Kelly A. Wong

A phase 1, randomized, placebo-controlled, 3-day, dose-ranging study of GS-5885, an NS5A inhibitor, in patients with genotype 1 hepatitis C

BACKGROUND & AIMS GS-5885 is an inhibitor of the hepatitis C virus (HCV) NS5A protein and exhibits potent suppression of genotype 1 HCV replicons. The safety, tolerability, pharmacokinetics, antiviral activity, and resistance profile of once-daily GS-5885 doses of 1-90 mg were evaluated in patients with chronic genotype 1 HCV. METHODS Genotype 1 HCV-infected patients were randomized to 3 days of once-daily (QD) dosing with placebo (n=12) or GS-5885 1 mg (n=10), 3 mg (n=10), 10 mg (n=20), 30 mg (n=10), or 90 mg (n=10). Plasma samples for pharmacokinetics, HCV RNA, and NS5A sequencing were collected through day 14. RESULTS GS-5885 was well tolerated and resulted in median maximal reductions in HCV RNA ranging from 2.3 log(10) IU/ml (1 mg QD) to 3.3 log(10) IU/ml (10 mg QD in genotype 1b and 30 mg QD). E(max) modeling indicated GS-5885 30 mg was associated with>95% of maximal antiviral response to HCV genotype 1a. HCV RNA reductions were generally more sustained among patients with genotype 1b vs. 1a. Three of 60 patients had a reduced response and harbored NS5A-resistant virus at baseline. NS5A sequencing identified residues 30 and 31 in genotype 1a, and 93 in genotype 1b as the predominant sites of mutation following GS-5885 dosing. Plasma pharmacokinetics was consistent with QD dosing. CONCLUSIONS During 3 days of monotherapy, low doses of GS-5885 demonstrated significant antiviral activity in genotype 1a and 1b HCV-infected patients. GS-5885 is currently being evaluated in combination with direct antiviral regimens with and without peginterferon.

Characterization of Hepatitis C Virus Resistance from a Multiple-Dose Clinical Trial of the Novel NS5A Inhibitor GS-5885

ABSTRACT GS-5885 is a novel hepatitis C virus (HCV) NS5A inhibitor. In a 3-day monotherapy study in treatment-naive genotype 1a (GT1a) and GT1b HCV-infected subjects, median viral load reductions ranged from 2.3 to 3.3 log10 HCV RNA IU/ml across dosing cohorts (1, 3, 10, 30, or 90 mg once daily). Here, we report viral sequencing and phenotypic analysis of clinical isolates from this study. Detection of baseline NS5A amino acid substitutions at positions 28, 30, 31, or 93 in GT1a was associated with a reduced treatment response. In the GT1b cohort, Y93H was detected in 100% of subjects at day 4 or 14. In the Gt1a cohort, population sequencing detected NS5A resistance-associated mutations at day 4 or 14 for 3/10 subjects at the 1-mg dose and for all subjects dosed at ≥3 mg. A subset of mutants that confer a low level of reduced susceptibility to GS-5885 was not detected by population sequencing at the 30- and 90-mg doses. Subject-derived M28T, Q30R, L31M, and Y93C mutations all conferred >30-fold reductions in GS-5885 and daclatasvir susceptibilities in vitro. Site-directed NS5A mutants also showed reduced susceptibility to GS-5885. However, all NS5A mutants tested remained fully susceptible to other classes of direct-acting antivirals (DAAs), interferon alpha, and ribavirin. Importantly, the nonoverlapping resistance profile and high potency of GS-5885 support its further development with other direct-acting antivirals for the treatment of chronic HCV. (This study has been registered at ClinicalTrials.gov under registration number NCT01193478.)

