Peter de Bruijn

Modulation of Irinotecan Metabolism by Ketoconazole

PURPOSE Irinotecan (CPT-11) is a prodrug of SN-38 and has been registered for the treatment of advanced colorectal cancer. It is converted by the cytochrome P450 3A4 isozyme (CYP3A4) into several inactive metabolites, including 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxycamptothecin (APC). To investigate the role of CYP3A4 in irinotecan pharmacology, we evaluated the consequences of simultaneous treatment of irinotecan with a potent enzyme inhibitor, ketoconazole, in a group of cancer patients. PATIENTS AND METHODS A total of seven assessable patients was treated in a randomized, cross-over design with irinotecan (350 mg/m(2) intravenously for 90 minutes) given alone and followed 3 weeks later by irinotecan (100 mg/m(2)) in combination with ketoconazole (200 mg orally for 2 days) or vice versa. Serial plasma, urine, and feces samples were obtained up to 500 hours after dosing and analyzed for irinotecan, metabolites (7-ethyl-10-hydroxycamptothecin [SN-38], SN-38 glucuronide [SN-38G], and APC), and ketoconazole by high-performance liquid chromatography. RESULTS With ketoconazole coadministration, the relative formation of APC was reduced by 87% (P =.002), whereas the relative exposure to the carboxylesterase-mediated SN-38 as expected on the basis of dose (area under the plasma concentration-time curve normalized to dose) was increased by 109% (P =.004). These metabolic alterations occurred without substantial changes in irinotecan clearance (P =.90) and formation of SN-38G (P =.93). CONCLUSION Inhibition of CYP3A4 in cancer patients treated with irinotecan leads to significantly increased formation of SN-38. Simultaneous administration of various commonly prescribed inhibitors of CYP3A4 can potentially result in fatal outcomes, and up to four-fold reductions in irinotecan dose are indicated.

Clinical pharmacokinetics of doxorubicin in combination with Gf120918, a potent inhibitor of Mdr1 P-glycoprotein

Previous clinical investigations with doxorubicin indicated that modulators of P-glycoprotein dramatically decrease the systemic clearance of the drug, which complicates the interpretation of toxicity and response data. In the present study, we examined the pharmacokinetics of doxorubicin and GF120918, a novel potent P-glycoprotein inhibitor, in cancer patients in a search for more selective modulation of multidrug resistance (MDR). Seven cohorts (46 patients) received sequential treatments with doxorubicin alone by a 5 min i.v. bolus (50-75 mg/m2), oral GF120918 alone (50 mg q.d.-400 mg b.i.d.), and the combination of doxorubicin and GF120918. Serial blood and urine samples were taken during both treatment courses and analyzed for doxorubicin and its metabolite doxorubicinol by a liquid chromatographic assay. The pharmacokinetic characteristics of doxorubicin in the presence or absence of GF120918 indicate a very minor overall effect of the modulator, except at the highest combined dose level (i.e. 75 mg/m2 plus 400 mg b.i.d.). A limited number of patients experienced significantly increased exposure to doxorubicinol upon combined treatment, which was associated with concomitantly higher plasma levels of GF120918. Sigmoidal maximum-effect models revealed significant correlations (p<0.02) between the area under the curve of doxorubicinol and the percent decrease in neutrophils and platelets. Sigmoidicity factors in the fitted Hill equation were similar between both treatment courses, suggesting no pharmacodynamic potentiation of doxorubicinol myelotoxicity by GF120918. Our data indicate that GF120918 at the tested doses of combination treatment achieves plasma concentrations that reverse MDR in experimental models and it lacks the significant kinetic interaction with doxorubicin observed previously with other modulators. Hence, it may be possible in future trials to assess the contribution of a potent inhibitor of P-glycoprotein activity to the toxicity and activity of doxorubicin with the knowledge that profound plasma pharmacokinetic interactions are unlikely.

