Elsevier

Antiviral Research

Volume 94, Issue 1, April 2012, Pages 103-110
Antiviral Research

Combinations of favipiravir and peramivir for the treatment of pandemic influenza A/California/04/2009 (H1N1) virus infections in mice

https://doi.org/10.1016/j.antiviral.2012.03.001Get rights and content

Abstract

Favipiravir, an influenza virus RNA polymerase inhibitor, and peramivir, an influenza virus neuraminidase inhibitor, were evaluated alone and in combination against pandemic influenza A/California/04/2009 (H1N1) virus infections in mice. Infected mice were treated twice daily for 5 d starting 4 h after virus challenge. Favipiravir was 40%, 70%, and 100% protective at 20, 40, and 100 mg/kg/d. Peramivir was 30% protective at 0.5 mg/kg/d, but ineffective at lower doses when used as monotherapy. Combinations of favipiravir and peramivir increased the numbers of survivors by 10–50% when the 0.025, 0.05, and 0.1 mg/kg/d doses of peramivir were combined with 20 mg/kg/d favipiravir and when all doses of peramivir were combined with 40 mg/kg/d favipiravir. Three-dimensional analysis of drug interactions using the MacSynergy method indicates strong synergy for these drug combinations. In addition, an increase in lifespan for groups of mice treated with drug combinations, compared to the most effective monotherapy group, was observed for the 0.025, 0.05, and 0.1 mg/kg/d doses of peramivir combined with favipiravir at the 20 mg dose level. Therefore, the 20 mg/kg/d dose of favipiravir was selected for further combination studies. Increased survival was exhibited when this dose was combined with peramivir doses of 0.1, 0.25 and 0.5 mg/kg/d (1 mg/kg/d of peramivir alone was 100% protective in this experiment). Improved body weight relative to either compound alone was evident using 0.25, 0.5, and 1 mg/kg/d of peramivir. Significant reductions in lung hemorrhage score and lung weight were evident on day 6 post-infection. In addition, virus titers were reduced significantly on day 4 post-infection by combination therapy containing favipiravir combined with peramivir at 0.25 and 0.5 mg/kg/d. These data demonstrate that combinations of favipiravir and peramivir perform better than suboptimal doses of each compound alone for the treatment of influenza virus infections in mice.

Highlights

Favipiravir and peramivir were evaluated against influenza A virus in mice. ► Combinations of favipiravir and peramivir increased survivors by 10–50%. ► Three-dimensional analyses of drug interactions indicate strong synergy.

Introduction

In 2009 a new strain of influenza A (H1N1) emerged in Mexico that soon was transmitted throughout the world (Centers for Disease Control and Prevention, 2009b).

Although the case mortality rate proved to be less than originally feared, the pandemic caused significant morbidity and economic burden (Shrestha et al., 2011). The recent H1N1 pandemic highlights the need for effective antiviral therapy for largely immune-naïve populations. This crisis is over, the population is largely immune to the virus, and the 2009 pandemic virus has now become the new seasonal H1N1 virus undergoing antigenic drift. The world will await the emergence of the next pandemic virus. But the burden of seasonal influenza caused by influenza A (H1N1 and H3N2) and influenza B, will ever be with us (Xue et al., 2010). Additionally, the possibility of avian H5N1, H7N7, and H9N2 viruses adapting more efficiently for infection and spread in humans should not be overlooked (Yen and Webster, 2009).

The pandemic virus that emerged is resistant to the antiviral drugs amantadine and rimantadine (Mossad, 2009), as are the majority of H3N2 viruses (Deyde et al., 2007, Hata et al., 2007, Centers for Disease Control and Prevention, 2009a, Zaraket et al., 2010, Puzelli et al., 2011) and most highly pathogenic H5N1 avian viruses (Cheung et al., 2006). By the end of the 2009–2010 season, almost all (98.9%) of tested 2009 pandemic H1N1 viruses were susceptible to oseltamivir (Fiore et al., 2011). It is fortunate that the 2009 pandemic virus was sensitive to the drug oseltamivir (Hall et al., 2009, Rungrotmongkol et al., 2009), because many seasonal H1N1 viruses in circulation just prior to that time were resistant (Besselaar et al., 2008, Dharan et al., 2009, Meijer et al., 2009). One of the most effective means of curtailing the emergence of resistant viruses as well as improving treatment efficacy is to use compounds in combination (Ilyushina et al., 2006). Compounds with different modes of action, given in combination, have proven to be beneficial at improving the outcome of infection. Over the years a number of combination chemotherapy studies have been performed against influenza viruses in mouse models (Govorkova and Webster, 2010). Studies from our laboratory have supported these conclusions (Smee et al., 2002, Smee et al., 2006, Smee et al., 2009, Smee et al., 2010a, Smee et al., 2010b). Combination chemotherapy studies in mice are generally performed using suboptimal doses of the inhibitors so that improvements can be measured. However, in a clinical setting the drugs will be used at their approved doses.

