This report concerns four double-blind, placebo-controlled, sequential-panel, single-ascending-dose (SAD) or multiple-ascending-dose (MAD) studies in healthy subjects and subjects with chronic HCV infection.
In the double-blind, placebo-controlled, sequential SAD study in healthy subjects (SAD-HS), eight healthy subjects were enrolled in each of seven dose panels (10, 50, 100, 200, 400, 600, and 1,200 mg). Subjects were admitted to the clinical facility 2 days prior to dosing (day −2). On day 1, subjects were randomly assigned in a ratio of 3:1 to receive a single oral dose of asunaprevir (n = 6/dose group) or placebo (n = 2). Subjects remained in the clinical facility for at least 72 h after dosing and were furloughed from the clinical facility on day 4 and discharged on day 7 following a safety assessment.
In the double-blind, placebo-controlled, sequential SAD study in subjects with chronic HCV genotype 1 (SAD-cHCV), six subjects with chronic HCV infection were enrolled in each of four sequential dose panels (10, 50, 200, and ≤600 mg asunaprevir). The 600-mg dose was selected as the final dose based upon data from the preceding dose panels. Subjects were admitted to the clinical facility on day −1 (the day prior to dosing). On day 1, subjects were randomly assigned in a ratio of 5:1 to receive a single oral dose of asunaprevir (n = 5/dose group) or placebo (n = 1). Forty-eight hours after dosing (day 3), subjects were furloughed from the facility; they had an outpatient visit on day 4 and were discharged on day 7 following a safety assessment.
In the double-blind, placebo-controlled, sequential MAD study in healthy subjects (MAD-HS), eight healthy subjects were enrolled in each of six dose panels (10, 50, 100, 200, 400, and 600 mg asunaprevir every 12 h). Subjects were admitted to the clinical facility on day −1. On day 1, participants were randomly assigned in a ratio of 3:1 to receive asunaprevir (n = 6/dose group) or placebo (n = 2) every 12 h for 14 days. Asunaprevir was dosed twice daily in both MAD studies on the basis of the frequent viral rebound prior to 24 h that was observed in the SAD-cHCV study (see Results and ). Subjects remained in the clinical facility for at least 72 h after the final dosing on the morning of day 14 and were furloughed on day 17 and discharged on day 21 following a safety assessment.
Fig 1 HCV RNA response, SAD-cHCV study. Changes from baseline are shown in serum hepatitis C virus (HCV) RNA following a single 10-mg to 600-mg dose of asunaprevir in the single-ascending-dose study in HCV-infected subjects. Dashed lines indicate data for individual (more ...) (iv) MAD-cHCV.
In the double-blind, placebo-controlled, sequential MAD study in subjects with chronic HCV genotype 1 (MAD-cHCV), five HCV-infected subjects were randomly assigned within each of three dose panels (200, 400, and 600 mg asunaprevir every 12 h). Subjects were admitted to the clinical facility on day −1. On day 1, subjects were randomly assigned in a ratio of 4:1 to receive asunaprevir (n = 4/dose group) or placebo (n = 1) every 12 h for 3 days. Subjects remained in the clinical facility from day −1 until the morning of day 4. Subjects returned for additional blood sampling for clinical laboratory tests, pharmacokinetic and/or viral RNA measurements, and resistance testing at approximately days 5 to 10, 14, 21, and 28. Additional longer-term follow-up visits were scheduled at days 42, 98, and 182.
In all studies, escalation to the subsequent dose panel took place only after safety data from the previous panel had been analyzed and dose escalation was deemed safe by the sponsor and the investigators. Subjects who did not complete the study (except those whose participation was discontinued for adverse events) could be replaced. The morning dose on the first and last days of dosing required an overnight fast; all other doses in every-12-h (Q12h) regimens (MAD studies) required fasting for at least 2 h before and approximately 2 h after dosing.
The number of subjects in each study was based on primary assessments in the respective studies: safety and tolerability in the two SAD studies and the MAD study in healthy subjects and changes in log10 HCV RNA from the baseline to day 3 in the MAD-cHCV study. Accordingly, five or six subjects administered active drug at each dose level in the two SAD studies and the MAD-HS study provided 80% probability of observing at least one occurrence at a dose level of any adverse event that would occur with 28% or 24% incidence, respectively, in the population from which the sample was drawn. Active drug was administered to four subjects per dose level in the MAD-cHCV study, providing probabilities of 0.01 and 0.94 to observe a mean decrease from the baseline of ≥1.5 log10 HCV RNA if the true mean decrease is 0.0 and 2.5 log10 HCV RNA, respectively. This calculation was based on the assumption that the decrease in log10 HCV RNA from the baseline to day 3 is normally distributed and that the standard deviation for the decrease in log10 HCV RNA from the baseline to day 3 is 1.3.
These studies were approved by institutional review boards in all study centers and conducted in accordance with good clinical practice, as defined by the International Conference on Harmonization, in accordance with the ethical principles underlying European Union Directive 2001/20/EC and the U.S. Code of Federal Regulations, Title 21, Part 50, and in accordance with the ethical principles that have their origin in the Declaration of Helsinki. Informed written consent was obtained from all subjects.
Within each dose panel for each study, patients were randomly assigned to receive asunaprevir or a placebo according to a computer-generated randomization scheme prepared by Bristol-Myers Squibb. An interactive voice response system was used to assign a unique subject number and a blinded container number, which was provided to the blinded study staff who supervised and recorded the drug administration.
