Metabolism and pharmacokinetics of the anti-hepatitis C virus nucleotide prodrug GS-6620

Abstract

The anti-hepatitis C virus nucleotide prodrug GS-6620 employs a double-prodrug approach, with l-alanine-isopropyl ester and phenol moieties attached to the 5'-phosphate that release the nucleoside monophosphate in hepatocytes and a 3'-isobutyryl ester added to improve permeability and oral bioavailability. Consistent with the stability found in intestinal homogenates, following oral administration, intact prodrug levels in blood plasma were the highest in dogs, followed by monkeys, and then were the lowest in hamsters. In contrast, liver levels of the triphosphate metabolite at the equivalent surface area-adjusted doses were highest in hamsters, followed by in dogs and monkeys. Studies in isolated primary hepatocytes suggest that relatively poor oral absorption in hamsters and monkeys was compensated for by relatively efficient hepatocyte activation. As intestinal absorption was found to be critical to the effectiveness of GS-6620 in nonclinical species, stomach pH, formulation, and food effect studies were completed in dogs. Consistent with in vitro absorption studies in Caco-2 cells, the absorption of GS-6620 was found to be complex and highly dependent on concentration. Higher rates of metabolism were observed at lower concentrations that were unable to saturate intestinal efflux transporters. In first-in-human clinical trials, the oral administration of GS-6620 resulted in poor plasma exposure relative to that observed in dogs and in large pharmacokinetic and pharmacodynamic variabilities. While a double-prodrug approach, including a 3'-isobutyryl ester, provided higher intrinsic intestinal permeability, this substitution appeared to be a metabolic liability, resulting in extensive intestinal metabolism and relatively poor oral absorption in humans.

FIG 1

Intracellular activation of GS-6620 in primary hepatocytes. (A) Intracellular metabolites, metabolite X (GS-566650) (○), GS-558272 (♢), GS-639477 (□), and GS-441326 (△) formed by GS-6620 in primary human hepatocytes. (B) Intracellular formation of the active triphosphates in primary hepatocytes from hamsters (▲), dogs (◆), monkeys (●), and humans (■). The cells were incubated with 10 μM GS-6620 for 2 h before the compound was removed by replacing the medium. The graphs were generated by averaging each time point from the number of experiments shown in Table 1. The C_max and t_1/2 in panel B may not match exactly with the numbers in Table 1, which are averages of the individually determined values.

FIG 2

Proposed metabolic activation pathway of GS-6620. The enzymes involved in the pathway are based on the results shown in Table 2. The primary intracellular activation pathway was proposed to proceed from GS-6620 to GS-465124, to metabolite X, and to GS-558272. The formation of metabolite B was not observed in our biochemical assays.

FIG 3

Effect of overexpression of enzymes involved in metabolism of GS-6620. CES1-, CES2-, or CatA-overexpressing cells were incubated with 10 μM [³H] GS-6620 for 45 and 90 min, and GS-6620 metabolites in medium and cells were analyzed by HPLC-LS. The efficiency of protein overexpression was monitored by quantitative Western blots.

FIG 4

Concentration and efflux transport inhibitor-dependent permeability of GS-6620 through monolayers of Caco-2 cells. (A) Concentration-dependent permeability was determined using 1, 10, and 100 μM GS-6620. (B) Effects of efflux inhibitors, cobicistat (30 μM), or cyclosporine (CsA) (10 μM) on permeability were assessed using 10 μM GS-6620. The white and black bars indicate forward (apical to basolateral) and reverse (basolateral to apical) permeability, respectively. The numbers above the bars indicate the efflux ratios.

FIG 5

Plasma and liver PK profiles of GS-6620. (A) Plasma PK following a single oral dose of GS-6620 to hamsters, dogs, and monkeys at 10.6, 2.5, and 3.86 mg/kg, respectively. (B) Effects of formulation, stomach pH, or feeding condition on GS-6620 plasma PK following a 100-mg oral dose of GS-6620 to dogs. (C) Liver PK of GS-441326 following a single oral dose of GS-6620 in a solution form to hamsters and dogs, a suspension form to monkeys, or a tablet form to dogs with or without food.