Results from two open-label, multidose studies in healthy adult subjects are reported. The first study evaluated the pharmacokinetics of telbivudine (200 mg/day) and lamivudine (100 mg/day), alone and in combination. The second study evaluated the pharmacokinetics of telbivudine (600 mg/day) and adefovir (10 mg/day), alone and in combination. These studies were conducted in accordance with Good Clinical Practice procedures as described in ICH Harmonized Tripartite Guidelines, the U.S. Code of Federal Regulations (CRF), the principles of the Declaration of Helsinki, and U.S. Food and Drug Administration regulations. Approval for the studies was obtained from the appropriate local Institutional Review Boards.
The telbivudine-lamivudine study was conducted at MDS Pharma Services in Phoenix, Ariz. The first subject was dosed on 7 May 2001, and the last subject completed the final dose on 22 May 2001. The telbivudine-adefovir study was conducted at MDS Pharma Services in Lincoln, Nebr. The first subject was dosed on 29 September 2003, and the last subject completed the final dose on 22 October 2003.
The telbivudine-lamivudine study enrolled only male subjects, while both male and female subjects participated in the telbivudine-adefovir study. All subjects gave written informed consent after the nature of the studies was fully explained. Healthy adults from the general population who had voluntarily consented to participate in the studies were included if they met the following major inclusion criteria: aged 18 to 65 years; body weight within 15% of normal for their size and frame; and no evidence of clinically significant abnormalities on medical history, physical examination, 12-lead electrocardiogram, or clinical laboratory testing during screening. Female subjects were required to be practicing a double-barrier method of contraception plus systemic contraceptives or be surgically incapable of pregnancy or postmenopausal for at least 1 year. Pregnant or lactating female subjects were excluded from the studies. Subjects were also excluded if they had a history of clinically significant disease that, in the opinion of the investigator, might put them at risk; tested positive for human immunodeficiency virus, hepatitis C virus, or HBV; tested positive for drugs of abuse; had a history of alcohol abuse; or had participated in a clinical study within 30 days prior to study drug administration.
Both studies used an open-label, multidose, randomized, parallel-group design.
In the telbivudine-lamivudine study, 16 male subjects were randomized to two parallel groups, with 8 subjects in each group. Subjects in group 1 received telbivudine (200 mg/day; two 100-mg tablets) as a single agent on days 1 to 7 and then in combination (telbivudine at 200 mg/day plus lamivudine at 100 mg/day) on days 8 to 14. Subjects in group 2 received lamivudine (100 mg/day) as a single agent on days 1 to 7 and then in combination (telbivudine at 200 mg/day plus lamivudine at 100 mg/day) on days 8 to 14. Lamivudine was taken at the same time with telbivudine. Similarly, the telbivudine-adefovir study enrolled 16 healthy male and female subjects who were randomized to two parallel groups of 8 subjects each (designated group 3 and group 4). Subjects randomized to group 3 received telbivudine as a single agent (600 mg/day; three 200-mg tablets) on day 1. This dosing was repeated on days 3 to 16, thus resulting in 14 consecutive daily doses. Adefovir (10 mg/day, taken at the same time with telbivudine) was dosed in combination with telbivudine on days 10 to 16 (telbivudine at 600 mg/day plus adefovir at 10 mg/day). Subjects randomized to group 4 received adefovir (10 mg/day) as a single agent on days 1 to 7 and then in combination (telbivudine at 600 mg/day plus adefovir at 10 mg/day) on days 8 to 14.
For both studies, the subjects discontinued over-the-counter medications (including aspirin, vitamins, and herbal supplements) and any prescription medications except for oral contraceptives within 2 days prior to reporting to the clinics. Study medications were given on an empty stomach after a fasting period of approximately 10 h prior to each dose, and for an additional 4 h postdosing. All meals and beverages served during the studies were xanthine-free and caffeine-free. Grapefruit juice was not allowed from 48 h before the first dose until discharge from the studies. For each dose, 240 ml (8 fluid oz.) of water were given with the study medication dose(s). No other liquid intake was permitted from 1 h prior to dosing until 2 h postdosing. Water was permitted ad libitum 2 h postdosing. Subjects were asked to remain upright (sitting or standing) for the first 4 h after dosing and not to engage in any strenuous activity during their stay at the study centers.
