Clinical and in vitro resistance to GS-9669, a thumb site II nonnucleoside inhibitor of the hepatitis C virus NS5B polymerase

Abstract

Treatment with GS-9669, a novel nonnucleoside inhibitor (site II) of hepatitis C virus (HCV) nonstructural 5B (NS5B) polymerase, resulted in significant antiviral activity in HCV genotype (GT) 1 patients dosed at 50 and 500 mg once daily (QD) and at 50, 100, and 500 mg twice daily (BID) for 3 days. This report characterizes the virologic resistance to GS-9669 in vitro and in GT1 HCV-infected patients from a phase I clinical study. An in vitro resistance selection study with GS-9669 revealed substitutions at several NS5B residues that conferred resistance. The M423 variants were selected at low drug concentrations (5× the 50% effective concentration [EC50]), and the L419, R422, and I482 variants were selected at higher drug concentrations (20× the EC50). During the phase I clinical study, substitutions at NS5B residues 419, 422, and 486 were the predominant changes associated with GS-9669 monotherapy. Substitutions at position 423 were observed only in GT1a patients in the low-dose groups (50 and 100 mg BID). Interestingly, four HCV patients had substitutions at position 423 at baseline. Consistent with the low resistance level at this position, three patients with M423I or M423V at baseline achieved >2-log10 reductions of HCV RNA when treated with 100 mg BID or with 500 mg QD or BID of GS-9669. The fourth patient, who had the M423V substitution at baseline, had a 4.4-log10 reduction of HCV RNA with 500 mg BID of GS-9669. Phenotypic analyses demonstrated that the viral isolates with multiple GS-9669 resistance-associated variants have reduced susceptibility to GS-9669 and lomibuvir (VX-222) but are not cross-resistant to other classes of HCV inhibitors. (This study has been registered at ClinicalTrials.gov under registration no. NCT01431898.).

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Figures

FIG 1

GS-9669 structure.

FIG 2

Influence of NNI site II on the formation of replicon colonies. A total of 75,000 GT1b replicon cells were treated for 18 to 21 days in the presence of drug in 6-well plates. The cell monolayers were then stained with crystal violet to visualize the resistant colonies (top). Colonies were counted using a Bio-Rad colony counter (bottom). The assays were performed at least three times for each compound. The average numbers of colonies per plate ± standard deviation (SD) are presented.

FIG 3

(A) Maximum change from baseline in HCV RNA. The maximum mean ± SD viral load reductions in patients dosed with GS-9669 for 3 days are shown. The P values compare the viral load reductions between the 500 mg QD GT1a and 500 mg QD GT1b groups and between 100 mg BID GT1a and 100 mg BID GT1b groups (two-tailed t test). The patients had the following resistance-associated variants (RAVs) at baseline: AH, M423V; EB, M423I; EH, M423I; and BC, M423V. (B) In vitro susceptibility of HCV genotype 1a and 1b clinical isolates to GS-9669 at baseline. The mean ± SD of the EC50s for GS-9669 inhibition of 30 GT1a and 16 GT1b treatment-naive patient isolates that were cloned in HCV replicons and tested in vitro are shown. The patients had the following RAVs detected at baseline: AH, M423V; EB, M423I; and BC, M423V.

FIG 4

In vitro susceptibility of patient isolates to GS-9669 and other HCV inhibitors. Phenotypic analysis of NS5B clinical isolates selected from those who had amino substitutions detected at 419, 423, 422, 482, 486, and 494 by population sequencing (n = 39) and the corresponding baseline samples for use as individual comparators are shown. The isolates were cloned into HCV replicons, and drug susceptibility was tested. The mean ± SD EC50 fold changes from baseline are shown. The P values compare the EC50 fold change from baseline between GS-9669 and lomibuvir (two-tailed t test). The EC50s for the baseline GT1a and GT1b isolates were as follows: GS-9669, 4.9 ± 2.4; lomibuvir, 10.4 ± 9.5; sofosbuvir, 77.3 ± 29.8; vedroprevir, 14.4 ± 6.7; ledipasvir, 0.005 ± 0.0016; ribavirin, 19.8 ± 5.1; and interferon, 1.6 ± 1.1.