Favipiravir (also referred to as T-705) was first reported in 2002 to be effective against influenza virus infections in cell culture and in mice (Furuta et al., 2002, Sidwell et al., 2007, Kiso et al., 2010). The compound inhibits influenza virus RNA polymerase (Furuta et al., 2005). Recently, favipiravir was shown to inhibit various isolates of the pandemic 2009 H1N1 virus, including some oseltamivir-resistant H275Y viruses, with 50% inhibitory concentrations generally ranging from 0.5 to 6 μM (Sleeman et al., 2010). In that study, there were some viral outliers, such as an A/Illinois/10/2009 isolate that was inhibited at 22.5 μM in a plaque reduction assay. However, the A/Illinois/10/2009 virus had a similar inhibition profile to other isolates when assayed by virus yield reduction. Favipiravir is currently in Phase 3 clinical trials in Japan, while Phase 2 trials in the US are underway. Among the viral neuraminidase inhibitors, peramivir is effective against various influenza virus strains in cell culture at 0.01–1 μM (influenza A viruses) and 0.1–2.3 μM (influenza B viruses) (Smee et al., 2001). It is also effective against influenza virus infections in mice (Sidwell et al., 2001, Bantia et al., 2001, Bantia et al., 2006), including the 2009 pandemic virus (Bantia et al., 2011). The poor oral bioavailability of peramivir in humans (Barroso et al., 2005) has hindered its clinical development, and recent studies have investigated its use by intramuscular administration (Bantia et al., 2006, Bantia et al., 2011, Boltz et al., 2008, Yun et al., 2008). In Japan, peramivir has been licensed under the trade name Rapiacta®, while in the US, peramivir is currently in Phase 3 clinical trials.

One combination chemotherapy study in mice has been published using favipiravir and oseltamivir to treat infections caused by seasonal H1N1 and H3N2 viruses, and a low-pathogenic avian influenza H5N1 virus (Smee et al., 2010a). In that report, synergistic improvements in survival were achieved using low doses of each inhibitor in combination. In addition, peramivir and ribavirin were combined for treatment of a seasonal H1N1 virus infection (Smee et al., 2002), as were combinations of oseltamivir and peramivir (Smee et al., 2010b). Improvements in survival and in body weight were evident at particular doses. The combination of peramivir and rimantadine was also shown to provide an improved treatment outcome against an amantadine-sensitive influenza A H3N2 virus infection in mice (Bantia et al., 2010).

The present studies investigated the use of favipiravir and peramivir in combination for treatment of pandemic H1N1 virus infections in mice. Survival, mean day of death, mean body weight, and lung parameters (lung score, lung weight, and virus titers) were evaluated following a lethal virus infection.

Section snippets

Compounds

Favipiravir and peramivir were provided by Toyama Chemical Co. (Tokyo, Japan) and BioCryst Pharmaceuticals (Birmingham, AL), respectively. Favipiravir was suspended in 0.4% carboxymethylcellulose, and peramivir was dissolved in sterile water. Favipiravir was administered in a 0.1 ml volume by oral gavage, whereas peramivir was injected (0.05 ml) intramuscularly. Routes of administration of each compound were selected based upon known bioavailabilities and preferred routes of administration (

Antiviral combination study to evaluate survival and antiviral drug synergy

Survival results following combination treatment using peramivir and favipiravir for a lethal infection with influenza H1N1 virus are reported in Table 1. The concentrations of peramivir used in this study appear to be suboptimal because even the highest dose, 0.5 mg/kg/d, produced only 30% protection following monotherapy. Monotherapy with favipiravir provided 40%, 70%, and 100% protection against lethal infection following administration of 20, 40, and 100 mg/kg/d, respectively. Nine groups

Discussion

These studies are the first, to our knowledge, to evaluate the effects of combination antiviral drug therapy using favipiravir and peramivir to treat an infection with pandemic H1N1 influenza virus. The animal studies presented here demonstrated that the combination of favipiravir and peramivir at suboptimal doses for each could provide significant improvements in survival and body weight. The estimated improvement in survival for the combination was about 10–50% compared to the use of

Acknowledgements

This work was supported by Contract N01-AI-30063 (awarded to Southern Research Institute, Birmingham, AL) 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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