Susceptibility of Treatment-Naive Hepatitis C Virus (HCV) Clinical Isolates to HCV Protease Inhibitors

ABSTRACT In order to assess the natural variation in susceptibility to hepatitis C virus (HCV) NS3 protease inhibitors (PIs) among untreated HCV patient samples, the susceptibilities of 39 baseline clinical isolates were determined using a transient-replication assay on a panel of HCV PIs, including two α-ketoamides (VX-950 and SCH-503034) and three macrocyclic inhibitors (MK-7009, ITMN-191, and TMC-435350). Some natural variation in susceptibility to all HCV PIs tested was observed among the baseline clinical isolates. The susceptibility to VX-950 correlated strongly with the susceptibility to SCH-503034. A moderate correlation was observed between the susceptibilities to ITMN-191 and MK-7009. In contrast, the phenotypic correlations between the α-ketoamides and macrocyclic inhibitors were significantly lower. This difference is partly attributable to reduced susceptibility of the HCV variants containing the NS3 polymorphism Q80K (existing in 47% of genotype 1a isolates) to the macrocyclic compounds but no change in the sensitivity of the same variants to the α-ketoamides tested. Our results suggest that the natural variation in baseline susceptibility may contribute to different degrees of antiviral response among patients in vivo, particularly at lower doses.

Characterization of Resistance to the Protease Inhibitor GS-9451 in Hepatitis C Virus-Infected Patients

ABSTRACT GS-9451, a novel hepatitis C virus (HCV) nonstructural 3/4a (NS3/4a) protease inhibitor, is highly active in patients infected with HCV genotype 1 (GT 1). The aim of this study is to characterize the clinical resistance profile of GS-9451 in GT 1 HCV-infected patients in a phase 1, 3-day monotherapy study. The full-length NS3/4A gene was population sequenced at baseline, on the final treatment day, and at follow-up time points. NS3 protease domains from patient isolates with emerging mutations were cloned into an NS3 shuttle vector, and their susceptibilities to GS-9451 and other HCV inhibitors were determined using a transient replication assay. No resistance mutations at NS3 position 155, 156, or 168 were detected in any of the baseline samples or in viruses from patients treated with 60 mg of GS-9451 once daily. Among patients who received 200 mg and 400 mg of GS-9451, viruses with mutations at position D168 (D168E/G/V) and R155 (R155K), which confer high-level resistance to GS-9451, were detected in those with GT 1b and GT 1a virus, respectively. Viruses with D168 mutations were no longer detected in any GT 1b patient at day 14 and subsequent time points. In GT 1a patients, R155K mutants were replaced by the wild type in 57% of patients at week 24. These NS3 clinical mutants were sensitive to NS5B and NS5A inhibitors, as well as alpha interferon (IFN-α) and ribavirin. The lack of cross-resistance between GS-9451 and other classes of HCV inhibitors supports the utility of combination therapy.

The Combination of Alisporivir plus an NS5A Inhibitor Provides Additive to Synergistic Anti-Hepatitis C Virus Activity without Detectable Cross-Resistance

ABSTRACT Alisporivir (ALV), a cyclophilin inhibitor, is a host-targeting antiviral (HTA) with multigenotypic anti-hepatitis C virus (HCV) activity and a high barrier to resistance. Recent advances have supported the concept of interferon (IFN)-free regimens to treat chronic hepatitis C. As the most advanced oral HTA, ALV with direct-acting antivirals (DAAs) represents an attractive drug combination for IFN-free therapy. In this study, we investigated whether particular DAAs exhibit additive, synergistic, or antagonistic effects when combined with ALV. Drug combinations of ALV with NS3 protease, NS5B polymerase, and NS5A inhibitors were investigated in HCV replicons from genotypes 1a, 1b, 2a, 3, and 4a (GT1a to -4a). Combinations of ALV with DAAs exerted an additive effect on GT1 and -4. A significant and specific synergistic effect was observed with ALV-NS5A inhibitor combination on GT2 and -3. Furthermore, ALV was fully active against DAA-resistant variants, and ALV-resistant variants were fully susceptible to DAAs. ALV blocks the contact between cyclophilin A and domain II of NS5A, and NS5A inhibitors target domain I of NS5A; our data suggest a molecular basis for the use of these two classes of inhibitors acting on two distinct domains of NS5A. These results provide in vitro evidence that ALV with NS5A inhibitor combination represents an attractive strategy and a potentially effective IFN-free regimen for treatment of patients with chronic hepatitis C. Due to its high barrier and lack of cross-resistance, ALV could be a cornerstone drug partner for DAAs.

