Effects of the combination of favipiravir (T-705) and oseltamivir on influenza a virus infections in mice

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Antimicrob Agents Chemother



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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.