Efficacy and safety of elbasvir/grazoprevir in treatment-naive Chinese adults with hepatitis C virus infection: A randomized trial.

BACKGROUND AND AIM: In China, clinical experience with direct-acting antiviral treatments for hepatitis C virus (HCV) infection is still emerging. C-CORAL is a phase 3, multinational, placebo-controlled, double-blind trial of elbasvir/grazoprevir (EBR/GZR) in participants with HCV infection from the Asia-Pacific region and Russia. Here, we report the data from participants enrolled in China. METHODS: Treatment-naive participants with chronic HCV genotype (GT) 1, GT4, or GT6 infection were randomly assigned to receive 50 mg EBR/100 mg GZR for 12 weeks (immediate-treatment group, ITG) or placebo followed by deferred treatment with EBR/GZR (deferred-treatment group, DTG). The primary efficacy end-point was sustained virologic response at 12 weeks after completing treatment (SVR12), and the primary safety end-point was a comparison of safety between participants receiving EBR/GZR and placebo (NCT02251990; Protocol PN-5172-067). RESULTS: A total of 152 participants in China were randomly assigned (ITG, n = 115; DTG, n = 37). SVR12 was achieved in 96.7% (146/151) participants overall and in 97.3% (142/146) of those with GT1b infection. Four participants relapsed (GT1b, n = 3; GT6a, n = 1). Drug-related AEs were reported in 25 (21.7%) and 9 (24.3%) participants receiving EBR/GZR and placebo, respectively; no drug-related serious adverse events (AEs) occurred. Two (1.7%) participants receiving EBR/GZR had late hepatic transaminase elevations. Patient-reported outcomes indicate improved quality of life at follow-up week 4 in participants receiving EBR/GZR compared to placebo. CONCLUSION: EBR/GZR administered for 12 weeks represents a highly effective and safe treatment option for Chinese individuals with HCV GT1 infection.

Interplay of Amino Acid Residues at Positions 28 and 31 in NS5A Defines Resistance Pathways in HCV GT2.

Hepatitis C virus (HCV) genotype (GT) 2 represents approximately 9% of all viral infections globally. While treatment outcomes for GT2-infected patients have improved substantially with direct-acting antiviral agents (DAAs) compared to interferon-α, the presence of polymorphisms in NS5A can impact efficacy of NS5A inhibitor-containing regimens. Thus, pathways of NS5A resistance were explored in GT2 subtypes using elbasvir, an NS5A inhibitor with broad genotype activity. Resistance selection studies, resistance analysis in NS5A-inhibitor treated virologic failures, antiviral activities in replicons bearing a panel of GT2 subtype sequences and amino acid substitutions introduced by site-directed mutagenesis were performed to define determinants of inhibitor susceptibility. Elbasvir showed differential antiviral activity in replicons bearing GT2 sequences. The EC50 values for replicons bearing reference NS5A sequences for GT2a and GT2b were 0.003 and 3.4 nanomolar (nM) respectively. Studies with recombinant replicons demonstrated crosstalk between amino acid positions 28 and 31. The combination of phenylalanine and methionine at positions 28 and 31 respectively, conferred the highest potency reduction for elbasvir in GT2a and GT2b. This combination was observed in failures from the C-SCAPE trial. Addition of grazoprevir, an NS3/4A protease inhibitor, to elbasvir more effectively suppressed the emergence of resistance in GT2 at modest inhibitor concentrations (3X EC90). Ruzasvir, a potent, pan-genotype NS5A inhibitor successfully inhibited replicons bearing GT2 resistance-associated substitutions (RASs) at positions 28 and 31. The studies demonstrate crosstalk between amino acids at positions 28 and 31 in NS5A modulate inhibitor potency and may impact treatment outcomes in some HCV GT2-infected patients.

Efficacy and safety of elbasvir/grazoprevir in participants with hepatitis C virus genotype 1, 4, or 6 infection from the Asia-Pacific region and Russia: Final results from the randomized C-CORAL study.

