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

Abstract

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. 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. 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. 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.