Anita Y. M. Howe

Boceprevir versus placebo with pegylated interferon alfa-2b and ribavirin for treatment of hepatitis C virus genotype 1 in patients with HIV: a randomised, double-blind, controlled phase 2 trial.

BACKGROUND Rates of sustained virological response (SVR) to peginterferon-ribavirin are low in patients with hepatitis C virus (HCV) genotype 1 and HIV. We aimed to assess efficacy and safety of triple therapy with boceprevir plus pegylated interferon alfa-2b (peginterferon) and ribavirin, which increases rates of SVR in patients with HCV alone. METHODS In our double-blind, randomised controlled phase 2 trial, we enrolled adults (18-65 years) with untreated HCV genotype 1 infection and controlled HIV (HIV RNA <50 copies per mL) at 30 academic and non-academic study sites. We randomly allocated patients (1:2) according to a computer generated sequence, stratified by Metavir score and baseline HCV RNA level, to receive peginterferon 1·5 μg/kg per week with weight-based ribavirin (600-1400 mg per day) for 4 weeks, followed by peginterferon-ribavirin plus either placebo (control group) or 800 mg boceprevir three times per day (boceprevir group) for 44 weeks. Non-nucleoside reverse-transcriptase inhibitors, zidovudine, and didanosine were not permitted. The primary efficacy endpoint was SVR (defined as undetectable plasma HCV RNA) at follow-up week 24 after end of treatment. We assessed efficacy and safety in all patients who received at least one dose of study drug. This study is registered with ClinicalTrials.gov, number NCT00959699. FINDINGS From Jan 15, 2010, to Dec 29, 2010, we enrolled 99 patients, 98 of whom received at least one treatment dose. 40 (63%) of 64 patients in the boceprevir group had an SVR at follow-up week 24, compared with ten (29%) of 34 control patients (difference 33·1%, 95% CI 13·7-52·5; p=0·0008). Adverse events were more common in patients who received boceprevir than in control patients: 26 (41%) versus nine (26%) had anaemia, 23 (36%) versus seven (21%) pyrexia, 22 (34%) versus six (18%) decreased appetite, 18 (28%) versus five (15%) dysgeusia, 18 (28%) versus five (15%) vomiting, and 12 (19%) versus two (6%) neutropenia. Three patients who received boceprevir plus peginterferon-ribavirin and four controls had HIV virological breakthrough. INTERPRETATION Boceprevir in combination with peginterferon-ribavirin could be an important therapeutic option for patients with HCV and HIV. FUNDING Merck.

HCV796: A selective nonstructural protein 5B polymerase inhibitor with potent anti‐hepatitis C virus activity In Vitro, in mice with chimeric human livers, and in humans infected with hepatitis C virus

Anti‐hepatitis C virus (HCV) drug development has been challenged by a lack of experience with inhibitors inclusive of in vitro, animal model, and clinical study. This manuscript outlines activity and correlation across such a spectrum of models and into clinical trials with a novel selective nonstructural protein 5B (NS5B) polymerase inhibitor, HCV796. Enzyme assays yielded median inhibitory concentration (IC50) values of 0.01 to 0.14 μM for genotype 1, with half maximal effective concentration (EC50s) of 5 nM and 9 nM against genotype 1a and 1b replicons. In the chimeric mouse model, a 2.02 ± 0.55 log reduction in HCV titer was seen with monotherapy, whereas a suboptimal dose of 30 mg/kg three times per day in combination with interferon demonstrated a 2.44 log reduction (P = 0.001 versus interferon alone) Clinical outcomes in combination with pegylated interferon and ribavirin have revealed additive efficacy in treatment naïve patients. Abnormal liver function test results were observed in 8% of HCV‐796 patients treated for over 8 weeks, resulting in suspension of further trial activity. Conclusion: The RNA‐dependent RNA polymerase inhibitor HCV796 demonstrated potent anti‐HCV activity consistently through enzyme inhibition assays, subgenomic replicon, and chimeric mouse studies. Strong correlations of outcomes in the mouse model were seen with subsequent clinical trials, including a plateau in dose‐related antiviral activity and additive impact from combination therapy with interferon. These outcomes demonstrate the utility of the range of in vitro and in vivo models now available for anti‐HCV drug development and support the potential utility of polymerase inhibitors in future combination therapies for HCV treatment. (HEPATOLOGY 2009.)

