Reverse-transcription of RNA was performed using a SuperScript III First Strand Synthesis Supermix kit (Invitrogen, Carlsbad, CA) with random hexamers according

to the manufacturer’s instructions. Polymerase chain reaction was conducted with a Platinum PCR SuperMix kit (Invitrogen). Reaction products were purified on a Biomek FX system (Beckman Coulter, Fullerton, CA) using a magnetic bead kit (Agencourt Bioscience Corporation, Beverly, MA). DNA sequencing of purified material was conducted on a 3730xl DNA Analyzer (Applied Biosystems). To investigate a potential correlation between exposure to narlaprevir and antiviral activity, plasma samples were collected over the course of the study for pharmacokinetic profiling of narlaprevir with or without ritonavir. In period 1, serial blood narlaprevir pharmacokinetic samples were LY2157299 cell line taken on days 1 and 7. Additional narlaprevir pharmacokinetic learn more sampling was performed on days 2, 5, 6, and 7 for trough level (Cmin) determination. In period 2, serial blood narlaprevir pharmacokinetic samples were collected on days 1, 7, and 14, and additional pharmacokinetic samples for Cmin determination were obtained on days 2, 8, 10, 12, and 14. Plasma

concentrations of narlaprevir were determined using a validated liquid chromatographic–tandem mass spectrometric method with a limit of quantification of 6.08 ng/mL. Patients were monitored for safety and tolerability at regular intervals from the start of dosing through a follow-up visit 24 weeks after completion of SOC. Safety assessments included physical examination, vital signs, clinical laboratory tests, electrocardiograms, and the recording of all adverse events. Forty-one patients (10 patients each in cohorts 1-3 and 11 patients in cohort 4) were enrolled in the study. One patient in cohort 4 discontinued

immediately after the first dose on day 1 because of intolerance to the drug suspension; study medication was stopped at the discretion of the investigator, and this find more patient was replaced. A total of 40 patients completed the narlaprevir treatment phase of the study and initiated SOC immediately after period 2. Treatment-experienced patients consisted of 12 relapse patients and eight nonresponders. Demographic and other baseline characteristics of the randomized patients are shown in Table 1. The pharmacokinetic profile of narlaprevir 800 mg three times daily or 400 mg with ritonavir two times daily was characterized (Table 2). Exposure to narlaprevir with and without coadministration of PEG-IFN-α-2b for both treatment-naïve and treatment-experienced patients was comparable. Narlaprevir was eliminated more slowly when coadministered with ritonavir than when administered alone. Dose-normalized daily exposures (area under the curve) on day 14 in the presence of PEG-IFN-α-2b and ritonavir increased 7.