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

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Antimicrob Agents Chemother



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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.

The effective treatment of influenza virus infections remains a public health priority. In the 2007-2008 influenza season there was a rise in the number of infected individuals, due to two of the vaccine virus strains being suboptimally matched with viruses circulating in nature (2). The afflicted individuals would have benefited from antiviral drug treatment. The threat of emerging highly pathogenic avian influenza A (H5N1) viruses for which no vaccines exist is also a concern (29). Recent data indicate the widespread viral resistance to the antiviral drug amantadine (6, 11) and the growing frequency of resistance to the other widely used antiviral drug oseltamivir (5, 19). Certain clades of highly pathogenic H5N1 viruses are resistant to amantadine, whereas other clades are not (3, 15).

Highly pathogenic H5N1 virus infections of humans have a high human mortality rate, exceeding 60% (18). Such severe infections are difficult to treat with oseltamivir (4). Thus, there is a need for more potent therapy, as well as for treatment that may decrease the frequency of the emergence of drug-resistant viruses (14). Combination chemotherapy with the right medications may be the answer to both problems. Investigators over the years have studied various compounds in combination in vitro (10, 13, 14, 21, 26) and in mouse models (8, 16, 17, 20, 22, 26, 28) against the H1N1, H3N2, H5N1, and H9N2 strains of influenza viruses. These include the testing of M2 channel blockers amantadine and rimantadine; the neuraminidase inhibitors oseltamivir carboxylate, peramivir, and zanamivir; and the nucleoside analog ribavirin (an inhibitor of influenza virus RNA polymerase [7]). Since the M2 channel blockers, neuraminidase inhibitors, and ribavirin all have separate modes of antiviral action, various combinations of these inhibitors have been more beneficial than monotherapy in treating infections in cell culture and in mice. Due to the widespread occurrence of viruses that are resistant to amantadine, a recent study has focused on the treatment of amantadine-resistant influenza virus infections (17).

We have developed a mouse model using a low-pathogenic North American strain, influenza A/Duck/MN/1525/81 (H5N1) virus for antiviral drug testing. The virus causes a severe lethal respiratory infection in mice that is treatable by antiviral therapy (25). The experimental influenza A/Duck mouse infection model described in the present set of experiments is not optimal, as it does not fully reflect the type of pathogenesis of the highly pathogenic avian influenza H5N1 viruses. The low-pathogenic A/Duck virus does not contain the multibasic amino acid R-X-R/K-R motif in the hemagglutinin protein, whereas the highly pathogenic avian viruses do (8). Having this motif allows for the highly pathogenic viruses to be proteolytically activated by ubiquitous subtilisin-like cellular proteases, favoring systemic spread in vivo beyond the respiratory tract, causing multiorgan failure. Indeed, Ilyushina et al. demonstrated spread of highly pathogenic H5N1 virus strains to other organs besides the lungs with a mouse model (16, 17). A high virus titer (approximately 104 cell culture 50% infective doses [CCID50] per mouse) is required to induce lethality with the wild-type A/Duck virus, compared to 1 to 4 PFU of A/Vietnam or A/Turkey viruses (16, 17). Thus, the highly pathogenic viruses are more virulent in mice. The A/Duck virus is sensitive to neuraminidase inhibitors (27) and to the RNA polymerase inhibitors ribavirin and T-705 (25, 27). It is also sensitive to inhibition by amantadine in vitro, as reported herein.

For these studies we also developed an amantadine-resistant A/Duck virus that is lethal to mice. Treatment of infections caused by this virus was compared with the treatment of wild-type virus infections, using the drugs amantadine, oseltamivir (or the cell culture-active form oseltamivir carboxylate), and ribavirin. The results with mice correlate with recent reports by Ilyushina et al. (16, 17) using highly pathogenic H5N1 amantadine-sensitive and amantadine-resistant viruses. In those studies, amantadine-oseltamivir and oseltamivir-ribavirin combinations were evaluated, but not amantadine-ribavirin combinations. An advantage to using the A/Duck virus mouse model is that studies can be conducted in a low-containment laboratory.

To our knowledge, the present investigation represents the first report of the use of amantadine and ribavirin in combination in vitro against an amantadine-resistant H5N1 virus and of oseltamivir carboxylate and ribavirin in combination against either sensitive or resistant H5N1 viruses in vitro. This is also the first report of treatment of an H5N1 virus infection in mice with the combination of amantadine plus ribavirin.