Absorption, Metabolism, and Excretion of C-Temozolomide following Oral Administration to Patients with Advanced Cancer

The purpose of this study is to characterize the absorption, metabolism, and excretion of carbon 14-labeled temozolomide (C-TMZ) administered p.o. to adult patients with advanced solid malignancies. On day 1 of cycle 1, six patients received a single oral 200-mg dose of C-TMZ (70.2 mCi). Whole blood, plasma, urine, and feces were collected from days 1–8 and on day 14 of cycle 1. Total radioactivity was measured in all samples. TMZ, 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide (MTIC), and 4-amino-5-imidazole-carboxamide (AIC) concentrations were determined in plasma, and urine and plasma samples were profiled for metabolite/degradation products. Maximum TMZ plasma concentrations were achieved between 0.33 to 2 h (mean, 1.2 h), and half-life, apparent volume of distribution, and oral clearance values averaged 1.9 h, 17 liters/m, and 104 ml/min/m, respectively. A firstorder absorption, one-compartment linear model, which included first-order formation of MTIC from TMZ and elimination of MTIC via degradation to AIC, and a peripheral distribution compartment for AIC, adequately described the plasma TMZ, MTIC, and AIC concentrations. MTIC systemic clearance was estimated to be 5384 ml/min/m, and the half-life was calculated to be 2.5 min. Metabolite profiles of plasma at 1 and 4 h after treatment showed that C-derived radioactivity was primarily associated with TMZ, and a smaller amount was attributed to AIC. Profiles of urine samples from 0–24 h revealed that C-TMZ-derived urinary radioactivity was primarily associated with unchanged drug (5.6%), AIC (12%), or 3-methyl-2,3-dihydro-4-oxoimidazo[5,1-d]tetrazine-8-carboxylic acid (2.3%). The recovered radioactive dose (39%) was principally eliminated in the urine (38%), and a small amount (0.8%) was excreted in the feces. TMZ exhibits rapid oral absorption and high systemic availability. The primary elimination pathway for TMZ is by pH-dependent degradation to MTIC and further degradation to AIC. Incomplete recovery of radioactivity may be explained by the incorporation of AIC into nucleic acids. INTRODUCTION TMZ is an alkylating agent of the imidazotetrazine series that has demonstrated notable antitumor activity in patients with recurrent and refractory high-grade glioma and melanoma in phase I and II trials (1–6). The results of both preclinical and phase I studies indicate that TMZ is completely bioavailable (F ; 100%) when administered p.o. (1) and that antitumor activity is schedule-dependent (7, 8). Prominent clinical activity was observed principally on frequent dosing schedules, particularly when TMZ is given p.o. once a day for 5 days every 4 weeks (1–6). Present clinical developmental efforts are defining the role for TMZ in treating patients with high-grade glioma, melanoma, and other various malignancies. It has been proposed that TMZ and DTIC, a structurally related alkylating agent that is used clinically to treat melanoma and lymphoma, exert their antitumor activity by generating the linear triazene MTIC (7, 9), which reportedly alkylates the O-position of guanine in DNA, with additional alkylation also occurring at the N-position (9–12). Unlike DTIC, which reReceived 9/23/98; accepted 11/17/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by a grant from Schering-Plough Research Institute and by Grant RR-00035 to the Adult General Clinical Research Center, The Johns Hopkins Hospital. 2 To whom requests for reprints should be addressed, at Department of Clinical Research, Cancer Therapy and Research Center, Institute for Drug Development, 8122 Datapoint, Suite 700, San Antonio, TX 78229. Phone: (210) 616-5850; Fax: (210) 692-7502; E-mail: [email protected]. 