BACKGROUND AND AIM: Although treatment with direct-acting antivirals has dramatically improved morbidity and mortality attributable to chronic hepatitis C virus infection, universal access to these medicines has been slow in the Asia-Pacific region and Russia. This study evaluated efficacy and safety of elbasvir/grazoprevir in participants with hepatitis C virus infection from Asia-Pacific countries and Russia (C-CORAL). METHODS: C-CORAL was a phase 3, randomized, placebo-controlled study (NCT02251990). Treatment-naive, HIV-negative, cirrhotic and non-cirrhotic participants with chronic hepatitis C genotype 1, 4, or 6 infection were randomized to elbasvir 50 mg/grazoprevir 100 mg once daily for 12 weeks (immediate-treatment group) or placebo followed by deferred treatment with elbasvir/grazoprevir (deferred-treatment group). The primary efficacy outcome was sustained virologic response at 12 weeks, and the primary safety outcome was a comparison between the immediate-treatment group and placebo phase of the deferred-treatment group. RESULTS: A total of 489 participants were randomized (immediate-treatment group, n = 366; deferred-treatment group, n = 123). Sustained virologic response at 12 weeks in the combined immediate/deferred-treatment groups was 94.4% (459/486; 95% confidence interval = 92.4-96.5%). Sustained virologic response at 12 weeks was 98.2% in participants with genotype 1b, 91.9% with genotype 1a, and 66.7% with genotype 6 infection. Similar rates of adverse events and drug-related adverse events were seen in the immediate-treatment group versus placebo phase of the deferred-treatment group (51.0% vs 50.4% and 21.4% vs 21.1%). CONCLUSIONS: Elbasvir/grazoprevir for 12 weeks represents an effective and well-tolerated treatment option for treatment-naive people with genotype 1 infection from Asia-Pacific countries and Russia.

Characterization of hepatitis C virus resistance to grazoprevir reveals complex patterns of mutations following on-treatment breakthrough that are not observed at relapse.

PURPOSE: A detailed analysis of hepatitis C virus (HCV) resistance-associated substitutions (RASs) is required to understand why people fail direct-acting antiviral therapies. This study was conducted to assess RASs in an analysis of 2 trials evaluating the second-generation NS3/4A protease inhibitor grazoprevir (GZR) in combination with peginterferon/ribavirin. PATIENTS AND METHODS: From a total of 113 participants with HCV genotype 1 infection, RASs were evaluated in 25 patients who relapsed and 6 patients with on-treatment virologic breakthrough using consensus Sanger and clonal sequence analysis of NS3/NS4a genes, with in vitro testing of replicon mutants. Next-generation sequencing (NGS) was used in a subset of participants to assess minority variants and the evolution of the whole viral genome. RESULTS: Baseline RASs did not predict treatment failure. Relapse was most commonly associated with RASs at D168, although additional RASs (Y56, R155 and A156) were also detected, particularly in participants with on-treatment breakthrough. Treatment-emergent RASs usually reverted to wild-type (WT), suggesting these mutations were associated with a negative fitness cost (confirmed using in vitro assays). NGS was the most sensitive assay for the detection of minor variants. Significant viral sequence divergence (up to 5.9% codons) was observed across whole genomes in association with the acquisition and reversion of RASs. CONCLUSION: Relapse with GZR and peginterferon/ribavirin is commonly associated with single RASs in NS3 that generally revert to WT, while breakthrough follows more complex patterns of viral resistance. NGS suggests that large diverse pools of viral quasispecies that emerge with RASs facilitate rapid viral evolution.

In Vitro Antiviral Profile of Ruzasvir, a Potent and Pangenotype Inhibitor of Hepatitis C Virus NS5A.

