Subjects and design.
Randomized, double-blind, placebo-controlled single-dose and multiple-dose, dose escalation studies were conducted with healthy subjects. For both trials, subjects were healthy (as judged by a physical exam, medical history, and laboratory testing) nonsmoking males or females without childbearing potential. Subjects testing positive for anti-HIV antibody, anti-hepatitis C antibody, or hepatitis B surface antigen were excluded. Subjects were not allowed to receive any prescription or nonprescription drugs, including vitamins and herbal products, within 7 days of dosing and throughout the studies, unless this restriction was deemed unlikely to interfere with the conduct of the study or the safety of the individual subject.
The single-dose study utilized an alternating panel of two cohorts with 10 subjects each, 8 receiving S/GSK1349572 and 2 receiving placebo. Subjects randomized to placebo received placebo on all dosing occasions. S/GSK1394572 was administered as a suspension and was given after a 10-h fast. Doses were selected based on allometric scaling and preclinical toxicology studies and were then adjusted based on real-time PK analysis. The doses administered in the study were 2, 5, 10, 25, 50, and 100 mg, with the first cohort receiving 2, 10, and 50 mg and the second cohort receiving 5, 25, and 100 mg. Suspensions of the active drug and placebo were prepared by the study site pharmacist.
In the repeat-dose study, three cohorts received S/GSK1394572 as a suspension at doses of 10, 25, and 50 mg in the fasting state, once daily for 10 days. The 10-mg and 50-mg dose cohorts consisted of 8 active and 2 placebo subjects. The 25-mg dose cohort consisted of 10 active and 2 placebo subjects. All subjects in this cohort received 3 mg midazolam (MDZ), a CYP3A4 metabolic probe, given orally as the commercially available syrup alone on day −1 and with S/GSK1394572 25 mg on day 10.
For both trials, all subjects were admitted to the research unit on the evening prior to dosing with the investigational product (IP) and remained there until the 72-h postdose assessments were completed. Vital signs, electrocardiograms (ECGs), and laboratory tests were performed prior to dosing and through 72 h postdosing. Adverse events (AEs) were collected by patient interviews at regular intervals. The severities of AEs and laboratory abnormalities were assessed using the National Institute of Allergy and Infectious Diseases, Division of AIDS (DAIDS), grading table (5
). Serial PK blood samples for plasma S/GSK1349572 concentrations were obtained predose and at 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 10, 12, 16, 24, 36, 48, and 72 h postdose in the single-dose study. For the multiple-dose study, serial PK blood samples were obtained on days −1 and 10 for plasma MDZ concentrations and on days 1 and 10 predose and at 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 h postdose for plasma S/GSK1349572 concentrations. Daily predose samples were also collected on days 3 through 9. All subjects provided informed written consent, and the protocol was approved by the Ohio Valley Institutional Review Board and the Covance Clinical Research Unit (CRU) Institutional Review Board.
Following extraction from plasma by protein precipitation, S/GSK1349572 and MDZ concentrations were determined by a validated high-performance liquid chromatography-tandem mass spectrometry method (PE Sciex Analyst, version 1.4.1; SMS2000, version 2.0) using a TurboIonSpray source and multiple reaction monitoring at GlaxoSmithKline. For analysis of S/GSK1349572, [2H7 15N]S/GSK1349572 was used as an internal standard. The validated linear concentration range was 5 to 5,000 ng/ml, and three concentrations of quality control (QC) samples were included in each run, at 20, 400, and 4,000 ng/ml. Based on the results of the analysis of these QC samples, the bias ranged from 0.0 to 3.5%, and the intra- and interassay coefficients of variation (CVs) were less than or equal to 7.2% and 5.4%, respectively. For MDZ, [13C3 2H3]midazolam was used as an internal standard. The validated linear concentration range was 0.35 to 72 ng/ml, and three concentrations of QC samples were included in each run, at 1.4, 7.0, and 58 ng/ml. Based on the results of the analysis of these QC samples, the bias ranged from 2.8 to 9.4%, and the intra- and interassay coefficients of variation were less than or equal to 6.8% and 4.9%, respectively.
A noncompartmental pharmacokinetic analysis of the concentration-time data was performed with WinNonlin (version 4.1; Pharsight Corporation, Mountain View, CA). Plasma PK parameters for S/GSK1349572 were calculated for each treatment as follows: for the single-dose study and day 1 in the repeat-dose study, the area under the concentration-time curve from time zero to the last measurable concentration (AUC0-t), the area under the concentration-time curve extrapolated to infinity (AUC0-∞), the maximum observed concentration of the drug in plasma (Cmax), the time of the maximum observed concentration of the drug in plasma (Tmax), the terminal elimination phase half-life (t1/2), the apparent oral clearance (CL/F), and the observed time point immediately prior to the first quantifiable concentration (Tlag) were assessed. In the repeat-dose study, the area under the concentration-time curve over a dosing interval (AUC0-τ), Cmax, Tmax, the predose concentration (C0), the concentration at the end of the dosing interval (Cτ), and the minimum observed concentration of the drug in plasma (Cmin) were assessed for day 10. The following PK parameters were calculated for MDZ: AUC0-t and AUC0-∞. The actual elapsed time from dosing was used to estimate all individual PK parameters.
Descriptive statistics, including geometric means and 95% confidence intervals (CIs), were calculated for all PK parameters and summarized by the type of treatment. The dose proportionality of S/GSK1349572 PK parameters (AUC0-t, AUC0-∞, Cmax) from the single-dose study or day 1 in the repeat-dose study and from day 10 (AUC0-τ, Cmax, Cmin, and Cτ) in the repeat-dose study was assessed using the power model y = α × doseβ, where y denotes the PK parameter being analyzed and α depends on the subject. Dose proportionality implies that β = 1 and was assessed by estimating β along with its 90% CI. For the repeat-dose escalation, the time invariance ratios (ratio of AUC0-τ on day 10 to AUC0-∞ on day 1) and accumulation ratios (ratios of the day 10 AUC0-τ to the day 1 AUC0-24, of the day 10 Cmax to the day 1 Cmax, and of the day 10 Cτ to the day 1 C24) were calculated as geometric least squares (GLS) means and assessed by analysis of variance with subjects as the random effect and day as the fixed effect. Achievement of steady state was assessed by visual inspection of plots and by calculation of the point estimate and 90% CI of the slope of the linear regression of the day 5 to 10 predose concentrations versus day by dose group. To support the claim that steady state was reached, the predose concentration slope estimate had to be close to zero or the 90% CI for the slope estimate had to include zero.
Following log transformation, the plasma midazolam AUC0-t was analyzed using a mixed-effects model with treatment as the fixed effect and subject as the random effect. Point estimates and their associated 90% CIs were constructed for the differences between treatments (MDZ plus S/GSK1349572 minus MDZ alone). The point estimates and their associated 90% CIs were then back-transformed to provide point estimates and 90% CIs for the treatment ratios (MDZ plus S/GSK1349572 versus MDZ alone).