3 The abbreviations used are: TMZ, temozolomide; MTIC, 5-(3-methyltriazen-1-yl)imidazole-4-carboxamide; AIC, and 4-amino-5-imidazole-carboxamide; TMA, 3-methyl-2,3-dihydro-4-oxoimidazo[5,1-d] tetrazine-8-carboxylic acid, temozolomide acid; C-TMZ, carbon 14labeled temozolomide; DTIC, dacarbazine; HPLC, high performance liquid chromatography; IS, internal standard; LOQ, lower limit of quantitation; Cmax, maximum concentration; Tmax, time of maximum concentration; AUC, area under the plasma or blood concentration-time curve; Cls/F, temozolomide systemic clearance; Varea/F, temozolomide apparent volume of distribution, Tlag, lag time; ka, first-order absorption rate constant; Vc/F, volume of distribution for TMZ and MTIC, and the central volume of distribution for AIC; Vp,AIC/F, peripheral volume of distribution for AIC; Cls,TMZ/F, clearance for the conversion of TMZ to MTIC; Cls,MTIC/F, clearance for the conversion of MTIC to AIC; Cld,AIC/F, AIC distribution clearance; Cls,AIC/F, AIC systemic clearance; elimination rate constant (ke); CV, coefficient of variation, tf, time of final quantifiable sample; AUC(0–24 h), AUC from time 0–24 h; AUC(I), AUC from time zero to infinity; t1/2, terminal half-life. 309 Vol. 5, 309–317, February 1999 Clinical Cancer Research Research. on October 24, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from quires oxidative N-demethylation by cytochrome P450 enzymes (8), TMZ is converted to MTIC under physiological conditions by a nonenzymatic, chemical degradation process (Fig. 1; Refs. 7–9). The conversion of TMZ to MTIC and the further breakdown of MTIC to AIC and a methyl-diazonium cation (Fig. 1), is irreversible and pH-dependent. In aqueous buffers, TMZ is stable at pH ,4, but rapidly decomposes to MTIC at pH .7; in contrast, MTIC is stable at alkaline pH, but rapidly breaks down to AIC at pH ,7 (9, 13, 14). TMZ has an in vitro half-life of 1.9 h in phosphate buffer at 37°C and pH 7.4, whereas MTIC placed in the same solution has a half-life of ;2 min (9). A small percentage of an administered TMZ dose is metabolized (2%) to TMA, the carboxylic acid analogue of TMZ (Fig. 1; Ref. 15). The oral bioavailability and plasma disposition of an early formulation of TMZ administered as a single dose of 200-1200 mg/m were characterized by Newlands et al. (1). At the 200 mg/m dose level, bioavailability of TMZ was complete in five patients who received TMZ both p.o. and i.v. in a crossover study design fashion; mean absolute bioavailability was 109%. Over the dose range studied, drug exposure measured as AUC increased linearly with dose, and the terminal half-life and clearance were dose-independent with mean values of 1.8 h and 196 ml/min, respectively. The plasma disposition and urinary excretion of TMZ and its degradation products were characterized in patients with solid malignancies treated with TMZ ranging from 50–250 mg/m/day given daily for 5 days (5, 13, 16). Concentrations of TMZ, MTIC, AIC, and TMA were determined in plasma, and concentrations of TMZ and TMA were determined in urine. TMZ was rapidly absorbed and eliminated, with a mean time to peak plasma concentration of 1 h and a mean terminal half-life of 1.8 h. Maximum plasma concentrations and AUC values increased proportionally with dose, and no accumulation of drug in plasma was observed over 5 days of TMZ administration. Mean oral clearance values ranged from 102–115 ml/min/m with a CV of ,20%. The appearance and disappearance of MTIC paralleled that of parent compound in plasma. On the basis of AUC values, MTIC, AIC, and TMA represented approximately 2.2%, 40%, and 0.8% of systemic exposure to TMZ, respectively. The mean urinary excretion of TMZ and TMA was minimal with values of 5.6% and 0.8%, respectively. Although the antitumor activities of TMZ in patients with melanoma and high-grade glioma have been well characterized and pivotal clinical efficacy evaluations are in progress, a comprehensive characterization of the absorption and systemic availability, metabolism/degradation and disposition in blood and plasma and total body excretion of TMZ and its degradation products after oral administration has not been performed. The principal objective of this study was to define the absorption, metabolism, and excretion of oral TMZ after administration of a single dose of C-TMZ to patients with advanced cancer. A secondary goal was to develop a pharmacokinetic model to: (a) describe the plasma disposition of TMZ and its degradation/ bioconversion products MTIC and AIC; and (b) characterize the kinetics of the conversion of TMZ to MTIC and the subsequent degradation of MTIC to AIC in these patients. PATIENTS AND METHODS Patient Selection. Male or female patients with histologically confirmed advanced solid malignancies who failed to respond to standard therapy or for whom adequate therapy was not available were eligible for this study. Eligibility criteria also included: age $18 years; an Eastern Cooperative Oncology Group Performance Status # 2 (ambulatory and capable of self-care); a life expectancy of $12 weeks; adequate hematopoietic (absolute neutrophil count $1,500/ml, platelet