Robert S. Jansen

Pharmacogenetic Screening of CYP3A and ABCB1 in Relation to Population Pharmacokinetics of Docetaxel

Purpose: Despite the extensive clinical experience with docetaxel, unpredictable interindividual variability in efficacy and toxicity remain important limitations associated with the use of this anticancer drug. Large interindividual pharmacokinetic variability has been associated with variation in toxicity profiles. Genetic polymorphisms in drug-metabolizing enzymes and drug transporters could possibly explain the observed pharmacokinetic variability. The aim of this study was therefore to investigate the influence of polymorphisms in the CYP3A and ABCB1 genes on the population pharmacokinetics of docetaxel. Experimental Design: Whole blood samples were obtained from patients with solid tumors and treated with docetaxel to quantify the exposure to docetaxel. DNA was collected to determine polymorphisms in the CYP3A and ABCB1 genes with DNA sequencing. A population pharmacokinetic analysis of docetaxel was done using nonlinear mixed-effect modeling. Results: In total, 92 patients were assessable for pharmacokinetic analysis of docetaxel. A three-compartmental model adequately described the pharmacokinetics of docetaxel. Several polymorphisms in the CYP3A and ABCB1 genes were found, with allele frequencies of 0.54% to 48.4%. The homozygous C1236T polymorphism in the ABCB1 gene (ABCB1*8) was significantly correlated with a decreased docetaxel clearance (−25%; P = 0.0039). No other relationships between polymorphisms and pharmacokinetic variables reached statistical significance. Furthermore, no relationship between haplotypes of CYP3A and ABCB1 and the pharmacokinetics could be identified. Conclusions: The polymorphism C1236T in the ABCB1 gene was significantly related to docetaxel clearance. Our current finding may provide a meaningful tool to explain interindividual differences in docetaxel treatment in daily practice.

ABCC6–Mediated ATP Secretion by the Liver Is the Main Source of the Mineralization Inhibitor Inorganic Pyrophosphate in the Systemic Circulation—Brief Report

Objective— Mutations in ABCC6 underlie the ectopic mineralization disorder pseudoxanthoma elasticum (PXE) and some forms of generalized arterial calcification of infancy, both of which affect the cardiovascular system. Using cultured cells, we recently showed that ATP-binding cassette subfamily C member 6 (ABCC6) mediates the cellular release of ATP, which is extracellularly rapidly converted into AMP and the mineralization inhibitor inorganic pyrophosphate (PPi). The current study was performed to determine which tissues release ATP in an ABCC6-dependent manner in vivo, where released ATP is converted into AMP and PPi, and whether human PXE ptients have low plasma PPi concentrations. Approach and Results— Using cultured primary hepatocytes and in vivo liver perfusion experiments, we found that ABCC6 mediates the direct, sinusoidal, release of ATP from the liver. Outside hepatocytes, but still within the liver vasculature, released ATP is converted into AMP and PPi. The absence of functional ABCC6 in patients with PXE leads to strongly reduced plasma PPi concentrations. Conclusions— Hepatic ABCC6-mediated ATP release is the main source of circulating PPi, revealing an unanticipated role of the liver in systemic PPi homeostasis. Patients with PXE have a strongly reduced plasma PPi level, explaining their mineralization disorder. Our results indicate that systemic PPi is relatively stable and that PXE, generalized arterial calcification of infancy, and other ectopic mineralization disorders could be treated with PPi supplementation therapy.

ABCC6 prevents ectopic mineralization seen in pseudoxanthoma elasticum by inducing cellular nucleotide release

