In these studies, different dosage combinations of oseltamivir and T-705 produced improvements in survival and body weight compared to those seen with either compound used alone. At the lower doses of T-705, the combinations containing oseltamivir produced statistically significant improvements in survival and in body weight. Analysis of the survival data demonstrated that certain low-dose combinations of T-705 and oseltamivir produced synergistic interactions. At higher doses, where greater protection was provided by treatment with oseltamivir or T-705 alone, increased survival was also demonstrated. The differences were not significant due to limitations in numbers of animals and because survival approached the limit of protection (100%). In the second H5N1 experiment, where a high degree of protection was afforded, the significant improvement in body weight was indicative that the combination was superior to T-705 alone (Fig. ), since improved body weight correlates with milder disease.
Differences were noted in the effectiveness of treatment of H1N1, H3N2, and H5N1 virus infections with oseltamivir and T-705. The A/NWS/33 (H1N1) virus infection responded most favorably to treatment with oseltamivir. A 5-day course of treatment with 1 mg/kg/day was sufficient to protect nearly all of the mice from death. In contrast, 50 mg/kg/day was only partially protective against A/Victoria/3/75 (H3N2). We had to extend treatment to 7 days to even get efficacy against the H3N2 virus. A 5-day course of oseltamivir treatment of this infection was ineffective (data not shown). Oseltamivir did not have significant protective activity against the H5N1 virus infection when treatments were initiated either 2 h before or 24 h after infection. T-705 in the range of 20 to 25 mg/kg/day was not effective or only weakly active against the three viruses. However, T-705 was more effective against the H5N1 infection when treatments started at −2 h than at +24 h.
The reasons why oseltamivir and T-705 were less effective against the A/Duck (H5N1) virus than against the other viruses in mice are not understood. The results of the in vitro studies indicate that the H5N1 viral neuraminidase was inhibited less by oseltamivir carboxylate than the H3N2 and H1N1 neuraminidases, but the differences among them were small. This might make a difference in terms of drug efficacy. Viral neuraminidase inhibition was more relevant than cell culture assays for demonstrating efficacy in the case of the A/NWS (H1N1) virus, since poor cell culture activity of oseltamivir carboxylate was observed against this virus. Although T-705 is less potent than oseltamivir in vitro, it was more effective than oseltamivir against the H5N1 virus infections in mice. T-705 was slightly less potent against the H5N1 virus than against the other viruses in cell culture, but this may not necessarily explain why the A/Duck virus infection is more difficult to treat in mice.
One reason that the A/Duck virus may be more difficult to treat than the other viruses in vivo is its very rapid replication in mouse lungs (data not shown). This translates into an early time to death (compare mean days of death of placebo groups in Tables to ). Compounds must be given early to combat such a rapidly evolving infection. Decreasing the A/Duck virus challenge dose does not appreciably lengthen the time to death but does cause some animals to survive the infection altogether (our unpublished data). Virulence factors (such as certain cytokines and chemokines) that could differ in their expression with infection with different virus strains may also be important in disease progression. Research in this area is in progress in our laboratory.
Another observation from the treatment of the A/Duck infection was the poor dose-responsive effect of T-705 seen in some studies, such as that whose results are shown in Table where the 100-mg/kg/day dose gave less survival benefit than the 50-mg/kg/day dose. In other studies, depending upon timing and dose, a better dose-responsive effect was achieved. It may be that some mice are more prone to die from infection than others (creating intragroup variability), but this would be difficult to assess.
The variability in daily body weights during the H5N1 infection was greater in the 40-mg/kg/day T-705 group than in either the 80- or 20-mg/kg/day group (Fig. and ). We attribute this to the efficacy of the compound at this particular dose. The 80-mg/kg/day dose controlled the infection better than the 40-mg/kg/day dose, and the 20-mg/kg/day dose was largely ineffective. Some of the mice treated with 40 mg/kg/day of T-705 were close to dying from the infection (and consequently had a lower body weight) but managed to survive the infection. Other animals fared better and, thus, had higher body weights. It was this range of protection at the 40-mg/kg/day dose that resulted in more variability in body weights.
In these experiments, we did not investigate the effects of treatment on lung virus titer production. This would have added considerable numbers of animals to the already large studies. We have previously reported the effects of drug combinations (amantadine, ribavirin, and oseltamivir) on A/Duck (H5N1) virus titers (26
). In these experiments, the combinations reduced lung virus production, but there were not necessarily differences seen in virus titers for the combinations versus monotherapy, in spite of a survival benefit with the combinations. The overall effect of treatment to reduce lung virus titers was small. Single time points (such as day 6 after infection) for assessing inhibition of lung virus production do not seem to reflect the benefit derived from the full course of treatment. Increases in numbers of survivors and improvements in body weight are more indicative of treatment efficacy.
Combination treatments may be necessary due to widespread emergence of drug-resistant viruses. Because of the high frequency of amantadine- (or rimantadine-) resistant viruses in nature, this may limit the use of either of these compounds as a combination agent. In support of this statement, treatment of amantadine-resistant virus infections with amantadine plus oseltamivir or ribavirin provided no improvement compared to using oseltamivir or ribavirin alone (15
). Although the combination of oseltamivir and ribavirin is effective, ribavirin has some undesirable toxic side effects. T-705 appears to be a safer compound to administer than ribavirin. As T-705 advances through clinical development, it may become a viable option for the treatment of seasonal (H1N1 and H3N2), pandemic (the recent 2009 emerging virus), and highly pathogenic (H5N1) influenza virus infections, either used alone or in combination with oseltamivir.