This was a phase I open-label, two-period, crossover, PK study conducted at a single center (St. Stephen's Centre, Chelsea and Westminster Hospital, London, United Kingdom) from 5 September 2005 until 22 May 2006. Men or women aged 18 to 65 years with HIV type 1 (HIV-1) infection documented by HIV-1 antibody enzyme-linked immunosorbent assay and confirmed by Western blot detection of HIV-1 antibody were eligible for this study if they had the following characteristics at screening: undetectable viral load (<400 copies/ml), being on an ABC-containing regimen for ≥8 weeks, CD4+ count of ≥250 cells/mm3, weight of 40 to 100 kg, body mass index of 19 to 29 kg/m2, and willingness to temporarily switch their ABC schedule from QD to BID, or vice versa, for 11 days. Females were to have no childbearing potential or were to agree to protocol-specified methods of contraception, including a double-barrier method, complete abstinence from intercourse from 2 weeks prestudy to 2 weeks poststudy, or use of an intrauterine device with a <1% failure rate.
Subjects were excluded if they were receiving tenofovir, hydroxyurea, mycophenolate, or ribavirin; were in another experimental drug trial within 30 days of screening; regularly consumed >83 ml of a 12% strength alcoholic beverage per day; had a medical condition that interfered with the absorption, distribution, metabolism, or excretion of drugs; regularly took drugs of abuse; had liver enzyme test results >3 times the upper limit of normal or bilirubin levels >2 times the upper limit of normal; had a hemoglobin level of <12 g/dl or a platelet count of <50,000/μl; were pregnant or nursing; were positive for hepatitis C virus antibody or hepatitis B virus surface antigen; or had a history of suspected ABC hypersensitivity reaction. All subjects provided written informed consent to participate in the study. The study protocol was approved by the ethics committee at the study site.
The primary objective of this study was to compare CBV-TP exposure values (area under the concentration-time curve from 0 to 24 h [AUC0-24], maximum concentration of drug in serum [Cmax], and concentration at the end of dosing interval [Cτ]) from ABC 600 mg QD and ABC 300 mg BID. The secondary objective included assessing gender differences in PKs of plasma ABC and intracellular CBV-TP and assessing the safety and tolerability of dosing with ABC 300 mg BID and ABC 600 mg QD during the study period.
A sample size of at least 24 evaluable subjects was deemed necessary based on experience with an earlier CBV-TP study, CNA10905, which had investigated the steady-state PKs of intracellular CBV-TP in HIV-infected subjects on an ABC 300 mg BID regimen (13
). In that study, intersubject coefficients of variation (CV) of CBV-TP PK parameters were between 62% and 73%. Assuming that intrasubject variability was lower than intersubject variability and that the intrasubject CV was at 40% and assuming two one-sided tests at α = 0.05 for each side and a sample size of 24, it was estimated that the range of 90% confidence interval (CI) for the treatment ratio was within 20% of the point estimates for the PK parameters AUC and Cmax
. Due to the potential for subject withdrawal, the complexity of sample processing, and the possibility that some samples may have been unusable, 30 subjects were to be enrolled. If the intrasubject CV was at 55% and the sample size was 24 evaluable subjects, it was estimated that the lower and upper bounds of the 90% CI would be within 28% of the point estimate for AUC and Cmax
. It was planned to enroll at least 12 women to assess gender differences.
Study drug dosing.
Subjects were screened between 30 and 8 days before the first study day. In period 1 (day −1 to day 1), subjects continued their usual ABC regimen. In period 2 (day 2 to day 12), subjects stabilized on ABC 300 mg BID were switched to ABC 600 mg QD and those on ABC 600 mg QD were switched to ABC 300 mg BID from days 2 to 11. Subjects continued receiving all other antiretroviral agents. Subjects were discharged on day 12 and were allowed to switch back to their original ABC regimen. Subjects attended a follow-up visit 7 to 10 days afterwards. On the days of PK sampling (day 1 and day 11), subjects underwent fasting for at least 10 h prior to the ABC dose, the doses of ABC were taken under the supervision of clinic staff, and then subjects underwent fasting for 2 h after dosing; when subjects received an ABC BID-containing regimen, the evening dose of ABC was skipped on day 1 or day 11 to better characterize the PKs of intracellular CBV-TP.
Blood or urine samples for laboratory safety assessments (clinical chemistry, hematology, immunology, urinalysis, and pregnancy test) were taken on day −1, day 5, and day 10 and at follow-up. On day −1 and day 10 and at follow-up, blood samples (approximately 14 ml total) were also collected for measurement of viral load by the HIV-1 Monitor Version 1.0 PCR assay (lower limit of quantitation, 400 copies/ml) (Roche, Nutley, NJ) and of CD4+ lymphocyte cell count by flow cytometry. Vital signs were measured on day 2 and day 11. Samples for viral genotyping were collected on day 2 and day 12 and at follow-up. Adverse events were assessed intermittently throughout the study.
PK sample collection and processing.
Blood samples (16 ml each) were collected in CPT tubes (two tubes, 8 ml per sample) at the time points specified (predose and 2, 4, 6, 8, 12, 16, and 24 h postdose) for measuring intracellular CBV-TP and plasma ABC on day 1 and day 11. In the morning on day 2, subjects had the last 24-hour PK sample on their current regimens collected; this was immediately followed by the ABC regimen switch. An additional 2-ml blood sample for plasma ABC only was collected in a blood collection tube with EDTA at 1 h postdose on day 1 and day 11.