Determination of paclitaxel in human plasma using single solvent extraction prior to isocratic reversed-phase high-performance liquid chromatography with ultraviolet detection

An isocratic reversed-phase high-performance liquid chromatographic method with ultraviolet detection at 230 nm has been developed for the determination of paclitaxel in human plasma. Plasma samples were prepared by a selective one-step liquid-liquid extraction involving a mixture of acetonitrile-n-butyl chloride (1:4, v/v). Paclitaxel and the internal standard docetaxel were separated using a column packed with ODS-80A material, and a mobile phase consisting of water-methanol-tetrahydrofuran-ammonium hydroxide (37.5:60:2.5:0.1, v/v). The calibration graph for paclitaxel was linear in the range 10-500 ng/ml, with a lower limit of quantitation of 10 ng/ml, using 1 ml plasma samples. The extraction recoveries of spiked paclitaxel and docetaxel to drug-free human plasma were 89.6+/-8.52 and 93.7+/-5.0%, respectively. Validation data showed that the assay for paclitaxel is sensitive, selective, accurate and reproducible. The assay has been used in a single pharmacokinetic experiment in a patient to investigate the applicability of the method in vivo.

Liquid chromatographic determination of irinotecan and three major metabolites in human plasma, urine and feces

A new simple reversed-phase high-performance liquid chromatographic method was developed for the determination of irinotecan (CPT-11) and three metabolites in human plasma, urine and feces homogenate. The metabolites of interest were 7-ethyl-10-hydroxycamptothecin (SN-38), its beta-glucuronide derivative (SN-38G) and 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecin (RPR 121056A; also referred to as APC). Sample pretreatment from the various biological matrices involved a rapid protein precipitation with simultaneous solvent extraction of 250-microl aliquots of sample with 500 microl of methanol-5% (w/v) aqueous perchloric acid (1:1, v/v). Separation of the compounds was achieved on an analytical column packed with Hypersil ODS material (100X4.6 mm I.D., 5 microm), and isocratic elution with a mixture of methanol-0.1 M ammonium acetate containing 10 mM tetrabutylammonium sulphate (30:70, v/v), pH 5.3 (hydrochloric acid). The column effluent was monitored at excitation and emission wavelengths of 355 and 515 nm, respectively. Results from a 4-day validation study indicated that this single-run determination allows for simple, simultaneous and rapid quantitation and identification of all analytes with excellent reliability. The described procedure permits the analysis of patient samples, and will be implemented in future studies to investigate the complete metabolic fate and disposition of CPT-11 in cancer patients.

Determination of irinotecan (CPT-11) and its active metabolite SN-38 in human plasma by reversed-phase high-performance liquid chromatography with fluorescence detection.

Sensitive high-performance liquid chromatographic assays have been developed to determine the levels of the lactone and lactone plus carboxylate (total) forms of the antitumor agent irinotecan (CPT-11) and its active metabolite SN-38, in human plasma. The related compound camptothecin was used as the internal standard. The selective sample pretreatment for the lactone forms involved a single solvent extraction with acetonitrile-n-butyl chloride (1:4, v/v), whereas the sample clean-up for the total forms was a simple protein precipitation with aqueous perchloric acid-methanol (1:1, v/v), which results in the conversion of the carboxylate to the lactone forms. Chromatography was carried out on a Hypersil ODS column, with detection performed fluorimetrically. The methods have been validated, and stability tests under various conditions have been performed. The lower limits of quantitation are 0.5 and 2.0 ng/ml for the lactone and total forms, respectively. The assays have been used in a single pharmacokinetic experiment in a patient to investigate the applicability of the method in vivo.

