Publications by authors named "Alfred H Schinkel"

171 Publications

Quantification of KRAS inhibitor sotorasib in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry.

J Chromatogr B Analyt Technol Biomed Life Sci 2021 Jun 20;1174:122718. Epub 2021 Apr 20.

Utrecht University, Faculty of Science, Department of Pharmaceutical Sciences, Division of Pharmacology, Universiteitsweg 99, 3584 CG Utrecht, the Netherlands. Electronic address:

Sotorasib is a KRAS inhibitor with promising anticancer activity in phase I clinical studies. This compound is currently under further clinical evaluation as monotherapy and combination therapy against solid tumors. In this study, a liquid chromatography-tandem mass spectrometric method to quantify sotorasib in mouse plasma and eight tissue-related matrices (brain, liver, spleen, kidney, small intestine, small intestine content, lung, and testis homogenates) was developed and validated. Protein precipitation using acetonitrile was utilized in 96-well format to extract sotorasib and erlotinib (internal standard) from mouse plasma and tissue homogenates. Separation of the analytes was performed on an Acquity UPLC® BEH C18 column by gradient elution of methanol and 0.1% formic acid in water at a flow rate of 0.6 ml/min. Sotorasib was detected by a triple quadrupole mass spectrometer with positive electrospray ionization in selected reaction monitoring mode. A linear calibration range of 2-2,000 ng/ml of sotorasib was achieved during the validation. Accuracy values were in the range of 90.7-111.4%, and precision values (intra- and interday) were between 1.7% and 9.2% for all tested levels in all investigated matrices. The method was successfully applied to investigate the plasma pharmacokinetics and tissue accumulation of sotorasib in female wild-type mice.
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http://dx.doi.org/10.1016/j.jchromb.2021.122718DOI Listing
June 2021

ABCB1 and ABCG2 Restrict Brain and Testis Accumulation and, Alongside CYP3A, Limit Oral Availability of the Novel TRK Inhibitor Selitrectinib.

Mol Cancer Ther 2021 Jun 30;20(6):1173-1182. Epub 2021 Mar 30.

Division of Pharmacology, The Netherlands Cancer Institute, Amsterdam, the Netherlands.

Selitrectinib (BAY2731954; LOXO-195) is a promising oral tropomyosin receptor kinase (TRK) inhibitor currently in phase I/II clinical trials for the treatment of histology-agnostic cancers positive for TRK fusions. With therapeutic resistance eventually developing with first-generation TRK inhibitors, selitrectinib was designed to overcome resistance mediated by acquired kinase domain mutations. Using genetically modified mouse models and pharmacological inhibitors, we investigated the roles of the multidrug efflux transporters ABCB1 and ABCG2, and the drug-metabolizing CYP3A enzyme complex in selitrectinib pharmacokinetics. , selitrectinib was markedly transported by mouse Abcg2 and human ABCB1, and modestly by human ABCG2. Following oral administration at 10 mg/kg, selitrectinib brain-to-plasma ratios were increased in (twofold) and (5.8-fold) compared with wild-type mice, but not in single mice. Testis distribution showed similar results. mAbcb1a/1b and mAbcg2 each restricted the plasma exposure of selitrectinib: With both systems absent oral availability increased by 1.7-fold. Oral administration of the ABCB1/ABCG2 inhibitor elacridar boosted plasma exposure and brain accumulation in wild-type mice to the same levels as seen in mice. In mice, plasma exposure of selitrectinib over 4 hours was increased by 1.4-fold and subsequently reduced by 2.3-fold upon transgenic overexpression of human CYP3A4 in liver and intestine. The relative tissue distribution of selitrectinib remained unaltered. Thus, selitrectinib brain accumulation and oral availability are substantially restricted by ABCB1 and ABCG2, and this can be reversed by pharmacological inhibition. Moreover, oral availability of selitrectinib is limited by CYP3A activity. These insights may be useful to optimize the clinical application of selitrectinib.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0705DOI Listing
June 2021

Pharmacokinetics of docetaxel and ritonavir after oral administration of ModraDoc006/r in patients with prostate cancer versus patients with other advanced solid tumours.

Cancer Chemother Pharmacol 2021 Jun 20;87(6):855-869. Epub 2021 Mar 20.

Department of Pharmacy and Pharmacology, The Netherlands Cancer Institute, Antoni van Leeuwenhoek, Plesmanlaan 121, Amsterdam, 1066CX, The Netherlands.

Purpose: ModraDoc006 is a novel oral formulation of docetaxel. The clearance of intravenous docetaxel is higher in medically castrated prostate cancer patients as compared to patients with other types of solid tumours. Oral docetaxel requires co-administration ritonavir (r), which might further impact the pharmacokinetics (PK). We now compare the PK of docetaxel and ritonavir between patients with Hormone Sensitive Prostate Cancer (HSPC), metastatic Castration-Resistant Prostate Cancer (mCRPC) and other metastatic solid tumours, treated on the same dose and weekly schedule of ModraDoc006/r.

Methods: The docetaxel and ritonavir PK were compared between four patient groups from three clinical phase I trials, including eight male and eight female patients with different types of solid tumours (study 1), seven patients with HSPC (study 2) and five patients with mCRPC (study 3). All patients were treated with ModraDoc006 30 mg and ritonavir 100 mg in the morning, followed by ModraDoc006 20 mg and ritonavir 100 mg in the evening (ModraDoc006/r 30-20/100-100). For comparative purposes, the PK of six mCRPC patients that received 30-20/200-100 in study 3 were also evaluated.

