1 Clinical and In Vitro Resistance to GS

AAC Accepts, published online ahead of print on 25 August 2014
Antimicrob. Agents Chemother. doi:10.1128/AAC.02815-14
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Clinical and In Vitro Resistance to GS-9669, a Thumb Site II Nonnucleoside Inhibitor of the
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Hepatitis C Virus NS5B Polymerase
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Hadas Dvory-Sobola#, Christian Voitenleitnera, Eric Maberya*, Taylor Skurnaca, Eric J. Lawitzb,
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John McHutchisona, Evguenia S. Svarovskaiaa, William Delaneya, Michael D. Millera, and
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Hongmei Moa
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Gilead Sciences, Foster City, California, USAa; Alamo Medical Research, San Antonio, Texas,
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USAb
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Running head: Resistance to the NNI Site II GS-9669 in HCV Patients
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#Address correspondence to Hadas Dvory-Sobol, [email protected]
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*
Present Address: Reset Therapeutics, Burlingame, California, USA
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Key words: hepatitis C virus (HCV), resistance, cross-resistance, nonnucleoside inhibitors
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(NNIs), direct-acting antivirals (DAAs), GS-9669
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1
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Abstract:
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Treatment with GS-9669, a novel nonnucleoside inhibitor (site II) of hepatitis C virus (HCV)
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NS5B polymerase, resulted in significant antiviral activity in HCV genotype (GT) 1 patients
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dosed at 50 and 500 mg once daily (QD), and 50, 100, and 500 mg twice daily (BID) for 3 days.
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This report characterizes virologic resistance to GS-9669 in vitro and in GT1 HCV-infected
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patients from a phase 1 clinical study. An in vitro resistance selection study with GS-9669
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revealed substitutions at several NS5B residues that conferred resistance. M423 variants were
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selected at low drug concentrations (5× EC50), and L419, R422, and I482 variants were selected
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at higher drug concentrations (20× EC50). During the phase 1 clinical study, substitutions at
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NS5B residues 419, 422, and 486 were the predominant changes associated with GS-9669
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monotherapy. Substitutions at position 423 were observed only in GT1a patients in the low-dose
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groups (50 and 100 mg BID). Interestingly, four HCV patients had substitutions at position 423
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at baseline. Consistent with the low resistance level at this position, three patients with M423I or
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M423V at baseline achieved >2 log10 reductions of HCV RNA, when treated with 100 mg BID,
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or 500 mg QD or BID of GS-9669. The fourth patient, with M423V at baseline, had a 4.4 log10
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reduction of HCV RNA with 500 mg BID. Phenotypic analyses demonstrated that viral isolates
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with multiple GS-9669 resistance-associated variants have reduced susceptibility to GS-9669 and
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lomibuvir (VX-222), but are not cross-resistant to other classes of HCV inhibitors.
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2
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Introduction
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Hepatitis C virus (HCV) infects an estimated 170 million people world-wide (1). Infection can
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lead to cirrhosis, to hepatocellular carcinoma, or other complications. Until recently, the standard
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of care for the treatment of chronic HCV infection consisted of 24-48 weeks of pegylated
42
interferon (PegIFN) and ribavirin (RBV) (2), which are associated with significant side effects
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including fever, fatigue, anemia, leucopenia, thrombocytopenia, and depression (3,4). Sustained
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virologic response (SVR) occurs in only 42% to 53% of patients with genotype (GT) 1 or GT4
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HCV, and up to 78% to 82% of patients infected with GT2 or GT3 HCV (5,6). Novel direct-
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acting antiviral agents (DAAs) are being developed in combination with PegIFN/RBV and are
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also being pursued as components of IFN-free and IFN/RBV-free regimens to improve efficacy
48
and shorten treatment duration. Two protease inhibitors (PIs) approved for treatment of HCV,
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telaprevir and boceprevir, have demonstrated significantly improved SVR rates when given in
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combination with PegIFN/RBV in GT1 patients (60-75% for combination compared with 38-
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46% for PegIFN/RBV only) (7,8). However, these new agents require thrice-daily dosing and are
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associated with more frequent and severe anemia and rash (9,10). Two HCV drugs received FDA
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approval at the end of 2013, Olysio (simeprevir), a NS3/4a protease inhibitor in combination
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with PegIFN/RBV, and Sovaldi (sofosbuvir), a nucleotide inhibitor, which is the first drug that
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has demonstrated safety and efficacy to treat non-genotype 1 HCV infection without the need for
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co-administration of PegIFN.
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GS-9669 (Fig. 1) is a novel, thumb site II nonnucleoside inhibitor (NNI) of the HCV NS5B RNA
58
polymerase, with a binding affinity of 1.4 nM for the GT1b NS5B protein. It is a selective
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inhibitor of HCV RNA replication with a mean 50% effective concentration (EC50) of ≤11 nM in
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GT1 and GT5 replicon assays (11). Other NNIs currently in phase 2 clinical studies include BI-
3
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207127 and BMS-791325 (binding to thumb site I), filibuvir and lomibuvir (binding to thumb
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site II), setrobuvir, ABT-072, and ABT-333 (binding to palm site I), and tegobuvir (also binding
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in the palm). (12). In a phase 1b study of filibuvir, resistance-associated variants (RAVs) at
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NS5B residue M423 (M423I/T/V) were observed in 76% of patients following treatment (13).
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The frequency of RAVs at this residue was similar between subtype 1a and subtype 1b viruses.
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RAVs at NS5B residues R422 (R422K), M426 (M426A), and V494 (V494A) were also detected
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in a small number of patients at baseline or end of therapy and mediate reductions in filibuvir
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susceptibility (13). GS-9669 has reduced in vitro activity against known resistance variants
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associated with thumb site II inhibitors (L419M, R422K, F429L, and I482L in GT1b and L419M
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and I482L in GT1a) (11). To further investigate the resistance profile of GS-9669, in vitro
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resistance selections were performed, and NS5B gene sequencing and phenotypic assessments
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were conducted for HCV patients treated with GS-9669 at multiple doses during a 3-day phase 1
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clinical study (ClinicalTrials.gov identifier: NCT01431898).
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4
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Materials and Methods:
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Compounds. IFN-αA human and RBV (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide)
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were purchased from Sigma Aldrich (St. Louis, MO). All other compounds (GS-9451
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[vedroprevir], GS-5885 [ledipasvir], GS-9190, GS-9669, sofosbuvir, filibuvir, and VX-222
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[lomibuvir]) were synthesized by Gilead Sciences (Foster City, CA).
