Effects of double combinations of amantadine, oseltamivir, and ribavirin on influenza A (H5N1) virus infections in cell culture and in mice

Abstract

An amantadine-resistant influenza A/Duck/MN/1525/81 (H5N1) virus was developed from the low-pathogenic North American wild-type (amantadine-sensitive) virus for studying treatment of infections in cell culture and in mice. Double combinations of amantadine, oseltamivir (or the cell culture-active form, oseltamivir carboxylate), and ribavirin were used. Amantadine-oseltamivir carboxylate and amantadine-ribavirin combinations showed synergistic interactions over a range of doses against wild-type virus in Madin-Darby canine kidney (MDCK) cell culture, but oseltamivir carboxylate-ribavirin combinations did not. Primarily additive interactions were seen with oseltamivir carboxylate-ribavirin combinations against amantadine-resistant virus. The presence of amantadine in drug combinations against the resistant virus did not improve activity. The wild-type and amantadine-resistant viruses were lethal to mice by intranasal instillation. The resistant virus infection could not be treated with amantadine up to 100 mg/kg body weight/day, whereas the wild-type virus infection was treatable with oral doses of 10 (weakly effective) to 100 mg/kg/day administered twice a day for 5 days starting 4 h prior to virus exposure. Drug combination studies showed that treatment of the amantadine-resistant virus infection with amantadine-oseltamivir or amantadine-ribavirin combinations was not significantly better than using oseltamivir or ribavirin alone. In contrast, the oseltamivir-ribavirin (25- and 75-mg/kg/day combination) treatments produced significant reductions in mortality. The wild-type virus infection was markedly reduced in severity by all three combinations (amantadine, 10 mg/kg/day combined with the other compounds at 20 or 40 mg/kg/day) compared to monotherapy with the three compounds. Results indicate a lack of benefit of amantadine in combinations against amantadine-resistant virus, but positive benefits in combinations against amantadine-sensitive virus.

FIG. 1.

Three-dimensional synergy plots of interactions of amantadine and oseltamivir carboxylate (Oselt. Carbox.) (top) and amantadine and ribavirin (bottom) on influenza A/Duck/MN/1525/81 (H5N1) virus yields from MDCK cells. The data used are those presented in Table 1. Plots were made at the 95% confidence level.

FIG. 2.

Effects of treatment with amantadine, oseltamivir, and ribavirin, used alone or in combination, on mean body weights during an influenza A/Duck/MN/1525/81 (H5N1) amantadine-sensitive virus infection in mice. Oral treatments were given twice a day for 5 days starting 4 h before virus exposure. Data represent mean values using 10 drug-treated mice and 20 placebos per group. By day 11 the monotherapy groups had 1 to 4 survivors and the combination treatment groups had 9 or 10 survivors (per Table 4).

FIG. 3.

Effects of treatment with amantadine, oseltamivir, and ribavirin, used alone or in combination, on lung virus titers determined 72 h after influenza A/Duck/MN/1525/81 (H5N1) amantadine-sensitive virus infection of mice. Oral treatments were given twice a day starting 4 h before virus exposure, with the last treatment administered 4 h prior to sacrifice of the mice. Data are mean values ± standard deviations using lungs from five mice per group. A, amantadine plus oseltamivir; B, amantadine plus ribavirin; C and D, oseltamivir plus ribavirin. Values are shown as mg/kg/day.