Inhibition of NS5A has emerged as an attractive strategy to intervene in hepatitis C virus (HCV) replication. Ruzasvir (formerly MK-8408) was developed as a novel NS5A inhibitor to improve upon the potency and barrier to resistance of early compounds. Ruzasvir inhibited HCV RNA replication with 50% effective concentrations (EC50s) of 1 to 4 pM in Huh7 or Huh7.5 cells bearing replicons for HCV genotype 1 (GT1) to GT7. The antiviral activity was modestly (10-fold) reduced in the presence of 40% normal human serum. The picomolar potency in replicon cells extended to sequences of clinical isolates available in public databases that were synthesized and tested as replicons. In GT1a, ruzasvir inhibited common NS5A resistance-associated substitutions (RASs), with the exception of M28G. De novo resistance selection studies identified pathways with certain amino acid substitutions at residues 28, 30, 31, and 93 across genotypes. Substitutions at position 93 were more common in GT1 to -4, while changes at position 31 emerged frequently in GT5 and -6. With the exception of GT4, the reintroduction of selected RASs conferred a ≥100-fold potency reduction in the antiviral activity of ruzasvir. Common RASs from other classes of direct-acting antiviral agents (DAAs) did not confer cross-resistance to ruzasvir. The interaction of ruzasvir with an NS3/4A protease inhibitor (grazoprevir) and an NS5B polymerase prodrug (uprifosbuvir) was additive to synergistic, with no evidence of antagonism or cytotoxicity. The antiviral profile of ruzasvir supported its further evaluation in human trials in combination with grazoprevir and uprifosbuvir.

Evolutionary pathways to NS5A inhibitor resistance in genotype 1 hepatitis C virus.

Direct-acting antivirals (DAAs) targeting NS5A are broadly effective against hepatitis C virus (HCV) infections, but sustained virological response rates are generally lower in patients infected with genotype (gt)-1a than gt-1b viruses. The explanation for this remains uncertain. Here, we adopted a highly accurate, ultra-deep primer ID sequencing approach to intensively study serial changes in the NS5A-coding region of HCV in gt-1a- and gt-1b-infected subjects receiving a short course of monotherapy with the NS5A inhibitor, elbasvir. Low or undetectable levels of viremia precluded on-treatment analysis in gt-1b-infected subjects, but variants with the resistance-associated substitution (RAS) Y93H in NS5A dominated rebounding virus populations following cessation of treatment. These variants persisted until the end of the study, two months later. In contrast, while Y93H emerged in multiple lineages and became dominant in subjects with gt-1a virus, these haplotypes rapidly decreased in frequency off therapy. Substitutions at Q30 and L31 emerged in distinctly independent lineages at later time points, ultimately coming to dominate the virus population off therapy. Consistent with this, cell culture studies with gt-1a and gt-1b reporter viruses and replicons demonstrated that Y93H confers a much greater loss of replicative fitness in gt-1a than gt-1b virus, and that L31M/V both compensates for the loss of fitness associated with Q30R (but not Y93H) and also boosts drug resistance. These observations show how differences in the impact of RASs on drug resistance and replicative fitness influence the evolution of gt-1a and gt-1b viruses during monotherapy with an antiviral targeting NS5A.

Elbasvir/grazoprevir in Asia-Pacific/Russian participants with chronic hepatitis C virus genotype 1, 4, or 6 infection.