count $100,000/ml, hemoglobin $10.0 g/dl), hepatic (total bilirubin within the upper limits of institutional laboratory normal, and aspartate aminotransferase or alanine aminotransferase and alkaline phosphatase #2 times the upper limit of institutional laboratory normal), and renal (serum creatinine within the upper limits of institutional laboratory normal) functions; no history or evidence of a medical condition that might affect gastrointestinal function; no surgical resection of the stomach or small bowel; no chemotherapy or biological therapy within 4 weeks of treatment (6 weeks if the previous chemotherapy regimen included mitomycin C or a nitrosourea); no radiation therapy to .50% of bone marrow; and no other coexisting medical conditions of sufficient severity to prevent full compliance with the study. Patients gave informed, written consent according to federal and institutional guidelines. Study Design. This single institution phase I study was designed to characterize the absorption, metabolism, and excretion of C-TMZ administered p.o. to six adult patients with advanced cancer. On day 1 of cycle 1, patients received a single dose of C-TMZ. Blood, urine, and feces samples were collected from days 1–8 and on day 14 of cycle 1 for pharmacokinetic studies. Patients were hospitalized for the intensive pharmacokinetic evaluation on days 1–8, after which they received the remainder of their TMZ (unlabeled) treatment on days 8–11 of cycle 1 at a dose of 150 mg/m/day for 4 consecutive days. After completion of cycle 1, patients were treated with TMZ (150 mg/m/day) for 5 consecutive days every 28 days, with dose adjustments made according to hematological tolerance. Treatment was continued in this phase of the study, which is not summarized in this study, as long as disease progression did not occur. Fig. 1 Chemical structure of TMZ and proposed metabolism and degradation pathways. p, the position of the C-labeled carbon atom. 310 Absorption, Metabolism, and Excretion of C-TMZ Research. on October 24, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Drug Dosage and Administration. Patients were administered a 200-mg dose of TMZ containing 70.2 mCi of C-TMZ with 8 ounces of noncarbonated water in the morning after an overnight fast. Patients continued to fast until 4 h after treatment. To ensure uniform hydration and adequate urine collection, patients were instructed to drink 8 ounces of noncarbonated water at 2, 6, 10, and 14 h after treatment. Approximately 1 h before administration of C-TMZ, patients were treated with i.v. ondansetron (32 mg) to prevent nausea and vomiting. Any medication that could potentially influence gastric pH or drug absorption (e.g., histamine-2 antagonists, omeprazole, sulcrafate) were withheld for a period of 48 h before treatment and 8 h after treatment. Patients were not treated with antacids for a period of 12 h before TMZ treatment and 8 h after treatment. Radiolabeled TMZ was supplied as capsules of 100 mg of C-TMZ (35.1 mCi; specific activity ;0.375 mCi/mg) by Schering-Plough Research Institute (Kenilworth, NJ). The C label was on the sixth position of the imidazole ring of TMZ, as shown in Fig. 1. Sample Collection and Processing. Blood samples were collected in prechilled heparinized tubes and immediately placed in an ice-water bath. Two blood samples were collected before treatment, at 20 and 40 min, and at 1, 1.5, 2, 3, 4, 8, 12, and 24 h after C-TMZ administration. The first blood sample (;1.5 ml) was drawn for MTIC plasma concentration determinations. Plasma was immediately separated by centrifugation (4°C), divided into two aliquots, frozen immediately in a dry iced-methanol bath, and stored at 280°C, pending analysis of MTIC. The second blood sample (;7.5 ml) was collected for blood and plasma radioactivity and TMZ and AIC plasma concentration determinations. Before centrifugation, 2 3 0.2-ml aliquots of whole blood were transferred to two combustion cones fitted with a combustion pad, placed into scintillation vials, and immediately frozen at 220°C. Within 20 min after collection, the remaining blood was centrifuged (4°C), plasma was placed in a plastic tube containing 0.2 ml of 8.5% phosphoric acid to chemically stabilize TMZ, mixed, the pH adjusted to pH 4 (with additional phosphoric acid, if necessary), and then frozen at 280°C. Blood samples were also collected at 48, 72, 96, 120, 144, and 168 h after C-TMZ administration and similarly processed