Oseltamivir-ribavirin combination therapy for highly pathogenic H5N1 influenza virus infection in mice

Abstract

We studied the effects of a neuraminidase inhibitor (oseltamivir) and an inhibitor of influenza virus polymerases (ribavirin) against two highly pathogenic H5N1 influenza viruses. In vitro, A/Vietnam/1203/04 virus (clade 1) was highly susceptible to oseltamivir carboxylate (50% inhibitory concentration [IC(50)] = 0.3 nM), whereas A/Turkey/15/06 virus (clade 2.2) had reduced susceptibility (IC(50) = 5.5 nM). In vivo, BALB/c mice were treated with oseltamivir (1, 10, 50, or 100 mg/kg of body weight/day), ribavirin (37.5, 55, or 75 mg/kg/day), or the combination of both drugs for 8 days, starting 4 h before virus inoculation. Monotherapy produced a dose-dependent antiviral effect against the two H5N1 viruses in vivo. Three-dimensional analysis of the drug-drug interactions revealed that oseltamivir and ribavirin interacted principally in an additive manner, with several exceptions of marginal synergy or marginal antagonism at some concentrations. The combination of ribavirin at 37.5 mg/kg/day and oseltamivir at 1 mg/kg/day and the combination of ribavirin at 37.5 mg/kg/day and oseltamivir at 10 mg/kg/day were synergistic against A/Vietnam/1203/04 and A/Turkey/15/06 viruses, respectively. These optimal oseltamivir-ribavirin combinations significantly inhibited virus replication in mouse organs, prevented the spread of H5N1 viruses beyond the respiratory tract, and abrogated the cytokine response (P < 0.01). Importantly, we observed clear differences between the efficacies of the drug combinations against two H5N1 viruses: higher doses were required for the protection of mice against A/Turkey/15/06 virus than for the protection of mice against A/Vietnam/1203/04 virus. Our preliminary results suggest that oseltamivir-ribavirin combinations can have a greater or lesser antiviral effect than monotherapy, depending on the H5N1 virus and the concentrations used.

FIG. 1.

Three-dimensional plots showing the interaction of oseltamivir and ribavirin on the effect of treatment on the number of survivors (A), the mean day of death (B), and the risk of death (C) for mice infected with influenza A/Vietnam/1203/04 (H5N1) virus. Treatments were given twice daily for 8 days starting 4 h before virus exposure. Light bars, synergy; dark bars, antagonism. The synergy or antagonism for the various drug combinations, although minor, was significant at the 95% confidence level.

FIG. 2.

Three-dimensional plots showing the interaction of oseltamivir and ribavirin on the effect of treatment on the number of survivors (A), the mean day of death (B), and the risk of death (C) for mice infected with influenza A/Turkey/15/06 (H5N1) virus. Treatments were given twice daily for 8 days starting 4 h before virus exposure. Light bars, synergy; dark bars, antagonism. The synergy or antagonism for several drug combinations, although minor, was significant at the 95% confidence level.

FIG. 3.

Effect of treatment with oseltamivir (Os), ribavirin (Rib), and their combinations on virus titers (A) and cytokine/chemokine expression (B) of mice infected with influenza A/Vietnam/1203/04 (H5N1) or A/Turkey/15/06 (H5N1) viruses on day 3 postinoculation. (A) Oseltamivir and ribavirin (alone or in combination) or PBS was administered by oral gavage twice daily, starting 4 h before inoculation of 6-week-old BALB/c mice with 5 MLD₅₀/mouse of H5N1 virus. Each data bar represents the mean virus titer ± SD (log₁₀ EID₅₀/ml) in the organs of three mice. *, P < 0.05, compared with the results for the placebo-treated control group, one-way ANOVA; **, P < 0.01 compared with the results for the placebo-treated control group, one-way ANOVA; °, P < 0.001 compared with the results for the group treated with oseltamivir alone, one-way ANOVA; †, P < 0.05, compared with the results for the group treated with ribavirin alone, one-way ANOVA; ††, P < 0.001 compared with the results for the group treated with ribavirin alone, one-way ANOVA. (B) The expression of cytokines (IL-1α, IL-6, TNF-α, IFN-α), and chemokines (MCP-1, IP-10) in lung homogenates was determined by enzyme-linked immunosorbent assay on day 3 postinoculation. The means and SDs of the results are based on experiments with five mice. The dashed line indicates the level of cytokine/chemokine expression in mock-infected mice treated with oseltamivir and ribavirin (alone or in combination) or PBS (placebo). The cytokine induction profile in completely clean mice was also measured and is comparable to that of mock-infected mice. *, P < 0.05 compared with the results for the placebo-treated infected control group, one-way ANOVA; **, P < 0.01 compared with the results for the placebo-treated infected control group, one-way ANOVA; °, P < 0.01 compared with the results for the treated mock-infected control groups, one-way ANOVA.