A (H1N1) virus infection in mice. Treatments with a low dose, one, two, three or four times daily, were highly inhibitory, unless therapy was terminated relatively early in the infection (days 2-3), in which case efficacy was curtailed. Single administrations at various times relative to virus exposure had essentially no effect. The 32 mg/kg/day dose was significantly inhibitory using all treatment schedules. These data indicated a requirement for the compound to be in the host when lung virus titres were reaching maximal levels and, for minimally effective doses, that at least continued daily therapy was needed to maintain adequate serum levels to achieve an appropriate antiviral effect. Twice daily p.o. treatment for 5 days with 20 mg/kg/day of GS 4104 totally prevented deaths in mice receiving high viral challenge doses that were sufficient to kill placebo-treated controls in less than 5 days. Other parameters of antiviral efficacy (lung consolidation, arterial oxygen saturation, lung virus titres) were also markedly inhibited regardless of viral challenge doses. These data provide further insights into how the maximum therapeutic benefit can be derived from use of this orally effective influenza virus neuraminidase inhibitor.     

Treatment of mannan-enhanced influenza B virus infections in mice with oseltamivir, ribavirin and viramidine

Mannan, a polysaccharide preparation from Saccharomyces cerevisiae, has previously been shown to enhance influenza virus replication in mice by inhibiting host defense collectins. The use of mannan in infections may serve to broaden the types of influenza viruses that can be studied in rodent infection models. When mannan was co-administered with influenza B/Sichuan/379/99 virus to mice, the animals died from the infection, whereas mice infected with only virus survived. Three types of influenza A (H1N1) and another influenza B (Hong Kong/330/01) virus infection were also enhanced by mannan, but not four types of influenza A (H3N2) viruses. Mannan was used at 0.16 or 0.5 mg/mouse for optimal disease-enhancing activity using influenza B/Sichuan/379/99 virus. Using this model, influenza B/Sichuan/379/99 infections were treated with oseltamivir, ribavirin or viramidine (the carboxamidine derivative of ribavirin). When oral gavage treatments started 4 h before virus and mannan challenge, oseltamivir was effective at 2.5, 5 and 10 mg/kg/day. Ribavirin was active at 20, 40 and 80 mg/kg/day. Viramidine was effective at 80 and 160 mg/kg/day but not at 40 mg/kg/day. Active drug doses improved lung consolidation scores and lung weights, with decreases in lung virus titres also noted. Arterial oxygen saturation values in treated groups were significantly better than those of the placebo group on days 7-11 of the infection. Oseltamivir (5 mg/kg/day) and ribavirin (40 mg/kg/day) were used alone and in combination to determine how late after infection they could be beneficially administered. Ribavirin alone was very effective (90-100% survival of mice) when treatments started as late as 3 days after infection. Forty percent survival was evident even when treatments started 4 days post-infection. Oseltamivir was active starting treatments 1 day after virus exposure, but lost considerable efficacy when treatments began after that time. The combination of ribavirin and oseltamivir appeared to be no better than ribavirin alone, due to the stronger beneficial effect of ribavirin in this model. The overall results demonstrate that mannan can be used to enhance certain non-lethal influenza virus infections sufficiently to allow antiviral studies.     

Amantadine-oseltamivir combination therapy for H5N1 influenza virus infection in mice

BACKGROUND: The clinical management of H5N1 influenza virus infection in humans remains unclear. Combination chemotherapy with drugs that target different viral proteins might be more effective than monotherapy. METHODS: BALB/c mice were treated by oral gavage for 5 days with amantadine (1.5, 15 or 30 mg/kg/day) and oseltamivir (1 or 10 mg/kg/day) separately or in combination. Mice were challenged 24 h after initiation of treatment with 10 mouse 50% lethal doses of either amantadine-sensitive (having S31 in the M2 protein) or amantadine-resistant (having N31 in the M2 protein) recombinant A/Vietnam/1203/04 (H5N1) virus. RESULTS: Combination treatment with amantadine (15 or 30 mg/kg/day) and oseltamivir (10 mg/kg/day) provided greater protection (60% and 90%, respectively) against lethal infection with amantadine-sensitive H5N1 virus than did monotherapy. Moreover, spread of the virus to the brain was prevented by both combination regimens. The efficacy of the drug combinations against amantadine-resistant H5N1 virus was comparable to that of oseltamivir alone. Oseltamivir produced a dose-dependent effect against both recombinant H5N1 viruses (P < 0.05) but did not provide complete protection against lethal infection. Importantly, no mutations in the HA, NA and M2 proteins were detected when the two drugs were used in combination. CONCLUSIONS: Combination chemotherapy provided a survival advantage over single-agent treatment of mice inoculated with neurotropic H5N1 influenza virus. This strategy might be an option for the control of pandemic influenza viruses that are sensitive to amantadine. Combinations that include other drugs should be explored.     

Mechanism of Action of T-705 Ribosyl Triphosphate against Influenza Virus RNA Polymerase

T-705 (favipiravir; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) selectively and strongly inhibits replication of the influenza virus in vitro and in vivo. T-705 has been shown to be converted to T-705-4-ribofuranosyl-5-triphosphate (T-705RTP) by intracellular enzymes and then functions as a nucleotide analog to selectively inhibit RNA-dependent RNA polymerase (RdRp) of the influenza virus. To elucidate these inhibitory mechanisms, we analyzed the enzyme kinetics of inhibition using Lineweaver-Burk plots of four natural nucleoside triphosphates and conducted polyacrylamide gel electrophoresis of the primer extension products initiated from ³²P-radiolabeled 5′Cap1 RNA. Enzyme kinetic analysis demonstrated that T-705RTP inhibited the incorporation of ATP and GTP in a competitive manner, which suggests that T-705RTP is recognized as a purine nucleotide by influenza virus RdRp and inhibited the incorporation of UTP and CTP in noncompetitive and mixed-type manners, respectively. Primer extension analysis demonstrated that a single molecule of T-705RTP was incorporated into the nascent RNA strand of the influenza virus and inhibited the subsequent incorporation of nucleotides. These results suggest that a single molecule of T-705RTP is incorporated into the nascent RNA strand as a purine nucleotide analog and inhibits strand extension, even though the natural ribose of T-705RTP has a 3′-OH group, which is essential for forming a covalent bond with the phosphate group.     

