Implications of finding synergic in vitro drug–drug interactions between interferon-{alpha} and ribavirin for the treatment of hepatitis C virus

Victor E. Buckwold*

Infectious Disease Research Department, Southern Research Institute, Frederick, MD 21701, USA


    Abstract
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Hepatitis C virus (HCV) infections are currently treated using a combination of interferon-{alpha} (IFN-{alpha}) and ribavirin (RBV). If IFN-{alpha} is utilized alone, the sustained virological response (SVR) rate is ~20%, whereas when RBV is used alone it does not lead to an SVR. However, when IFN-{alpha} and RBV are used together, the combination leads to an SVR rate of ~40%. This clinical synergy is thought to be due to the direct antiviral effects of RBV, or to indirect effects of RBV that stimulate the immune response. Evidence for either hypothesis is limited. Recently, we undertook an in vitro drug–drug combination analysis using surrogate model systems of HCV replication and found a reproducible synergy of antiviral effects between the two drugs at physiologically relevant drug concentrations. Our findings provide experimental support for the contention that the direct effects of these drugs’ antiviral activity are responsible for the clinical synergy observed in patients.

Keywords: interferon-{alpha}, combination analysis, HCV, ribavirin

Hepatitis C virus (HCV) is estimated to infect over 170 million people worldwide, and infection with this agent is associated with an increased risk of the development of liver cirrhosis and hepatocellular carcinoma. The current standard of care for the treatment of HCV infections is a combination therapy of both interferon-{alpha} (IFN-{alpha}) and the purine nucleoside analogue ribavirin (RBV). IFN-{alpha} is an FDA-licensed antiviral agent with activity against a wide variety of RNA and DNA viruses, including hepatitis B virus (HBV) and HCV. If IFN-{alpha} is utilized alone as a monotherapy in chronically infected HCV patients, the success rate is ~20%.1 Treatment success with HCV infection is generally defined as the rate of sustained virological response (SVR), which indicates that HCV RNA remains undetectable in patients 6 months (24 weeks) following the cessation of therapy. Somewhat higher rates of SVR are now being achieved using pegylated IFN-{alpha}, which has a much improved half-life over that of standard IFN-{alpha}.2 The mechanism of action of IFN-{alpha} is unclear, but both direct and immune-mediated mechanisms seem to be involved.3,4

RBV is another FDA-licensed antiviral agent that is active against a wide variety of RNA and DNA viruses. However, when RBV was tested alone as a possible antiviral therapy against HCV, it did not reduce viral loads or lead to an SVR (the SVR rate for RBV is 0%).57 Some of these early reports likened the reduction in some patient liver enzyme alanine aminotransferase (ALT) levels to an antiviral effect of the drug. This seemed like a reasonable interpretation at the time, since ALT level stabilization is a good indicator of effective antiviral therapy in the case of HBV infection. Unfortunately, a large amount of clinical experience gained since that time indicates clearly that ALT levels fluctuate over time in HCV-infected patients, and this marker does not provide a good indication of drug efficacy. The mechanism of action of RBV is controversial, and both direct and indirect mechanisms have been proposed.8,9

When IFN-{alpha} and RBV are used together, the combination therapy is much more clinically effective than either drug alone, leading to rates of SVR of ~40%.1 Again, the recent introduction of pegylated IFNs has resulted in an increase in the drug combination’s effectiveness.2 However, if RBV is not active against HCV in vivo, how does the use of RBV with IFN-{alpha} lead to the clinical synergy? Two explanations, that are not mutually exclusive, are possible.

(i) Indirect effects: RBV stimulates the immune response, leading to an increased effectiveness of IFN-{alpha} as an antiviral agent.

(ii) Direct effects: the direct antiviral activity of RBV is accentuated by IFN-{alpha}, leading to increased effectiveness of RBV as an antiviral agent.