Significance Pseudoxanthoma elasticum (PXE) is a heritable disease characterized by mineralization of the skin, eyes, and arteries, for which no effective treatment exists. PXE is caused by inactivating mutations in the gene encoding the transporter ABCC6. PXE is a metabolic disease caused by the absence of an unknown factor in the circulation. Our study indicates that the factor that normally prevents PXE is pyrophosphate, which is provided to the circulation in the form of nucleoside triphosphates via an ABCC6-dependent mechanism. Our findings provide leads for the treatment of this intractable disease. Pseudoxanthoma elasticum (PXE) is an autosomal recessive disease characterized by progressive ectopic mineralization of the skin, eyes, and arteries, for which no effective treatment exists. PXE is caused by inactivating mutations in the gene encoding ATP-binding cassette sub-family C member 6 (ABCC6), an ATP-dependent efflux transporter present mainly in the liver. Abcc6−/− mice have been instrumental in demonstrating that PXE is a metabolic disease caused by the absence of an unknown factor in the circulation, the presence of which depends on ABCC6 in the liver. Why absence of this factor results in PXE has remained a mystery. Here we report that medium from HEK293 cells overexpressing either human or rat ABCC6 potently inhibits mineralization in vitro, whereas medium from HEK293 control cells does not. Untargeted metabolomics revealed that cells expressing ABCC6 excrete large amounts of nucleoside triphosphates, even though ABCC6 itself does not transport nucleoside triphosphates. Extracellularly, ectonucleotidases hydrolyze the excreted nucleoside triphosphates to nucleoside monophosphates and inorganic pyrophosphate (PPi), a strong inhibitor of mineralization that plays a pivotal role in several mineralization disorders similar to PXE. The in vivo relevance of our data are demonstrated in Abcc6−/− mice, which had plasma PPi levels <40% of those found in WT mice. This study provides insight into how ABCC6 affects PXE. Our data indicate that the factor that normally prevents PXE is PPi, which is provided to the circulation in the form of nucleoside triphosphates via an as-yet unidentified but ABCC6-dependent mechanism.

Multidrug Resistance Protein 2 Is an Important Determinant of Paclitaxel Pharmacokinetics

Purpose: P-glycoprotein (P-gp; ABCB1) efficiently transports lipophilic amphipathic drugs, including the widely used anticancer drug paclitaxel (Taxol). We found previously that human multidrug resistance protein 2 (MRP2; ABCC2) also transports paclitaxel in vitro, and although we expected that paclitaxel pharmacokinetics would be dominated by P-gp, the effect of Mrp2 was tested in vivo. Experimental Design: We generated and characterized Mdr1a/1b/Mrp2−/− mice, allowing assessment of the distinct roles of Mrp2 and Mdr1a/1b P-gp in paclitaxel pharmacokinetics. Results: Surprisingly, the effect of Mrp2 on i.v. administration of paclitaxel was as great as that of P-gp. The area under plasma concentration-time curve (AUC)i.v. in both Mrp2−/− and Mdr1a/1b−/− mice was 1.3-fold higher than in wild-type mice, and in Mdr1a/1b/Mrp2−/− mice, a 1.7-fold increase was found. In spite of this similar effect, Mrp2 and P-gp had mostly complementary functions in paclitaxel elimination. Mrp2 dominated the hepatobiliary excretion, which was reduced by 80% in Mrp2−/− mice. In contrast, P-gp dominated the direct intestinal excretion, with a minor role for Mrp2. The AUCoral of paclitaxel was 8.5-fold increased by Mdr1a/1b deficiency but not affected by Mrp2 deficiency. However, in the absence of Mdr1a/1b P-gp, additional Mrp2 deficiency increased the AUCoral another 1.7-fold. Conclusions: Thus far, Mrp2 was thought to mainly affect organic anionic drugs in vivo. Our data show that Mrp2 can also be a major determinant of the pharmacokinetic behavior of highly lipophilic anticancer drugs, even in the presence of other efficient transporters. Variation in MRP2 activity might thus directly affect the effective exposure to paclitaxel, on i.v. administration, but also on oral administration, especially when P-gp activity is inhibited.

Contribution of the drug transporter ABCG2 (breast cancer resistance protein) to resistance against anticancer nucleosides

We have studied the potential contribution of ABCG2 (breast cancer resistance protein) to resistance to nucleoside analogues. In cells transfected with DNA constructs resulting in overexpression of human or mouse ABCG2, we found resistance against cladribine, clofarabine, fludarabine, 6-mercaptopurine, and 6-mercaptopurine riboside in both MDCKII and HEK293 cells and against gemcitabine only in HEK293 cells. With Transwell studies in MDCK cells and transport experiments with vesicles from Sf9 and HEK293 cells, we show that ABCG2 is able to transport not only the nucleotide CdAMP, like several other ATP-binding cassette transporters of the ABCC (multidrug resistance protein) family, but also the nucleoside cladribine itself. Expression of ABCG2 in cells results in a substantial decrease of intracellular CdATP, explaining the resistance against cladribine. The high transport rate of cladribine and clofarabine by ABCG2 deduced from Transwell experiments raises the possibility that this transporter could affect the disposition of nucleoside analogues in patients or cause resistance in tumors. [Mol Cancer Ther 2008;7(9):3092–102]

