In vitro activity and preclinical profile of TMC435350, a potent hepatitis C virus protease inhibitor

Abstract

The hepatitis C virus (HCV) NS3/4A serine protease has been explored as a target for the inhibition of viral replication in preclinical models and in HCV-infected patients. TMC435350 is a highly specific and potent inhibitor of NS3/4A protease selected from a series of novel macrocyclic inhibitors. In biochemical assays using NS3/4A proteases of genotypes 1a and 1b, inhibition constants of 0.5 and 0.4 nM, respectively, were determined. TMC435350 inhibited HCV replication in a cellular assay (subgenomic 1b replicon) with a half-maximal effective concentration (EC(50)) of 8 nM and a selectivity index of 5,875. The compound was synergistic with alpha interferon and an NS5B inhibitor in the replicon model and additive with ribavirin. In rats, TMC435350 was extensively distributed to the liver and intestinal tract (tissue/plasma area under the concentration-time curve ratios of >35), and the absolute bioavailability was 44% after a single oral administration. Compound concentrations detected in both plasma and liver at 8 h postdosing were above the EC(99) value measured in the replicon. In conclusion, given the selective and potent in vitro anti-HCV activity, the potential for combination with other anti-HCV agents, and the favorable pharmacokinetic profile, TMC435350 has been selected for clinical development.

FIG. 1.

Structure of TMC435350.

FIG. 2.

Inhibition of HCV replication in genotype 1b replicon cells treated with TMC435350. (A) Huh7-Luc replicon cells were treated for 72 h with a dilution range of TMC435350 concentrations relative to control conditions (no compound). The dose-response curve of % inhibition of luciferase signal is shown; each point represents the mean and standard deviation of four independent experiments. The EC₅₀ is indicated. (B) Dose-dependent reduction of HCV protein expression in TMC435350-treated replicon cells using an immunoblot. Equal amounts of each cell lysate were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent immunoblot analysis with specific antibodies against NS5B and α-tubulin. Molecular masses are given in kDa.

FIG. 3.

Mutual influence between TMC435350 and IFN-α as analyzed by the Loewe additivity model. Huh7-Luc replicon cells were treated for 72 h with compound combinations. The combination index value for an effective dose of 50%, 75%, or 90% inhibition (ED₅₀, ED₇₅, and ED₉₀, respectively) was calculated. The graph is a representative example of the data in Table 2.

FIG. 4.

Nine-day incubation of genotype 1b replicon cells with TMC435350 alone, IFN-α alone, and TMC435350 in combination with IFN-α. Huh7-Luc replicon cells were harvested after 3, 6, or 9 days of treatment, and RNA levels were assessed by qRT-PCR. The normalized (host RPL13A) HCV RNA levels of treated replicon cells relative to untreated cells are plotted on a log₁₀ scale. □, 0.5 U/ml IFN-α; ▵, 5 nM TMC435350; ▴, 5 nM TMC435350 plus 0.5 U/ml IFN-α; ○, 50 nM TMC435350; •, 50 nM TMC435350 plus 0.5 U/ml IFN-α. Each point represents the mean value of three independent experiments.

FIG. 5.

Tissue distribution of TMC435350 after a single oral administration of 40 mg/kg in Sprague-Dawley rats. AUC_{0-31 h} (except for muscle, AUC_{0-8 h}) tissue/plasma ratios of TMC435350 are shown. Each bar represents the mean value for three rats.

FIG. 6.

Time-dependent exposure after single oral administration of 40 mg/kg TMC435350 in plasma and liver tissue of Sprague-Dawley rats. Plasma (⧫) and liver (▴) tissue concentrations of TMC435350 are plotted over time, and EC₅₀, EC₉₀, and EC₉₉ values as determined for TMC435350 in Huh7-Luc replicon cells are indicated. Each point represents the mean value for three rats.