In the telbivudine-lamivudine study, intensive pharmacokinetic sampling was performed over a period of 24 h after dosing on day 1 (group 1) and on days 7 and 14 (both groups). Serial blood samples (5 ml at each time point) for measuring plasma telbivudine and/or lamivudine were drawn into heparin-containing Vacutainer tubes at the following time points: 0 (predose), 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 10, 12, 16, and 24 h. Blood samples for monitoring trough levels of telbivudine and/or lamivudine in plasma were collected prior to daily dosing on days 5, 6, 12, and 13 for both groups. In the telbivudine-adefovir study, intensive pharmacokinetic sampling was performed over a period of 48 h after dosing on days 1 and 16 in group 3 and on day 14 in group 4 and over 24 h on day 9 in group 3 and on day 7 in group 4. Serial blood samples (5 ml at each time point) for measuring plasma telbivudine and/or adefovir were drawn into heparin-containing Vacutainer tubes at the following time points: 0 (predose), 0.5, 1, 2, 3, 4, 6, 8, 12, 16, 20, and 24 h (group 3 on day 9 and group 4 on day 7) and 28, 36, and 48 h (group 3 on days 1 and 16 and group 4 on day 14). Blood samples for monitoring trough plasma levels of telbivudine and/or adefovir were drawn prior to daily dosing on days 4 to 8 and 11 to 15 for group 3 and on days 1 to 6 and 9 to 13 for group 4, respectively.
Plasma was obtained by centrifugation and stored frozen at −20°C or below until analysis. The total durations encompassing sample collection and completion of bioanalysis were 35 days in the telbivudine-lamivudine study and up to 182 days in the telbivudine-adefovir study. All three drugs have been shown to remain stable during storage and assay. The short-term stability of the analytes in plasma has been documented when spiked samples were subjected to two to three freeze-thaw cycles (−22°C to room temperature) and storage at ambient temperature for 19 to 26 h.
Plasma sample analysis.
Plasma samples were analyzed for telbivudine, lamivudine, and adefovir by using validated high-performance liquid chromatography and tandem mass spectrometry (MS/MS) methodologies.
Study drugs were assessed simultaneously in the telbivudine-lamivudine study. The same assay was also used to measure plasma telbivudine in the telbivudine-adefovir study. Briefly, to 100 μl of calibration standards (10 to 5,000 ng/ml for telbivudine and 10 to 2,519 ng/ml for lamivudine), quality controls (QCs; 30 to 4,000 ng/ml for telbivudine and 30 to 2,000 ng/ml for lamivudine) and unknown plasma samples were added 40 μl of internal standard (β-l-2′-deoxyadenosine [LdA] at 650 ng/ml) containing thymidine phosphorylase (EC 184.108.40.206 [Sigma Chemical Co., St. Louis, Mo.]; >1 U/μl). The mixture was vortexed thoroughly and incubated at 37°C for 1 h to digest any endogenous thymidine that may interfere with telbivudine assay. After incubation, acetonitrile (200 μl) was added to precipitate protein. Samples were centrifuged, and the supernatant was transferred to injection vials. Extraction recovery for both analytes was nearly complete. Chromatography was performed on a TSK-GEL Amide-80 column (4.6 by 150 mm, 5 μm; Tosoh Bioscience, Montgomeryville, Pa.). Elution was carried out isocratically at 1 ml/min with a mobile phase of 90:10 (vol/vol) methanol-25 mM ammonium formate (pH 3.5). Under these conditions, the retention times were approximately 1.68, 1.73, and 1.73 min for telbivudine, lamivudine, and LdA, respectively. Telbivudine, lamivudine, and LdA were monitored by using a PE Sciex API 3000 MS/MS mass analyzer at mass transition of 243.0 to 127.1 m/z, 230.0 to 112.1 m/z, and 252.0 to 136.0 m/z, respectively. There was no interference at the specific mass transitions indicating the absence of matrix effect and lack of influence from concomitant drug. The mass analyzer was operated under positive mode using atmospheric pressure chemical ionization. This assay has a lower limit of quantitation of 10 ng/ml for both drugs with calibration curve ranging from 10 to 5,000 ng/ml for telbivudine and from 10 to 2,519 ng/ml for lamivudine. The intra- and interday precisions (CVs) and accuracies (percent deviation) were from 2.3 to 5.6% and −4.2 to 1.4%, respectively, based on QC samples with spiked concentrations ranging from 30 to 4,000 ng/ml for telbivudine, and were from 1.5 to 4.2% and −1.1 to 8.1%, respectively, based on QC samples with spiked concentrations ranging from 30 to 2,000 ng/ml for lamivudine.