Oral Administration of Gemcitabine in Patients with Refractory Tumors : A Clinical and Pharmacologic Study

Purpose: To determine the toxicity, tolerability, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of oral gemcitabine (2′,2′-difluorodeoxycytidine; dFdC) in patients with cancer. Experimental Design: Patients with advanced or metastatic cancer refractory to standard therapy were eligible. Gemcitabine was administered p.o. starting at 1 mg once daily using dose escalation with three patients per dose level. Patients received one of two dosing schemes: (a) once daily dosing for 14 days of a 21-day cycle or (b) every other day dosing for 21 days of a 28-day cycle. Pharmacokinetics were assessed by measuring concentrations of dFdC and 2′,2′-difluorodeoxyuridine (dFdU) in plasma and gemcitabine triphosphate in peripheral blood mononuclear cells, and pharmacodynamics by measuring the effect on T-cell proliferation. Results: Thirty patients entered the study. Oral gemcitabine was generally well-tolerated. The maximum tolerated dose was not reached. Mainly moderate gastrointestinal toxicities occurred except for one patient who died after experiencing grade 4 hepatic failure during cycle two. One patient with a leiomyosarcoma had stable disease during 2 years and 7 months. Systemic exposure to dFdC was low with an estimated bioavailability of 10%. dFdC was highly converted to dFdU, probably via first pass metabolism and dFdU had a long terminal half-life (∼89 h). Concentrations of dFdCTP in peripheral blood mononuclear cells were low, but high levels of gemcitabine triphosphate, the phosphorylated metabolite of dFdU, were detected. Conclusions: Systemic exposure to oral gemcitabine was low due to extensive first-pass metabolism to dFdU. Moderate toxicity combined with hints of activity warrant further investigation of the concept of prolonged exposure to gemcitabine.

Coadministration of Ritonavir Strongly Enhances the Apparent Oral Bioavailability of Docetaxel in Patients with Solid Tumors

Purpose: To enhance the systemic exposure to oral docetaxel by coadministration of ritonavir, an efficacious inhibitor of CYP 3A4 with minor P-glycoprotein inhibiting effects, in patients with cancer. Experimental Design: A proof-of-concept study was carried out in 12 patients with solid tumors. The first cohort of patients (n = 4) received 10 mg and the subsequent cohort (n = 8) 100 mg of oral docetaxel, coadministered with 100 mg oral ritonavir randomized simultaneously or ritonavir given 60 minutes before docetaxel on days 1 and 8. On day 15 or 22, patients received 100 mg i.v. docetaxel. Results: The area under the plasma concentration-time curve in patients who received 10 mg oral docetaxel in combination with ritonavir was low, and the dose could safely be increased to 100 mg. The area under the plasma concentration-time curve in patients who received 100 mg oral docetaxel combined with ritonavir simultaneously or ritonavir given 60 minutes before docetaxel was 2.4 ± 1.5 and 2.8 ± 1.4 mg/h/L, respectively, compared with 1.9 ± 0.4 mg/h/L after i.v. docetaxel. The apparent oral bioavailability of docetaxel combined with ritonavir simultaneously or ritonavir given 60 minutes before docetaxel was 131% ± 90% and 161% ± 91%, respectively. The oral combination of docetaxel and ritonavir was well tolerated. Conclusion: Coadministration of ritonavir significantly enhanced the apparent oral bioavailability of docetaxel. These data are promising and form the basis for further development of a clinically applicable oral formulation of docetaxel combined with ritonavir.

Phase I Study of Oral Gemcitabine Prodrug (LY2334737) Alone and in Combination with Erlotinib in Patients with Advanced Solid Tumors

