Triple combination of oseltamivir, amantadine, and ribavirin displays synergistic activity against multiple influenza virus strains in vitro

Abstract

The recurring emergence of influenza virus strains that are resistant to available antiviral medications has become a global health concern, especially in light of the potential for a new influenza virus pandemic. Currently, virtually all circulating strains of influenza A virus in the United States are resistant to either of the two major classes of anti-influenza drugs (adamantanes and neuraminidase inhibitors). Thus, new therapeutic approaches that can be rapidly deployed and that will address the issue of recurring resistance should be developed. We have tested double and triple combinations of the approved anti-influenza drugs oseltamivir and amantadine together with ribavirin against three influenza virus strains using cytopathic effect inhibition assays in MDCK cells. We selected A/New Caledonia/20/99 (H1N1) and A/Sydney/05/97 (H3N2) as representatives of the wild-type versions of the predominant circulating seasonal influenza virus strains and A/Duck/MN/1525/81 (H5N1) as a representative of avian influenza virus strains. Dose-response curves were generated for all drug combinations, and the degree of drug interaction was quantified using a model that calculates the synergy (or antagonism) between the drugs in double and triple combinations. This report demonstrates that a triple combination of antivirals was highly synergistic against influenza A virus. Importantly, the synergy of the triple combination was 2- to 13-fold greater than the synergy of any double combination depending on the influenza virus subtype. These data support the investigation of a novel combination of oseltamivir, amantadine, and ribavirin as an effective treatment for both seasonal and pandemic influenza virus, allowing the efficient use of the existing drug supplies.

FIG. 1.

Inhibition of A/New Caledonia/20/99 (H1N1)-induced cytopathic effect in MDCK cells treated with oseltamivir carboxylate (red squares), amantadine (green circles), and ribavirin (blue triangles) as determined by NR assay. Data are presented as the means from three replicates with standard deviations.

FIG. 2.

Inhibition of A/New Caledonia/20/99 (H1N1)-induced cytopathic effect in MDCK cells treated with double and triple combinations of amantadine (AMT), oseltamivir carboxylate (OSC), and ribavirin (RBV) as determined by NR assay. (A) Dose response for OSC alone (red circles), with 1.0 μg/ml RBV (black squares), with 0.032 μg/ml AMT (blue triangles), and with 1.0 μg/ml RBV plus 0.032 μg/ml AMT (green triangles). (B) Dose response for AMT alone (red circles), with 1.0 μg/ml RBV (black squares), with 0.01 μg/ml OSC (blue triangles), and with 1.0 μg/ml RBV plus 0.01 μg/ml OSC (green triangles). (C) Dose response for RBV alone (red circles), with 0.032 μg/ml AMT (black squares), with 0.032 μg/ml OSC (blue triangles), and with 0.032 μg/ml AMT plus 0.032 μg/ml OSC (green triangles). Data are presented as the means from three independent experiments (three replicates each) for each condition with standard deviations.

FIG. 3.

Synergistic inhibition of A/New Caledonia/20/99 (H1N1) replication as determined by NR assay in MDCK cells. Calculated additive interactions were subtracted from the experimentally determined inhibition to reveal regions of synergy (inhibition above the expected level) or antagonism (inhibition below the expected level). Values were derived from mean triplicate data and are presented at 95% confidence. This experiment was repeated five times with similar results. Areas in blue indicate doses of each drug that are synergistic, gray areas indicate doses that are additive, and red areas indicate doses that are antagonistic. The intensity of the color (blue or red) corresponds to the percent inhibition above or below the expected level. (A) Double combinations of amantadine and oseltamivir carboxylate (top), amantadine and ribavirin (middle), and ribavirin and oseltamivir carboxylate (bottom). Concentrations of each drug are indicated on the axes. (B) Triple combinations of oseltamivir carboxylate, amantadine, and ribavirin. Concentrations of each drug are indicated on the axes, with each plane representing a different concentration of amantadine. Data for the triple combination at amantadine concentrations above the EC₅₀ of amantadine (0.21 μg/ml) (Table 1) were excluded due to the efficacy of amantadine alone, which precluded the assessment of synergy.

FIG. 4.

