No abstract

In a sentinel cohort, hepatitis C virus (HCV) patients (primarily genotype [GT] 1a) were treated with daclatasvir (NS5A inhibitor) and asunaprevir (NS3 protease inhibitor). Preexistence, emergence, and persistence of resistance variants in patients who failed this treatment are described. HCV-infected null responders received daclatasvir (60 mg once daily) and asunaprevir (600 mg twice daily) alone (Group A, 11 patients) or with peginterferon alfa-2a and ribavirin (Group B, 10 patients) for 24 weeks. Resistance testing was performed on baseline samples and samples with HCV RNA 1,000 IU/mL at Week 1 through posttreatment Week 48. Resistance substitution susceptibility to inhibition by asunaprevir and daclatasvir was assessed using HCV replicon assays. In Group A, six GT1a patients experiencing viral breakthrough and one GT1a patient who relapsed had detectable NS5A (Q30E/R, L31V/M, Y93C/N) and NS3 (R155K, D168A/ E/V/Y) resistance-associated variants at failure. Two of six viral breakthrough patients achieved SVR48 after treatment intensification with peginterferon alfa-2a and ribavirin. For 2/4 viral breakthrough patients not responding to treatment intensification, NS3 resistance variants changed (D168Y to D168T; R155K to V36M-R155K). At posttreatment Week 48, daclatasvir-resistant variants persisted while asunaprevir-resistant variants were generally replaced by wild-type sequences. The NS3 sequence remained unchanged in the one patient with NS3-R155K at baseline, relapse, and posttreatment Week 48. In Group B, no viral breakthrough was observed. Conclusion: The treatment failure of daclatasvir and asunaprevir in HCV GT1a patients was associated with both NS5A and NS3 resistance variants in prior null responders. NS5A resistance variants persisted while NS3 resistance variants generally decayed, suggesting a higher relative fitness of NS5A variants. (HEPATOLOGY 2013;58:902-911) T he investigational direct-acting antivirals, daclatasvir and asunaprevir, are currently in clinical development for treating hepatitis C virus (HCV). Daclatasvir is a first-in-class, highly selective NS5A replication complex inhibitor with picomolar potency and broad HCV genotypic coverage. 1 Asunaprevir is a selective NS3 protease inhibitor with antiviral activity in vitro against HCV genotype (GT) 1 and GT4. 2 These direct-acting antivirals have demonstrated efficacy when individually combined with peginterferon alfa-2a and ribavirin to treatmentnaive GT1 patients. [3][4][5][6] These regimens were well tolerated.When peginterferon alfa-2a and ribavirin were added to the dual combination of daclatasvir and asunaprevir, all patients experienced sustained virologic response (SVR) at 48 weeks posttreatment. 7 The combination of daclatasvir and asunaprevir alone resulted in rapid suppression of HCV RNA levels in GT1 null responder patients. 7 This proof-of-concept study was the first to show that null responder HCV-infected patients could be cured with 24 weeks of an Abbreviations: HCV, hepatitis C virus; GT, genotype; LLOQ, lower lim...

The discovery of asunaprevir (BMS-650032, 24) is described. This tripeptidic acylsulfonamide inhibitor of the NS3/4A enzyme is currently in phase III clinical trials for the treatment of hepatitis C virus infection. The discovery of 24 was enabled by employing an isolated rabbit heart model to screen for the cardiovascular (CV) liabilities (changes to HR and SNRT) that were responsible for the discontinuation of an earlier lead from this chemical series, BMS-605339 (1), from clinical trials. The structure-activity relationships (SARs) developed with respect to CV effects established that small structural changes to the P2* subsite of the molecule had a significant impact on the CV profile of a given compound. The antiviral activity, preclincial PK profile, and toxicology studies in rat and dog supported clinical development of BMS-650032 (24).

b Asunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) NS3 protease inhibitor demonstrating efficacy in alfa interferonsparing, direct-acting antiviral dual-combination regimens (together with the NS5A replication complex inhibitor daclatasvir) in patients chronically infected with HCV genotype 1b. Here, we describe a comprehensive in vitro genotypic and phenotypic analysis of asunaprevir-associated resistance against genotypes 1a and 1b using HCV replicons and patient samples obtained from clinical studies of short-term asunaprevir monotherapy. During genotype 1a resistance selection using HCV replicons, the primary NS3 protease substitutions identified were R155K, D168G, and I170T, which conferred low-to moderate-level asunaprevir resistance (5-to 21-fold) in transient-transfection susceptibility assays. For genotype 1b, a higher level of asunaprevirassociated resistance was observed at the same selection pressures, ranging from 170-to 400-fold relative to the wild-type control. The primary NS3 protease substitutions identified occurred predominantly at amino acid residue D168 (D168A/G/H/V/Y) and were associated with high-level asunaprevir resistance (16-to 280-fold) and impaired replication capacity. In asunaprevir single-ascending-dose and 3-day multiple-ascending-dose studies in HCV genotype 1a-or 1b-infected patients, the predominant pre-existing NS3 baseline polymorphism was NS3-Q80K. This substitution impacted initial virologic response rates in a singleascending-dose study, but its effects after multiple doses were more ambiguous. Interestingly, for patient NS3 protease sequences containing Q80 and those containing K80, susceptibilities to asunaprevir were comparable when tested in an enzyme assay. No resistance-associated variants emerged in these clinical studies that significantly impacted susceptibility to asunaprevir. Importantly, asunaprevir-resistant replicons remained susceptible to an NS5A replication complex inhibitor, consistent with a role for asunaprevir in combination therapies. Hepatitis C virus (HCV), a positive-strand RNA virus that belongs to the family Flaviviridae, infects an estimated 170 million individuals worldwide, causing over 350,000 deaths annually (47). Progression of chronic HCV infection can gradually evolve into cirrhosis, liver failure, and/or hepatocellular carcinoma. In the United States, these clinical manifestations are the leading indication for liver transplantation and account for significant liver-related morbidity and mortality each year (14). Although a combination regimen of pegylated alfa interferon and ribavirin remains a vital therapeutic option against chronic HCV infection, various host and viral factors are believed to influence the outcome of treatment, and different genotypes are also associated with variable responses to this regimen. Sustained virologic response (SVR) rates of just 40 to 50% are achieved in treatmentnaïve patients with HCV genotype 1, whereas higher rates (78 to 86%) have been reported during the course of therapy against HCV...

The discovery of BMS-605339 (35), a tripeptidic inhibitor of the NS3/4A enzyme, is described. This compound incorporates a cyclopropylacylsulfonamide moiety that was designed to improve the potency of carboxylic acid prototypes through the introduction of favorable nonbonding interactions within the S1' site of the protease. The identification of 35 was enabled through the optimization and balance of critical properties including potency and pharmacokinetics (PK). This was achieved through modulation of the P2* subsite of the inhibitor which identified the isoquinoline ring system as a key template for improving PK properties with further optimization achieved through functionalization. A methoxy moiety at the C6 position of this isoquinoline ring system proved to be optimal with respect to potency and PK, thus providing the clinical compound 35 which demonstrated antiviral activity in HCV-infected patients.