Utilization of alpha-1-acid glycoprotein levels in the serum as a parameter for in vivo assay of influenza virus inhibitors

Alpha-1-acid glycoprotein (AGP), an acute phase protein in serum assayed by single radial immunodiffusion using a commercially available kit, was found to significantly increase in mice infected with influenza A and B viruses. Experiments were run to determine the rate of increase of serum AGP and its relation to other influenza disease parameters, including lung consolidation, development of lung virus titres, decline in arterial oxygen saturation (SaO2), histopathological changes in the lung, and death of the animal. Maximal AGP levels occurred by day 3 in the animals, at about the same time lung virus titres reached their peak and inflammatory effects were evident in the lung. Serum levels of AGP were then compared with other disease parameters in the evaluation of the anti-influenza A and B virus efficacy of oseltamivir and ribavirin in mice. Treatment was by oral gavage twice daily for 5 days, beginning 4 h before virus exposure using doses of 100, 10, and 1 mg/kg per day of oseltamivir and 75 mg/kg per day of ribavirin. Against the influenza A infection, significant inhibition of death, SaO2 decline, and lung consolidation was seen at all doses of each compound; day-6 AGP levels were reduced in a dose-responsive manner. Lung virus titres were lessened at this time, but to a significant degree only at the high dose of oseltamivir and by ribavirin. The influenza B virus infection, which appeared more severe than the influenza A infection, was also significantly inhibited by both compounds, but to a lesser extent. The serum AGP levels were again lessened by therapy with both compounds. The influence of challenge dose of influenza A virus on AGP level and on the antiviral activity of 20 mg/kg per day of oseltamivir, administered by oral gavage, was determined in mice. The AGP level was in proportion to the viral challenge dose; oseltamivir significantly inhibited AGP levels and all other disease parameters regardless of size of viral inoculum. These data indicate murine AGP levels to be markedly stimulated by infection with influenza A and B viruses, and the level of the protein to be an additional measure of antiviral efficacy.     

Comparison of efficacies of RWJ-270201, zanamivir, and oseltamivir against H5N1, H9N2, and other avian influenza viruses

The orally administered neuraminidase (NA) inhibitor RWJ-270201 was tested in parallel with zanamivir and oseltamivir against a panel of avian influenza viruses for inhibition of NA activity and replication in tissue culture. The agents were then tested for protection of mice against lethal H5N1 and H9N2 virus infection. In vitro, RWJ-270201 was highly effective against all nine NA subtypes. NA inhibition by RWJ-270201 (50% inhibitory concentration, 0.9 to 4.3 nM) was superior to that by zanamivir and oseltamivir carboxylate. RWJ-270201 inhibited the replication of avian influenza viruses of both Eurasian and American lineages in MDCK cells (50% effective concentration, 0.5 to 11.8 microM). Mice given 10 mg of RWJ-270201 per kg of body weight per day were completely protected against lethal challenge with influenza A/Hong Kong/156/97 (H5N1) and A/quail/Hong Kong/G1/97 (H9N2) viruses. Both RWJ-270201 and oseltamivir significantly reduced virus titers in mouse lungs at daily dosages of 1.0 and 10 mg/kg and prevented the spread of virus to the brain. When treatment began 48 h after exposure to H5N1 virus, 10 mg of RWJ-270201/kg/day protected 50% of mice from death. These results suggest that RWJ-270201 is at least as effective as either zanamivir or oseltamivir against avian influenza viruses and may be of potential clinical use for treatment of emerging influenza viruses that may be transmitted from birds to humans.     

Activity of T-705 in a hamster model of yellow fever virus infection in comparison with that of a chemically related compound, T-1106

Treatment with the nucleoside analog T-1106 was previously shown to be effective in a hamster model of yellow fever virus (YFV) disease, even though it had only slight activity in cell culture. In the study described in this report, the activity of T-705, a chemically related compound currently undergoing clinical trials for the treatment of influenza (FDANews 4:1, 2007), was tested against YFV in cell culture and in the hamster model. The antiviral efficacy of T-705 in cell culture occurred at a concentration of 330 μM, which was more than threefold lower than the concentration at which T-1106 had antiviral efficacy, as determined by a virus yield reduction assay and confirmed by a luciferase-based ATP detection assay. Time-of-addition studies revealed that addition of T-705, T-1106, or ribavirin at 0, 4, 8, or 12 h after virus challenge was effective in inhibiting virus in Vero cells, suggesting that these three agents have similar mechanisms of action in cell culture. Because of its more potent activity in cell culture, it was anticipated that T-705 treatment of hamsters infected with YFV would result in protection from disease. Significant improvements in survival and disease parameters were seen in infected animals when T-705 was administered orally at a dose of 200 or 400 mg/kg of body weight per day when it was given twice a day for 8 days. Significant improvements were also observed with a dose of 400 mg/kg/day when treatment initiation was delayed as late as 3 days after virus inoculation. Although the dose of T-705 required for efficacy in hamsters is higher than that of T-1106 required for efficacy, T-705 treatment is effective in significantly improving disease parameters in YFV-infected hamsters, which may indicate its potential utility in the treatment of YFV disease in humans.