Results: The maximum plasma concentration (C) was significantly lower for both docetaxel and ritonavir in the prostate cancer patients as compared to the patients with other types of solid tumours treated at ModraDoc006/r 30-20/100-100. The docetaxel area under the plasma concentration versus time curve (AUC) was significantly different at this dose, with a mean AUC of 1359 ± 374 ng/mL*h (N = 8) in female patients and 894 ± 223 ng/mL*h (N = 8) in male patients with different solid tumours (study 1), 321 ± 81 (N = 7) in HSPC (study 2) and 367 ± 182 ng/mL*h (N = 5) in mCRPC (study 3). A similar pattern was observed for ritonavir. ModraDoc006/r 30-20/200-100 in six mCRPC patients led to a comparable ritonavir exposure as compared to the patients at 30-20/100-100 in study 1 and increased the docetaxel AUC to 1266 ± 473 ng/mL*h (N = 6).

Conclusion: The exposure to docetaxel and ritonavir was significantly lower in prostate cancer patients as compared to patients with other types of solid tumours, treated on ModraDoc006/r 30-20/100-100. An increase of the ritonavir dose increased the docetaxel exposure in mCRPC patients. Therefore, a different RP2D of ModraDoc006/r is pursued in castrated prostate cancer patients as compared to patients with other types of solid tumours.

Trial Registration: Study 1: ClinicalTrials.gov Identifier NCT01173913, date of registration August 2, 2010. Study 2: ClinicalTrials.gov Identifier NCT03066154, date of registration February 28, 2017. Study 3: ClinicalTrials.gov Identifier NCT03136640, date of registration May 2, 2017.
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http://dx.doi.org/10.1007/s00280-021-04259-5DOI Listing
June 2021

The role of drug efflux and uptake transporters ABCB1 (P-gp), ABCG2 (BCRP) and OATP1A/1B and of CYP3A4 in the pharmacokinetics of the CDK inhibitor milciclib.

Eur J Pharm Sci 2021 Apr 30;159:105740. Epub 2021 Jan 30.

Division of Pharmacology, The Netherlands Cancer Institute, Amsterdam, The Netherlands. Electronic address:

The promising anticancer drug milciclib potently inhibits cyclin-dependent kinase (CDK) 2 and tropomyosin receptor kinase (TRK) A, and is currently in phase II clinical studies. To characterize factors affecting milciclib pharmacokinetics, we investigated whether milciclib is a substrate of the multidrug efflux and uptake transporters ABCB1 (P-gp), ABCG2 (BCRP), and OATP1A/1B, and the drug-metabolizing enzyme CYP3A, using genetically-modified mouse models and Madin-Darby Canine Kidney (MDCK-II) cells. In vitro, milciclib was transported by mAbcg2, and this was inhibited by the ABCG2 inhibitor Ko143. Upon oral administration of milciclib, its plasma exposure in Abcb1a/1b, Abcg2, and Abcb1a/1b;Abcg2 mice was similar to that found in wild-type mice. Milciclib showed good brain penetration even in wild-type mice (brain-to-plasma ratio of 1.2), but this was further increased by 5.2-fold when both Abcb1 and Abcg2 were ablated, and to a lesser extent in single Abcb1- or Abcg2-deficient mice. Oatp1a/1b deficiency had only a minor impact on the milciclib plasma AUC and C. The milciclib AUC increased 1.9-fold in Cyp3a mice but decreased only 1.3-fold upon overexpression of human CYP3A4. Thus, ABCB1 and ABCG2 cooperatively limit milciclib brain penetration. The low impact of OATP1 and CYP3A could be clinically favorable for milciclib, reducing the risks of unintended drug-drug interactions or interindividual variation in CYP3A4 activity.
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http://dx.doi.org/10.1016/j.ejps.2021.105740DOI Listing
April 2021

Clinical Pharmacokinetics and Pharmacodynamics of the Cyclin-Dependent Kinase 4 and 6 Inhibitors Palbociclib, Ribociclib, and Abemaciclib.

Clin Pharmacokinet 2020 12;59(12):1501-1520

Division of Medical Oncology, Department of Clinical Pharmacology, The Netherlands Cancer Institute-Antoni van Leeuwenhoek, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.

Palbociclib, ribociclib, and abemaciclib are inhibitors of the cyclin-dependent kinases 4 and 6 approved for the treatment of locally advanced or metastatic breast cancer. In this review, we provide an overview of the available clinical pharmacokinetic and pharmacodynamic characteristics of these novel drugs, summarize the results of food-effect and drug-drug interaction studies, and highlight exposure-response and exposure-toxicity relationships. All three drugs exhibit a large inter-individual variability in exposure (coefficient of variation range 40-95% for minimum plasma concentration), are extensively metabolized by cytochrome P450 3A4, and have their brain penetration limited by efflux transporters. Abemaciclib has three active metabolites with similar potency that are clinically relevant (i.e., M2, M20, M18), whereas the metabolites of palbociclib and ribociclib are not of clinical significance. Pharmacokinetic exposure increases in a dose-proportional manner for palbociclib, whereas exposure increases under- and over-proportionally with an increasing dose for abemaciclib and ribociclib, respectively. High exposure is associated with an increased risk of neutropenia, and for ribociclib also to corrected QT prolongation. For abemaciclib, a clear exposure-efficacy relationship has been described, while for palbociclib and ribociclib exposure-response analyses remain inconclusive. Future studies are needed to address exposure-efficacy relationships to further improve dosing.
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http://dx.doi.org/10.1007/s40262-020-00930-xDOI Listing
December 2020

Development and validation of an LC-MS/MS method for the quantitative analysis of milciclib in human and mouse plasma, mouse tissue homogenates and tissue culture medium.