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In vitro resistance selection in replicons. Resistance selections were performed as previously
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described (14). Briefly, GT1a- or GT1b-containing replicon cells were cultured in the presence
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of 5× or 20× EC50 of GS-9669 until small colonies formed. These colonies were expanded and
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characterized by sequence analysis.
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Transient transfection of replicon RNA into Huh7 Cells and EC50 determination. Resistance
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mutations were introduced into the GT1a (15) or GT1b replicon (16) by site-directed
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mutagenesis and tested in transient transfections as previously described (14). Briefly, NS5B
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mutations were introduced into a plasmid encoding the PI-hRluc replicon using a QuikChange II
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XL mutagenesis kit, following the manufacturer’s instructions (Stratagene, La Jolla, CA).
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Mutations were confirmed by DNA sequencing. Replicon RNAs were transcribed in vitro from
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replicon-encoding plasmids using a MEGAscript kit (Ambion, Austin, TX). RNA was
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transfected into Huh-lunet cells using the method of Lohmann et al (16). Briefly, cells were
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trypsinized and washed twice with PBS. A suspension of 4 × 106 cells in 400 μL of PBS was
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mixed with 5 μg of RNA and subjected to electroporation using settings of 960 μF and 270 V.
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Cells were transferred into 40 ml of pre-warmed culture medium and then seeded into 96-well
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plates (100 μL/well). Compounds were 3-fold serially diluted in 100% DMSO and added to cells
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at a 1:200 dilution, achieving a final DMSO concentration of 0.5% in a total volume of
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200 μL/well. Cells were treated for 3 days, after which culture media were removed, cells were
5
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lysed, and Renilla luciferase activity was quantified using a commercially available assay
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(Promega, Madison, WI) and a Top Count instrument (Perkin Elmer, Waltham, MA). EC50
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values were calculated as the compound concentration at which a 50% reduction in the level of
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Renilla reporter activity was observed when compared with control samples with DMSO. Dose-
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response curves and EC50 values were generated using GraphPad Prism software package
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(GraphPad Software, La Jolla, CA) by nonlinear regression analysis. The replication level of
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either reference strains (1b-Con1 or 1a-H77) or chimera replicons derived transiently from
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clinical isolates was determined as the ratio of the Renilla luciferase signal at Day 4 to that at 4 h
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post-electroporation, to normalize for transfection efficiency. The replication capacity of each
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replicon was expressed as their normalized replication efficiency compared with that of the
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reference strain (1b-Con1 or 1a-H77) within the same experiment.
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Colony reduction assays. Colony reduction assays were performed by incubating GT1b cells at
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different multiples of the EC50 as indicated for 21 days in the presence of 0.5 mg/ml of G418 and
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drug in 6-well plates. The surviving colonies were stained with Crystal Violet and counted using
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a Colony Counter (Bio-Rad, Hercules, CA).
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Clinical trial population and study design. A total of 70 patients were enrolled in 1 of 7 cohorts
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of 10 patients each randomized 8:2 to treatment with GS-9669 or matching placebo for 3 days.
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All patients completed dosing with study drug. GS-9669 was administered once daily (QD) at 50
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mg and 500 mg in GT1a patients and 500 mg in GT1b patients, or twice daily (BID) at 50 mg,
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100 mg, and 500 mg in GT1a patients and 100 mg in GT1b patients. Patients had plasma HCV
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RNA levels >5 log10 IU/ml at screening. Of the 70 patients in the study, 49 were HCV GT1a, 20
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were HCV GT1b, and 1 was HCV GT3a (placebo). The study was conducted in compliance with
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the Declaration of Helsinki. The study protocol and informed consent documents were reviewed
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and approved by the institutional review board of the participating institution, and informed
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consent was obtained from all patients before any study-specified procedures.
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Plasma samples were collected from all patients before dosing on Day 1 (baseline), at Day 4 (or
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earlier timepoint if the viral load was less than 1000 IU/ml), and Day 17 and stored at -80°C for
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NS5B sequencing and phenotypic analyses.
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Antiviral activity. Plasma samples were obtained at baseline, Day 1 (6 and 12 hours post-dose),
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Days 2, 3, and 5 (AM and PM), and Days 7, 10, and 17. HCV RNA levels were quantified using
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Taqman version 2.0 assay (Roche Molecular Systems, Inc., Branchburg, NJ; lower limit of
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quantification, 25 IU/ml). Change from baseline in HCV RNA was determined for each
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timepoint.
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Amplification and population sequencing of the HCV NS5B gene. HCV genotypic analyses
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were performed at Monogram Biosciences, Inc (South San Francisco, CA). Briefly, virus
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particles were disrupted by addition of lysis buffer and genomic viral RNA (vRNA) was
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extracted. Purified vRNA was used as a template for cDNA synthesis in a reverse transcriptase
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reaction and then was used as the template for the first round of a two-round nested polymerase
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chain reaction (PCR) that results in the amplification of the entire NS5B region. The inner
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(nested) primers contain restriction endonuclease recognition/cleavage sites that enable cloning
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of NS5B amplification products into an HCV replicon test vector for phenotypic drug
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susceptibility analysis. PCR products were purified and were used for the template in each of 12
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sequencing reactions using either subtype 1a- or 1b-specific sequencing primers. The sequencing
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assay was validated and can detect quasispecies in 20% and 10% of mixtures in 100% and 75%
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of assays, respectively (17).
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Sequences were automatically trimmed and assembled based on homology to a subtype-specific
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reference sequence (H77 for 1a, Con1 for 1b). The final consensus sequences were exported
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along with a list of amino acid differences from the reference. All produced NS5B sequences
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were submitted to the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/index.html) and
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assigned accession numbers KM215799-KM216009
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HCV NS5B phenotypic assay. NS5B amplification products were ligated into a bacterial
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plasmid cloning vector and then used to transform competent Escherichia coli. After ligation and
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transformation to competent E. coli, plasmid DNA was purified from bacterial cultures, and then
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linearized by restriction endonuclease digestion. In vitro transcribed RNA was then
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electroporated into a Huh7 cell line, and electroporated cells were incubated in the absence and
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presence of serially diluted inhibitors. Luciferase activity was expressed as relative light units.
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Inhibitor susceptibility was determined by evaluating the ability of patient isolates to replicate in
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the absence and presence of inhibitor at 72-96 hours post-electroporation. The percentage
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inhibition at each serial diluted inhibitor concentration was derived as follows: [1 – (luciferase
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activity in the presence of inhibitor ÷ luciferase activity in the absence of inhibitor)] × 100
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inhibitor susceptibility profiles (curves) were derived from these values, and inhibition data (e.g.,
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EC50; the inhibitor concentration required to reduce virus replication by 50%) was extrapolated
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from fitted curves. Inhibition data were reported as fold change relative to that of a reference
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vector (e.g., EC50 (sample)/ EC50 (reference)) processed in the same assay batch (e.g., EC50 fold-
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change from reference).