Pharmacokinetic, Metabolic, and Pharmacodynamic Profiles in a Dose-Escalating Study of Irinotecan and Cisplatin

PURPOSE To investigate the pharmacokinetics and pharmacodynamics of irinotecan and cisplatin administered once every 3 weeks in a dose-escalating study in patients with solid tumors. PATIENTS AND METHODS Fifty-two cancer patients were treated with irinotecan administered as a 90-minute infusion at doses ranging from 175 to 300 mg/m(2) followed by cisplatin administered as a 3-hour intravenous infusion at doses ranging from 60 to 80 mg/m(2). After reaching the maximum-tolerated dose, the sequence of drug administration was revised. For pharmacokinetic analysis, serial plasma samples were obtained on days 1 through 3 of the first cycle. Forty-five patients were assessable for irinotecan pharmacokinetics, and 46 were assessable for cisplatin pharmacokinetics. RESULTS Irinotecan and cisplatin demonstrated linear pharmacokinetics comparable to that observed with single-agent administration, which suggests an absence of pharmacokinetic interaction. SN-38G constituted the major plasma metabolite of irinotecan, whereas 7-ethyl-10-[4-N-(1-piperidino)1-amino]-carbonyloxycamptothecine (NPC) was only a minor metabolite in plasma, possibly indicating a rapid conversion of NPC to SN-38. The terminal elimination phases of SN-38 and SN-38G were similar and relatively delayed when compared with the elimination of irinotecan. Maximal DNA adduct formation did not significantly differ from that observed with single-agent administration. The percentage decrease in WBC was significantly related to the areas under the plasma concentration-time curve (AUCs) of the lactone form of irinotecan (P =.0245) and SN-38 (P =. 0123). The severity of diarrhea was not significantly related to the AUCs of irinotecan and SN-38, nor to the systemic glucuronidation rate of SN-38. CONCLUSION There was no apparent pharmacokinetic interaction between irinotecan and cisplatin in this study. Reversion of the administration sequence of the drugs did not seem to have any influence on the pharmacokinetics. The incidence and severity of delayed-type diarrhea was not related to any of the studied parameters.

Influence of Polymorphic OATP1B-Type Carriers on the Disposition of Docetaxel

Purpose: Docetaxel is extensively metabolized by CYP3A4 in the liver but mechanisms by which the drug is taken up into hepatocytes remain poorly understood. We hypothesized that (i) liver uptake of docetaxel is mediated by the polymorphic solute carriers OATP1B1 and OATP1B3 and (ii) inherited genetic defects in this process may impair systemic drug elimination. Experimental Design: Transport of docetaxel was studied in vitro using various cell lines stably transfected with OATP1B1*1A (wild-type), OATP1B1*5 [c.521T>C (V174A); rs4149056], OATP1B3, or the mouse transporter Oatp1b2. Docetaxel clearance was evaluated in wild-type and Oatp1b2-knockout mice as well as in two cohorts of patients with multiple variant transporter genotypes (n = 213). Results: Docetaxel was found to be a substrate for OATP1B1, OATP1B3, and Oatp1b2 but was not transported by OATP1B1*5. Deficiency of Oatp1b2 in mice was associated with an 18-fold decrease in docetaxel clearance (P = 0.0099), which was unrelated to changes in intrinsic metabolic capacity in mouse liver microsomes. In patients, however, none of the studied common reduced function variants in OATP1B1 or OATP1B3 were associated with docetaxel clearance (P > 0.05). Conclusions: The existence of at least two potentially redundant uptake transporters in the human liver with similar affinity for docetaxel supports the possibility that functional defects in both of these proteins may be required to confer substantially altered disposition phenotypes. In view of the established exposure–toxicity relationships for docetaxel, we suggest that caution is warranted if docetaxel has to be administered together with agents that potently inhibit both OATP1B1 and OATP1B3. Clin Cancer Res; 18(16); 4433–40. ©2012 AACR.

CYP3A4*22 genotype and systemic exposure affect paclitaxel-induced neurotoxicity.