J Pharm Biomed Anal 2020 Oct 1;190:113516. Epub 2020 Aug 1.

Department of Pharmacy & Pharmacology, The Netherlands Cancer Institute, Amsterdam, the Netherlands; Division of Pharmacology, The Netherlands Cancer Institute, Amsterdam, the Netherlands; Division of Pharmacoepidemiology and Clinical Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands.

Milciclib is a promising cyclin-dependent kinase inhibitor currently in phase II clinical trials to treat several types of cancer. The first bioanalytical method for the quantitative analysis of milciclib in several biomatrices using liquid chromatography-tandem mass spectrometry is described here. This method was fully validated in human plasma according to FDA and EMA guidelines, and partially validated in mouse plasma, homogenates of mouse brain, kidney, liver, small intestine, spleen, and tissue culture medium. Palbociclib, an analog compound, was used as internal standard. A simple and fast sample pre-treatment by protein precipitation with acetonitrile was used, leading to efficient extraction of the analyte with recoveries between 95-100%. Chromatographic separation was achieved with a C analytical column and a gradient elution using 10 mM ammonium bicarbonate in water and 10 mM ammonium bicarbonate in water-methanol (1:9, v/v). This assay was selective, accurate, precise and linear in the concentration range of 1-1000 ng/mL. Moreover, samples above the upper limit of quantification can be integrally diluted up to 10-fold prior to analysis. The use of human plasma as a surrogate matrix to quantify milciclib in tissue culture medium and mouse matrices resulted in acceptable accuracy and precision, however tissue culture medium samples required a dilution with human plasma prior the pre-treatment. All performance parameters of the method complied with the acceptance criteria recommended by the guidelines, except for the carry-over, which was slightly above (22.9% of the lower limit of quantification) the recommended percentage (20%). Therefore, additional measures were taken to assure data integrity. Stability of milciclib in all matrices was evaluated, and in some matrices the analyte was unstable under the tested conditions. It is therefore recommended to keep these samples as briefly as possible at room temperature during the pre-treatment, and to store them at -70 °C to avoid analyte degradation. This method was successfully applied to support preclinical pharmacokinetic studies of milciclib.
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http://dx.doi.org/10.1016/j.jpba.2020.113516DOI Listing
October 2020

No relation between docetaxel administration route and high-grade diarrhea incidence.

Pharmacol Res Perspect 2020 08;8(4):e00633

Department of Clinical Pharmacology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Oral administration of docetaxel in combination with the CYP3A4 inhibitor ritonavir is used in clinical trials to improve oral bioavailability of docetaxel. Diarrhea was the most commonly observed and dose-limiting toxicity. This study combined preclinical and clinical data and investigated incidence, severity and cause of oral docetaxel-induced diarrhea. In this study, incidence and severity of diarrhea in patients were compared to exposure to orally administered docetaxel. Intestinal toxicity after oral or intraperitoneal administration of docetaxel was further explored in mice lacking Cyp3a and mice lacking both Cyp3a and P-glycoprotein. In patients, severity of diarrhea increased significantly with an increase in AUC and C (P = .035 and P = .025, respectively), but not with an increase in the orally administered dose (P = .11). Furthermore, incidence of grade 3/4 diarrhea after oral docetaxel administration was similar as reported after intravenous docetaxel administration. Intestinal toxicity in mice was only observed at high systemic exposure to docetaxel and was similar after oral and intraperitoneal administration of docetaxel. In conclusion, our data show that the onset of severe diarrhea after oral administration of docetaxel in humans is similar after oral and intravenous administration of docetaxel and is caused by the concentration of docetaxel in the systemic blood circulation. Mouse experiments confirmed that intestinal toxicity is caused by a high systemic exposure and not by local intestinal exposure. Severe diarrhea in patients after oral docetaxel is reversible and is not related to the route of administration of docetaxel.
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http://dx.doi.org/10.1002/prp2.633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7387127PMC
August 2020

Extrahepatic metabolism of ibrutinib.

Invest New Drugs 2021 Feb 4;39(1):1-14. Epub 2020 Jul 4.

Division of Pharmacoepidemiology & Clinical Pharmacology, Faculty of Science, Department of Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 CG, Utrecht, The Netherlands.

Ibrutinib is a first-in-class Bruton's kinase inhibitor used in the treatment of multiple lymphomas. In addition to CYP3A4-mediated metabolism, glutathione conjugation can be observed. Subsequently, metabolism of the conjugates and finally their excretion in feces and urine occurs. These metabolites, however, can reach substantial concentrations in human subjects, especially when CYP3A4 is inhibited. Ibrutinib has unexplained nephrotoxicity and high metabolite concentrations are also found in kidneys of Cyp3a knockout mice. Here, a mechanism is proposed where the intermediate cysteine metabolite is bioactivated. The metabolism of ibrutinib through this glutathione cycle was confirmed in cultured human renal proximal tubule cells. Ibrutinib-mediated toxicity was enhanced in-vitro by inhibitors of breast cancer resistance protein (BCRP), P-glycoprotein (P-gp) and multidrug resistance protein (MRP). This was a result of accumulating cysteine metabolite levels due to efflux inhibition. Finally, through inhibition of downstream metabolism, it was shown now that direct conjugation was responsible for cysteine metabolite toxicity.
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http://dx.doi.org/10.1007/s10637-020-00970-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851014PMC
February 2021

Human organic anion transporting polypeptide (OATP) 1B3 and mouse OATP1A/1B affect liver accumulation of Ochratoxin A in mice.