for determination of blood and plasma radioactivity and TMZ and AIC concentrations in plasma. Additional blood samples (;22 ml) were collected at 1 and 4 h after C-TMZ administration to assess ex vivo protein binding and for metabolite profiling. Blood samples were centrifuged (4°C) and 3 3 1-ml aliquots of plasma were transferred to the sample reservoir of three separate ultrafiltration devices. The ultrafiltration devices were centrifuged at 1000–2000g for 20 min at 37°C, and the filtrate cup was removed and frozen at 220°C. The remaining plasma was divided into two equal portions, acidified with 0.2 ml of 8.5% phosphoric acid, adjusted to pH 4, if necessary, and frozen at 220°C until assayed for a metabolite profile(s). Urine was collected in plastic containers containing 2 ml of 8.5% phosphoric acid solution at baseline and then continuously during the following timed collections: 0–4, 4–8, 8–12, 12–24, 48–72, 72–96, 96–120, 120–144, and 144–168 h after CTMZ administration. An additional 24-h urine collection was obtained between days 13 and 14. After each void, the pH was determined and adjusted to pH 4 with additional 8.5% phosphoric acid, if necessary. For the first 48 h, the entire volume of urine collected during each interval was retained; for all other intervals, the total volume of urine was recorded at the end of the collection period, and 2 3 50-ml aliquots were frozen at 220°C until assayed for radioactivity. A fecal sample was collected before drug administration, and then all feces were collected continuously until 168 h after administration of C-TMZ. An additional fecal sample was collected on day 14. The fecal samples were transferred to plastic containers and immediately frozen at 220°C until assayed for radioactivity. Sample Preparation and Analysis. All samples were assayed for total radioactivity using liquid scintillation spectrometry with external standardization methods. Plasma samples also were assayed for TMZ, MTIC, and AIC using HPLC and UV detection with modifications of previously described procedures (13, 14, 17). Briefly, plasma samples were enriched with ethazolastone solution (IS) and extracted with ethyl acetate. Samples were centrifuged, and the organic layer was removed and completely evaporated under a stream of nitrogen. The final extract was reconstituted with HPLC mobile phase [0.1% acetic acid and acetonitrile (90/10, v/v)] and analyzed by reversedphase HPLC. Samples were injected onto an Ultrasphere octadecyl silane column equipped with a silica guard column, and column eluate was monitored at 316 nm. Over the range of 0.100–20.0 mg/ml, the TMZ assay precision (percentage of CV) was 6.7%, accuracy (percentage of bias) was 2.8%, and the LOQ in plasma was 0.100 mg/ml. For MTIC determinations, plasma samples were enriched with ice-cold metronidazole solution (IS), and plasma proteins were precipitated with isopropyl alcohol. Samples were vortex mixed, centrifuged (4°C), and a portion of the resultant supernatant was diluted in mobile phase (50 mM ammonium acetate containing 2% isopropyl alcohol). Extracts were analyzed using a C8 reversed-phase column protected by a C18 guard column, and MTIC was detected at 316 nm. Over the range of 25–1000 ng/ml, assay precision and accuracy was 14% and 5.1%, respectively, and the LOQ for MTIC in plasma was 25 ng/ml. To determine AIC plasma concentrations, samples were enriched with AICO solution (IS) and extracted by solid-phase extraction using a strong cation-exchange column. AIC was eluted from the cation-exchange resin using methanol containing 0.6% ammonium hydroxide. The resulting eluate was dried under a stream of nitrogen, and reconstituted with methanol. The mobile phase consisted of 0.1% trifluoroacetic acid (adjusted to pH 2.5 with triethylamine) and methanol. Separations were performed on an octadecyl silane reversed-phase column using a gradient elution system, and AIC was detected at 265 nm. AIC assay precision (10%) and accuracy (4.5%) were acceptable over the concentration range of 20–2000 ng/ml, and the LOQ for AIC in plasma was 20 ng/ml. Plasma samples, collected at 1 and 4 h after treatment for metabolite profiles, were pooled from all six patients at each time point, and ;1% of the total urine volume excreted from each patient within each of the 0–4, 4–8, 8–24, 24–48, and 48–72-h collection intervals was pooled for determination of metabolite profiles. Pooled plasma samples were extracted by protein precipitation