Elbasvir-grazoprevir to treat hepatitis C virus infection in persons receiving opioid agonist therapy a randomized trial

Background Hepatitis C virus (HCV) infection is common in persons who inject drugs (PWID). Objective To evaluate elbasvir-grazoprevir in treating HCV infection in PWID. Design Randomized, placebo-controlled, double-blind trial. (ClinicalTrials.gov: NCT02105688). Setting Australia, Canada, France, Germany, Israel, the Netherlands, New Zealand, Norway, Spain, Taiwan, the United Kingdom, and the United States. Patients 301 treatment-naive patients with chronic HCV genotype 1, 4, or 6 infection who were at least 80% adherent to visits for opioid agonist therapy (OAT). Intervention The immediate-treatment group (ITG) received elbasvir-grazoprevir for 12 weeks; the deferred-treatment group (DTG) received placebo for 12 weeks, no treatment for 4 weeks, then open-label elbasvir-grazoprevir for 12 weeks. Measurements The primary outcome was sustained virologic response at 12 weeks (SVR12), evaluated separately in the ITG and DTG. Other outcomes included SVR24, viral recurrence or reinfection, and adverse events. Results The SVR12 was 91.5% (95% CI, 86.8% to 95.0%) in the ITG and 89.5% (95% CI, 81.5% to 94.8%) in the active phase of the DTG. Drug use at baseline and during treatment did not affect SVR12 or adherence to HCV therapy. Among 18 patients with posttreatment viral recurrence through 24-week follow-up, 6 had probable reinfection. If the probable reinfections were assumed to be responses, SVR12 was 94.0% (CI, 89.8% to 96.9%) in the ITG. One patient in the ITG (1 of 201) and 1 in the placebo-phase DTG (1 of 100) discontinued treatment because of an adverse event. Limitation These findings may not be generalizable to PWID who are not receiving OAT, nor do they apply to persons with genotype 3 infection, a common strain in PWID. Conclusion Patients with HCV infection who were receiving OAT and treated with elbasvir-grazoprevir had high rates of SVR12, regardless of ongoing drug use. These results support the removal of drug use as a barrier to interferon-free HCV treatment for patients receiving OAT. Primary Funding Source Merck & Co.

Molecular Mechanism of Hepatitis C Virus Replicon Variants with Reduced Susceptibility to a Benzofuran Inhibitor, HCV-796

ABSTRACT HCV-796 selectively inhibits hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. In hepatoma cells containing a genotype 1b HCV replicon, HCV-796 reduced HCV RNA levels by 3 to 4 log10 HCV copies/μg total RNA (the concentration of the compound that inhibited 50% of the HCV RNA level was 9 nM). Cells bearing replicon variants with reduced susceptibility to HCV-796 were generated in the presence of HCV-796, followed by G418 selection. Sequence analysis of the NS5B gene derived from the replicon variants revealed several amino acid changes within 5 Å of the drug-binding pocket. Specifically, mutations were observed at Leu314, Cys316, Ile363, Ser365, and Met414 of NS5B, which directly interact with HCV-796. The impacts of the amino acid substitutions on viral fitness and drug susceptibility were examined in recombinant replicons and NS5B enzymes with the single-amino-acid mutations. The replicon variants were 10- to 1,000-fold less efficient in forming colonies in cells than the wild-type replicon; the S365L variant failed to establish a stable cell line. Other variants (L314F, I363V, and M414V) had four- to ninefold-lower steady-state HCV RNA levels. Reduced binding affinity with HCV-796 was demonstrated in an enzyme harboring the C316Y mutation. The effects of these resistance mutations were structurally rationalized using X-ray crystallography data. While different levels of resistance to HCV-796 were observed in the replicon and enzyme variants, these variants retained their susceptibilities to pegylated interferon, ribavirin, and other HCV-specific inhibitors. The combined virological, biochemical, biophysical, and structural approaches revealed the mechanism of resistance in the variants selected by the potent polymerase inhibitor HCV-796.

Grazoprevir and elbasvir plus ribavirin for chronic HCV genotype-1 infection after failure of combination therapy containing a direct-acting antiviral agent

BACKGROUND & AIMS The Phase-2 C-SALVAGE study evaluated an investigational interferon-free combination of grazoprevir (a NS3/4A protease inhibitor) and elbasvir (a NS5A inhibitor) with ribavirin for patients with chronic HCV genotype-1 infection who had failed licensed DAA-containing therapy. METHODS C-SALVAGE was an open-label study of grazoprevir 100 mg and elbasvir 50 mg QD with weight-based ribavirin BID for 12 weeks in cirrhotic and non-cirrhotic patients with chronic HCV genotype-1 infection who had not attained SVR after ⩾4 weeks of peginterferon and ribavirin plus either boceprevir, telaprevir, or simeprevir. Exclusion criteria included decompensated liver disease, hepatocellular carcinoma, and HIV or HBV co-infection. The primary efficacy outcome was SVR12 defined as a HCV RNA level below the assay limit of quantification 12 weeks after the end of treatment. RESULTS Of the 79 patients treated with ⩾1 dose of study drug, 66 (84%) patients had a history of virologic failure on a regimen containing a NS3/4A protease inhibitor; 12 of the other 13 patients discontinued prior treatment because of adverse experiences. At entry, 34 (43.6%) of 78 evaluable patients harbored NS3 RAVs. SVR12 rates were 76/79 (96.2%) overall, including 28/30 (93.3%) patients with genotype 1a infection, 63/66 (95.5%) patients with prior virologic failure, 43/43 (100%) patients without baseline RAVs, 31/34 (91.2%) patients with baseline NS3 RAVs, 6/8 (75.0%) patients with baseline NS5A RAVs, 4/6 (66.7%) patients with both baseline NS3 and RAVs, and 32/34 (94.1%) cirrhotic patients. None of the five reported serious adverse events were considered drug-related. CONCLUSIONS Grazoprevir and elbasvir plus ribavirin for 12 weeks provides a promising new treatment option for patients after failure of triple therapy containing an earlier-generation protease inhibitor.