Toxicol Appl Pharmacol 2020 08 26;401:115072. Epub 2020 May 26.

The Netherlands Cancer Institute, Division of Pharmacology, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands. Electronic address:

Ochratoxin A (OTA) is a dietary mycotoxin that can cause nephrotoxicity, hepatotoxicity, neurotoxicity and carcinogenicity. We found that in mice OTA is transported by the drug transporters mouse (m)ABCB1 and/or mABCG2, mOATP1A/1B, and human (h)OATP1B3. The complete deletion of mABCB1 and mABCG2 resulted in ~2-fold higher OTA liver and kidney accumulation upon intravenous injection. Upon oral administration, absence of mOATP1A/1B led to a substantial (>3-fold) decrease in hepatic and small intestinal exposure of OTA. Furthermore, in humanized mouse strains, hepatic expression of transgenic hOATP1B3, but not hOATP1B1, partly reversed the reduced liver concentration of OTA in mOATP1A/1B knockout mice. These data indicate that transgenic hOATP1B3 can significantly transport OTA into the liver, and can at least partly compensate for the loss of the mOATP1A/1B transporters. This study shows that some ABC and OATP transporters can substantially affect the pharmacokinetics of OTA, which might have implications for its toxicity behavior.
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http://dx.doi.org/10.1016/j.taap.2020.115072DOI Listing
August 2020

Bioanalytical method for the simultaneous quantification of irinotecan and its active metabolite SN-38 in mouse plasma and tissue homogenates using HPLC-fluorescence.

J Chromatogr B Analyt Technol Biomed Life Sci 2020 Jul 19;1149:122177. Epub 2020 May 19.

Department of Pharmacy & Pharmacology, The Netherlands Cancer Institute, Amsterdam, the Netherlands; Division of Pharmacology, The Netherlands Cancer Institute, Amsterdam, the Netherlands; Division of Pharmacoepidemiology and Clinical Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands.

A simple and rapid bioanalytical method was developed for the simultaneous quantification of irinotecan and SN-38 in mouse plasma and tissue homogenates using High-Performance Liquid Chromatography with Fluorescence detection (HPLC-FL). Camptothecin was used as internal standard and protein precipitation with acetonitrile-methanol (1:1, v/v) followed by acidification with 0.5 M hydrochloric acid was used for sample pre-treatment. The analytes and the internal standard were detected using an excitation and emission wavelength of 368 and 515 nm, respectively. The linearity, selectivity, accuracy and precision, carry-over, limit of detection and lower limit of quantification of the method are described. The method was linear from 7.5 to 1500 ng/mL for irinotecan and from 5 to 1000 ng/mL for SN-38. For all matrices, the accuracy bias and precision variation were within ±15% and ≤15%, respectively. This method was successfully applied to study the pharmacokinetics of irinotecan and SN-38 using in vivo mouse models.
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http://dx.doi.org/10.1016/j.jchromb.2020.122177DOI Listing
July 2020

Quantitative bioanalytical assay for the selective RET inhibitors selpercatinib and pralsetinib in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry.

J Chromatogr B Analyt Technol Biomed Life Sci 2020 Jun 1;1147:122131. Epub 2020 May 1.

Utrecht University, Faculty of Science, Department of Pharmaceutical Sciences, Division of Pharmacology, Universiteitsweg 99, 3584 CG Utrecht, the Netherlands. Electronic address:

Selpercatinib and pralsetinib are potent and selective tyrosine kinase inhibitors targeting the rearranged during transfection (RET) receptor in various types of cancer. In this study, a bioanalytical assay was developed and fully validated for selpercatinib and pralsetinib in mouse plasma and partially in eight mouse tissue homogenates using liquid chromatograph-tandem mass spectrometry. Samples were pre-treated by protein precipitation with acetonitrile using erlotinib as internal standard. Separation of the analytes was performed on an ethylene bridged octadecyl silica C18 column by gradient elution using ammonium hydroxide (in water) and methanol. Analytes were detected by positive electrospray ionization in selected reaction monitoring mode. A linear concentration range of 2-2000 ng/ml was used for the validation of the assay for both inhibitors. The precision values (within-day and between-day) ranged between 3.4 and 10.2% for selpercatinib and 3.1-14.6% for pralsetinib in all matrices. Furthermore, data obtained for accuracy were between 91.7 and 109.3% and 85.1-114.1% for selpercatinib and pralsetinib, respectively. No significant matrix effects or extraction losses were observed and both analytes were stable under all investigated conditions. Finally, a pilot study for selpercatinib in mice was conducted employing this method, followed by a successful incurred sample reanalysis.
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http://dx.doi.org/10.1016/j.jchromb.2020.122131DOI Listing
June 2020

Inhibition of Hepatic Bile Acid Uptake by Myrcludex B Promotes Glucagon-Like Peptide-1 Release and Reduces Obesity.

Cell Mol Gastroenterol Hepatol 2020 21;10(3):451-466. Epub 2020 Apr 21.

Tytgat Institute for Liver and Intestinal Research, Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, the Netherlands; Department of Gastroenterology and Hepatology, Amsterdam Gastroenterology Endocrinology Metabolism, Amsterdam University Medical Center, University of Amsterdam, Amsterdam, the Netherlands. Electronic address:

Background & Aims: Bile acids are important metabolic signaling molecules. Bile acid receptor activation promotes body weight loss and improves glycemic control. The incretin hormone GLP-1 and thyroid hormone activation of T4 to T3 have been suggested as important contributors. Here, we identify the hepatic bile acid uptake transporter Na taurocholate co-transporting polypeptide (NTCP) as target to prolong postprandial bile acid signaling.