using methanol acidified with 70% per311 Clinical Cancer Research Research. on October 24, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from chloric acid, mixed, and centrifuged. This and all subsequent centrifugation steps were performed at ambient temperature. The supernatant was filtered (0.45 mm) and transferred into a glass tube, reduced in volume to 250–500 ml under a stream of nitrogen (30°C), and centrifuged. The clarified supernatant was transferred to a microcentrifuge tube equipped with a filter (0.2 mm), centrifuged at 15,800 3 g for 5 min, and hexane sulfonic acid was added to achieve a 50-mM concentration. A standard solution containing 500 mg/ml each of TMZ, TMA, and AIC was prepared in 50 mM hexane sulfonic acid in 0.5% acetic acid, and a 30-ml aliquot of this standard solution was added to each sample and mixed before chromatographic analysis. Pooled urine from each collection time interval was freezedried for ;16 h (lyophilized volume: #8.4 ml for the 0–4-, 4–8-, and 8–24-h collection interval; $20 ml for the 24–48and 48–72-h collection period). Lyophilized residues from the 0–4-, 4–8-, and 8–24-h samples were each reconstituted into 100 ml of the standard solution along with an additional 100 ml of 50 mM hexane sulfonic acid in 0.5% acetic acid, and then centrifuged at 2000 3 g for 5 min. Twenty (20) and 150-ml aliquots of the clarified supernatant were used to determine total radioactivity and profile metabolites, respectively. Due to the high endogenous UV background that was observed with smaller volumes of lyophilized urine, the 24–48and 48–72-h samples were expected to saturate detector response at 254 nm. Therefore, lyophilized residues from these collection periods were not enriched with standard mixture, but were instead reconstituted into 500 ml of 50 mM hexane sulfonic acid in 0.5% acetic acid and processed in the same manner as those from the earlier collection time points. Plasma and urine samples were not evaluated for the presence of conjugated metabolites because previous studies had shown that TMZ and/or its metabolites were unstable under conditions normally used for enzymatic hydrolysis (data not shown). Chromatographic analysis for metabolite profiling was performed using an HPLC system consisting of a Waters 600E System Controller, a Waters Model 717 plus Autosampler, a Supelco LC-DB C18 (4.6 3 250 mm) HPLC column, a Waters 18 Symmetry guard column, and a Waters Model 486 Tunable Absorbance Detector at 254 nm. In-line detection of radioactive effluent was performed on a Packard Instruments Radiomatic A250 Flo-One radiometric flow detector. Packard Ultima-Flo liquid scintillation mixture was mixed post-column with the eluent at a 3:1 ratio (v/v). For plasma samples that contained concentrations of radioactivity too low for in-line detection, 0.5-min fractions were collected postradioactive detector using an ISCO Foxy fraction collector; fractions were then mixed with 5 ml of Ultima Gold XR liquid scintillation fluid, which in turn were analyzed using a Packard Tri-Carb Model 2550 TR/LL Liquid Scintillation Analyzer. A linear gradient elution program using 50 nM hexane sulfonic acid prepared in methanol and 50 mM hexane sulfonic acid in 0.5% acetic acid was generated by the Waters 600E System Controller set at Gradient #6. Separations were accomplished at 31°C using an Eppendorf CH-30 Column Heater; and the mobile phase flow rate was maintained at 1 ml/min. The signal outputs from the UV and radiometric detectors were collected using the Flo-One software. Additionally, plasma samples obtained at 1 and 4 h were assayed by an ultrafiltration procedure to determine the extent of protein binding. Three 1-ml aliquots of plasma were centrifuged (37°C) at 1000–2000 3 g for 20 min using Centrifree micropartition tubes (Amicon). Because of the known instability of TMZ in plasma at physiological pH (7–9, 14), protein binding due to total drug-derived radioactivity was evaluated rather than that of TMZ. Unfiltered plasma and ultrafiltrate radioactivity concentrations were used to determine the percentage of protein-bound radioactivity and the free fraction of radioactivity. Unfiltered radioactivity concentrations were adjusted by a dilution factor, which corrected for the addition of phosphoric acid to the plasma samples. The percentage of protein-bound radioactivity was calculated by subtracting the ultrafiltrate concentration from the calculated unfiltered concentration, dividing