Establishment of a robust hepatitis C virus replicon cell line over-expressing P-glycoprotein that facilitates analysis of P-gp drug transporter effects on inhibitor antiviral activity

Abstract

P-glycoprotein (P-gp) is an active efflux pump affecting the pharmacokinetic (PK) profiles of drugs that are P-gp substrates. The Caco-2 bi-directional assay is widely used to identify drug-P-gp interactions in vitro. For molecules exhibiting non-classical drug properties however, ambiguous results limit its use in lead optimization. The goal of this study was to develop a robust cell-based assay system to directly measure the role of P-gp-driven efflux in reducing the potency of hepatitis C virus (HCV) replication inhibitors. Vinblastine (Vin) was employed to select for a Vin-resistant HCV replicon (313-11) from the parental cell line (377-2). The 313-11 cell line was >50-fold resistant to Vin and over-expressed P-gp, as determined by Western immunoblots. Increased expression of P-gp was mediated by up-regulation of the MDR1 transcript. The reduced potency of different classes of HCV replication inhibitors in the 313-11 P-gp cell line was restored in the presence of known P-gp inhibitors. Addition of the P-gp inhibitor, tariquidar, increased the uptake of a radiolabeled HCV replication inhibitor by 14-fold in the 313-11 replicon cell line. Finally, a positive correlation was demonstrated between potency in the 313-11 replicon and the bi-directional Caco-2 efflux ratio for a panel of HCV protease inhibitors. In conclusion, a robust P-gp HCV replicon cell-based assay has been developed to measure the effect of the P-gp efflux pump on the potency of different classes of HCV replication inhibitors. This system establishes a direct correlation between antiviral activity and the effect of P-gp efflux in a single cell line.