Methods: Organic anion transporting polypeptide (OATP)1a/1b KO mice with or without reconstitution with human OATP1B1 in the liver were treated with the NTCP inhibitor Myrcludex B for 3.5 weeks after the onset of obesity induced by high fat diet-feeding. Furthermore, radiolabeled T4 was injected to determine the role of NTCP and OATPs in thyroid hormone clearance from plasma.

Results: Inhibition of NTCP by Myrcludex B in obese Oatp1a/1b KO mice inhibited hepatic clearance of bile acids from portal and systemic blood, stimulated GLP-1 secretion, reduced body weight, and decreased (hepatic) adiposity. NTCP inhibition did not affect hepatic T4 uptake nor lead to increased thyroid hormone activation. Myrcludex B treatment increased fecal energy output, explaining body weight reductions amongst unaltered food intake and energy expenditure.

Conclusions: Pharmacologically targeting hepatic bile acid uptake to increase bile acid signaling is a novel approach to treat obesity and induce GLP1- secretion.
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http://dx.doi.org/10.1016/j.jcmgh.2020.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363705PMC
April 2020

Bioanalytical assay for the new-generation ROS1/TRK/ALK inhibitor repotrectinib in mouse plasma and tissue homogenate using liquid chromatography-tandem mass spectrometry.

J Chromatogr B Analyt Technol Biomed Life Sci 2020 May 1;1144:122098. Epub 2020 Apr 1.

Utrecht University, Faculty of Science, Department of Pharmaceutical Sciences, Division Pharmacology, Universiteitsweg 99, 3584 CG Utrecht, the Netherlands. Electronic address:

Repotrectinib, a next-generation ROS1/TRK/ALK tyrosine kinase inhibitor, overcomes resistance due to acquired solvent-front mutations involving ROS1, NTRK1-3, and ALK. A bioanalytical assay for quantification of repotrectinib in mouse plasma and seven tissue-related matrices (brain, liver, spleen, kidney, small intestinal tissue, small intestinal content, and testis homogenates) was developed and validated using liquid chromatography with tandem mass spectrometric detection in a high-throughput 96-well format. Protein precipitation was performed by adding acetonitrile, also containing the internal standard axitinib, to 10-µl samples for all matrices. Chromatographic separation of analytes was done on an ACQUITY UPLC® BEH C18 column by gradient elution using ammonium hydroxide in water and methanol. Compounds were monitored with positive electrospray ionization using a triple quadruple mass spectrometer in selected reaction monitoring mode. The method was successfully validated in the 1-1000 ng/ml calibration range. Precisions (intra- and interday) were in the range of 1.3-8.7% and accuracies were in between 90.5% and 107.3% for all levels in all matrices. The developed method was successfully applied to investigate the plasma pharmacokinetics and tissue accumulation of repotrectinib in wild-type mice.
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http://dx.doi.org/10.1016/j.jchromb.2020.122098DOI Listing
May 2020

Brain accumulation of tivozanib is restricted by ABCB1 (P-glycoprotein) and ABCG2 (breast cancer resistance protein) in mice.

Int J Pharm 2020 May 28;581:119277. Epub 2020 Mar 28.

Division of Pharmacology, The Netherlands Cancer Institute, Amsterdam, The Netherlands. Electronic address:

Tivozanib is a potent and selective inhibitor of VEGFR1-3, recently approved by the EMA for first-line treatment of renal cell carcinoma. We used wild-type, knockout, and transgenic mouse strains to study the effects of the drug transporters ABCB1, ABCG2, and OATP1A/1B, and of the CYP3A enzymes on the oral availability and tissue distribution of tivozanib. Tivozanib was transported by human ABCB1 and mouse Abcg2 in polarized MDCK-II cells. Upon oral administration, tivozanib showed rapid absorption and the plasma concentration-time curves showed secondary peaks in all mouse strains, suggesting enterohepatic recirculation. The brain-to-plasma ratios were significantly increased in Abcb1a/1b (2.2-fold) and Abcb1a/1b;Abcg2 (2.6-fold) mice compared to wild-type mice, indicating a modest protective role of these transporters in the blood-brain barrier. Slco1a/1b mice showed a 1.2-fold lower liver-to-plasma ratio than wild-type mice, suggesting a minor role of mOatp1a/1b in tivozanib liver distribution. Oral plasma pharmacokinetics of tivozanib was not significantly altered in these mouse strains, nor in Cyp3a knockout and CYP3A4-humanized mice. The modest effect of ABC transporters on tivozanib brain accumulation, if also true in humans, might mean that this drug is not strongly limited in its therapeutic efficacy against malignant lesions situated partly or completely behind the blood-brain barrier.
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http://dx.doi.org/10.1016/j.ijpharm.2020.119277DOI Listing
May 2020

OATP1A/1B, CYP3A, ABCB1, and ABCG2 limit oral availability of the NTRK inhibitor larotrectinib, while ABCB1 and ABCG2 also restrict its brain accumulation.

Br J Pharmacol 2020 07 12;177(13):3060-3074. Epub 2020 Apr 12.

Division of Pharmacology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Background And Purpose: Larotrectinib is a FDA-approved oral small-molecule inhibitor for treatment of neurotrophic tropomyosin receptor kinase fusion-positive cancer. We here investigated the functions of the multidrug efflux transporters ABCB1 and ABCG2, the SLCO1A/1B (OATP1A/1B) uptake transporters, and the multispecific drug-metabolizing enzyme CYP3A in larotrectinib pharmacokinetic behaviour.