The Flaviviridae family of viruses contains three separate genera: (i) hepaciviruses, comprised entirely of the various HCV genotypes; (ii) pestiviruses, which have bovine viral diarrhoea virus (BVDV) as the type member; and (iii) flaviviruses, with yellow fever virus (YFV) as the type member.

Cultured cells cannot be infected reproducibly with HCV. HCV RNA replicons are robust experimental systems10 that have shown utility in the evaluation of antiviral agents; however, since replicons do not faithfully reproduce all steps of the HCV replication cycle, surrogate viruses are often utilized for the identification and characterization of anti-HCV antiviral agents. BVDV is most often utilized for this purpose.11 YFV is seldom employed individually as a surrogate model of HCV, since it is further removed phylogenetically from HCV than is BVDV;11 however, it is useful in studies of broad-acting antiviral compounds such as IFN-{alpha} and RBV, which have antiviral activity against both viruses, presumably through the same or a similar mechanism of action.

Recently, we undertook an in vitro drug–drug combination analysis in order to determine whether the direct antiviral activity of RBV might account for the effectiveness of the IFN-{alpha} and RBV combination. In our study,12 we found that both IFN-{alpha} and RBV had antiviral activity against the BVDV and YFV surrogate models of HCV. We tested the antiviral effects of a wide range of concentrations of the two drugs, both with each drug used alone and with the two drugs mixed together. We then used the MacSynergy II program13 to calculate the theoretical additive effects of combining the drugs based on the dose–response curves of the individual drugs. The calculated additive surface, representing the predicted additive interaction, was then subtracted from the observed data to reveal regions of statistically significant greater than expected (synergy) or less than expected (antagonism) drug–drug interactions. These data were used to create difference plots to indicate and statistically evaluate any potential effects on drug antiviral activity or cytotoxicity that were greater than (synergy) or less than (antagonism) those predicted, by simple additive drug effects as a quantitative measure of these drug–drug interactions.

When the drug–drug combination analysis was performed using BVDV, we found a reproducible synergy of antiviral effects between the two drugs at physiologically relevant drug concentrations. A clear antagonism of antiviral effects between the drugs was also observed, but only when very high concentrations of RBV, that are not reached in vivo in HCV-infected patients,1417 were employed. The combination of IFN-{alpha} and RBV did not have any statistically significant effects on cytotoxicity that were not expected from simple additive drug effects.

A nearly identical result was observed when the drug–drug combination analysis was performed using YFV. A synergy of antiviral activity between the drugs was apparent, as well as strong antagonism of antiviral effects when very high (physiologically irrelevant) concentrations of both drugs were used. As with BVDV, there was no significant synergy of drug cytotoxic effects. We did observe a reproducible antagonism of the cytotoxic effects of RBV against Vero cells by IFN-{alpha}, but this effect was only apparent at very high concentrations of RBV that are probably not relevant in vivo.1417

In summary, we found significant synergic drug–drug interactions between IFN-{alpha} and RBV in terms of antiviral effects against both BVDV and YFV at physiologically relevant drug concentrations. While our findings do not discount the possibility that indirect, immune system-mediated phenomena contribute to the clinical synergy of IFN-{alpha} and RBV in HCV-infected patients, they do provide direct experimental support for the contention that the direct effects of these drugs’ antiviral activity may be responsible for this drug synergy. This provides an explanation for the known clinical synergy observed when IFN-{alpha} and RBV are used in patients. It is not yet clear whether IFN-{alpha} stimulation of the cells renders them susceptible to the antiviral effects of RBV or vice versa. The exact mechanism by which this occurs is not known. Nonetheless, our observations provide some insight into the manner by which IFN-{alpha} and RBV work well together clinically. Further experimental work utilizing these experimental systems may elucidate the mechanism(s) by which these favourable drug–drug interactions occur.


    Acknowledgements
 
This project was funded in part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services (contract N01-AI-30047, grant AI-53574).


    Footnotes
 
* Tel: +1-301-694-3232; Fax: +1-301-694-7223; E-mail: Buckwold{at}sri.org Back


    References
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