Preclinical Profile and Characterization of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir (BMS-650032)

Abstract

Asunaprevir (ASV; BMS-650032) is a hepatitis C virus (HCV) NS3 protease inhibitor that has demonstrated efficacy in patients chronically infected with HCV genotype 1 when combined with alfa interferon and/or the NS5A replication complex inhibitor daclatasvir. ASV competitively binds to the NS3/4A protease complex, with K(i) values of 0.4 and 0.24 nM against recombinant enzymes representing genotypes 1a (H77) and 1b (J4L6S), respectively. Selectivity was demonstrated by the absence of any significant activity against the closely related GB virus-B NS3 protease and a panel of human serine or cysteine proteases. In cell culture, ASV inhibited replication of HCV replicons representing genotypes 1 and 4, with 50% effective concentrations (EC(50)s) ranging from 1 to 4 nM, and had weaker activity against genotypes 2 and 3 (EC(50), 67 to 1,162 nM). Selectivity was again demonstrated by the absence of activity (EC(50), >12 μM) against a panel of other RNA viruses. ASV exhibited additive or synergistic activity in combination studies with alfa interferon, ribavirin, and/or inhibitors specifically targeting NS5A or NS5B. Plasma and tissue exposures in vivo in several animal species indicated that ASV displayed a hepatotropic disposition (liver-to-plasma ratios ranging from 40- to 359-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥110-fold above the inhibitor EC(50)s observed with HCV genotype-1 replicons. Based on these virologic and exposure properties, ASV holds promise for future utility in a combination with other anti-HCV agents in the treatment of HCV-infected patients.

Fig 1

Chemical structure of ASV (BMS-650032): N-(tert-butoxycarbonyl)-3-methyl-l-valyl-(4R)-N-((1R,2S)-1{[(cyclopropylsulfonyl)amino]carbonyl}-2-vinylcyclopropyl) −4-[(4-methoxy-7-chloroisoquinolin-1-yl)oxy]-l-prolinamide (45). P and P′ labeling indicates the ASV moieties that correspond to the natural HCV substrate positions occupying the NS3 protease active site.

Fig 2

Lineweaver-Burk plot and K_i determination for asunaprevir (ASV). (A) Lineweaver-Burk plot of 1/V (reciprocal of rate of change of fluorescence) versus 1/S (reciprocal of the substrate concentration). (B) K_i determination plot of the apparent K_m value (slope obtained from the Lineweaver-Burk plot) versus ASV concentration.