Experimental Approach: In vitro, transepithelial drug transport and uptake assays were performed. In vivo, larotrectinib (10 mg·kg ) was administered orally to relevant genetically modified mouse models. Cell medium, plasma samples, and organ homogenates were measured by a sensitive and specific LC-MS/MS larotrectinib assay.

Key Results: In vitro, larotrectinib was avidly transported by human (h) ABCB1 and mouse (m) Abcg2 efficiently by hABCG2 and modestly by hOATP1A2. In vivo, both mAbcb1a/1b and mAbcg2 markedly limited larotrectinib oral availability and brain and testis accumulation (by 2.1-fold, 10.4-fold, and 2.7-fold, respectively), with mAbcb1a/1b playing a more prominent role. mOatp1a/1b also restricted larotrectinib oral availability (by 3.8-fold) and overall tissue exposure, apparently by mediating substantial uptake into the liver, thus likely facilitating hepatobiliary excretion. Additionally, larotrectinib is an excellent substrate of CYP3A, which restricts the oral availability of larotrectinib and hence its tissue exposure.

Conclusions And Implications: ABCG2 and especially ABCB1 limit the oral availability and brain and testis penetration of larotrectinib, while OATP1A/1B transporters restrict its systemic exposure by mediating hepatic uptake, thus allowing hepatobiliary excretion. CYP3A-mediated metabolism can strongly limit larotrectinib oral availability and hence its tissue concentrations. These insights may be useful in the further clinical development of larotrectinib.
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http://dx.doi.org/10.1111/bph.15034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7279963PMC
July 2020

P-glycoprotein (ABCB1/MDR1) limits brain accumulation and Cytochrome P450-3A (CYP3A) restricts oral availability of the novel FGFR4 inhibitor fisogatinib (BLU-554).

Int J Pharm 2020 Jan 20;573:118842. Epub 2019 Nov 20.

Division of Pharmacology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands. Electronic address:

Fisogatinib (BLU-554) is a highly selective and potent oral fibroblast growth factor receptor 4 (FGFR4) inhibitor currently in Phase I clinical trials for treatment of hepatocellular carcinoma (HCC). Using (male) genetically modified mouse models, we investigated the roles of the multidrug efflux transporters ABCB1 and ABCG2, the OATP1A/1B uptake transporters, and the drug-metabolizing CYP3A complex in fisogatinib pharmacokinetics. In vitro, fisogatinib was modestly transported by hABCB1. Upon oral administration of 10 mg/kg fisogatinib, its brain accumulation was substantially increased in Abcb1a/1b (6.3-fold) and Abcb1a/1b;Abcg2 mice (7.2-fold) compared to wild-type mice, but not in single Abcg2 mice. The oral plasma pharmacokinetics and liver distribution of fisogatinib were not significantly affected by the absence of Oatp1a/1b drug uptake transporters. We further found that plasma exposure of fisogatinib in Cyp3a mice increased by 1.4-fold, and was subsequently 1.6-fold decreased upon transgenic overexpression of human CYP3A4 in liver and intestine. However, the relative tissue distribution of fisogatinib remained unaltered. In summary, in mice, fisogatinib brain accumulation is substantially limited by ABCB1 P-glycoprotein in the blood-brain barrier, and oral availability of fisogatinib is markedly restricted by CYP3A activity. The obtained insights may be useful for optimizing the clinical efficacy and safety of fisogatinib.
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http://dx.doi.org/10.1016/j.ijpharm.2019.118842DOI Listing
January 2020

Metabolome Analysis Reveals Dermal Histamine Accumulation in Murine Dermatitis Provoked by Genetic Deletion of P-Glycoprotein and Breast Cancer Resistance Protein.

Pharm Res 2019 Sep 11;36(11):158. Epub 2019 Sep 11.

Faculty of Pharmacy, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan.

Purpose: P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are xenobiotic transporters which pump out variety types of compounds, but information on their interaction with endogenous substrates in the skin is limited. The purpose of the present study was to clarify possible association of these transporters in dermal accumulation of inflammatory mediators.

Methods: Dermatitis model was constructed by repeated topical application of oxazolone in wild-type, and P-gp and BCRP gene triple knockout (Mdr1a/1b/Bcrp) mice to observe difference in phenotype. Target metabolome analysis of 583 metabolites was performed using skin and plasma.

Results: Dermatitis and scratching behavior in dermatitis model of Mdr1a/1b/Bcrp mice were more severe than wild-type mice, suggesting protective roles of these transporters. This hypothesis was supported by the metabolome analysis which revealed that concentration of histamine and other dermatitis-associated metabolites like urate and serotonin in the dermatitis skin, but not normal skin, of Mdr1a/1b/Bcrp mice was higher than that of wild-type mice. Gene expression of P-gp and BCRP was reduced in oxazolone-treated skin and the skin of patients with atopic dermatitis or psoriasis.

Conclusions: These results suggest possible association of these efflux transporters with dermal inflammatory mediators, and such association could be observed in the dermatitis skin.
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http://dx.doi.org/10.1007/s11095-019-2695-3DOI Listing
September 2019

P-glycoprotein Limits Ribociclib Brain Exposure and CYP3A4 Restricts Its Oral Bioavailability.

Mol Pharm 2019 09 5;16(9):3842-3852. Epub 2019 Aug 5.

Division of Pharmacology , The Netherlands Cancer Institute , Amsterdam , The Netherlands.