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To assess the effect of baseline polymorphs on antiviral activity, the fold change in EC50 values
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were plotted against change in HCV RNA from baseline to Day 17.
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Results
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Selection of in vitro resistance to GS-9669 using HCV replicons. To characterize the resistance
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profile of GS-9669, we selected resistance in GT1 HCV using replicon cell lines. Specifically,
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GT1a and GT1b replicon cell lines were treated with GS-9669 at 5× or 20× EC50 and G418 for
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several weeks until resistant colonies formed. Approximately 25 individual colonies from each
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selection condition were isolated, expanded, and analyzed for changes in the HCV NS5B gene.
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At the lower concentration of GS-9669 (5× EC50), resistant variants were detected only at residue
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423 of NS5B (M423I/T/V) (Table 1). M423T was the dominant variant (50% in GT1a and 60%
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in GT1b). In addition, M423I was found in GT1a and M423V in GT1b. At the higher
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concentration of GS-9669 (20× EC50), changes at residues 419, 422, and 482 were detected, with
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I482L and R422K being the most frequently observed variants in GT1a and GT1b, respectively
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(Table 1). Phenotypic analyses of the variants observed during in vitro resistance selections
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confirmed that they reduced GS-9669 susceptibility in vitro (Table 2). In both genotypes, M423
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RAVs conferred low to moderate resistance levels (4.6- to 19.3-fold), while L419M, R422K, and
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I482L conferred higher levels of resistance (26- to 815-fold).
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Replicon colony reduction assay. The in vitro resistance barrier for an HCV inhibitor is
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influenced by various factors: first, the “genetic barrier to resistance,” which is the number of
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amino acid substitutions needed for a viral variant to confer resistance to the drug (if a single
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substitution is sufficient to confer high-level resistance, then the drug is generally considered to
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have a low genetic barrier to resistance); and second, the fitness of the resistance viral variant,
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which enables it to grow in a replicative environment (18).
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Resistance barriers can be assessed using the HCV replicon system in colony-formation assays;
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upon treatment of a replicon cell culture with an inhibitor and G418, the number of surviving
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replicon colonies at different drug conditions will reflect the resistance barrier of the inhibitor.
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GS-9669 was thus tested in a colony formation assay in GT1b replicon cells at 1×, 5×, 10×, 20×,
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and 40× EC50 (Fig. 2). After 21 days, DMSO-treated control wells contained confluent
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monolayers of cells. The number of colonies present in GS-9669-treated wells decreased with
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increasing drug concentration (Fig. 2). Only a few colonies remained in the highest dose
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treatment group (40× EC50). GS-9669 treatment resulted in fewer colonies at 5× and 10× EC50
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(25 nM and 50 nM, respectively) compared with filibuvir and lomibuvir. This result suggests that
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GS-9669 presents a higher barrier to resistance in HCV GT1b replicon cells at the low multiples
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of EC50 (5× and 10× EC50) compared with filibuvir and lomibuvir.
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Antiviral response to GS-9669 in GT1 HCV-infected patients. The samples analyzed in the
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phase 1 study were obtained from 49 GT1a and 20 GT1b HCV patients who were dosed with
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GS-9669 or placebo for 3 days. Among the QD-dosed cohorts, mean maximum reductions in
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HCV RNA levels through Day 4 were -2.18, -3.36, and -3.45 log10 IU/ml for the GS-9669 50 mg
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GT1a, 500 mg GT1a, and 500 mg GT1b groups, respectively (Table 3, Fig. 3). Among the BID-
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dosed cohorts, median maximum reductions in HCV RNA levels through Day 4 were -3.35,
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-3.40, -4.02, and -3.47 log10 IU/ml in the GS-9669 50 mg GT1a, 100 mg GT1a, 500 mg GT1a,
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and 100 mg GT1b groups, respectively (Table 3, Fig 3). Maximum reductions in HCV RNA
205
levels were comparable among patients with GT1a and GT1b HCV infection dosed with 500 mg
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QD or 100 mg BID (P = 0.75 and 0.8, respectively).
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NS5B polymerase polymorphism present at baseline. NS5B polymorphism present at baseline
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included variants associated with reduced susceptibility to GS-9669. Substitutions at positions
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previously shown to have reduced susceptibilities to NNIs were observed in four patients dosed
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with GS-9669 (Table 4, Fig. 3). Four patients had RAVs at amino acid residue 423 (M423V,
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M423I) which confer low-level reduced susceptibility to GS-9669 in vitro (Table 2). Despite the
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presence of M423V/I, three of four patients showed partial antiviral responses with >2 log10
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reductions in HCV RNA after receiving GS-9669 100 mg BID or 500 mg QD, and the remaining
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patient had a 4.44 log10 HCV RNA reduction after receiving GS-9669 500 mg BID. Other NS5B
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variants at positions M426, Y448, and V494, previously shown to affect susceptibility to site II
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and III NNIs (19-21), were observed at baseline either alone or in combination with M423
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substitutions (M426L, n= 8; Y448H, n= 1; V494I, n = 3). A previous study found that Y448H
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does not confer cross-resistance to GS-9669 (22), and drug susceptibility assays conducted here
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indicated that neither M426L nor V494I confer reduced susceptibility to GS-9669 (Table 2).
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Patients with these variants showed similar response to other patients in the same treatment
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group. These three positions are highly polymorphic in NS5B, and the substitutions observed at
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baseline do not appear to affect GS-9669 susceptibility in vitro or in vivo. A mixture of the
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variant S556S/G that was previously shown to reduce susceptibility to site III NNIs was detected
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in one patient at baseline. This patient had a 3.38 log10 HCV RNA reduction after receiving GS-
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9669 500 mg QD, suggesting this variant did not affect the antiviral response to GS-9669; NS5B
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residue 556 also appears to be highly polymorphic.
