INX-08189, a phosphoramidate prodrug of 6-O-methyl-2'-C-methyl guanosine, is a potent inhibitor of hepatitis C virus replication with excellent pharmacokinetic and pharmacodynamic properties

Abstract

INX-08189 is an aryl-phosphoramidate of 6-O-methyl-2'-C-methyl guanosine. INX-08189 was highly potent in replicon assays, with a 50% effective concentration of 10±6 nM against hepatitis C genotype 1b at 72 h. The inhibitory effect on viral replication was rapid, with a 50% effective concentration (EC50) of 35±8 nM at 24 h. An intracellular 2'-C-methyl guanosine triphosphate (2'-C-MeGTP) concentration of 2.43±0.42 pmol/10(6) cells was sufficient to achieve 90% inhibition of viral replication. In vitro resistance studies confirmed that the S282T mutation in the NS5b gene conferred an approximately 10-fold reduction in sensitivity to INX-08189. However, the complete inhibition of S282T mutant replicons still could be achieved with an EC90 of 344±170 nM. Drug combination studies of INX-08189 and ribavirin indicated significant synergy in antiviral potency both in wild-type and S282T-expressing replicons. Genotype 1b replicons could be cleared after 14 days of culture when exposed to as little as 20 nM INX-08189. No evidence of mitochondrial toxicity was observed after 14 days of INX-08189 exposure in both HepG2 and CEM human cell lines. In vivo studies of rats and cynomolgus monkeys demonstrated that 2'-C-MeGTP concentrations in liver equivalent to the EC90 could be attained after a single oral dose of INX-08189. Rat liver 2'-C-MeGTP concentrations were proportional to dose, sustained for greater than 24 h, and correlated with plasma concentrations of the nucleoside metabolite 2'-C-methyl guanosine. The characteristics displayed by INX-08189 support its continued development as a clinical candidate for the treatment of chronic HCV infection.

Fig. 1.

Structure of INX-08189: (2S)-neopentyl 2-(2R,3R,4R)-5-(2-amino-6-methoxy-9H-purin-9-yl)-3,4-dihydroxy-4-methyltetrahydrofuran-2-yl)(methoxy)(naphthalen-1-yloxy)(phosphorylamino) propanoate.

Fig. 2.

Determination of 2′-C-MeGTP intracellular half-life in genotype 1b replicon. Genotype 1b replicon cells were incubated with 1 μM INX-08189 for 8 h, at which point the drug was removed. Intracellular 2′-C-MeGTP concentrations were measured by LC-MS/MS. Symbols are means ± SD for triplicate determinations in a single experiment.

Fig. 3.

Inhibition of HCV replication. Representative inhibition curves are plotted for replicons expressing wild-type (•), S282T (Δ), I585T (■), A540T (▾), S282T/I585T (♢), and S96T/N142T () NS5B sequences. Concentrations of INX-08189 are indicated on the x axis, and the measured luminescent signals are expressed as a percentage of signal obtained in no-treatment controls on the y axis. Symbols are means ± SD for triplicate determinations in a single experiment.

Fig. 4.

Clearance of wild-type replicons in vitro with INX-08189. (A) Huh-7 genotype 1b replicons cells were cultured in the presence of INX-08189 without G418 for 14 days at concentrations of 5 (•), 10 (♢), 20 (■), 40 (○), and 80 nM (). Luciferase expression was monitored in the cultures at the indicated time points and expressed as log₁₀ change in luminescence. (B) Cells were subcultured under G418 selection for up to 5 weeks, and surviving colonies were fixed and stained with crystal violet.

Fig. 5.

Concentrations of 2′-C-methyl GTP in rat liver following oral dosing with INX-08189. Each line represents a separate dose level: 25 mg/kg (), 10 mg/kg (♢), 5 mg/kg (■), and 3 mg/kg (○). Symbols represent averages from two rats with the exception of the 3-mg/kg dose group, which included data from six rats. The EC₉₀ determined for Huh-7 cells (243 pmol/g) is indicated by the dotted line.

Fig. 6.

Concentrations of INX-08189 and 2′-C-methyl guanosine in plasma after the oral dosing of INX-08189 in cynomolgus monkeys. Cynomolgus monkeys surgically fitted with portal vein cannulas were dosed orally with INX-08189 at 25 mg/kg. The analytes measured were 2′-C-methyl guanosine concentration in systemic plasma (•), INX-08189 in portal plasma (♢), and INX-08189 in systemic plasma (■).