Forty males and females of ages between 18 and 65 years with body mass indices ranging from 18 to 36 kg/m2 were enrolled in the study. Females were to be surgically sterile, postmenopausal for at least 12 months at screening, or taking protocol-specified contraceptive measures. Only treatment-naïve, noncirrhotic patients with chronic HCV genotype 1 infection and HCV RNA levels of ≥50,000 IU/ml were eligible for inclusion in the study. Patients were in good health, with no significant comorbidities. Patients were excluded if they were positive for anti-hepatitis A virus immunoglobulin M (IgM) antibodies, hepatitis B surface antigen, anti-hepatitis B core protein IgM antibodies, or anti-human immunodeficiency virus antibodies. No medication associated with QT interval prolongation was permitted within 30 days prior to dosing or during the study, and any other concurrent medication required approval by the investigator and the sponsor. Patients who had received any systemic antineoplastic or immunomodulatory treatment within 6 months prior to the first dose of study drug or who might have needed such treatments at any time during the study were ineligible.
The study was conducted from May 2009 to September 2009. All patients provided informed consent prior to screening. The protocol was approved by the investigational review board (Western International Review Board, Olympia, WA), and the study (protocol number P7851-1102) was conducted according to good clinical practice and the Declaration of Helsinki.
The study followed a double-blind, parallel-group, randomized, placebo-controlled, multiple-ascending-dose design with oral administration of once-daily doses of either GS-9851 or placebo for 3 days. Patients were allocated to one of four groups, each of which consisted of 10 patients who were randomized to receive either active GS-9851 or placebo (active, 8; placebo, 2). The starting dose of GS-9851 was 50 mg, followed by 100 mg, 200 mg, and 400 mg. Following the completion of each dose level, pharmacokinetic, pharmacodynamic (i.e., plasma HCV RNA), and safety data were examined by the sponsor and investigator to determine whether escalation to the next dose level was warranted.
Patients who had not undergone a liver biopsy within 3 years of dosing were asked to provide a biopsy sample to rule out cirrhosis or advanced fibrosis. Clinical laboratory tests were performed within 30 days of dosing to confirm patient eligibility for the study. The day before the first dose (day −1), patients were admitted to the study unit for baseline assessments that included vital signs, hematology, chemistry, and urinalysis. Patients received an oral dose of GS-9851 or placebo in the morning after a 10-h fast for 3 consecutive days. Study medication was administered with 240 ml of water. Patients were allowed to drink water freely throughout the study period except for a 3-h window that began 1 h prior to dosing until 2 h after each morning dose, during which the only water allowed was that taken with the study drug. Safety, pharmacokinetic, and pharmacodynamic assessments were performed from day 1 to the morning of day 4, after which the patients were discharged from the study unit provided they did not exhibit any ongoing safety issues. Patients returned to the study site on the mornings of days 5, 6, 7, 10, and 14 to undergo follow-up assessments.
Blood samples for quantification of HCV RNA and viral genotyping were collected at screening and in the morning (predose on dosing days) on days 1 to 7, 10, and 14. Blood samples for pharmacokinetic analysis were drawn at predefined time points from day 1 until day 6. On day 1, blood samples were collected predose and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, and 16 h postdose. On day 2, blood was collected predose only, and on day 3, it was collected predose and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, 24, 48, and 72 h following the last dose of study medication. In the 200-mg and 400-mg GS-9851 dose groups, urine was collected predose on day 1 and at the following intervals: 0 to 6, 6 to 12, and 12 to 24 h postdosing. On day 3, urine samples were collected at the following intervals following the last dose of study medication: 0 to 6, 6 to 12, 12 to 24, 24 to 48, and 48 to 72 h for analysis of the urinary concentrations of GS-9851 and its metabolites, GS-566500 and GS-331007.
Plasma concentrations of GS-9851, GS-566500, and GS-331007 were determined using a validated high-performance liquid chromatography method utilizing tandem mass spectroscopy (LC-MS/MS) (QPS, LLC, Newark, DE). The linear range of the plasma assay was 5 to 5,000 ng/ml for GS-9851 and 10 to 5,000 ng/ml for GS-566500 and GS-331007. Urine samples were analyzed using a validated LC-MS/MS method to determine concentrations of GS-9851, GS-566500, and GS-331007 (QPS, Newark, DE). The lower limit of quantitation (LLQ) of the assay was 10 ng/ml for each compound, with a linear quantifiable range of 10 to 10,000 ng/ml.