The prevalence of hepatitis C virus (HCV) infection in Asian countries is high. This study assessed the efficacy and safety of elbasvir/grazoprevir (EBR/GZR) in participants with HCV infection from Asia-Pacific countries and Russia. In this phase 3, randomized, placebo-controlled, double-blind study, treatment-naive participants with HCV genotype (GT) 1, 4, or 6 infection were randomized to EBR 50 mg/GZR 100 mg (immediate-treatment group [ITG]) or placebo (deferred-treatment group [DTG]) once daily for 12 weeks (Protocol PN-5172-067, NCT02251990). The primary efficacy variable was a nonrandomized comparison of sustained virologic response at 12 weeks after the end of therapy (SVR12) for the ITG with a historical control. The primary safety outcome was a randomized comparison between the ITG and DTG. Three hundred thirty-seven participants were randomized to the ITG (n = 251) or DTG (n = 86); 199 (59.2%) participants were Asian, and 250 (74.4%) had HCV GT1b infection. Overall, 232/250 (92.8%) participants in the ITG achieved SVR12 (97.5% confidence interval, 89.1, 96.5). Of the 18 participants who failed to attain SVR12, 1 was lost to follow-up and 17 had virologic failure, 13 of whom had HCV GT6 infection. The incidence of adverse events was similar between participants receiving EBR/GZR and placebo (50.8% versus 51.2%; difference, -0.3%; 95% confidence interval, -12.3, 11.9). Conclusion: EBR/GZR for 12 weeks provides an effective and well-tolerated regimen for chronic HCV GT1 infection in treatment-naive people from Asia-Pacific countries and Russia, particularly for the large population with GT1b infection. EBR/GZR is not recommended for the treatment of individuals with HCV GT6 infection. (Hepatology Communications 2018;2:595-606).

MK-8325: A silyl proline-containing NS5A inhibitor with pan-genotype activity for treatment of HCV.

HCV NS5A inhibitors have shown impressive in vitro potency profiles in HCV replicon assays thus making them attractive components for inclusion in an all oral fixed dose combination regimen. Herein, we describe the discovery and characterization of silyl proline-containing HCV NS5A inhibitor MK-8325 with good pan-genotype activity and acceptable pharmacokinetic properties.

Discovery of MK-6169, a Potent Pan-Genotype Hepatitis C Virus NS5A Inhibitor with Optimized Activity against Common Resistance-Associated Substitutions.

We describe the discovery of MK-6169, a potent and pan-genotype hepatitis C virus NS5A inhibitor with optimized activity against common resistance-associated substitutions. SAR studies around the combination of changes to both the valine and aminal carbon region of elbasvir led to the discovery of a series of compounds with substantially improved potency against common resistance-associated substitutions in the major genotypes, as well as good pharmacokinetics in both rat and dog. Through further optimization of key leads from this effort, MK-6169 (21) was discovered as a preclinical candidate for further development.

Antiviral Activity and Resistance Analysis of NS3/4A Protease Inhibitor Grazoprevir and NS5A Inhibitor Elbasvir in Hepatitis C Virus GT4 Replicons.

Although genotype 4 (GT4)-infected patients represent a minor overall percentage of the global hepatitis C virus (HCV)-infected population, the high prevalence of the genotype in specific geographic regions coupled with substantial sequence diversity makes it an important genotype to study for antiviral drug discovery and development. We evaluated two direct-acting antiviral agents-grazoprevir, an HCV NS3/4A protease inhibitor, and elbasvir, an HCV NS5A inhibitor-in GT4 replicons prior to clinical studies in this genotype. Following a bioinformatics analysis of available GT4 sequences, a set of replicons bearing representative GT4 clinical isolates was generated. For grazoprevir, the 50% effective concentration (EC50) against the replicon bearing the reference GT4a (ED43) NS3 protease and NS4A was 0.7 nM. The median EC50 for grazoprevir against chimeric replicons encoding NS3/4A sequences from GT4 clinical isolates was 0.2 nM (range, 0.11 to 0.33 nM; n = 5). The difficulty in establishing replicons bearing NS3/4A resistance-associated substitutions was substantially overcome with the identification of a G162R adaptive substitution in NS3. Single NS3 substitutions D168A/V identified from de novo resistance selection studies reduced grazoprevir antiviral activity by 137- and 47-fold, respectively, in the background of the G162R replicon. For elbasvir, the EC50 against the replicon bearing the reference full-length GT4a (ED43) NS5A gene was 0.0002 nM. The median EC50 for elbasvir against chimeric replicons bearing clinical isolates from GT4 was 0.0007 nM (range, 0.0002 to 34 nM; n = 14). De novo resistance selection studies in GT4 demonstrated a high propensity to suppress the emergence of amino acid substitutions that confer high-potency reductions to elbasvir. Phenotypic characterization of the NS5A amino acid substitutions identified (L30F, L30S, M31V, and Y93H) indicated that they conferred 15-, 4-, 2.5-, and 7.5-fold potency losses, respectively, to elbasvir. The activity profiles of grazoprevir and elbasvir supported the testing of the direct-acting antivirals in clinical studies.