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The effect of baseline polymorphism on GS-9669 in vitro activity was further assessed in a
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transient replicon assay in which the NS5B regions derived from patient isolates were introduced
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into a GT1b replicon backbone. Average EC50 values for GS-9669 at baseline for GT1a and
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GT1b were 3.8 ± 1.3 and 6.8 ± 2.7 nM, respectively (Fig. 3b). The only patient isolates with
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significantly decreased susceptibility to GS-9669 at baseline were from patients AH and EB,
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who had M423Vor M423I at baseline (4.5- and 9.3-fold reduced, respectively, compared with
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the reference wild-type replicon). The other patient (BC) with M423V at baseline had only a 1.4-
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234
fold change from reference. This patient had a 4.44 log10 HCV RNA reduction after receiving
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GS-9669 500 mg BID. No phenotypic data were obtained for the fourth patient who had M423I
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at baseline (EH). No changes in GS-9669 activity were observed when baseline isolates with the
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M426L or S556S/G polymorphs were tested (<2.5 change from reference).
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Substitutions selected in HCV of patients following GS-9669 treatment. HCV NS5B amino
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acid substitutions were identified by comparing sequences obtained during or after completion of
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dosing with the baseline population sequence for each patient. Amino acid changes at positions
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not known to be associated with resistance to HCV NNIs were considered potential resistance
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mutations if they developed in multiple patients at conserved sites. NS5B conserved sites were
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defined as amino acid positions that are >99% conserved in Gilead’s virology database
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containing 639 GT1a and 406 GT1b sequences from treatment-naïve patients. Conservation of
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these amino acids was also confirmed using publically available sequences from the European
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Hepatitis C Virus Database (euHCVdb, http://euhcvdb.ibcp.fr). NS5B positions 419, 422, 482,
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486, and 494 were >99% conserved in GT1a and GT1b. M423 was also conserved in GT1b,
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whereas M423I was found in 2% of GT1a HCV sequences.
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Analyses of on-treatment samples or post-treatment samples when viral load was >1000 IU/ml
250
on Day 4 (Day 5 or Day 7) indicated that drug RAVs were present in 23/32 (71.9%) GT1a
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patients and 14/16 (87.5%) GT1b patients who received GS-9669 BID or >50 mg QD (Table 5).
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Patients treated with the lowest dose of GS-9669, 50 mg QD, had the least resistance detected
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(1/8, 12.5%). HCV from most patients had multiple NNI RAVs detected that appeared as
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mixtures with wild-type at each position. Substitutions at residues 419, 422, and 486 were the
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predominant changes associated with GS-9669 therapy and were observed in 22/48 (46%), 22/48
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(46%), and 31/48 (65%) patients, respectively, following 3 days of GS-9669 >50 mg QD
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treatment. Substitutions at position 423 were observed only in GT1a patients who received lower
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doses of GS-9669 (50 mg BID and 100 mg BID). NNI RAVs were not detected in the HCV
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samples of any placebo patients.
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The stability of RAVs after the cessation of treatment was also assessed. Except for one GT1a
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patient in the 500 mg QD cohort, the NNI RAVs were no longer detected by population
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sequencing in any of GT1a patients at Day 17 (Table 6). For GT1b patients with RAVs detected
263
at earlier timepoints, they were still detected in 9 of 13 patients (69%) on Day 17. No other
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substitutions were observed in multiple patients across all dose groups.
265
Phenotypic and cross-resistance analyses. To determine whether the sequence changes
266
described above are associated with reduced susceptibility, phenotypic analyses were performed
267
for samples from patients with amino substitutions detected at NS5B positions 419, 422, 423,
268
482, 486, or 494. Phenotypic analyses were also performed for corresponding baseline samples
269
for use as individual comparators. GS-9669 EC50 values were obtained for viruses from 39
270
patients both at baseline and either Day 4 or Day 17 (Fig. 4 and Table S1).
271
Phenotypic analyses demonstrated that 26/39 samples analyzed with multiple RAVs at positions
272
A486 and/or L419 and/or R422 and/or M423 had reduced susceptibility to GS-9669 with >3-fold
273
EC50 changes from baseline. Thirteen of 39 patient isolates had RAVs detected, but did not have
274
a change in susceptibility (fold change from baseline <3); this is most likely due to the
275
recombinant mutant virus analyzed having a mixture with wild-type and the more efficient
276
replication of the wild-type replicon compared with the variants during the assay. Only two
277
patients, BB and EG, had full single substitutions detected (L419M or L419S), and these had 48-
278
and >317-fold changes from baseline, respectively, for GS-9669. All other substitutions were
279
detected as mixtures alone or with other mutations (mixtures of 2 or 3 positions) and showed low
13
280
to high levels of resistance to GS-9669. Almost all patient isolates with GS-9669 RAVs also had
281
reduced susceptibility to lomibuvir, another site II NNI. All GS-9669-resistant mutants
282
maintained wild-type susceptibility to other tested classes of HCV inhibitors, including
283
sofosbuvir (NI), GS-9451 (PI), ledipasvir (NS5A), and ribavirin. In addition, phenotypic
284
analyses for 3 patients with no RAVs detected but other NS5B substitutions (M173I, V329V/I,
285
G/S543G, G66D/G, V/I116V, and R300R/Q) showed that these isolates were fully susceptible to
286
GS-9669 and other tested HCV inhibitors.
287
To evaluate resistance levels of single RAVs that were observed as mixtures alone or with other
288
RAVs in the clinical samples, the predominant RAVs detected in GT1a or GT1b isolates were
289
introduced into the wild-type replicon by site-directed mutagenesis and phenotyped (Table 2).
290
All substitutions at positions 419 and 422 confer high levels of resistance to GS-9669 (>90-fold).
291
The V482I, A486V, and V494V variants confer more moderate levels of resistance to GS-9669
292
in GT1a and GT1b (20- to 50-fold). M423 substitutions confer the lowest levels of resistance to
293
GS-9669 (5- to 20-fold). Most GS-9669 RAVs conferred comparable resistance levels to
294
lomibuvir.
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295
Discussion
296
Treatment with GS-9669, a novel NNI site II NS5B inhibitor resulted in a significant antiviral
297
effect in GT1 HCV-infected patients in a phase 1 monotherapy study (12). Our current study
298
analyzed drug-resistant HCV variants selected in vitro, using the replicon system, and in patients
299
from the phase 1 study. Our in vitro results indicated that NS5B residues L419, R422, M423, and
300
I482 are major resistance loci for GS-9669, with M423 RAVs selected at low drug
301
concentrations and L419, R422, and I482 RAVs selected at higher drug concentrations. In
302
agreement with these genotypic observations, phenotypic analyses of the RAVs indicated that
303
M423 RAVs conferred low to moderate levels of GS-9669 resistance, whereas the remaining
304
variants conferred higher degrees of resistance. Colony-reduction assays indicated that GS-9669
305
selected fewer resistant colonies at higher doses, suggesting that variants conferring low-level
306
resistance may be suppressed at high doses. We noted that at 5× and 10× EC50 concentrations,
307
there were significantly fewer colonies selected by GS-9669 compared with filibuvir and
308
lomibuvir, potentially due to enhanced suppression of M423V/T mutants by GS-9669 versus
309
these other inhibitors. Interestingly, it has been previously shown that filibuvir has high levels of
310
resistance to the M423 mutants (>560-fold) and R422K (>340-fold) (13) compared with 8.5- to
311
15.8- and 144.7-fold for GS-9669 for the M423V/T/I and R422K mutants, respectively.