Purpose: Paclitaxel is used for the treatment of several solid tumors and displays a high interindividual variation in exposure and toxicity. Neurotoxicity is one of the most prominent side effects of paclitaxel. This study explores potential predictive pharmacokinetic and pharmacogenetic determinants for the onset and severity of neurotoxicity. Experimental Design: In an exploratory cohort of patients (n = 261) treated with paclitaxel, neurotoxicity incidence, and severity, pharmacokinetic parameters and pharmacogenetic variants were determined. Paclitaxel plasma concentrations were measured by high-performance liquid chromatography or liquid chromatography/tandem mass spectrometry, and individual pharmacokinetic parameters were estimated from previously developed population pharmacokinetic models by nonlinear mixed effects modeling. Genetic variants of paclitaxel pharmacokinetics tested were CYP3A4*22, CYP2C8*3, CYP2C8*4, and ABCB1 3435 C>T. The association between CYP3A4*22 and neurotoxicity observed in the exploratory cohort was validated in an independent patient cohort (n = 239). Results: Exposure to paclitaxel (logAUC) was correlated with severity of neurotoxicity (P < 0.00001). Female CYP3A4*22 carriers were at increased risk of developing neurotoxicity (P = 0.043) in the exploratory cohort. CYP3A4*22 carrier status itself was not associated with pharmacokinetic parameters (CL, AUC, Cmax, or T>0.05) of paclitaxel in males or females. Other genetic variants displayed no association with neurotoxicity. In the subsequent independent validation cohort, CYP3A4*22 carriers were at risk of developing grade 3 neurotoxicity (OR = 19.1; P = 0.001). Conclusions: Paclitaxel exposure showed a relationship with the severity of paclitaxel-induced neurotoxicity. In this study, female CYP3A4*22 carriers had increased risk of developing severe neurotoxicity during paclitaxel therapy. These observations may guide future individualization of paclitaxel treatment. Clin Cancer Res; 19(12); 3316–24. ©2013 AACR.

Therapeutic Drug Monitoring for the Individualization of Docetaxel Dosing: A Randomized Pharmacokinetic Study

Purpose: Docetaxel pharmacokinetic (PK) parameters, notably clearance and exposure (AUC), are characterized by large interindividual variability. The purpose of this study was to evaluate the effect of PK-guided [area under the plasma concentration versus time curve (AUC) targeted], individualized docetaxel dosing on interindividual variability in exposure. Experimental Design: A limited sampling strategy in combination with a validated population PK model, Bayesian analysis, and a predefined target AUC was used. Fifteen patients were treated for at least 2 courses with body surface area–based docetaxel and 15 with at least 1 course of PK-guided docetaxel dosing. Results: Interindividual variability (SD of ln AUC) was decreased by 35% (N = 15) after 1 PK-guided course; when all courses were evaluated, variability was decreased by 39% (P = 0.055). PK-guided dosing also decreased the interindividual variability of percentage decrease in white blood cell and absolute neutrophil counts by approximately 50%. Conclusions: Further research is required to determine whether the decrease in PK variability can contribute to a reduction in interindividual variability in efficacy and toxicity. Clin Cancer Res; 17(2); 353–62. ©2011 AACR.

Liquid chromatographic determination of ketoconazole, a potent inhibitor of CYP3A4-mediated metabolism.

A high-performance liquid chromatographic assay with UV detection has been developed for the determination of ketoconazole in human plasma. Quantitative extraction was achieved by a single solvent extraction involving a mixture of acetonitrile-n-butyl chloride (1:4, v/v). Ketoconazole and the internal standard (clotrimazole) were separated on a column packed with Inertsil ODS-80A material and a mobile phase composed of water-acetonitrile-tetrahydrofuran-ammonium hydroxide-triethylamine (45:50.2:2.5:0.1:0.1, v/v). The column effluent was monitored at a wavelength of 206 nm with a detector range set at 0.5. The calibration graph was linear in the range of 20-2000 ng/ml, with a lower limit of quantitation of 20.0 ng/ml. The extraction recoveries for ketoconazole and clotrimazole in human plasma were 93+/-9.7% and 83+/-10.0%, respectively. The developed method has been successfully applied to a clinical study to examine the pharmacokinetics of ketoconazole in a cancer patient.