Inhibition of Hepatitis C Virus Replication by GS-6620, a Potent C-Nucleoside Monophosphate Prodrug

ABSTRACT As a class, nucleotide inhibitors (NIs) of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase offer advantages over other direct-acting antivirals, including properties, such as pangenotype activity, a high barrier to resistance, and reduced potential for drug-drug interactions. We studied the in vitro pharmacology of a novel C-nucleoside adenosine analog monophosphate prodrug, GS-6620. It was found to be a potent and selective HCV inhibitor against HCV replicons of genotypes 1 to 6 and against an infectious genotype 2a virus (50% effective concentration [EC50], 0.048 to 0.68 μM). GS-6620 showed limited activities against other viruses, maintaining only some of its activity against the closely related bovine viral diarrhea virus (EC50, 1.5 μM). The active 5′-triphosphate metabolite of GS-6620 is a chain terminator of viral RNA synthesis and a competitive inhibitor of NS5B-catalyzed ATP incorporation, with Ki/Km values of 0.23 and 0.18 for HCV NS5B genotypes 1b and 2a, respectively. With its unique dual substitutions of 1′-CN and 2′-C-Me on the ribose ring, the active triphosphate metabolite was found to have enhanced selectivity for the HCV NS5B polymerase over host RNA polymerases. GS-6620 demonstrated a high barrier to resistance in vitro. Prolonged passaging resulted in the selection of the S282T mutation in NS5B that was found to be resistant in both cellular and enzymatic assays (>30-fold). Consistent with its in vitro profile, GS-6620 exhibited the potential for potent anti-HCV activity in a proof-of-concept clinical trial, but its utility was limited by the requirement of high dose levels and pharmacokinetic and pharmacodynamic variability.

Alisporivir plus ribavirin, interferon free or in combination with pegylated interferon, for hepatitis C virus genotype 2 or 3 infection

Alisporivir is a cyclophilin inhibitor with pan‐genotypic anti–hepatitis C virus (HCV) activity and a high barrier to viral resistance. The VITAL‐1 study assessed alisporivir as interferon (IFN)‐free therapy in treatment‐naïve patients infected with HCV genotype 2 or 3. Three hundred forty patients without cirrhosis were randomized to: arm 1, alisporivir (ALV) 1,000 mg once‐daily (QD); arm 2, ALV 600 mg QD and ribavirin (RBV); arm 3, ALV 800 mg QD and RBV; arm 4, ALV 600 mg QD and pegylated IFN (Peg‐IFN); or arm 5, Peg‐IFN and RBV. Patients receiving IFN‐free ALV regimens who achieved rapid virological response (RVR) continued the same treatment throughout, whereas those with detectable HCV RNA at week 4 received ALV, RBV, and Peg‐IFN from weeks 6 to 24. Overall, 300 patients received ALV‐based regimens. In arm 1 to arm 4, the intent‐to‐treat rates of sustained virological response (SVR) 24 weeks after treatment (SVR24) were from 80% to 85%, compared with 58% (n = 23 of 40) with Peg‐IFN/RBV. Per‐protocol analysis showed higher SVR24 rates in patients who received ALV/RBV, IFN‐free after RVR (92%; n = 56 of 61) than with ALV alone after RVR (72%; n = 13 of 18) or with Peg‐IFN/RBV (70%; n = 23 of 33). Both RVRs and SVRs to ALV IFN‐free regimens were numerically higher in genotype 3– than in genotype 2–infected patients. Viral breakthrough was infrequent (3%; n = 7 of 258). IFN‐free ALV treatment showed markedly better safety/tolerability than IFN‐containing regimens. Conclusions: ALV plus RBV represents an effective IFN‐free option for a proportion of patients with HCV genotype 2 or 3 infections, with high SVR rates for patients with early viral clearance. Further investigations of ALV in IFN‐free combination regimens with direct‐acting antiviral drugs deserve exploration in future trials. (Hepatology 2015;62:1013‐1023)

Tegobuvir (GS-9190) potency against HCV chimeric replicons derived from consensus NS5B sequences from genotypes 2b, 3a, 4a, 5a, and 6a