Unraveling the structural basis of grazoprevir potency against clinically relevant substitutions in hepatitis C virus NS3/4A protease from genotype 1a.

Grazoprevir is a potent pan-genotype and macrocyclic inhibitor of hepatitis C virus (HCV) NS3/4A protease and was developed for treating chronic HCV infection. In HCV genotype (GT) 1a, grazoprevir maintains potent activity against a majority of NS3 resistance-associated amino acid substitutions, including the highly prevalent and naturally occurring Q80K polymorphism that impacts simeprevir, another NS3/4A protease inhibitor. The basis for an unexpected difference in the clinical impact of some NS3 substitutions was investigated. Phenotypic analysis of resistance-associated substitutions identified in NS3 from GT1a-infected patients who failed therapy with grazoprevir (in combination with elbasvir, an inhibitor of HCV NS5A protein) showed that positions 56, 156, and 168 in NS3 were most impactful because they diminished protein-inhibitor interactions. Although an amino acid substitution from aspartic acid to alanine at position 168 (D168A) reduced the potency of grazoprevir, its combination with R155K unexpectedly nullified this effect. Molecular dynamics and free-energy surface studies indicated that Asp-168 is important in anchoring Arg-155 for ligand binding but is not critical for Lys-155 because of the inherent flexibility of its side chain. Moreover, modeling studies supported a strong direct cation-heterocycle interaction between the Lys-155 side chain of the double substitution, R155K/D168A, and the lone pair on the quinoxaline in grazoprevir. This unique interaction provides a structural basis for grazoprevir's higher potency than simeprevir, an inhibitor to which the double substitution confers a significant reduction in potency. Our findings are consistent with the detection of R155K/D168A in NS3 from virologic failures treated with simeprevir but not grazoprevir.

Discovery of Ruzasvir (MK-8408): A Potent, Pan-Genotype HCV NS5A Inhibitor with Optimized Activity against Common Resistance-Associated Polymorphisms.

We describe the research that led to the discovery of compound 40 (ruzasvir, MK-8408), a pan-genotypic HCV nonstructural protein 5A (NS5A) inhibitor with a "flat" GT1 mutant profile. This NS5A inhibitor contains a unique tetracyclic indole core while maintaining the imidazole-proline-valine Moc motifs of our previous NS5A inhibitors. Compound 40 is currently in early clinical trials and is under evaluation as part of an all-oral DAA regimen for the treatment of chronic HCV infection.

Discovery of Chromane Containing Hepatitis C Virus (HCV) NS5A Inhibitors with Improved Potency against Resistance-Associated Variants.

The discovery of potent and pan-genotypic HCV NS5A inhibitors faces many challenges including the significant diversity among genotypes, substantial potency shift conferred on some key resistance-associated variants, inconsistent SARs between different genotypes and mutants, and the lacking of models of inhibitor/protein complexes for rational inhibitor design. As part of ongoing efforts on HCV NS5A inhibition at Merck, we now describe the discovery of a novel series of chromane containing NS5A inhibitors. SAR studies around the "Z" group of the tetracyclic indole scaffold explored fused bicyclic rings as alternates to the phenyl group of elbasvir (1, MK-8742) and identified novel chromane and 2,3-dihydrobenzofuran derivatives as "Z" group replacements offered good potency across all genotypes. This effort, incorporating the C-1 fluoro substitution at the tetracyclic indole core, led to the discovery of a new series of NS5A inhibitors, such as compounds 14 and 25-28, with significantly improved potency against resistance-associated variants, such as GT2b, GT1a Y93H, and GT1a L31V. Compound 14 also showed reasonable PK exposures in preclinical species (rat and dog).

