Eligible patients included men and women (ages, 18 to 55 years, inclusive) in whom hepatitis B surface antigen (HBsAg) was detectable in serum for at least 3 months or who were positive for HBsAg and negative for immunoglobulin M antibody to hepatitis B core antigen. In addition, detectable HBV DNA (determined by the Quantiplex HBV DNA assay [Chiron, Emeryville, Calif.]) and aspartate aminotransferase or alanine aminotransferase (ALT) levels no more than 2.5 times the upper limit of normal (ULN) were required for inclusion in the study. Patients were not eligible for the study if they had hepatitis C or D or HIV type 1 infection or decompensated liver disease (defined as a bilirubin level more than 1.5 times the ULN, a prothrombin time less than 60% of that for the control or more than 1.25 times the ULN, or a history of ascites, variceal hemorrhage, or hepatic encephalopathy). Patients were also excluded if they had received interferon within the past 3 months or nucleoside analogues or other antivirals for a duration of more than 3 months or within 60 days of screening or were receiving antiviral, immunomodulatory, or corticosteroid therapy.
The study was approved by the institutional review board or independent ethics committee at the participating centers, and all the patients gave written informed consent before enrollment in the study.
The study described here was a phase I-II dose escalation cohort study. Following a 30-day screening period, the patients were sequentially enrolled into five cohorts (each cohort contained a minimum of eight patients) for q.d. dosing for 8 weeks, with a follow-up period extending 28 days after the treatment period concluded. Patients returned at the baseline and treatment days 7, 14, 28, 42, and 56. Serum was assayed for HBV DNA level and hepatitis B e antigen (HBeAg) at the time of screening, at the baseline, and at each on-treatment visit. During the follow-up period, HBV DNA levels were measured 24 and 48 h after the administration of the last dose and again after 28 days of follow-up. Physical examination, evaluation of vital signs, and laboratory hematology, biochemistry, and urinalysis studies for the assessment of safety were performed at all visits except for the follow-up visits 24 and 48 h after administration of the final FTC dose.
Initiation of treatment in subsequent dose cohorts occurred after the preceding cohort was filled and after it was determined that the FTC safety profile was acceptable for the previous cohort. A cohort size of 8 to 12 patients was allowed per protocol. The dose levels evaluated were 25, 50, 100, 200, and 300 mg (all doses were given q.d.).
Drug assay and pharmacokinetic evaluations.
The pharmacokinetics of FTC were evaluated following the administration of a single dose (the first dose) and at steady state. On day 1 and on day 28, patients took FTC following an overnight fast and did not eat until 3 h after they had ingested that dose. Blood or plasma samples were collected prior to administration of the dose and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 h after administration. Samples for determination of trough concentrations were obtained just prior to dosing on the morning of days 7, 14, 42, and 56.
Samples of blood (7 ml) were collected in tubes with potassium EDTA and were processed to provide plasma for FTC assays. The samples were analyzed by a validated liquid chromatography-mass spectrometry-mass spectrometry methodology with a quantitation range of 10 to 2,500 ng/ml. FTC was resolved with an aqueous-organic mobile phase on a reversed-phase column and was detected by mass spectrometry-mass spectrometry with atmospheric pressure chemical ionization. The interassay variability ranged from 3.7 to 9.2%, and the intraday assay variability ranged from 4.1 to 7.1%. The assay method was accurate, with the difference from the nominal value ranging from −1.3 to 8.1%.
Pharmacokinetic parameters were derived by noncompartmental methods. The principal parameters of interest were the maximum drug concentration in plasma (Cmax), the time to Cmax, the minimum drug concentration, the area under the concentration-time curve (AUC), and the apparent total body clearance.
The samples were assayed for HBV DNA at Triangle Pharmaceuticals, Inc., by use of the Digene HBV Hybrid Capture II assay. This is a signal amplification hybridization microplate assay that uses chemiluminescence for the detection and quantitation of HBV DNA in human serum and that has a limit of detection (LOD) of 4,700 copies/ml (0.017 pg/ml). The acceptability criterion for interassay and intra-assay variability was 20%. No genotypic analysis was performed.
All analyses of data related to the HBV DNA level were conducted with log10 transformed data, with comparisons made to the baseline levels.
The log10 HBV DNA levels and the change in log10 HBV DNA levels from the baseline levels for each patient were summarized by cohort for each on-treatment visit and were plotted over time.
The average area under the curve minus baseline (AAUCMB) for HBV DNA obtained by the Digene assay was calculated, tabulated, and plotted for all patients. The AAUCMB was derived from the area under the response-time curve (AUC) by the trapezoid method divided by the time from the baseline to the last available on-treatment value minus the baseline value, i.e.,
where C0,i is the measurement for patient i at the baseline, Cj,i is the measurement for patient i at week tj, tn is the last time point for which data on the HBV DNA level were available, tj is the actual day of the jth visit, and t0 is time zero. Data for patients for whom at least one evaluation was conducted after the baseline were included in the AAUCMB analysis.
The dose-response relationship of the anti-HBV activity of FTC was also evaluated by use of a pharmacological (Emax) model, as described by the following equation: antiviral activity = (Emax × dose)/(ED50 + dose), where Emax is the maximal anti-HBV activity, and EC50 is the dose required to produce 50% of the maximal anti-HBV activity. This equation contains two unknown parameters, Emax and ED50. The known parameters, dose and antiviral activity at each dose, are plotted, and the curve with the best fit is applied by use of this model. In the equation, antiviral activity was assessed by using two parameters, AAUCMB for the HBV DNA level and the level of suppression of the viral load from that at the baseline to that on day 56. The parameter that provided the best overall correlation with anti-HBV activity was AAUCMB because of the averaging effect over the entire dosing period versus a single measure of viral load suppression on the last day of dosing (the last on-treatment measurement).