Synergistic inhibition of A/Duck/MN/1525/81 (H5N1) replication as determined by NR assay in MDCK cells. Values were derived from mean triplicate data and are presented at 95% confidence. This experiment was repeated four times with similar results. Areas in blue indicate doses of each drug that are synergistic, gray areas indicate doses that are additive, and red areas indicate doses that are antagonistic. (A) Double combinations of amantadine and oseltamivir carboxylate (top), amantadine and ribavirin (middle), and ribavirin and oseltamivir carboxylate (bottom). Concentrations of each drug are indicated on the axes. (B) Triple combinations of oseltamivir carboxylate, amantadine, and ribavirin. Concentrations of each drug are indicated on the axes, with each plane representing a different concentration of amantadine. Data for the triple combination at amantadine concentrations above the EC₅₀ of amantadine (0.54 μg/ml; data not shown) were excluded due to the efficacy of amantadine alone, which precluded the assessment of synergy.

FIG. 5.

Synergistic inhibition of A/Sydney/05/97 (H3N2) replication as determined by NR assay in MDCK cells. Values were derived from mean duplicate data. Areas in blue indicate doses of each drug that are synergistic, gray areas indicate doses that are additive, and red areas indicate doses that are antagonistic. (A) Double combinations of amantadine and oseltamivir carboxylate (top), amantadine and ribavirin (middle), and ribavirin and oseltamivir carboxylate (bottom). Concentrations of each drug are indicated on the axes. (B) Triple combinations of oseltamivir carboxylate, amantadine, and ribavirin. Concentrations of each drug are indicated on the axes, with each plane representing a different concentration of amantadine. Data for the triple combination at amantadine concentrations above the EC₅₀ of amantadine (0.72 μg/ml; data not shown) were excluded due to the efficacy of amantadine alone, which precluded the assessment of synergy.

FIG. 6.

Plot of synergy volume for double combinations and the triple combination at 0.1 μg/ml amantadine (AMT) against influenza H1N1, H5N1, and H3N2 as determined by NR assay. Amantadine plus oseltamivir carboxylate (OSC) (white bars), amantadine plus ribavirin (RBV) (gray bars), oseltamivir carboxylate plus ribavirin (hatched bars), and the triple combination (black bars) are shown. Five independent experiments with 13 total replicates were conducted for the H1N1 virus, four independent experiments with 12 total replicates were conducted for the H5N1 virus, and one experiment with 2 replicates was conducted for the H3N2 virus. Synergy volumes are presented as the mean between experiments with standard deviations for the H1N1 and H5N1 viruses and the mean between replicates with standard deviations for the H3N2 virus.

FIG. 7.

Synergistic inhibition of A/Sydney/05/97 (H3N2) replication as determined by TCID₅₀ assay in MDCK cells. Values were derived from pooled replicate wells. Areas in blue indicate doses of each drug that are synergistic, gray areas indicate doses that are additive, and red areas indicate doses that are antagonistic. (A) Double combinations of amantadine and oseltamivir carboxylate (top), amantadine and ribavirin (middle), and ribavirin and oseltamivir carboxylate (bottom). Concentrations of each drug are indicated on the axes. (B) Triple combinations of oseltamivir carboxylate, amantadine, and ribavirin. Concentrations of each drug are indicated on the axes, with each plane representing a different concentration of amantadine. Data for the triple combination at amantadine concentrations above the EC₅₀ of amantadine (0.72 μg/ml; data not shown) were excluded due to the efficacy of amantadine alone, which precluded the assessment of synergy.

FIG. 8.

Synergistic inhibition of A/Sydney/05/97 (H3N2) replication as determined by qPCR assay. Values were derived from mean duplicate data. Areas in blue indicate doses of each drug that are synergistic, gray areas indicate doses that are additive, and red areas indicate doses that are antagonistic. (A) Double combinations of amantadine and oseltamivir carboxylate (top), amantadine and ribavirin (middle), and ribavirin and oseltamivir carboxylate (bottom). Concentrations of each drug are indicated on the axes. (B) Triple combinations of oseltamivir carboxylate, amantadine, and ribavirin. Concentrations of each drug are indicated on the axes, with each plane representing a different concentration of amantadine. Data for the triple combination at amantadine concentrations above the EC₅₀ of amantadine (0.72 μg/ml; data not shown) were excluded due to the efficacy of amantadine alone, which precluded the assessment of synergy.

FIG. 9.

Plot of synergy volume for double combinations and the triple combination at 0.32 μg/ml amantadine (AMT) against influenza H3N2 as determined by NR, TCID₅₀, and qPCR assays. OSC, oseltamivir carboxylate; RBV, ribavirin; CPE, cytopathic effect. The percent inhibition above the expected level as determined by the NR assay was converted to a log₁₀ scale in order for the synergy volume to be expressed in the same units as synergy volumes determined by TCID₅₀ and qPCR assays. Data are presented as the means of duplicates with standard deviation for NR and qPCR and from pooled duplicate samples for TCID₅₀.