Plasma adefovir was quantitated by using a proprietary high-pressure liquid chromatography-MS/MS methodology developed and validated at MDS Pharma Services (Saint Laurent, Montréal, Quebec, Canada). The adefovir assay has a lower limit of quantitation of 1 ng/ml, a calibration curve ranging from 1 to 200 ng/ml, and intra- and interday precisions (CVs) and accuracies (percent deviation) ranging from 1.9 to 6.6% and −1.2 to 5.0%, respectively, as well as an extraction recovery from 92.6 to 98.6%, based on QC samples ranging from 3 to 160 ng/ml. Adefovir was monitored at a mass transition of 274.1 to 162.1 m/z. This method is highly specific with no matrix effect or influence from coadministered telbivudine.
The plasma concentration time data were analyzed by using model-independent approaches with the WinNonlin software (version 4.1; Pharsight Corp., Mountain View, Calif.). The maximum plasma drug concentration (Cmax) and time to Cmax (Tmax) were directly obtained from the plasma concentration time profiles. The observed elimination half-life (t1/2) was calculated as 0.693/lz, where lz is the slope of the apparent elimination phase of the natural logarithmic (ln) transformation of the plasma drug concentration time curve estimated by using linear regression. The area under the plasma concentration-time curve from time zero to t (AUC0−t), where t is the time of last measurable sample, was calculated according to the linear trapezoidal rule. The AUC from time zero to infinity (AUC0−∞) was estimated as AUC0−t + Ct/lz, where Ct is the plasma concentration of the last measurable sample. AUCτ, a measure of the extent of exposure over the dose interval (τ = 24 h), was also calculated. Oral apparent total plasma clearance (CL/F) was calculated as dose/AUC0−∞ after a single dose and as dose/AUCτ at steady state.
In the telbivudine-lamivudine study, attainment of steady state for telbivudine and lamivudine on days 7 and 14 was evaluated by regressing the ln-transformed trough levels on days 5, 6, and 7 and on days 12, 13, and 14 over time, respectively. Similarly, in the telbivudine-adefovir study, attainment of telbivudine steady state on days 9 and 16 in group 3 and on day 14 in group 4 was evaluated by regressing the ln-transformed trough levels on days 8 to 10 and days 11 to 17 (group 3) and on days 13 to 15 (group 4) over time. Attainment of adefovir steady state on day 16 in group 3 and on days 7 and 14 in group 4 was evaluated by regressing the ln-transformed trough levels on days 14 to 17 (group 3) and on days 5 to 7 and days 9 to 15 (group 4) over time. Steady state was concluded if the slope was not statistically different from zero (P > 0.05).
Plasma pharmacokinetic drug-drug interaction was assessed by comparing combination with a single agent for the principal parameters underlying plasma exposure (AUCτ and Cmax). A parametric (normal-theory) general linear model was applied to the ln-transformed AUCτ and Cmax. An analysis of variance with subject and treatment as factors was performed by using the GLM procedure in SAS (SAS Institute, Inc., Cary, NC). The results for AUCτ and Cmax were reported as 90% confidence intervals (CI) surrounding the ratio (percentage) of the geometric means of the pharmacokinetic measures with or without the second drug. It was concluded that no statistically significant plasma drug-drug interaction exists if the 90% CI of the ratio of geometric means were to lie within 80 and 125% for AUCτ and Cmax.
The safety analysis included all subjects who received at least one dose of study medication. Safety measurements included clinical history, physical examination, laboratory measurements (hematology, serum chemistries, and urinalysis), vital signs, and assessments of adverse events (AEs). AEs, listed by preferred term and body system, were coded by using MedDRA version 3.3 or higher.