A novel HCV NS3 protease mutation selected by combination treatment of the protease inhibitor boceprevir and NS5B polymerase inhibitors

Boceprevir (SCH 503034) is an orally active novel inhibitor of the hepatitis C virus (HCV) NS3 protease currently in clinical development for the treatment of hepatitis C. In this in vitro study, we demonstrate that combination of boceprevir with a nucleoside analog or a non-nucleoside HCV NS5B polymerase inhibitor was superior to treatment by single agents in inhibiting viral RNA replication in replicon cells. In the presence of boceprevir (at 5×EC90), the addition of 2′-C-methyl-adenosine or an indole-N-acetamide targeting the polymerase finger-loop site (at 1×EC90) significantly reduced the emergence of resistant replicon colonies. A higher dose (5×EC90) of either of the polymerase inhibitors in combination with boceprevir suppressed replicon resistance further to below detectable levels. Sequencing analysis of replicon cells selected by the combination treatment revealed known resistance mutations to the two polymerase inhibitors but no previously reported resistance mutations to boceprevir. Interestingly, a novel mutation (M175L) in the protease domain was identified. The dually resistant replicon cells were monitored for over 30 passages and sensitivity to polymerase inhibitors was found to decrease over time in a manner that correlated with the increasing prevalence of specific resistance mutations. Importantly, these cells remained sensitive to interferon-α and different classes of polymerase inhibitors. These findings support the rationale for clinical evaluation of combination treatment of HCV protease and polymerase inhibitors.