Ribociclib is a CDK4/6 inhibitor recently approved for the treatment of some types of breast cancer in combination with an aromatase inhibitor. It is currently investigated in the clinic to treat other malignancies, including brain tumors. Using and genetically modified mouse models, we investigated the effect of the multidrug efflux transporters ABCB1 and ABCG2, and the drug-metabolizing CYP3A enzymes on ribociclib pharmacokinetics and tissue distribution. , ribociclib was avidly transported by human ABCB1, but not by human ABCG2 and only modestly by mouse Abcg2. Upon oral administration at 20 mg/kg, the plasma AUC of ribociclib was increased by 2.3-fold, and its terminal elimination was delayed in compared to wild-type mice. The brain-to-plasma ratios of ribociclib were increased by at least 23-fold relative to wild-type mice in and mice, but not noticeably in mice. Oral coadministration of elacridar, an ABCB1 and ABCG2 inhibitor, increased the brain penetration of ribociclib in wild-type mice to the same level as seen in mice. Plasma exposure of ribociclib further decreased by 3.8-fold when transgenic human CYP3A4 was overexpressed in -deficient mice. Ribociclib penetration into the brain is thus drastically limited by ABCB1 in the blood-brain barrier, but coadministration of elacridar can fully reverse this process. Moreover, human CYP3A4 can extensively metabolize ribociclib and strongly restrict its oral bioavailability. The insights obtained from this study may be useful to further optimize the clinical application of ribociclib, especially for the treatment of (metastatic) brain tumors.
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http://dx.doi.org/10.1021/acs.molpharmaceut.9b00475DOI Listing
September 2019

Cytochrome P450 3A4, 3A5, and 2C8 expression in breast, prostate, lung, endometrial, and ovarian tumors: relevance for resistance to taxanes.

Cancer Chemother Pharmacol 2019 09 15;84(3):487-499. Epub 2019 Jul 15.

Department of Pharmacy and Pharmacology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.

Enzymes of the cytochrome P450 (CYP) subfamily 3A and 2C play a major role in the metabolism of taxane anticancer agents. While their function in hepatic metabolism of taxanes is well established, expression of these enzymes in solid tumors may play a role in the in situ metabolism of drugs as well, potentially affecting the intrinsic taxane susceptibility of these tumors. This article reviews the available literature on intratumoral expression of docetaxel- and paclitaxel-metabolizing enzymes in mammary, prostate, lung, endometrial, and ovarian tumors. Furthermore, the clinical implications of the intratumoral expression of these enzymes are reviewed and the potential of concomitant treatment with protease inhibitors (PIs) as a method to inhibit CYP3A4-mediated metabolism is discussed.
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http://dx.doi.org/10.1007/s00280-019-03905-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6682574PMC
September 2019

P-glycoprotein (MDR1/ABCB1) controls brain accumulation and intestinal disposition of the novel TGF-β signaling pathway inhibitor galunisertib.

Int J Cancer 2020 03 15;146(6):1631-1642. Epub 2019 Jul 15.

The Netherlands Cancer Institute, Division of Pharmacology, Amsterdam, The Netherlands.

Galunisertib (LY2157299), a promising small-molecule inhibitor of the transforming growth factor-beta (TGF-β) receptor, is currently in mono- and combination therapy trials for various cancers including glioblastoma, hepatocellular carcinoma and breast cancer. Using genetically modified mouse models, we investigated the roles of the multidrug efflux transporters ABCB1 and ABCG2, the OATP1A/1B uptake transporters and the drug-metabolizing CYP3A complex in galunisertib pharmacokinetics. In vitro, galunisertib was vigorously transported by human ABCB1, and moderately by mouse Abcg2. Orally administered galunisertib (20 mg/kg) was very rapidly absorbed. Galunisertib brain-to-plasma ratios were increased by ~24-fold in Abcb1a/1b and Abcb1a/1b;Abcg2 mice compared to wild-type mice, but not in single Abcg2 mice, whereas galunisertib oral availability was not markedly affected. However, recovery of galunisertib in the small intestinal lumen was strongly reduced in Abcb1a/1b and Abcb1a/1b;Abcg2 mice. Oral coadministration of the ABCB1/ABCG2 inhibitor elacridar boosted galunisertib brain accumulation in wild-type mice to equal the levels seen in Abcb1a/1b;Abcg2 mice. Oatp1a/1b deficiency did not alter oral galunisertib pharmacokinetics or liver distribution. Cyp3a mice showed a 1.9-fold higher plasma AUC than wild-type mice, but this difference disappeared over 8 hr. Also, transgenic human CYP3A4 overexpression did not significantly alter oral galunisertib pharmacokinetics. Abcb1 thus markedly restricts galunisertib brain penetration and affects its intestinal disposition, possibly through biliary excretion. Elacridar coadministration could fully inhibit both processes, without causing acute toxicity. Moreover, mouse Cyp3a, but not human CYP3A4, may eliminate galunisertib at high plasma concentrations. These insights may help to guide the further clinical development and application of galunisertib.
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http://dx.doi.org/10.1002/ijc.32568DOI Listing
March 2020

Development and validation of a bioanalytical method for the quantification of the CDK4/6 inhibitors abemaciclib, palbociclib, and ribociclib in human and mouse matrices using liquid chromatography-tandem mass spectrometry.

Anal Bioanal Chem 2019 Aug 17;411(20):5331-5345. Epub 2019 Jun 17.

Department of Pharmacy & Pharmacology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.