Kinetic Analyses Reveal Potent and Early Blockade of Hepatitis C Virus Assembly by NS5A Inhibitors

BACKGROUND & AIMS All-oral regimens combining different classes of direct-acting antivirals (DAA) are highly effective for treatment of patients with chronic hepatitis C. NS5A inhibitors will likely form a component of future interferon-sparing treatment regimens. However, despite their potential, the detailed mechanism of action of NS5A inhibitors is unclear. To study their mechanisms, we compared their kinetics of antiviral suppression with those of other classes of DAA, using the hepatitis C virus genotype 1a cell culture-infectious virus H77S.3. METHODS We performed detailed kinetic analyses of specific steps in the hepatitis C virus life cycle using cell cultures incubated with protease inhibitors, polymerase inhibitors, or NS5A inhibitors. Assays were designed to measure active viral RNA synthesis and steady-state RNA abundance, polyprotein synthesis, virion assembly, and infectious virus production. RESULTS Despite their high potency, NS5A inhibitors were slow to inhibit viral RNA synthesis compared with protease or polymerase inhibitors. By 24 hours after addition of an NS5A inhibitor, polyprotein synthesis was reduced <50%, even at micromolar concentrations. In contrast, inhibition of virus release by NS5A inhibitors was potent and rapid, with onset of inhibition as early as 2 hours. Cells incubated with NS5A inhibitors were rapidly depleted of intracellular infectious virus and RNA-containing hepatitis C virus particles, indicating a block in virus assembly. CONCLUSIONS DAAs that target NS5A rapidly inhibit intracellular assembly of genotype 1a virions. They also inhibit formation of functional replicase complexes, but have no activity against preformed replicase, thereby resulting in slow shut-off of viral RNA synthesis.

Grazoprevir, Elbasvir, and Ribavirin for Chronic Hepatitis C Virus Genotype 1 Infection After Failure of Pegylated Interferon and Ribavirin With an Earlier-Generation Protease Inhibitor: Final 24-Week Results From C-SALVAGE

BACKGROUND The phase 2 C-SALVAGE study (Hepatitis C-Salvage Study for Patients who Failed DAA/PR Therapy) demonstrated a 96.2% sustained virologic response at 12 weeks (SVR12) rate using the NS3/4A protease inhibitor grazoprevir and the NS5A inhibitor elbasvir together with ribavirin in treatment-experienced patients with chronic hepatitis C virus (HCV) genotype 1 infection. METHODS C-SALVAGE was a prospective open-label trial of grazoprevir 100 mg once daily and elbasvir 50 mg once daily coadministered with weight-based ribavirin twice daily for 12 weeks in genotype 1-infected cirrhotic and noncirrhotic patients who had failed treatment with ≥ 4 weeks of pegylated interferon and ribavirin plus either boceprevir, telaprevir, or simeprevir. Although the primary efficacy outcome was SVR12, patients were also evaluated 24 weeks after cessation of study therapy. Population sequencing was performed at baseline and periodically in virologic failures throughout the 24-week posttherapy follow-up period. RESULTS SVR24 rates were 76 of 79 (96.2%) overall, with all 3 relapses occurring by posttherapy week 8. Every NS3 and NS5A variant detected at baseline reappeared at the time of relapse and persisted throughout the available follow-up period. NS3_A156T emerged in virus from each patient at relapse, but rapidly disappeared over the ensuing 2 weeks in 2 patients. NS5A_Y93H emerged in virus from 2 patients at relapse and persisted for the entire follow-up period. CONCLUSIONS Grazoprevir and elbasvir with ribavirin for 12 weeks maintained HCV suppression for at least 24 weeks posttherapy without late relapses. Baseline resistance-associated variants (RAVs) stably reappeared at relapse in all 3 patients with virologic failure. NS5A_RAVs emerging at relapse persisted for the full 24-week follow-up period. If confirmed, this finding could complicate retreatment of the small number of patients failing regimens containing an NS5A inhibitor. CLINICAL TRIALS REGISTRATION NCT02105454.