the result by the calculated unfiltered concentration, and multiplying the results by 100. The free fraction was calculated by subtracting the percentage bound from 100. Pharmacokinetic Analysis. Individual concentrations above the individual matrix LOQs for radioactivity, TMZ, MTIC, and AIC in plasma and radioactivity in blood were analyzed using model-independent methods (18). The Cmax and Tmax were the observed values on inspection of the concentration-time curves. The terminal rate constant, k, was calculated as the negative of the slope of the log-linear terminal portion of the plasma concentration-time curve using linear regression. The terminal half-life, t1/2, was calculated as 0.693/k. The AUC from time zero to the time of the final quantifiable sample, AUC(tf), was calculated using the linear trapezoidal method and was extrapolated to infinity (I) according to the following equation: AUC~I! 5 AUC~tf! 1 C~tf!/K where C(tf) was the estimated concentration at time tf. When the concentration of TMZ, MTIC, or AIC at 24 h was below the LOQ, and the k was determinable, AUC(I) was used to approximate the AUC from time 0–24 h, AUC(0–24 h). Otherwise, AUC(0–24 h) was also calculated using the linear trapezoidal method. TMZ Cls/F was calculated as the dose divided by AUC(I). TMZ Varea/F was calculated by dividing Cls/F by k. Relative systemic exposure of MTIC and AIC to that of TMZ was calculated as the AUC(0–24 h) ratio of MTIC:TMZ and AIC:TMZ, respectively. Plasma concentration data for TMZ, MTIC, and AIC were also analyzed using model-dependent methods. A first-order absorption, one-compartment linear model, which included first-order metabolite formation and elimination and a peripheral distribution compartment for AIC (Fig. 2), was used to simultaneously describe TMZ, MTIC, and AIC plasma disposition. The following assumptions were included: (a) the absorbed drug was in the form of TMZ, where TMZ is assumed to be stable at the acidic pH of the stomach and small bowel; (b) clearance of TMZ from plasma was due to pH-dependent, chemical conversion of parent compound to MTIC and subsequent breakdown of MTIC to AIC (7–9, 13, 14), although a small percentage of TMZ is excreted unchanged in the urine (5–10%; Ref. 5) and metabolized to TMA (1–2%; Refs. 5 and 15); (c) because TMZ degrades in all tissues by pH-dependent hydrolysis, MTIC and AIC are formed within the same volume as that for TMZ, and MTIC has the same volume of distribution as TMZ; and (d) AIC has an additional distribution compart312 Absorption, Metabolism, and Excretion of C-TMZ Research. on October 24, 2017. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from ment that is consistent with the partitioning of AIC into erythrocytes, where AIC is known to undergo phosphorylation (19). The compartmental model consisted of the following eight structural parameters: Tlag, ka, Vc/F, Vp,AIC/F, Cls,TMZ/F, Cls,MTIC/F, Cld,AIC/F and Cls,AIC/F. Individual pharmacokinetic parameters were estimated by the method of weighted least-squares regression as implemented in Adapt II release 3 (20). Initially, values for Tlag, ka, Vc/F, and Cls,TMZ/F were estimated by fitting a first-order absorption, one-compartment model to TMZ concentrations. These values were then fixed for each patient, and the complete model was fit to MTIC and AIC concentrations to estimate values for Vp,AIC/F, Cls,MTIC/F, Cld,AIC/F, and Cls,AIC/F. The elimination rate constant, ke, was calculated for both TMZ and MTIC by dividing Cls/F (for each moiety) by Vc/F, and the elimination t1/2 was calculated as 0.693/ke. Systemic exposure for MTIC and AIC to that of TMZ was calculated as the ratio of Cls,TMZ/ F:Cls,MTIC/F and Cls,TMZ/F:Cls,AIC/F, respectively (21). Urinary excretion of TMZ, TMA, and AIC, expressed as a percentage of dose, were calculated for each collection interval by dividing the amount of excreted radioactivity that coeluted with unlabeled standard (TMZ, TMA, and AIC) by the total administered radioactive TMZ dose. These calculations were based on the following assumptions: (a) the recovery of all radioactivity was constant throughout sample processing (e.g., there was no disproportionate loss of any single metabolite); and (b) the absolute response of the radiometric detector did not change throughout HPLC analysis using gradient elution. In addition, all drug-related radioactivity in urine after 24 h was assumed to be associated with an unidentified polar metabolites(s).