Eligible patients were randomly assigned to Group A or B. Group A patients received dual therapy consisting of daclatasvir (60 mg, orally, once daily) and asunaprevir (600 mg, orally, twice daily) for 24 weeks. Group A patients were eligible to have peginterferon alfa-2a and ribavirin added to their regimens for an additional 48 weeks (as indicated) if viral breakthrough occurred. Group B patients received quadruple therapy consisting of daclatasvir (60 mg, orally, once daily), asunaprevir (600 mg, orally, twice daily, dose
adjustment was not permitted), peginterferon alfa-2a 180 μg per week subcutaneously, and ribavirin, oral twice daily, with doses determined according to body weight (1,000 PD-0332991 order mg daily in patients with body weight of <75 kg, and 1,200 mg daily in patients with body weight of ≥75 kg) for 24 weeks. The primary endpoint of the study was undetectable HCV RNA at posttreatment Week 12. SVR was defined as continued undetectable HCV RNA 12 weeks after cessation of treatment (SVR12). HCV RNA was determined at a central laboratory using the Roche COBAS TaqMan v. 2 assay (Roche Molecular Diagnostics) with a lower limit of quantitation (LLOQ) of 25 IU/mL. HCV genotype was determined using VERSANT
HCV JQ1 Amplification 2.0 Kit (LiPA) (Siemens) and IL28B genotype (rs12979860 SNP) was determined by polymerase chain reaction (PCR) amplification and sequencing. Plasma samples for resistance testing were collected at baseline, Days 1 to 7, 14, and at Week 3, and every 2 weeks from weeks 4-12. After Week 12, for Group A, samples were collected every 2 weeks until Week 24 unless peginterferon alfa-2a
and ribavirin was added, in which case samples were collected every 4 weeks. For Group B, samples were collected every 4 weeks from weeks 12-24. Posttreatment samples were collected at weeks 4, 12, 24, 36, and 48. Resistance testing was performed on available baseline samples and samples with HCV RNA ≥1,000 IU/mL at Week 1 through posttreatment Week 48. Population sequencing was performed using methods as described.[8-10] Baseline sequences have been deposited in GenBank under accession numbers KC591725-KC591768. PCR amplification was performed on ≥2 PCR reactions Carnitine palmitoyltransferase II per sample where possible to assess for primer bias. For clonal analysis, PCR amplicons were cloned into the TOPO vector and transformed into TOP10 E. coli using a commercially available kit (TOPO TA cloning kit, Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions, with ≥30 individual colonies expanded and sequenced for each analysis. Resistance-associated NS5A and NS3 substitutions were introduced into HCV GT1a (H77c) replicon with adaptive variants P1495L and S2204I. These replicons were monitored for phenotypic changes to asunaprevir and daclatasvir as described.