Inclusion and exclusion criteria.
Healthy subjects were identified by medical history, physical examination, 12-lead electrocardiogram (ECG), and clinical laboratory evaluations. Men and women ages 18 to 49 years with a body mass index of 18 to 32 kg/m2 were eligible to participate. Female participants could not be nursing, pregnant, or of childbearing potential. Eligible patients with chronic HCV infection were men or women aged 18 to 60 years with a body mass index of 18 to 35 kg/m2 and chronic infection with HCV genotype 1, either treatment naive, treatment nonresponders (including relapsers), or treatment intolerant. Additional inclusion criteria were plasma HCV RNA levels of ≥100,000 IU/ml, a documented Fibrotest score of ≤0.72 or ≤0.59, and an aspartate aminotransferase platelet ratio index of ≤2 or the absence of cirrhosis based on liver biopsy within 12 months. Main exclusion criteria included previous exposure to another NS3 protease inhibitor, coinfection with human immunodeficiency virus or hepatitis B virus, or being women of childbearing potential.
Safety and antiviral evaluations.
Blood and urine samples for clinical laboratory evaluations, single 12-lead ECG, and vital sign measurements were collected at specified time points throughout all studies. Safety assessments were based on reported adverse events (AEs) and the results of vital sign measurements, physical examinations, ECGs, and clinical laboratory tests. The incidences of AEs were tabulated and reviewed for potential significance and clinical importance.
In the SAD studies, 12-lead ECGs were recorded at screening, 24 h predosing, and at 0 (predose), 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 48, and 72 h postdosing and prior to discharge. In the MAD-HS study, ECGs were recorded at screening, day −1 and 1 h after morning dosing on days 2, 4, 6, 9, 11, 13, 17, and 21. Additional serial ECGs were recorded 0 (predose), 1, 2, 4, 6, 8, and 12 h postdosing on days −1, 1, and 14 and 0, 2, and 4 h after morning dosing on days 3 and 7. In the MAD-cHCV study, ECGs were recorded at screening and at 0 (predose), 2, 4, 8, and 12 h postdosing on day 1, and 1 h after morning dosing on days 2, 3, 4, 7, 14, 21, and 28.
Antiviral response was assessed by the magnitude of change in plasma HCV RNA levels from baseline. The primary assessment for antiviral activity in the MAD study was a decrease in plasma HCV RNA levels from baseline to day 3. HCV RNA levels were determined using the Roche Cobas TaqMan HCV test, v2.0 (lower limit of quantification, 25 IU/ml; lower limit of detection, 10 IU/ml).
In studies of HCV-infected subjects, treatment-emergent HCV variants were analyzed genotypically by population sequencing of the NS3 protease domain; samples with sequences that changed during treatment or contained variants predicted to confer resistance were subsequently phenotyped if HCV RNA was ≥1,000 IU/ml. In the SAD-cHCV study, genotypic analysis was conducted on samples collected at baseline and 1, 2, and 6 days postdosing from subjects receiving 200-mg or 600-mg doses of asunaprevir and at baseline only in subjects receiving 10-mg or 50-mg doses of asunaprevir. In the MAD-cHCV study, genotypic analysis was conducted on samples collected at baseline, day 4 (12 h after the final dose) and day 6 from all asunaprevir recipients. The 4-h sample was used as a baseline for two subjects with missing baseline samples in the 400-mg cohort. For subjects with emergent polymorphisms detected at day 6, genotypic analyses were also conducted at days 42 and 182.
HCV RNA was isolated from subject serum with a QIAamp MinElute viral vacuum kit (Qiagen, Inc., Valencia, CA). First-strand cDNA was synthesized from random hexamer primers with a SuperScript III first-strand synthesis system for reverse transcriptase PCR (Invitrogen Corp., Carlsbad, CA). The NS3 protease-coding region was amplified with genotype-specific primers. At least two independent PCR products were amplified with different primer sets for each sample. A second PCR with the same primers or a nested PCR with internal primers was performed when required to obtain sufficient NS3 protease cDNA for sequence analysis. Sequences covering both strands were obtained for purified PCR products and compared with reference HCV genotype 1a (H77c) or genotype 1b (Con1) sequences.
For clonal analysis, PCR products were cloned using the Topo TA cloning kit (Invitrogen) as per the manufacturer's recommendation. The NS3 protease domain sequences were obtained for >30 individual clones using M13 universal forward and reverse and NS3 protease domain internal primers. Alignments of the individual sequences were performed with the Sequencer 4.6 software program (Gene Codes, Ann Arbor, MI). Phenotypic analyses of NS3 protease sequences containing genotypic polymorphisms were conducted using an HCV NS3 chimeric replicon system as described previously (17
All recorded adverse events were listed and tabulated by system organ class, preferred term, and treatment. Vital signs and clinical laboratory tests were listed and summarized by treatment. Any significant physical exam findings and clinical laboratory results were listed. The effects of asunaprevir on ECG parameters were explored graphically and by summary statistics.
The magnitude of changes in plasma HCV RNA levels was assessed by summarizing changes from baseline (day −1) by time (or study day) and treatment, summarizing maximum observed changes from baseline by treatment; the primary assessment of antiviral activity was based on the change from baseline to day 3 for the MAD-cHCV study. All statistical analyses were carried out using the software program SAS/STAT version 8.2 (SAS Institute Inc., Cary, NC).