In vitro activity: Asunaprevir (ASV) inhibits the NS3 proteolytic activity of genotype 1a (H77 strain) and genotype 1b (J4L6S strain), with IC50s of 0.7 and 0.3 nM, respectively. The EC50s of ASV against replicons encoding the NS3 protease domains representing genotypes 1a, 1b, and 4a, range from 1.2 to 4.0 nM. Replicon cells are maintained under selective pressure with asunaprevir at concentrations of 10 and 30 times the EC50 values (50 or 150 nM final concentrations, respectively). For genotype 1b resistance selection, replicon cells are maintained in the presence of asunaprevir at 10 or 30 times the EC50 values (30 or 90 nM final concentrations, respectively). Asunaprevir, administered at single or multiple doses of 200 to 600 mg twice daily, is generally well tolerated, achieving rapid and substantial decreases in HCV RNA levels in subjects chronically infected with genotype 1 HCV.

Kinase Assay: Asunaprevir (ASV) inhibits the NS3 proteolytic activity of genotype 1a (H77 strain) and genotype 1b (J4L6S strain), with IC50s of 0.7 and 0.3 nM, respectively. The EC50s of ASV against replicons encoding the NS3 protease domains representing genotypes 1a, 1b, and 4a, range from 1.2 to 4.0 nM.

Cell Assay: Cytotoxicity is determined by incubating cells (3,000 to 10,000 cells/well) with serially diluted test compounds or DMSO for 5 days (MT-2 cells) or 4 days (all other cell types). Cell viability is quantitated using an MTS assay for MT-2 or a Cell-Titer Blue reagent assay for HEK-293, HuH-7, HepG2, and MRC5 cells, and 50% cytotoxic concentrations (CC50s) are calculated. For the HCV and BVDV replicon assays, CC50s are determined from the same wells that are later used to determine EC50s.