The plasma pharmacokinetic profiles of GS-9851, GS-566500, and GS-331007 were determined for each patient by noncompartmental analysis of individual plasma concentration-time data using the software program WinNonlin, Professional, version 5.2 (Pharsight Corporation, Mountain View, CA). The following pharmacokinetic parameters were determined: maximum concentration in plasma (Cmax), time of maximum concentration (tmax), half-life (t1/2), area under the plasma concentration-time curve to the last measurable concentration (AUC0–t), AUC from time zero to 24 h (AUC0–24), AUC from time zero and extrapolated to infinity (AUC0–∞), percentage of AUC extrapolated (AUC extrapolated), amount excreted in urine from time zero to 24 h (Ae0–24), renal clearance (CLrenal), and percentage of study drug excreted in urine (urine excreted). The percentages of urine excreted for GS-566500 and GS-331007 were adjusted for molecular weight differences between GS-9851 and the respective metabolite. Molecular-weight-adjusted GS-331007/GS-9851 AUC ratios were calculated.
Clinical virology assessments.
Hepatitis C virus genotyping was performed by Cenetron Central Laboratories, Ltd. (Austin, TX), using the Siemens Versant Inno-LiPA HCV genotyping assay, version 2.0 (Tarrytown, NY). This assay was validated independently using NS5B sequencing as a standard, and the concordance obtained between the two methods at the genotype and subtype levels was greater than 99%. Quantification of plasma HCV RNA was performed by Cenetron Central Laboratories, Ltd., using Cobas TaqMan HCV (Roche, Indianapolis, IN). The established lower limit of this assay is 15 HCV IU/ml, which is defined by a 95% hit rate according to World Health Organization standards. Hepatitis C virus genotypic monitoring was performed using population sequencing of the HCV NS5B-encoding region of the polymerase at baseline and end of treatment for all patients by DDL Diagnostic Laboratory (Voorburg, The Netherlands). Any amino acid substitutions found in samples after GS-9851 exposure were compared with those in the respective baseline sequence for each patient.
Safety and tolerability assessments.
Safety and tolerability were assessed by the reporting of adverse events, including time of onset, duration, intensity, and potential causal relationship with the study drug, from first dose until day 14. Vital signs, including blood pressure and heart rate, were recorded at screening, on the day of admission, and on days 1, 2, 3, 4, and 14. On day 1, vital signs were recorded predose and at 1, 2, 3, 4, and 12 h postdose. On day 2 and day 3, data were collected predose and at 1, 2, 3, and 4 h postdose, and on day 4, data were collected prior to patient discharge. Twelve-lead electrocardiograms (ECGs) were performed at screening, on days 1, 2, and 3, at 1 h predose and at 1, 2, 3, and 4 h postdose, and on day 14. Samples for clinical laboratory testing, including hematology, chemistry, and urinalysis, were obtained at screening, day −1, and days 3, 6, and 14. Clinical laboratory changes were graded according to criteria specified in the protocol using the 2004 version of the DAIDS Table for Grading the Severity of Adult and Pediatric Adverse Events (http://www.mtnstopshiv.org/sites/default/files/attachments/Table_for_Grading_Severity_of_Adult_Pediatric_Adverse_Events.pdf
). Urinalysis included a qualitative dipstick test and quantitative spot measurements of albumin, creatinine, protein, and microscopic analysis.
No formal sample size calculations were performed. Pharmacokinetic parameters, except tmax, underwent log transformation prior to statistical analysis. The pharmacokinetic parameters AUC0--t, AUC0–24, AUC0–∞, and Cmax were also dose normalized. Standard summary statistics were determined for all pharmacokinetic parameters.
Dose proportionality was evaluated based on AUC0–24 and Cmax on day 1 and day 3. Dose proportionality was determined by utilizing a power model and analysis of variance (ANOVA) using log-transformed pharmacokinetic data for each dose normalized to a 100-mg reference dose. Accumulation was evaluated by comparing AUC0–24 on day 3 with AUC0–24 on day 1. A mixed-effects model with ln(AUC) as the dependent variable, a fixed effect for day, and a random patient effect was used to estimate the difference between AUC0–24 for day 3 and AUC0–24 for day 1 on the log scale. A 90% confidence interval (CI) was also estimated for each dose group. Steady state was determined through examination of time-concentration plots of plasma trough concentration data.
Hepatitis C virus RNA data were summarized using log10-transformed data. Summary statistics for HCV RNA values over time and change from the baseline over time were generated. For categorical analysis of virologic response, HCV data were summarized with respect to the number and percentage of patients experiencing an HCV RNA decrease of ≥1.0 log10. Percentages were based on the number of patients within the data set.