312
Analysis of GS-9669 susceptibility of baseline clinical NS5B isolates indicated potent (low
313
nanomolar) antiviral activity against both GT1a and GT1b in the vast majority of patient isolates.
314
In general, there was limited variation in baseline susceptibility (mean EC50 of 3.8 ± 1.3 and 6.8
315
± 2.7 nM, for GT1a and GT1b, respectively) of the different GT1 patient isolates to GS-9669.
316
This limited variation was consistent with the limited variability in virologic response to GS-
317
9669 in patients during the study. However there were a few outlier isolates with lower in vitro
15
318
susceptibility to GS-9669. These patients had known NS5B NNI RAVs at position M423 prior to
319
treatment with GS-9669. Overall, four analyzed patients had NS5B NNI RAVs at baseline
320
associated with reduced in vitro susceptibility to GS-9669. Greater than 2 log10 reductions in
321
HCV RNA were observed for three of these patients, but these antiviral responses were reduced
322
relative to patients without the M423 mutant in the same dosing group; no reduction in response
323
was observed for the fourth patient with M423V who received the highest dose of GS-9669 (>4
324
log log10 reductions in HCV RNA). No other NS5B variants observed at baseline showed
325
reduced susceptibility in vitro or reduced responses in vivo to GS-9669.
326
Similar to other NNIs, RAVs were detected shortly after suppression of the wild-type virus by
327
GS-9669. Resistance variants were detected in patients at NS5B positions 419, 422, 423, 482,
328
486, and 494. These results are in good agreement with findings from the in vitro resistance
329
selections where substitutions were also detected at positions 419, 422, 423, and 482. The variety
330
of amino acid substitutions observed in vivo at position 419 was greater than observed in vitro,
331
with L419 substitutions to M/S/P/T/V or I observed in vivo. Although we did not observe
332
resistance at positions 486 and 494 during in vitro resistance selections, this may be due to the
333
limited sequence diversity represented by lab HCV strains compared with the quasispecies
334
present in different HCV patients. A486V, R422K, and L419M were the predominant NS5B
335
RAVs observed in viruses from GT1a and GT1b patients. Interestingly, M423 variants were only
336
observed at lower GS-9669 doses (50 mg BID and 100 mg BID), agreeing with our in vitro
337
resistance selections where M423 RAVs were observed with the 5× EC50 treatment, but not at
338
the higher 20× EC50 treatment. The lack of clinical appearance of M423 RAVs at higher GS-
339
9669 doses also agrees with the low to moderate (<20-fold) resistance levels that M423 variants
340
confer to GS-9669; collectively these data imply clinical suppression of M423 variants by higher
16
341
doses of GS-9669 as also evidenced by the antiviral responses observed among the four patients
342
with these mutants at baseline. However, we note that results from ongoing deep sequencing
343
analysis of representative samples showed that M423 variants were detected at low frequencies
344
(<3.1%) in about 40% of patients treated with doses of 500 mg QD and BID in GT1a patients,
345
and 500 mg QD in GT1b patients (other groups were not tested). Nine of 12 GT1a patients and 7
346
of 7 GT1b patients still had detectable variants on Day 17 (23), and L419I/M, M423T and
347
A486T/V appeared to persist longer as minority variants. R422K, with the lowest in vitro
348
replication capacity, was not detected on Weeks 24 and 48 in most patients.
349
RAVs at positions 419 and 422 that were selected with higher concentrations of GS-9669 in vitro
350
were a good predictor of the major variants in vivo and were observed in GT1a and GT1b
351
patients dosed with GS-9669. In contrast, in a monotherapy study with filibuvir, RAVs at residue
352
M423 were the predominant change associated with filibuvir therapy (76% of patients treated),
353
and a small number of patients had RAVs at positions 422 and 426 (13). Overall, most
354
substitutions at positions 423 confer a low level of resistance to GS-9669 and lomibuvir, while
355
higher levels of resistance to all three NNIs (GS-9669, filibuvir, and lomibuvir) are conferred by
356
RAVs at positions 419, 422 and 486.
357
NS5B RAVs were observed by population sequencing in only one patient who received 50 mg
358
QD GS-9669, but in the majority of patients who received higher doses. This finding is likely
359
explained by the degree of antiviral suppression at different GS-9669 doses. More substantial
360
suppression of wild-type virus consequently resulted in more frequent detection of resistance
361
variants (higher dose and greater wild-type HCV viral suppression). Consistent with other phase
362
1 HCV monotherapy studies, this observation suggests that RAVs pre-exist at low levels prior to
363
treatment and become detectable once the wild-type population is sufficiently inhibited.
17
364
Drug-resistant variants were no longer detected by population sequencing in any of the GT1a
365
patients at Day 17 except for one GT1a patient in the 500 QD cohort. For GT1b patients with
366
RAVs detected at earlier timepoints, RAVs were either no longer detectable in 4/13 patients at
367
Day 17 or were detected at a significantly lower percentage of the viral population in 9/13
368
patients. These results suggest a decreased fitness of these mutants in vivo which is more readily
369
observed in GT1a. Interestingly, in vitro replication capacity of these mutants did not suggest
370
any fitness differences between GT1a and GT1b RAVs, perhaps illustrating the limitations of
371
assessing viral fitness in vitro using replicons.
372
Phenotypic analyses demonstrated that most viral isolates with multiple RAVs had reduced
373
susceptibility to GS-9669 and lomibuvir, but wild-type susceptibility to other classes of HCV
374
inhibitors including sofosbuvir, GS-9451, ledipasvir, and ribavirin. These results are consistent
375
with phenotypic analyses of site-directed recombinant replicons at positions 419, 422, 423, 482,
376
486, and 494, which displayed low to high levels of resistance to GS-9669 and lomibuvir, but
377
remained sensitive to other classes of HCV inhibitors. Previous data from our group also showed
378
similar results using different assays (11).