With the exception of nucleoside analogs, few direct acting antivirals in clinical development are active across the six major hepatitis C virus genotypes. We report novel consensus sequence chimeras for genotypes 2b, 3a, 4a, 5a, and 6a NS5B and show variable susceptibilities over a panel of NS5B inhibitors. Tegobuvir (GS-9190) had EC(50)s of <16 nM against genotype 1 and >100 nM for other genotypes tested here. An NS5B F445C mutation engineered into the GT3a, 4a, and 6a chimeric replicons lowered the tegobuvir EC(50) to levels comparable to those for genotype 1a, but did not considerably alter the EC(50) of site 2 or nucleoside analog inhibitors. These data support the use of HCV chimeras in profiling direct acting antivirals across genotypes and specifically determines the impact of the C445F NS5B polymorphism on tegobuvir potency against genotypes 3a, 4a, and 6a.

Preclinical and Clinical Resistance Profile of EDP-239, a Novel Hepatitis C Virus NS5A Inhibitor

ABSTRACT EDP-239, a potent and selective hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor developed for the treatment of HCV infection, has been investigated in vitro and in vivo. This study sought to characterize genotypic changes in the HCV NS5A sequence of genotype 1 (GT1) replicons and to compare those changes to GT1 viral RNA mutations isolated from clinical trial patients. Resistance selection experiments in vitro using a subgenomic replicon identified resistance-associated mutations (RAMs) at GT1a NS5A amino acid positions 24, 28, 30, 31, and 93 that confer various degrees of resistance to EDP-239. Key RAMs were similarly identified in GT1b NS5A at amino acid positions 31 and 93. Mutations F36L in GT1a and A92V in GT1b do not confer resistance to EDP-239 individually but were found to enhance the resistance of GT1a K24R and GT1b Y93H. RAMs were identified in GT1 patients at baseline or after dosing with EDP-239 that were similar to those detected in vitro. Baseline RAMs identified at NS5A position 93 in GT1, or positions 28 or 30 in GT1a only, correlated with a reduced treatment response. RAMs at additional positions were also detected and may have contributed to reduced EDP-239 efficacy. The most common GT1a and GT1b RAMs found to persist up to weeks 12, 24, or 48 were those at NS5A positions 28, 30, 31, 58 (GT1a only), and 93. Those RAMs persisting at the highest frequencies up to weeks 24 or 48 were L31M and Q30H/R for GT1a and L31M and Y93H for GT1b. (This study has been registered at ClinicalTrials.gov under identifier NCT01856426.)

1214 MULTIPLE MUTATIONS IN DOMAIN II OF NS5A IDENTIFIED IN GENOTYPE 2/3 VITAL-1 STUDY BREAKTHROUGH PATIENTS ARE REQUIRED TO REDUCE SUSCEPTIBILITY TO ALISPORIVIR IN VITRO

the ribavirin-sparing arm were excluded because of their high virological failure rates. Separate logistic regression models were obtained for FDV and BI207127 for plasma concentrations obtained during different time windows in the first month of therapy. Results: Patients with GT1b (any IL28B genotype) had flat or shallow relationships between trough concentrations of FDV and BI207127 and predicted SVR12. The predicted SVR12 was high throughout the concentration ranges examined. For both drugs there was a significantly greater effect of trough concentration on SVR12 in GT1a-CC patients compared with GT1b patients; GT1a-CC patients had predicted SVR12 >70% only at higher concentrations. Conclusions: GT1b-infected patients carrying any IL28B genotype are predicted to achieve high SVR rates across broad plasma concentration ranges of FDV and BI207127 given in combination. The results suggest that the daily doses tested in SOUND-C2 give adequate plasma concentrations in GT1b-infected patients. GT1aCC patients are predicted to require higher levels of FDV and BI207127. Further investigations of plasma concentration-response relationships in various patient subgroups, factors contributing to low plasma levels, and potential confounders, should be performed. Phase III trials of FDV 120mg QD plus BI207127 600mg BID plus RBV for 16 or 24 weeks are ongoing in GT1b-infected patients.