Evaluation of the efficacy and safety of a statin/caffeine combination against H5N1, H3N2 and H1N1 virus infection in BALB/c mice

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Abstract

The development of novel antiviral drugs is necessary for the prevention and treatment of a potential avian influenza pandemic. The aim of this study was to evaluate the efficacy and safety of a novel statin/caffeine combination against H5N1, H3N2 and H1N1 virus infection in a murine model. In H5N1-, H3N2- and H1N1-infected BALB/c mice, 50 μg statin/200 μg caffeine effectively ameliorated lung damage and inhibited viral replication and was at least as effective as oseltamivir and ribavirin. The statin/caffeine combination also appeared to be more effective when administered preventatively rather than as treatment. These findings provide justification for further research into this novel antiviral formulation.

Introduction

Humans have been afflicted by seasonal influenza epidemics for hundreds of years. Outbreaks in temperate countries occur annually and are often mild and transient; however, severe influenza outbreaks are capable of causing high morbidity and mortality among infants, the elderly, and immunocompromised patients (Lamb and Takeda, 2001).

In the last decade, the circulation of highly pathogenic avian influenza (H5N1) has been increasingly detected in poultry and wild birds (Alexander, 2007). Avian influenza H5N1 can cause severe, lethal human infections. As of April 2009, 421 human cases of avian influenza have been officially reported by the World Health Organization, of which 257 were fatal (WHO, 2009a, WHO, 2009b). There are fears that avian influenza H5N1 could cause an influenza pandemic in humans.

An outbreak of human cases of influenza A(H1N1) first appeared in south and central areas of Mexico in late April, 2009. As at 2 May, 16 countries have officially reported 658 cases of influenza A(H1N1) infection, and the situation continues to evolve (WHO, 2009a, WHO, 2009b).

Oseltamivir (Tamiflu®) and zanamivir (Relenza®) are recommended for both the treatment and prophylaxis of influenza, although whether either can effectively ameliorate avian influenza H5N1 human infections is uncertain, and clinical data are limited (Democratis et al., 2006, de Jong et al., 2005). Ribavirin is a broad-spectrum antiviral drug that effectively inhibits influenza A and B in vitro and in animal models (Sidwell et al., 1972, Khare et al., 1973), but clinical efficacy is equivocal (Rodriguez et al., 1994, Bernstein et al., 1988, Stein et al., 1987). Passive immunotherapy against H5N1 may be a viable therapeutic option; research with equine anti-H5N1 immunoglobulin G (IgG) is promising, but is currently still in an experimental stage (Lu et al., 2006). Consequently, it is necessary to develop other preventative and therapeutic antiviral drugs against avian influenza virus for use in the event of an avian influenza pandemic in humans.

Statins are a group of cholesterol-lowering agents and are one of the most widely prescribed drugs in the world. Statins inhibit the enzyme 3-hydroxy-3-methylglutarylcoenzyme-A (HMG CoA) reductase, which catalyses the rate-limiting step in cholesterol biosynthesis. The beneficial cardiovascular effects of statins are well documented (Armitage, 2007); however, recent reports suggest that statins (administered either therapeutically or prophylactically) might also have beneficial effects on influenza outcomes (Fedson, 2005, Fedson, 2006, Terblanche et al., 2006). Experimental and clinical evidence suggests that statins directly reduce inflammation, improve endothelial function, and stabilise atherosclerotic plaques independently of their lipid-lowering effects (Jain and Ridker, 2005). Statins can also interfere with cell structure and the environment necessary for viral replication (Sun and Whittaker, 2003, Rajendran and Simons, 2005).

Caffeine (1,3,7-trimethylxanthine), a member of the methylxanthine family of drugs, is widely consumed via coffee and tea, but is also used in a number of pain medications as an adjuvant analgesic (Sawynok and Yaksh, 1993, Forbes et al., 1990). Many studies have been conducted to investigate the physiological effects associated with caffeine and both positive and negative outcomes have been documented (Daly, 2007). Caffeine inhibits the action of adenosine, which interferes with cellular concentrations of cyclic adenosine monophosphate (cAMP) (Benowitz, 1990). cAMP is a potent immunomodulator, and a review of many in vitro and in vivo studies has suggested that caffeine exerts potential anti-inflammatory actions via the cAMP pathway and others (Horrigan et al., 2006) and may be beneficial for the heart (Bonita et al., 2007). Because caffeine can modulate various aspects of both innate and adaptive immunity by affecting cytokine production (Horrigan et al., 2006), it also has potential as an antiviral agent (Prahoveanu and Esanu, 1985).

Further to caffeine's use as an adjuvant treatment, the synergistic effect of caffeine when added to different compounds has also been explored both in animal models (Moo-Puc et al., 2003, López et al., 2006, Fernández-Dueñas et al., 2008) and clinical situations (Lev et al., 2007, Renner et al., 2007). For example, in a model of peripheral inflammation in rat, nociception was inhibited to a greater extent by a combination of acetylsalicylic acid (ASA) and caffeine than by ASA alone, while caffeine alone had no effect on nociception (Fernández-Dueñas et al., 2008). Therefore, in the search for a more effective antiviral drug, a novel statin (PTPM7) and caffeine (C53) formulation has been developed by Canopus BioPharma.

The research objective of this study was to compare the efficacy and safety of the statin/caffeine formulation with oseltamivir, ribavirin and equine anti-H5N1 IgG, against H5N1, H3N2, and H1N1 virus infection in a murine model.

Section snippets

Compounds

The statin (PTPM7, lovastatin) was provided by LGC Ltd., UK, and the caffeine (C53) was provided by Sigma–Aldrich, UK. Ribavirin was provided by Guangzhou Baiyunshan Pharmaceutical Co. Ltd. Oseltamivir (Tamiflu®) was provided by Roche Laboratories Inc. Horse anti-H5N1 immunoglobulin was provided by Sun Yat-sen University, PR China.

Viruses

Avian influenza A/Duck/Beijing/2000 (H5N1) virus, a highly pathogenic avian influenza (HPAI), was isolated and provided by the National Research Center for Wildlife

H5N1 symptoms

All mice were considered normal on days 1 and 2. Group L (virus group) demonstrated obvious onset of symptoms (sluggish behaviour, horripilation) at day 3. Horripilation was also observed in groups B, D, F, G, I and J from day 3 to day 8. Hair returned to normal in these groups from day 9 onwards. From day 11 to day 13, all groups showed signs of recovery apart from group L, which experienced continued symptoms of horripilation.

Deaths

Two mice in group E died on day 2, and one died on day 3. One mouse

Discussion

A novel combination of statin/caffeine has been shown to demonstrate preventative and therapeutic effects against H5N1, H3N2 and H1N1 influenza viruses in this study using experimentally infected mice. To our knowledge these are the first experiments to investigate such a combination formulation in a murine model of avian influenza, and there is sparse associated literature. The antiviral activity of caffeine alone has been published in two older small studies (Prahoveanu and Esanu, 1985,

Conclusions

This study suggests that a statin/caffeine compound may be a viable treatment or prophylactic against influenza virus infections, and certainly provides justification for further research. The statin/caffeine compound investigated here effectively ameliorated lung damage and inhibited viral replication in a murine model of H5N1, H3N2, and H1N1 influenza virus.

Acknowledgement

We thank Professor Liyan Liu of Henan Vocation-Technical Teachers College for her assistance with this study.

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