379
In summary, highly effective inhibition of wild-type HCV by the site II NNI GS-9669 revealed
380
variants that confer resistance at NS5B positions 419, 422, 423, 482, 486, and 494. Patients with
381
substitutions at position 423 at baseline achieved >2 log10 reduction in HCV RNA after 100 mg
382
BID to 500 mg BID GS-9669 treatment. This result is consistent with in vitro phenotypic
383
analyses indicating that substitutions at position 423 confer only low to moderate levels of
384
resistance to GS-9669 and is also consistent with the selection of these mutants at low but not
385
high levels of GS-9669 in vitro. The frequency of GS-9669 RAVs declined over 14 days off
386
treatment, indicating reduced fitness of these RAVs compare with wild-type. The lack of cross-
18
387
resistance between GS-9669-resistant mutants and sofosbuvir, ledipasvir, GS-9451, and
388
ribavirin, makes GS-9669 a candidate for use in combination with these inhibitors in GT1 HCV-
389
infected patients.
19
390
Acknowledgements
391
This work was supported by Gilead Sciences, Inc.
392
We gratefully acknowledge the patients who participated in the study, the investigators, nursing
393
staff, and research support staff involved in the study, and members of the project teams at
394
Gilead Sciences.
395
20
396
References
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
1. Lavanchy D. 2011. Evolving epidemiology of hepatitis C virus. Clin Microbiol Infect.
17:107-115.
2. Ghany MG, Strader DB, Thomas DL Seeff LB. 2009. Diagnosis, management, and
treatment of hepatitis C: an update. Hepatology. 49:1335-1374.
3. Di Bisceglie AM, McHutchison J Rice CM. 2002. New therapeutic strategies for hepatitis C.
Hepatology. 35:224-231.
4. Manns MP, Wedemeyer H Cornberg M. 2006. Treating viral hepatitis C: efficacy, side
effects, and complications. Gut. 55:1350-1359.
5. Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Goncales FL, Jr., Haussinger
D, Diago M, Carosi G, Dhumeaux D, Craxi A, Lin A, Hoffman J Yu J. 2002. Peginterferon
alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med. 347:975-982.
6. Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R,
Goodman ZD, Koury K, Ling M Albrecht JK. 2001. Peginterferon alfa-2b plus ribavirin
compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a
randomised trial. Lancet. 358:958-965.
7. Hezode C, Forestier N, Dusheiko G, Ferenci P, Pol S, Goeser T, Bronowicki JP,
Bourliere M, Gharakhanian S, Bengtsson L, McNair L, George S, Kieffer T, Kwong A,
Kauffman RS, Alam J, Pawlotsky JM Zeuzem S. 2009. Telaprevir and peginterferon with or
without ribavirin for chronic HCV infection. N. Engl. J. Med. 360:1839-1850.
8. Kwo PY, Lawitz EJ, McCone J, Schiff ER, Vierling JM, Pound D, Davis MN, Galati JS,
Gordon SC, Ravendhran N, Rossaro L, Anderson FH, Jacobson IM, Rubin R, Koury K,
Pedicone LD, Brass CA, Chaudhri E, Albrecht JK investigators S-. 2010. Efficacy of
boceprevir, an NS3 protease inhibitor, in combination with peginterferon alfa-2b and ribavirin in
treatment-naive patients with genotype 1 hepatitis C infection (SPRINT-1): an open-label,
randomised, multicentre phase 2 trial. Lancet. 376:705-716.
9. Imhof I Simmonds P. 2011. Genotype differences in susceptibility and resistance
development of hepatitis C virus to protease inhibitors telaprevir (VX-950) and danoprevir
(ITMN-191). Hepatology. 53:1090-1099.
10. Foster GR, Hezode C, Bronowicki JP, Carosi G, Weiland O, Verlinden L, van Heeswijk
R, van Baelen B, Picchio G Beumont M. 2011. Telaprevir alone or with peginterferon and
ribavirin reduces HCV RNA in patients with chronic genotype 2 but not genotype 3 infections.
Gastroenterology. 141:881-889 e881.
11. Fenaux M, Eng S, Leavitt SA, Lee YJ, Mabery EM, Tian Y, Byun D, Canales E, Clarke
MO, Doerffler E, Lazerwith SE, Lew W, Liu Q, Mertzman M, Morganelli P, Xu L, Ye H,
Zhang J, Matles M, Murray BP, Mwangi J, Hashash A, Krawczyk SH, Bidgood AM,
Appleby TC Watkins WJ. 2013. Preclinical characterization of GS-9669, a thumb site II
inhibitor of the hepatitis C virus NS5B polymerase. Antimicrob. Agents Chemother. 57:804-810.
12. Lawitz E, Hazan L, Gruener D, Hack H, Backonja M, Hill J, German P, Dvory-Sobol
H, Jain A, Arterburn S, Watkins W, Rossi S, McHutchinson J Rodriguez-Torres M. GS9669, A Novel NS5B Non-Nucleoside Thumb Site II Inhibitor, Demonstrates Potent Antiviral
Activity, Favorable Safety Profi le a nd Potential for Once-Daily Dosing [Poster 1189]. In. 47th
Annual Meeting of the European Association for the Study of the Liver (EASL); 2012;
Barcelona, Spain; April 18 - 22.
21
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
13. Troke PJ, Lewis M, Simpson P, Gore K, Hammond J, Craig C Westby M. 2012.
Characterization of resistance to the nonnucleoside NS5B inhibitor filibuvir in hepatitis C virusinfected patients. Antimicrob. Agents Chemother. 56:1331-1341.
14. Shih I-h, Vliegen I, Peng B, Yang H, Hebner C, Paeshuyse J, Purstinger G, Fenaux M,
Tian Y, Mabery E, Qi X, Bahador G, Paulson M, Lehman LS, Bondy S, Tse W, Reiser H,
Lee WA, Schmitz U, Neyts J Zhong W. 2011. Mechanistic characterization of GS-9190
(tegobuvir), a novel non-nucleoside inhibitor of hepatitis C virus NS5B polymerase. Antimicrob.
Agents Chemother. 55:4196-4203.
15. Robinson M, Yang H, Sun SC, Peng B, Tian Y, Pagratis N, Greenstein AE Delaney WE.
2010. Novel HCV Reporter Replicon Cell Lines Enable Efficient Antiviral Screening against
Genotype 1a. Antimicrob. Agents Chemother. 54:3099-3106.
16. Lohmann V, Korner F, Koch J, Herian U, Theilmann L Bartenschlager R. 1999.
Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science. 285:110113.
17. Choe S, Han D, Cheng M, Anton E, Stawiski E, Penuel E, Parkin N, Petropoulos CJ,
Reeves JD. 2010. Validation of the GeneSeq™ HCV NS5B Sequencing Assay for PatientDerived HCV Subtypes 1a and 1b. Abstr. 45th Annual Meeting of the European Association for
the Study of the Liver. http://www.natap.org/2010/EASL/EASL_67.htm.
