Eligibility
Eligible subjects had a diagnosis of a primary CNS tumor (excluding CNS lymphoma), were at least 18 years of age and had a Karnofsky performance score of 70 of higher. All subjects were required to have adequate hematologic, hepatic and renal function. Subjects were excluded if they had impaired gastrointestinal absorption, vomiting or any other medical condition that would compromise the intake of oral medication. Subjects were excluded if they had received chemotherapy or biologic anticancer therapy within 4 weeks before study entry, or mitomycin C or nitrosourea therapy within 6 weeks before study entry. This study was conducted in accordance with good clinical practice (GCP) and in compliance with the World Medical Association Declaration of Helsinki with respect to written informed consent and the protection of rights of human subjects.
Study design
This multicenter, open-label, randomized, crossover study compared the PK of intravenous and oral temozolomide. As this study was conducted in the context of treating subjects with primary CNS malignancies, oral temozolomide was administered at the highest approved dose (200 mg/m2) on days 1, 2 and 5. On days 3 and 4, temozolomide was administered orally on one day and by 90-min intravenous infusion on an alternate day at a dose of 150 mg/m2 (the approved dose of temozolomide for the first cycle of treatment). Subjects were assigned, according to a computer-generated random code, to receive intravenous temozolomide either on day 3 or day 4 with oral temozolomide on an alternate day. All daily oral doses were rounded down to the nearest 5 mg. The doses to be administered on days 3 and 4, intravenous versus oral, were identical. If vomiting occurred during oral dosing, the subject was not redosed. For PK sampling on days 3 and 4, subjects were to fast for a minimum of 8 h before each dose of temozolomide and to continue fasting for 4 h afterward.
The primary objective was to evaluate exposure equivalence of a 90-min intravenous infusion to an equivalent oral dose of temozolomide based on the ratio of the log-transformed means for AUC and
C
max for both temozolomide and MTIC. Based on regulatory guidelines, exposure equivalence was defined as a 90% confidence interval (CI) for the ratio of the means based on log-transformed data within the range of 80–125% [
9,
10]. Secondary end points included local tolerability and safety. Adverse events were graded according to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 3.0 over a 28-day period beginning from administration of the first dose of temozolomide.
The PK of temozolomide and MTIC following intravenous and oral administration was determined from serial blood samples taken on days 3 and 4, just before dosing (0 h), and at 0.25, 0.50, 1.00, 1.25, 1.5 (for intravenous dose, within 5 min after the end of infusion), 1.75, 2.00, 2.50, 3.00, 4.00, 6.00 and 8.00 h after initiation of infusion or administration of the oral dose. Plasma temozolomide and MTIC samples were collected and procured as previously described [
4,
11,
12]. Briefly, blood samples for MTIC were collected in prechilled heparinized tubes and immediately centrifuged for 10 min at 3,000 rpm at 4°C. The resulting plasma was immediately frozen in a dry ice methanol bath and then stored at -70°C until assayed. Blood samples for temozolomide were collected in prechilled heparinized tubes and then centrifuged for 10 min at 3,000 rpm at 4°C. Immediately following centrifugation, 50 μL of 8.5% phosphoric acid was added to each mL of plasma. Samples were then vortexed and stored at −20°C until assayed. Plasma concentrations of temozolomide and MTIC were determined by liquid chromatography, followed by tandem mass spectroscopy (LC-MS/MS). The lower limit of quantitation (LLOQ) for temozolomide and MTIC were 20 and 5 ng/mL, respectively. These methods were validated for selectivity, sensitivity, precision and accuracy. The stability of temozolomide in plasma after the addition of phosphoric acid and the stability of MTIC in plasma without phosphoric acid under various conditions were established. The temozolomide plasma assay was linear over the range of 20–30,000 ng/mL. The accuracy ranged from −6.8 to −2.1%, and the precision was 9.1–10%. The internal standard was ethazolastone. The MTIC plasma assay was linear over the range of 5–4,000 ng/mL. The accuracy ranged from –3.3 to 0.8% and the precision was 3.1–9.4%. The internal standard was dacarbazine.
The study protocol was written such that data from subjects/samples could be prospectively excluded from the primary analysis in case of protocol violations, unsuccessful dosing or possible sample procurement errors. For instance, subjects who vomited within 4 h of oral dosing on pharmacokinetic days, whose dose on days 3 and 4 were not within 10% of the recommended dose, or subjects whose intravenous infusion duration was not within 10% of 90 min were excluded. Additionally, if anomalous temozolomide or MTIC concentrations were observed (i.e., concentrations below LLOQ, a zero concentration between two non-LLOQ concentrations, or an LLOQ between two non-zero concentrations), the pH of the respective PK sample was checked to ensure that the sample was properly procured. If the sample was not at the recommended pH necessary to stabilize the analyte, the sample was excluded from analyses.
Noncompartmental analyses were conducted on individual concentration–time data. Log-transformed PK parameters (AUC and C
max) for temozolomide and MTIC were subjected to a crossover analysis of variance (ANOVA) model, extracting the effects due to treatment, sequence, subject within sequence and period. Assuming an intrasubject variability (coefficient of variation) of 20% and modeling and simulation results, a target enrollment of 20 subjects was selected to provide a minimum of 90% power for the 90% CI of the ratio of the treatment means for derived AUC and C
max for temozolomide and MTIC to fall within the 80–125% confidence range.