The CPT tubes were centrifuged within 60 min of blood collection at controlled room temperature (18 to 25°C) in a horizontal rotor (swing-out bucket) for 20 min at a relative centrifugal force of 1,500 to 1,800. A 1-ml aliquot of plasma was collected from the tube, transferred to a 1.8-ml Nunc polypropylene storage tube, and stored at ≤−20°C till assayed for ABC concentrations. The peripheral blood mononuclear cell (PBMC) layer was suspended in the remaining plasma, transferred from both CPT tubes to a single graduated 50-ml conical tube, and mixed with isotonic saline (0.9%) to achieve a total volume of 30 ml. A 500-μl aliquot was removed from the suspension for cell counting using a KOVA slide by trained personnel following a validated standard procedure. Cell counting was performed in duplicate and was repeated until cell counts from duplicates were within 25% of each other. The total number of PBMC counts from each PBMC sample (PBMCCT) was calculated and reported in the unit of million cells. The volume of remaining suspension was recorded (accurate to 0.1 ml), and the suspension was centrifuged for 15 min at a relative centrifugal force of 400 to pellet the cells. The supernatant was aspirated and discarded (plasma and platelets), and the cell pellet was well mixed with 1 ml ice-cold 70% methanol solution to lyse and completely redissolve the cell pellet. The lysed PBMC extract was completely transferred to a 1.8-ml Nunc cryostorage vial and stored at −70°C until assayed for CBV-TP concentrations.
PBMC extract samples were analyzed for CBV-TP by Taylor Technology (Princeton, NJ) using a validated analytical method based on anion exchange on Waters Accell QMA solid-phase extraction plates followed by enzymatic hydrolysis to CBV using alkaline phosphatase. The sample was cleaned up on a Varian C18 solid-phase extraction plate and reconstituted with water followed by high-performance liquid chromatography-tandem mass spectrometry (MS/MS) analysis using a YMC octyldecyl silane AQ 2.0- × 50-mm column and positive-ion MS/MS using a TurboIonSpray (MDS Sciex [Mississauga, Canada]) interface and multiple reaction monitoring. This method had a lower limit of quantification of 0.05 ng/ml with a 500-μl aliquot of human PBMC extracts. Bias and precision were calculated using interpolated concentrations of quality control samples at three concentration levels over a calibration range of 0.05 to 10 ng/ml. The interassay precision (CV) and interassay bias were 5.91% and −1.87%, respectively, with CBV-TP at 0.15 ng/ml; 5.58% and −1.39%, respectively, with CBV-TP at 1 ng/ml; and 3.54% and −1.41%, respectively, with CBV-TP at 7.5 ng/ml.
Plasma samples were analyzed for ABC concentration by GlaxoSmithKline (Research Triangle Park, NC) by protein precipitation and high-performance liquid chromatography-MS/MS in the positive-ion TurboIonSpray mode. The calibration curve range for ABC in human plasma was 2.5 to 2,500 ng/ml using a 50-μl sample aliquot of human plasma.
For each analytical method, quality control samples, containing the relevant analytes at three different concentrations and stored with study samples, were analyzed with each batch of samples against separately prepared calibration standards.
CBV-TP concentrations (C), in fmol/million cells, were calculated based on the CBV-TP concentration in PBMC extracts reported in ng/ml and cell counts (PBMCCT) using the formula C (fmol/million cells) = C (ng/ml) × 106/(molecular mass × PBMCCT/1 ml), where molecular mass is that of CBV-TP, 487.3 Da.
The following PK parameters for plasma ABC and intracellular CBV-TP were estimated using the noncompartmental Model 200 (for extravascular administration) of WinNonlin Professional Edition version 4.1 (Pharsight Corporation, Mountain View, CA) and actual elapsed time from dosing: AUC0-24, area under the drug concentration-time curve over a dosing interval at steady state (AUC0-τ), Cmax at steady state, time of maximum concentration at steady state, Cτ at steady state, and terminal t1/2. AUC0-24 for the BID regimen was calculated as 2 × AUC0-τ. In addition, the ratio of CBV-TP AUC0-24 and ABC AUC0-24 (AUCCBV/AUCABC) was calculated for each subject.
As female subjects enrolled in this study had lower body weights than did male subjects, in order to accurately evaluate the gender difference, weight-normalized (to a 70-kg person) PK parameters, including AUC0-24, Cmax, and Cτ, were estimated for plasma ABC and PBMC CBV-TP using the formula weight-normalized PK = PK × weight (in kg)/70 kg.
Summary statistics were provided for PK parameters by treatment and gender. Statistical analysis was performed to compare exposure differences in ABC and CBV-TP between ABC BID and ABC QD regimens and to assess gender effect. Analysis of variance, considering treatment arm, period, treatment, and gender as fixed effects and subject within treatment arm as a random effect, was performed using the SAS (version 8.2) mixed linear model procedure. The treatment-by-gender interaction was also included as a fixed effect in the model. Ratios of geometric least square means and associated 90% CIs were estimated for the key PK parameters: AUC0-24, Cmax, and Cτ (both original and weight normalized) for intracellular CBV-TP and plasma ABC and AUCCBV/AUCABC. A linear regression analysis, considering intracellular CBV-TP AUC0-24 as the response variable and plasma ABC AUC0-24 as the predictive variable, was also performed by treatment and overall to assess the relationship between exposures of plasma ABC and intracellular CBV-TP. The PK parameters were log transformed before the primary analyses, and treatment comparisons were expressed as ratios on the original scale. There was no formal statistical analysis of safety data.