18. Pawlotsky JM. 2012. New antiviral agents for hepatitis C. F1000 biology reports. 4:5.
19. Cooper C, Lawitz EJ, Ghali P, Rodriguez-Torres M, Anderson FH, Lee SS, Bedard J,
Chauret N, Thibert R, Boivin I, Nicolas O Proulx L. 2009. Evaluation of VCH-759
monotherapy in hepatitis C infection. J. Hepatol. 51:39-46.
20. Shi ST, Herlihy KJ, Graham JP, Fuhrman SA, Doan C, Parge H, Hickey M, Gao J, Yu
X, Chau F, Gonzalez J, Li H, Lewis C, Patick AK Duggal R. 2008. In vitro resistance study of
AG-021541, a novel nonnucleoside inhibitor of the hepatitis C virus RNA-dependent RNA
polymerase. Antimicrob. Agents Chemother. 52:675-683.
21. Shi ST, Herlihy KJ, Graham JP, Nonomiya J, Rahavendran SV, Skor H, Irvine R,
Binford S, Tatlock J, Li H, Gonzalez J, Linton A, Patick AK Lewis C. 2009. Preclinical
characterization of PF-00868554, a potent nonnucleoside inhibitor of the hepatitis C virus RNAdependent RNA polymerase. Antimicrob. Agents Chemother. 53:2544-2552.
22. Dvory-Sobol H, Xu S, Goodwin H, Goodman D, Svarovskaia ES, Miller MD Mo H.
2011. Replication Fitness and Drug Susceptibility of HCV Triple Class Drug-Resistant Mutants
[Abstract R_13]. Presented at the 6th International Workshop on Clinical Pharmacology of
Hepatitis Therapy ; 2011 June 22-23; Cambridge, MA.
23. Dvory-Sobol H, Gontcharova V, Martin R, Lawitz EJ, McHutchison JG, Svarovskaia
ES, Miller MD, Mo H. 2014. Low persistence of resistance-associated variants after 3 days of
monotherapy with NS5B NNI site II inhibitor GS-9669 in genotype 1 HCV patients. Abstr. 49th
Annual Meeting of the European Association for the Study of the Liver.
http://www.natap.org/2014/EASL/EASL_74.htm.
22
481
Figure Legends
482
FIG. 1. GS-9669 structure.
483
484
FIG. 2. Influence of NNI site II on the formation of replicon colonies. 75,000 GT1b replicon
485
cells were treated for 18-21 days in the presence of drug in 6-well plates. Cell monolayers were
486
then stained with Crystal Violet to visualize resistant colonies. Colonies were counted using a
487
Bio-Rad Colony Counter. Assays were performed at least three times for each compound. The
488
average number of colonies per plate ± SD is presented.
489
490
FIG. 3. (A) Maximum change from baseline in HCV RNA. Maximal viral load reduction in
491
patients dosed with GS-9669 for 3 days. Means ± standard deviations are shown. P values
492
compare viral load reduction between the 500 mg QD GT1a and 500 mg QD GT1b groups and
493
between 100 mg BID GT1a and 100 mg BID GT1b groups (two-tailed t test). Patients had the
494
following resistance-associated variants (RAVs) at baseline: AH - M423V; EB - M423I; EH -
495
M423I; BC - M423V.
496
(B) In vitro susceptibility of HCV genotype 1a and 1b clinical isolates at baseline to GS-
497
9669. EC50 values for GS-9669 inhibition of 30 GT1a and 16 GT1b treatment- naïve patient
498
isolates that were cloned in HCV replicons and tested in vitro. Means ± standard deviations are
499
shown. Patients had the following RAVs detected at baseline: AH - M423V; EB - M423I; BC-
500
M423V.
501
502
FIG. 4. In vitro susceptibility of patient isolates to GS-9669 and other HCV inhibitors.
503
Phenotypic analysis of NS5B clinical isolates selected from those who had amino substitutions
23
504
detected at 419, 423, 422, 482, 486, and 494 by population sequencing (n = 39) and
505
corresponding baseline samples for use as individual comparators. Isolates were cloned into
506
HCV replicons, and drug susceptibility was tested. EC50 fold change from baseline is shown.
507
Means ± standard deviations are shown. P values compare EC50 fold change from baseline
508
between GS-9669 and lomibuvir (two-tailed t test). EC50 for the baseline GT1a and GT1b
509
isolates were as follows: GS-9669 (4.9 ± 2.4), lomibuvir (10.4 ± 9.5), sofosbuvir (77.3 ± 29.8),
510
vedroprevir (14.4 ± 6.7), ledipasvir (0.005 ± 0.0016), ribavirin (19.8 ± 5.1), and interferon (1.6 ± 1.1).
511
24
512
TABLE 1.
513
Summary of in vitro resistance selection with GS-9669 in GT1a and GT1b
replicon systems
Clonal frequency of amino acid substitutions in the presence of GS-9669 (%)a
Amino acid GT1a
substitutions 5× EC50 [45 nM]
WT
GT1b
20× EC50 [180nM] 5× EC50 [25 nM]
20
L419M
10
10
6
32
L419W
12
R422K
10
M423I
30
M423T
50
I482L
514
GT, genotype; WT, wild type
515
a
50
60
M423V
516
20× EC50 [100nM]
30
80
HCV replicon cells were incubated with compound for 30-40 days (GT1a) and 20-30 days
(GT1b). At least 25 isolated colonies were sequenced from each selection condition.
517
25
518
TABLE 2.
