Combinations of 2'-<i>C</i>-Methylcytidine Analogues with Interferon-α2b and Triple Combination with Ribavirin in the Hepatitis C Virus Replicon System

Abstract

BACKGROUND: Hepatitis C virus (HCV) polymerase is an essential enzyme for HCV replication and has multiple inhibitor binding sites making it a major target for antiviral intervention. It is apparent that no single drug can inhibit HCV replication in humans. Hence, combinations of nucleoside analogues beta-D-2'-C-methylcytidine (2'-C-MeC; NM-107) or beta-D-2'-deoxy-2'-fluoro-2'-C-methyleytidine (2'-F-C-MeC; PSI-6130) with interferon-alpha2b (IFN-alpha2b) or triple combination with ribavirin (RBV) were evaluated. METHODS: Huh-7 cells containing the self-replicating subgenomic HCV replicon (Clone B) were used for drug combination studies. After drug treatment for 5 days, total cellular RNA was then extracted and both ribosomal RNA and HCV replicon RNA were amplified in a single-step multiplex real-time PCR assay. Drug interaction analyses were performed using the CalcuSyn program. RESULTS: Double combinations of 2'-C-MeC or 2'-F-C-MeC with IFN-alpha2b at all ratios tested had weighted average combination index (Cl(wt)) values <1 indicating synergistic inhibition of HCV replication in the replicon system. For the triple combinations of IFN-alpha2b plus RBV with either 2'-C-MeC or 2'-F-C-MeC, the Cl(wt) values at 1:1:1 ratio tested were 0.5 and 0.8, respectively, indicating synergistic antiviral effects. No apparent cytotoxicity effects were observed with any of the combinations tested. CONCLUSION: These promising in vitro data warrant clinical investigation of the nucleosides analogues such as 2'-C-MeC or 2'-F-C-MeC in their prodrug forms, together with IFN-alphac2b and RBV, for successful treatment of HCV infections.