Alternative core development around the tetracyclic indole class of HCV NS5A inhibitors.

Herein, we describe our research efforts to develop unique cores in molecules which function as HCV nonstructural protein 5A (NS5A) inhibitors. In particular, various fused tetracyclic cores were identified which showed genotype and mutant activities comparable to the indole-based tetracyclic core.

Structure-activity relationships of proline modifications around the tetracyclic-indole class of NS5A inhibitors.

We describe the impact of proline modifications, in our tetracyclic-indole based series of nonstructural protein 5A (NS5A) inhibitors, to their replicon profiles. This work identified NS5A inhibitors with an improved and flattened resistance profiles.

Substituted tetracyclic indole core derivatives of HCV NS5A inhibitor MK-8742.

As part of an ongoing effort in NS5A inhibition at Merck we now describe our efforts for introducing substitution around the tetracyclic indole core of MK-8742. Fluoro substitution on the core combined with the fluoro substitutions on the proline ring improved the potency against GT1a Y93H significantly. However, no improvement on GT2b potency was achieved. Limiting the fluoro substitution to C-1 of the tetracyclic indole core had a positive impact on the potency against the resistance associated variants, such as GT1a Y93H and GT2b, and the PK profile as well. Compounds, such as 62, with reduced potency shifts between wild type GT1a to GT2b, GT1a Y93H, and GT1a L31V were identified.

Aryl or heteroaryl substituted aminal derivatives of HCV NS5A inhibitor MK-8742.

Herein we describe our research efforts around the aryl and heteroaryl substitutions at the aminal carbon of the tetracyclic indole-based HCV NS5A inhibitor MK-8742. A series of potent NS5A inhibitors are described, such as compounds 45-47, 54, 56, and 65, which showed improved potency against clinically relevant and resistance associated HCV variants. The improved potency profiles of these compounds demonstrated an SAR that can improve the potency against GT2b, GT1a Y93H, and GT1a L31V altogether, which was unprecedented in our previous efforts in NS5A inhibition.

Matched and mixed cap derivatives in the tetracyclic indole class of HCV NS5A inhibitors.

A matched and mixed capping SAR study was conducted on the tetracyclic indole class of HCV NS5A inhibitors to examine the influence of modifications of this region on the overall HCV virologic resistance profiles.

Alkyl substituted aminal derivatives of HCV NS5A inhibitor MK-8742.

HCV NS5A inhibitors have demonstrated impressive in vitro potency profiles in HCV replicon assays and robust HCV RNA titer reduction in the clinic making them attractive components for inclusion in an all oral fixed dose combination regimen for the treatment of HCV infection. Herein we describe our continued research efforts around the alkyl "Z group" modification of the tetracyclic indole-based NS5A inhibitor MK-8742, which led to the discovery of a series of potent NS5A inhibitors. Compounds 10 and 19 are of particular interests since they are as potent as our previous leads and have much improved rat pharmacokinetic profiles.

Discovery of potent macrocyclic HCV NS5A inhibitors.

HCV NS5A inhibitors have demonstrated impressive in vitro virologic profiles in HCV replicon assays and robust HCV RNA titer reduction in the clinic making them attractive components for inclusion in an all oral fixed-dose combination (FDC) regimen for the treatment of HCV infection. Merck's effort in this area identified MK-4882 and MK-8325 as early development leads. Herein, we describe the discovery of potent macrocyclic NS5A inhibitors bearing the MK-8325 or MK-4882 core structure.