Purpose: LY2334737 is an orally available prodrug of gemcitabine. The objective of this study was to determine the maximum tolerated dose (MTD) and dose limiting toxicities (DLT) of daily administration of LY2334737 with or without erlotinib. Experimental Design: Patients with advanced or metastatic cancer were treated with escalating doses of LY2334737 monotherapy or in combination with continuous daily administration of 100 mg erlotinib. LY2334737 was given once daily for 14 days of a 21-day cycle. The study was extended with a bioequivalence trial to investigate a novel LY2334737 drug formulation. Results: A total of 65 patients were treated in this study. The MTD was 40 mg LY2334737. Fatigue was the most frequent DLT for LY2334737 monotherapy (4 patients) followed by elevated transaminase levels (2 patients), both observed at the 40- to 50-mg dose levels. Among the 10 patients in the combination arm, 2 had DLTs at the 40-mg dose level. These were fatigue and elevated liver enzyme levels. The most common adverse events were fatigue (n = 38), nausea (n = 27), vomiting (n = 24), diarrhea (n = 23), anorexia (n = 20), pyrexia (n = 18), and elevated transaminase levels (n = 14). The pharmacokinetics showed dose proportional increase in LY2334737 and gemcitabine exposure. The metabolite 2′,2′-difluorodeoxyuridine accumulated with an accumulation index of 4.3 (coefficient of variation: 20%). In one patient, complete response in prostate-specific antigen was observed for 4 cycles, and stable disease was achieved in 22 patients overall. Pharmacokinetic analysis showed that the 2 investigated LY2334737 drug formulations were bioequivalent. Conclusions: LY2334737 displays linear pharmacokinetics and the MTD is 40 mg with or without daily administration of 100 mg erlotinib. Signs of antitumor activity warrant further development. Clin Cancer Res; 17(18); 6071–82. ©2011 AACR.

Incorporation of concentration data below the limit of quantification in population pharmacokinetic analyses

Handling of data below the lower limit of quantification (LLOQ), below the limit of quantification (BLOQ) in population pharmacokinetic (PopPK) analyses is important for reducing bias and imprecision in parameter estimation. We aimed to evaluate whether using the concentration data below the LLOQ has superior performance over several established methods. The performance of this approach (“All data”) was evaluated and compared to other methods: “Discard,” “LLOQ/2,” and “LIKE” (likelihood‐based). An analytical and residual error model was constructed on the basis of in‐house analytical method validations and analyses from literature, with additional included variability to account for model misspecification. Simulation analyses were performed for various levels of BLOQ, several structural PopPK models, and additional influences. Performance was evaluated by relative root mean squared error (RMSE), and run success for the various BLOQ approaches. Performance was also evaluated for a real PopPK data set. For all PopPK models and levels of censoring, RMSE values were lowest using “All data.” Performance of the “LIKE” method was better than the “LLOQ/2” or “Discard” method. Differences between all methods were small at the lowest level of BLOQ censoring. “LIKE” method resulted in low successful minimization (<50%) and covariance step success (<30%), although estimates were obtained in most runs (~90%). For the real PK data set (7.4% BLOQ), similar parameter estimates were obtained using all methods. Incorporation of BLOQ concentrations showed superior performance in terms of bias and precision over established BLOQ methods, and shown to be feasible in a real PopPK analysis.

Mass Balance Study of [14C]Eribulin in Patients with Advanced Solid Tumors

This mass balance study investigated the metabolism and excretion of eribulin, a nontaxane microtubule dynamics inhibitor with a novel mechanism of action, in patients with advanced solid tumors. A single approximately 2 mg (approximately 80 μCi) dose of [14C]eribulin acetate was administered as a 2 to 5 min bolus injection to six patients on day 1. Blood, urine, and fecal samples were collected at specified time points on days 1 to 8 or until sample radioactivity was ≤1% of the administered dose. Mean plasma eribulin exposure (627 ng · h/ml) was comparable with that of total radioactivity (568 ng Eq · h/ml). Time-matched concentration ratios of eribulin to total radioactivity approached unity in blood and plasma, indicating that unchanged parent compound constituted almost all of the eribulin-derived radioactivity. Only minor metabolites were detected in plasma samples up to 60 min postdose, pooled across patients, each metabolite representing ≤0.6% of eribulin. Elimination half-lives for eribulin (45.6 h) and total radioactivity (42.3 h) were comparable. Eribulin-derived radioactivity excreted in feces was 81.5%, and that of unchanged eribulin was 61.9%. Renal clearance (0.301 l/h) was a minor component of total eribulin clearance (3.93 l/h). Eribulin-derived radioactivity excreted in urine (8.9%) was comparable with that of unchanged eribulin (8.1%), indicating minimal excretion of metabolite(s) in urine. Total recovery of the radioactive dose was 90.4% in urine and feces. Overall, no major metabolites of eribulin were detected in plasma. Eribulin is eliminated primarily unchanged in feces, whereas urine constitutes a minor route of elimination.