Mechanism by which mutations at his274 alter sensitivity of influenza a virus n1 neuraminidase to oseltamivir carboxylate and zanamivir.

Oseltamivir carboxylate is a potent and specific inhibitor of influenza neuraminidase (NA). An influenza A/H1N1 variant selected in vitro with reduced susceptibility to oseltamivir carboxylate contains a His274Tyr mutation. To understand the mechanism by which a His274Tyr mutation gives rise to drug resistance, we studied a series of NA variant proteins containing various substitutions at position 274. Replacement of His274 with larger side chain residues (Tyr or Phe) reduced the NA sensitivity to oseltamivir carboxylate. In contrast, replacement of His274 with smaller side chain residues (Gly, Asn, Ser, and Gln) resulted in enhanced or unchanged sensitivity to oseltamivir carboxylate. Previous studies have suggested that the slow-binding inhibition of NA by oseltamivir carboxylate is a result of the reorientation of Glu276. Loss of this slow-binding inhibition in the His274Tyr and His274Phe mutant NA but not in His274Asn, His274Gly, His274Ser, or His274Gln supports the conclusion that the conformational change of Glu276 is restricted in the His274Tyr and His274Phe mutant NA upon oseltamivir carboxylate binding. Interestingly, His274Asn, as well as His274Gly, His274Ser, and His274Gln, also displayed reduced sensitivity to zanamivir and its analogue, 4-amino-Neu5Ac2en. Substitution of His274 with Tyr in influenza A/Tokyo/3/67 (H3N2) recombinant NA did not affect the susceptibility to oseltamivir carboxylate. These data indicate that the volume occupied by the amino acid side chain at position 274 can influence the sensitivities of influenza N1 NA but not of N2 NA to both oseltamivir carboxylate and zanamivir.

Skin reactions in patients with influenza treated with oseltamivir: a retrospective cohort study.

Oseltamivir phosphate is an FDA-approved treatment for influenza that has been available for prescription use in the USA since 1999. The present report describes findings from a post-marketing safety study of skin reactions associated with oseltamivir use. All patients in the claims-derived Ingenix Research Database with a physician diagnosis of influenza and/or a dispensing of oseltamivir between 1 December 1999 and 31 March 2002 were identified. Cohort eligibility criteria included minimum baseline enrolment duration of 3 months, age of at least 1 year and no influenza vaccination on the date of influenza diagnosis or oseltamivir dispensing. Patients were classified into two primary cohorts, influenza diagnosis and oseltamivir dispensing on the same day, and influenza diagnosis but no oseltamivir at any time, and a cohort included for secondary analyses comprising patients who received an oseltamivir dispensing without an influenza diagnosis on the same day. Outcomes included general skin reactions and several specific skin reactions. Events occurring during the 30 days following the date of influenza diagnosis or oseltamivir dispensing were examined using Cox proportional hazards models. Model covariates included age, use of another influenza drug, month and year of index date, and use of antitussives. Adjusted rate ratios for the general class of skin reactions among the primary cohort of oseltamivir users versus non-users were 1.05 (95% CI: 0.88-1.24) for incident cases and 0.98 (95% CI: 0.77-1.24) among patients with a history of a skin reaction. Similar results were seen for the other skin reaction categories, and secondary analyses investigating the oseltamivir users without influenza revealed no elevation in risk. It is concluded that oseltamivir use does not appear to be associated with an increased risk of skin reactions.



Treatment of mannan-enhanced influenza B virus infections in mice with oseltamivir, ribavirin and viramidine.

Mannan, a polysaccharide preparation from Saccharomyces cerevisiae, has previously been shown to enhance influenza virus replication in mice by inhibiting host defense collectins. The use of mannan in infections may serve to broaden the types of influenza viruses that can be studied in rodent infection models. When mannan was co-administered with influenza B/Sichuan/379/99 virus to mice, the animals died from the infection, whereas mice infected with only virus survived. Three types of influenza A (H1N1) and another influenza B (Hong Kong/330/01) virus infection were also enhanced by mannan, but not four types of influenza A (H3N2) viruses. Mannan was used at 0.16 or 0.5 mg/mouse for optimal disease-enhancing activity using influenza B/Sichuan/379/99 virus. Using this model, influenza B/Sichuan/379/99 infections were treated with oseltamivir, ribavirin or viramidine (the carboxamidine derivative of ribavirin). When oral gavage treatments started 4 h before virus and mannan challenge, oseltamivir was effective at 2.5, 5 and 10 mg/kg/day. Ribavirin was active at 20, 40 and 80 mg/kg/day. Viramidine was effective at 80 and 160 mg/kg/day but not at 40 mg/kg/day. Active drug doses improved lung consolidation scores and lung weights, with decreases in lung virus titres also noted. Arterial oxygen saturation values in treated groups were significantly better than those of the placebo group on days 7-11 of the infection. Oseltamivir (5 mg/kg/day) and ribavirin (40 mg/kg/day) were used alone and in combination to determine how late after infection they could be beneficially administered. Ribavirin alone was very effective (90-100% survival of mice) when treatments started as late as 3 days after infection. Forty percent survival was evident even when treatments started 4 days post-infection. Oseltamivir was active starting treatments 1 day after virus exposure, but lost considerable efficacy when treatments began after that time. The combination of ribavirin and oseltamivir appeared to be no better than ribavirin alone, due to the stronger beneficial effect of ribavirin in this model. The overall results demonstrate that mannan can be used to enhance certain non-lethal influenza virus infections sufficiently to allow antiviral studies.