Combinations of oseltamivir and peramivir for the treatment of influenza A (H1N1) virus infections in cell culture and in mice
Introduction
Combinations of antiviral agents are being explored for the treatment of influenza virus infections as a means to improve efficacy and to help suppress the emergence of drug-resistant viruses. The recent H1N1 pandemic starting in the spring of 2009 (Centers for Disease Control and Prevention, 2009) highlights the need for effective antiviral therapy in a largely naïve population. Three classes of anti-influenza virus compounds with different modes of action have been identified. They are viral neuraminidase inhibitors (i.e. oseltamivir carboxylate, zanamivir and peramivir), viral M2 channel blockers (i.e. amantadine and rimantadine), and viral RNA polymerase inhibitors (i.e. ribavirin, viramidine and T-705 [favipiravir]). Amantadine and rimantadine have been rendered largely ineffective due to the high prevalence of resistant viruses in nature (Cheung et al., 2006, Ilyushina et al., 2006, Deyde et al., 2007, Hata et al., 2007, Mossad, 2009). After many years of only low prevalence of oseltamivir resistance, a transmissible seasonal H1N1 virus variant carrying the H275Y resistance mutation emerged in 2007 in Europe and spread worldwide until early 2009 (Besselaar et al., 2008, Dharan et al., 2009, Meijer et al., 2009). This seasonal H1N1 virus disappeared during 2009, concomitantly with the emergence of the 2009 pandemic H1N1 virus, sensitive to oseltamivir (Wang et al., 2010). Other drug-resistant virus variants may continue to emerge in the future, requiring new treatment strategies that may include combination treatments. Studies have been reported using combinations of compounds from these different classes either as double drug (Galabov et al., 2006, Ilyushina et al., 2007, Ilyushina et al., 2008, Smee et al., 2002, Smee et al., 2009) or, more recently, triple drug (Nguyen et al., 2009, Nguyen et al., 2010) combinations. Treatment of influenza virus infections with these combinations has generally resulted in additive to synergistic interactions.
Because oseltamivir carboxylate and peramivir are both viral neuraminidase inhibitors, the use of these two agents together would not be anticipated to produce synergistic interactions. Additivity would be more likely. Peramivir has a tighter binding affinity to the neuraminidase than oseltamivir carboxylate (Bantia et al., 2006). Oseltamivir and peramivir both bind to the neuraminidase active site. Contributions of active site residues to binding affinity for both compounds differ slightly, which results in a slightly different resistance profile. Compound specific resistance mutations have been observed, e.g. E119 V in N2 neuraminidase conferred resistance to oseltamivir, but not peramivir, whereas E119D conferred higher resistance to peramivir (Mishin et al., 2005, Hurt et al., 2006). Other viral isolates resistant to both inhibitors have been identified (Gubareva et al., 2001, Memoli et al., 2010, Okomo-Adhiambo et al., 2010). Most importantly, H275Y mutations in N1 neuraminidase confer resistance to both oseltamivir and peramivir.
A clear advantage to the use of oseltamivir to treat patients is its oral bioavailability (Li et al., 1998). Peramivir is orally active in mice (Bantia et al., 2001, Sidwell et al., 2001), but proved to be very poorly absorbable by this route in humans (Barroso et al., 2005, Bantia et al., 2006). For this reason, recent studies have focused on treatment with peramivir by intramuscular injection (Bantia et al., 2006, Boltz et al., 2008, Yun et al., 2008). Intravenous studies with peramivir in hospitalized patients are underway.
The purpose of the present investigation was to explore whether the use of oseltamivir combined with peramivir would prove to be adverse or beneficial in treating influenza virus infections. Studies were conducted in vitro and in mice infected with an oseltamivir-sensitive influenza A (H1N1) virus.
Section snippets
Compounds
Oseltamivir carboxylate was kindly provided by Roche, Basel, Switzerland. Oseltamivir phosphate (as Tamiflu® capsules, here referred to merely as oseltamivir), the orally active prodrug form of oseltamivir carboxylate, was purchased from a local pharmacy. Peramivir was provided by Biocryst Pharmaceuticals (Birmingham, AL). Oseltamivir carboxylate and peramivir were dissolved in cell culture medium for in vitro studies. Oseltamivir was used for animal studies. Because oseltamivir was obtained
Antiviral activity in cell culture
Oseltamivir carboxylate and peramivir were evaluated in combination for inhibition of virus yield in MDCK cell cultures using doses of 0.32–100 μM (Table 1). Oseltamivir carboxylate alone reduced virus yield by 4.4 log10 at 100 μM. Peramivir at 32 and 100 μM reduced virus yield by ≥5 log10 below the detection limit of the assay. Greater than 10-fold inhibition of virus titer from expected was found at three specific conditions, when 10 μM oseltamivir carboxylate was combined with either 3.2 or 10 μM
Discussion
The primary assumption that was made when beginning this research was that the combination of these two neuraminidase inhibitors, oseltamivir and peramivir, would lead to additive effects. This was based upon combining two compounds with the same mode of action. Synergy is generally expected for compounds having different modes of antiviral action. Because of the tighter binding of peramivir to the neuraminidase (Bantia et al., 2006), antagonistic interactions were also possible at specific
Acknowledgements
This work was supported by contracts N01-AI-30048 and N01-AI-30063 (awarded to Southern Research Institute) from the Virology Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, USA. The contents of this article do not necessarily reflect the position or policy of the government and no official endorsement should be inferred. The animal experiments were conducted in accordance with the approval of the Institutional Animal Care and Use Committee of Utah
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