Replicon resistance of NS5B Site II RAVs to GS-9669a,b
GT1a
NS5B site II
GS-9669 fold
GT1b
Lomibuvir fold
Replication
c
GS-9669 fold
Lomibuvir fold
Replication
change
change
capacity (%)c
RAVs
change
change
capacity (%)
L419M
87.3 ± 25.2
50.9 ± 17.5
115 ± 27.5
123.4 ± 37.6
127.7 ± 49.7
77.2 ± 20.6
L419S
197 ± 95.9
134.6 ± 44.3
0.95 ± 0.46
789.8 ± 354.3
346.9 ± 262
11.4 ± 1.9
R422K
144.7 ± 56.5
97.8 ± 5.7
2.53 ± 1.16
814.6 ± 476.9
545.8 ± 305
44.0 ± 14.6
M423V
8.5 ± 3.8
17.9 ± 9.0
51.9 ± 30.5
7.0 ± 3.5
18.0 ± 8.9
37.1 ± 13.4
M423T
15.8 ± 4.8
28.3 ± 9.1
82.4 ± 19.5
19.3 ± 4.0
49.6 ± 6.7
56.8 ± 21.9
M423I
10.6 ± 2.5
10.5 ± 1.6
106 ± 19.8
4.6 ± 0.4
5.6 ± 1.4
59.3 ± 26.1
M426L
1.1 ± 0.1
ND
71.6 ± 17.3
ND
ND
ND
I482L
26.1 ± 5.3
34.2 ± 9.0
127 ± 41.0
51.4 ± 8.5
101.2 ± 21.0
75.6 ± 23.0
A486I
ND
ND
ND
48.7 ± 15.5
102.1 ± 31.3
23.2 ± 9.2
A486T
ND
ND
ND
31.1 ± 8.4
55.7 ± 16.2
57.6 ± 24.3
A486V
39.6 ± 11.8
48.5 ± 17.2
84.6 ± 27.8
49.8 ±19.3
77.5 ± 31.5
83.7 ± 17.8
V494A
17.4 ± 4.2
27.2 ± 11.1
32.1 ± 15.6
18.1 ± 5.1
32.5 ± 4.0
51.5 ± 16.0
26
V494I
0.55 ± 0.05
ND
6.8 ± 2.1
ND
ND
ND
519
GT, genotype; ND, not determined.
520
a
Values are the results of 2 or more independent experiments.
521
b
Mean fold change in EC50 for the mutant replicon compared with the wild-type replicon determined in each experiment ± SD.
522
c
Replication capacity was normalized with that of the reference strain (1b-Con1 or 1a-H77) within the same experiment and
523
expressed as % ± SD.
27
524
TABLE 3.
Antiviral response to GS-9669 monotherapy
GS-9669 dose, HCV
No. of patients in
Mean maximal HCV reduction ± SD
genotype of patients
group
(range) (log10 IU/ml)a
50 mg QD, GT1a
8
-2.18 ± 0.60 (-2.97, -1.22)
500 mg QD, GT1a
8
-3.36 ± 0.69 (-4.04, -2.20)
500 mg QD, GT1b
8
-3.45 ± 0.47 (-4.06, -2.89)
50 mg BID, GT1a
8
-3.35 ± 0.37 (-3.79, -2.87)
100 mg BID, GT1a
8
-3.40 ± 0.59 (-3.98, -2.19)
500 mg BID, GT1a
8
-4.02 ± 0.33 (-4.44, -3.31)
100 mg BID, GT1b
8
-3.47 ± 0.37(-4.11, -3.03)
525
BID, twice daily; GT, genotype; QD, once daily.
526
a
Mean maximal viral load reduction at any time point during the first 7 days.
527
28
528
TABLE 4.
Patients with NS5B NNI RAVs detected at baseline
Mean max VL
529
Patient
Treatment
Known NNI
Max VL
reduction in the
ID
genotype
group
RAVsa
reduction
treatment group
EH
1a
500 mg QD
M423I
-2.20
-3.36
EB
1a
500 mg QD
M423I
-2.30
-3.36
AH
1a
100 mg BID
M423V
-2.19
-3.41
BC
1a
500 mg BID
M423V
-4.44
-4.02
BID, twice daily; max, maximum; NNI, nonnucleoside inhibitor; QD, once daily; RAVS,
530
531
Patient
resistance-associated variants; VL, viral load
a
Frequency analyses from a total of 639 GT1a and 405 GT1b NS5B gene sequences obtained
532
from Gilead databases showed that at position M423, 98% of the sequences contain the wild-
533
type amino acid residue Met, 0.6% contain Ile, and 0.5% contain Val.
29
534
TABLE 5.
NS5B drug-resistance variants at Days 2 to 7a
No. of
GT1a
GT1a
GT1a
GT1a
GT1a
GT1b
GT1b
GT1a with GT1b with No. of
100 mg
500 mg
500 mg
500 mg
100 mg
RAVs
50 mg QD 50 mg BID BID
b
No. of
BID
QD
QD
BID
(%)
c
RAVs
patients
(%)
with RAVs
RAVs
N=8
N=8
N=8
N=8
N=8
N=8
N=8
N = 32
N = 16
(%)c
V494V/A
—
—
—
1
—
—
—
1 (3.1%)
—
1 (2.1%)
I482I/L
—
1
—
—
—
—
1
1 (3.1%)
1 (7.7%)
2 (4.2%)
M423M/A/I/T/V
—
2
4
—
—
—
—
6 (18.75%) —
L419L/S/M/P/T/V/I —
3
3
3
3
4
6
12 (37.5%) 10 (62.5%) 22 (45.8%)
R422R/K
—
4
4
2
1
7
4
11 (34.4%) 11 (68.75%) 22 (45.8%)
A486A/V/I/T/M
1
7
4
4
2
8
5
17 (53.1%) 13 (81.2%) 30 (62.5%)
6 (86%)
5 (62.5%) 4 (50%)
No. of patients with 1 (12.5%) 8 (100%)
8 (100%) 6 (75%)
6 (12.5%)
23 (71.9%) 14 (87.5%) 37 (77.1%)
RAVs (%)
535
BID, twice daily; GT, genotype; QD, once daily; RAVs, resistance-associated variants.
536
a
Any patient who had mutant virus at Day 2 and/or Day 3 and/or Day 4 and/or Day 5 and/or Day 7 was counted.
537
b
RAVs detected as full mutant or mixture with wild-type.
538
c
Patients from the 50 mg QD cohort were not included because of the suboptimal antiviral response in this treatment group.
30
539
TABLE 6.
NS5B drug resistance variants observed at Day 17
GT1a
GT1a
GT1a
GT1a
GT1a
GT1b
GT1b
No. of
50 mg QD
50 mg BID 100 mg BID 500 mg BID 500 mg QD 500 mg QD 100 mg BID patients
RAVs
N=8
N=8
N=8
N=8
N=8
N=8
N=5
with RAVs
V494V/A
—
—
—
—
—
—
—
—
I482I/L
—
—
—
—
—
—
—
—
M423M/A/I/T/V
—
—
—
—
—
—
—
—
L419L/S/M/P/T V/I
—
—
—
—
1
1
3
5 (11.1%)
R422R/K
—
—
—
—
—
3
1
4 (8.9%)
A486A/V/I/T/M
—
—
—
—
—
6
3
9 (20.0%)
No. of patients with
0
0
0
0
1 (12.5%)
6 (75%)
3 (60%)
—
RAVs in each arm (%)
540
BID, twice daily; GT, genotype; QD, once daily; RAVs, resistance-associated variants.
31

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