Simeprevir (TMC435) is a potent HCV NS3/4A protease inhibitor, and inhibits HCV replication with EC50 of 8 nM.

HCV:8 nM (EC50)

在Huh7-Luc细胞中，Simeprevir的抗病毒活性与剂量呈依赖关系，其EC50和EC90值分别为8 nM和24 nM。Simeprevir对NS3/4A蛋白酶的抑制作用随时间依赖性增强，针对基因型1a和1b的总Kis估计值分别为0.5 nM和0.4 nM。Simeprevir是一种强效的HCV NS3/4A蛋白酶抑制剂（Ki=0.36 nM）以及病毒复制的抑制剂（复制子EC50=7.8 nM）。

在大鼠中，TMC435350（40 mg/kg，p.o.）广泛分布于肝脏和肠道（组织/血浆浓度-时间曲线下面积比值>35），且绝对生物利用度为44%。

In vitro inhibition of NS3/4A activity is determined using a fluorescence resonance energy transfer cleavage assay with the RetS1 peptide substrate, derived from the genotype 1a NS4A-4B junction, and bacterially expressed full-length NS3 protease domain, supplemented with an NS4A peptide. Briefly, NS3/4A is preincubated in the presence of TMC435350 for 10 min, and then the RetS1 substrate is added and fluorescence is continuously measured for 20 min (excitation, 355 nm; emission, 500 nm). Cleavage of the substrate is expressed as a percentage of the cleavage seen with the vehicle control.

Huh7-Luc cells are seeded at a density of 2,500 cells/well in a 384-well plate in Dulbecco's modified Eagle's medium plus 10% fetal calf serum and incubated with a range of concentrations of serially diluted simeprevir, in a final DMSO concentration of 0.5% in the absence of G418. After 72 h of incubation, Steady Lite reagent is added in a 1:1 ratio to the medium, and luciferase signal is measured using a ViewLux reader.

Twenty-four male specific-pathogen-free Sprague-Dawley rats, weighing between 200 and 300 g at the time of dosing, are divided into eight groups of three rats each. Seven groups are dosed orally (p.o.) by gastric intubation of a vitamin E acetate-d-α-tocopheryl polyethylene glycol 1000 succinate-polyethylene glycol 400 solution of Simeprevir (TMC435350) at 2 mL/kg body weight to provide a dose of 40 mg/kg. One group is dosed intravenously (i.v.) by slow bolus injection in a tail vein of a 20% 2-hydroxypropyl-β-cyclodextrin formulation of TMC435350 (containing TMC435350, 100 mg/mL 2-hydroxypropyl-β-cyclodextrin, 0.1 N NaOH to pH 8.0±0.1, and mannitol-and pyrogen-free water) at 2 mL/kg body weight to provide a dose of 4 mg/kg. Water and food are available ad libitum during the study.