Molecular Mechanism of a Thumb Domain Hepatitis C Virus Nonnucleoside RNA-Dependent RNA Polymerase Inhibitor

ABSTRACT A new pyranoindole class of small-molecule inhibitors was studied to understand viral resistance and elucidate the mechanism of inhibition in hepatitis C virus (HCV) replication. HCV replicon variants less susceptible to inhibition by the pyranoindoles were selected in Huh-7 hepatoma cells. Variant replicons contained clusters of mutations in the NS5B polymerase gene corresponding to the drug-binding pocket on the surface of the thumb domain identified by X-ray crystallography. An additional cluster of mutations present in part of a unique β-hairpin loop was also identified. The mutations were characterized by using recombinant replicon variants engineered with the corresponding amino acid substitutions. A single mutation (L419M or M423V), located at the pyranoindole-binding site, resulted in an 8- to 10-fold more resistant replicon, while a combination mutant (T19P, M71V, A338V, M423V, A442T) showed a 17-fold increase in drug resistance. The results of a competition experiment with purified NS5B enzyme with GTP showed that the inhibitory activity of the pyranoindole inhibitor was not affected by GTP at concentrations up to 250 μM. Following de novo initiation, the presence of a pyranoindole inhibitor resulted in the accumulation of a five-nucleotide oligomer, with a concomitant decrease in higher-molecular-weight products. The results of these studies have confirmed that pyranoindoles target the NS5B polymerase through interactions at the thumb domain. This inhibition is independent of GTP concentrations and is likely mediated by an allosteric blockade introduced by the inhibitor during the transition to RNA elongation after the formation of an initiation complex.

Selection and Characterization of Hepatitis C Virus Replicons Dually Resistant to the Polymerase and Protease Inhibitors HCV-796 and Boceprevir (SCH 503034)

ABSTRACT HCV-796 is a nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) polymerase, and boceprevir is an inhibitor of the NS3 serine protease. The emergence of replicon variants resistant to the combination of HCV-796 and boceprevir was evaluated. Combining the inhibitors greatly reduced the frequency with which resistant colonies arose; however, some resistant replicon cells could be isolated by the use of low inhibitor concentrations. These replicons were approximately 1,000-fold less susceptible to HCV-796 and 9-fold less susceptible to boceprevir. They also exhibited resistance to anthranilate nonnucleoside inhibitors of NS5B but were fully sensitive to inhibitors of different mechanisms: a pyranoindole, Hsp90 inhibitors, an NS5B nucleoside inhibitor, and pegylated interferon (Peg-IFN). The replicon was cleared from the combination-resistant cells by extended treatment with Peg-IFN. Mutations known to confer resistance to HCV-796 (NS5B C316Y) and boceprevir (NS3 V170A) were present in the combination-resistant replicons. These changes could be selected together and coexist in the same genome. The replicon bearing both changes exhibited reduced sensitivity to inhibition by HCV-796 and boceprevir but had a reduced replicative capacity.

Antiviral activity of boceprevir monotherapy in treatment-naive subjects with chronic hepatitis C genotype 2/3

BACKGROUND & AIMS To examine the antiviral activity of boceprevir, a hepatitis C virus (HCV) protease inhibitor, in HCV genotype (G) 2/3-infected patients. METHODS We assessed boceprevir and telaprevir activity against an HCV G2 and G3 isolates enzyme panel, in replicon, and in phenotypic cell-based assays. Additionally, a phase I study evaluated the antiviral activity of boceprevir monotherapy (200mg BID, 400mg BID, or 400mg TID) vs. placebo for 14 days in HCV G2/3 treatment-naive patients. RESULTS Boceprevir and telaprevir similarly inhibited G1 and G2 NS3/4A enzymes and replication in G1 and G2 replicon and cell-based assays. However, telaprevir demonstrated lower potency than boceprevir against HCV G3a enzyme (Ki=75 nM vs. 17 nM), in the G3a replicon assay (EC₅₀=953 nM vs. 159 nM), and against HCV G3a NS3 isolates (IC₅₀=3312 nM vs. 803 nM) in the cell-based assay. In HCV G2/3-infected patients, boceprevir (400 mg TID) resulted in a maximum mean decrease in HCV RNA of -1.60 log vs. -0.21 log with placebo. CONCLUSIONS In vitro, boceprevir is more active than telaprevir against the HCV G3 NS3/4A enzyme in cell-based and biochemical assays and against G3 isolates in replicon assays. In HCV G2/3-infected treatment-naive patients, decreases in HCV RNA levels with boceprevir (400 mg TID) were comparable to those observed with the same dose in HCV treatment-experienced G1-infected patients.