A novel method was developed and validated for the quantification of the three approved CDK4/6 inhibitors (abemaciclib, palbociclib, and ribociclib) in both human and mouse plasma and mouse tissue homogenates (liver, kidney, spleen, brain, and small intestine) using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). For all matrices, pretreatment was performed using 50 μL of sample by protein precipitation with acetonitrile, followed by dilution of the supernatant. Chromatographic separation of the analytes was done on a C18 column using gradient elution. A full validation was performed for human plasma, while a partial validation was executed for mouse plasma and mouse tissue homogenates. The method was linear in the calibration range from 2 to 200 ng/mL, with a correlation coefficient (r) ≥0.996 for each analyte. For both human and mouse plasma, the accuracy and precision were within ±15% and ≤15%, respectively, for all concentrations, except for the lower limit of quantification, where they were within ±20% and ≤20%, respectively. A fit-for-purpose strategy was followed for tissue homogenates, and the accuracy and precision were within ±20% and ≤20%, respectively, for all concentrations. Stability of all analytes in all matrices at different processing and storage conditions was tested; ribociclib and palbociclib were unstable in most tissue homogenates and conditions were modified to increase the stability. The method was successfully applied for the analysis of mouse samples from preclinical studies. A new ribociclib metabolite was detected in mouse plasma samples with the same m/z transition as the parent drug.
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http://dx.doi.org/10.1007/s00216-019-01932-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6647725PMC
August 2019

Brain accumulation of osimertinib and its active metabolite AZ5104 is restricted by ABCB1 (P-glycoprotein) and ABCG2 (breast cancer resistance protein).

Pharmacol Res 2019 08 5;146:104297. Epub 2019 Jun 5.

Division of Pharmacology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands. Electronic address:

Osimertinib is an irreversible EGFR inhibitor registered for advanced NSCLC patients whose tumors harbor recurrent somatic activating mutations in EGFR (EGFRm) or the frequently occurring EGFR-T790M resistance mutation. Using in vitro transport assays and appropriate knockout and transgenic mouse models, we investigated whether the multidrug efflux transporters ABCB1 and ABCG2 transport osimertinib and whether they influence the oral availability and brain accumulation of osimertinib and its most active metabolite, AZ5104. In vitro, human ABCB1 and mouse Abcg2 modestly transported osimertinib. In mice, Abcb1a/1b, with a minor contribution of Abcg2, markedly limited the brain accumulation of osimertinib and AZ5104. However, no effect of the ABC transporters was seen on osimertinib oral availability. In spite of up to 6-fold higher brain accumulation, we observed no acute toxicity signs of oral osimertinib in Abcb1a/1b;Abcg2 knockout mice. Interestingly, even in wild-type mice the intrinsic brain penetration of osimertinib was already relatively high, which may help to explain the documented partial efficacy of this drug against brain metastases. No substantial effects of mouse Cyp3a knockout or transgenic human CYP3A4 overexpression on oral osimertinib pharmacokinetics were observed, presumably due to a dominant role of mouse Cyp2d enzymes in osimertinib metabolism. Our results suggest that pharmacological inhibition of ABCB1 and ABCG2 during osimertinib therapy might potentially be considered to further benefit patients with brain (micro-)metastases positioned behind an intact blood-brain barrier, or with substantial expression of these transporters in the tumor cells, without invoking a high toxicity risk.
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http://dx.doi.org/10.1016/j.phrs.2019.104297DOI Listing
August 2019

Bioanalytical assay for the novel TRK inhibitor selitrectinib in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry.

J Chromatogr B Analyt Technol Biomed Life Sci 2019 Aug 27;1122-1123:78-82. Epub 2019 May 27.

Utrecht University, Faculty of Science, Department of Pharmaceutical Sciences, Division of Pharmacoepidemiology & Clinical Pharmacology, Universiteitsweg 99, 3584 CG Utrecht, the Netherlands; The Netherlands Cancer Institute, Department of Pharmacy & Pharmacology, Louwesweg 6, 1066 EC Amsterdam, the Netherlands. Electronic address:

Selitrectinib is a next generation tropomyosin receptor kinase (TRK) inhibitor developed to overcome acquired resistance to first generation TRK inhibitors. The drug is a cyclic analogue of larotrectinib. An existing bioanalytical assay for larotrectinib was therefore redesigned for selitrectinib. The assay used liquid chromatography-electrospray tandem mass spectrometry in positive selected reaction monitoring mode. Mouse plasma and tissue homogenates of brain, heart, kidney, liver, lung, small intestine, spleen, and testis were pretreated using acetonitrile protein precipitation with larotrectinib added as internal standard. Successful validation using current guidelines was obtained in the range 0.5-1000 ng/ml. Precision was within 5-12% and accuracy within 91-108% for all matrices investigated. The drug was stable in all matrices under the relevant storage conditions. Pharmacokinetics and tissue distribution of selitrectinib were monitored in a pilot study in mice demonstrating the applicability of the presented assay.
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http://dx.doi.org/10.1016/j.jchromb.2019.05.026DOI Listing
August 2019

Quantification of FGFR4 inhibitor BLU-554 in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry.

J Chromatogr B Analyt Technol Biomed Life Sci 2019 Mar 16;1110-1111:116-123. Epub 2019 Feb 16.

Utrecht University, Faculty of Science, Department of Pharmaceutical Sciences, Division of Pharmacoepidemiology & Clinical Pharmacology, Universiteitsweg 99, 3584 CG Utrecht, the Netherlands; Utrecht University, Faculty of Science, Department of Pharmaceutical Sciences, Division of Chemical Biology & Drug Development, Universiteitsweg 99, 3584 CG Utrecht, the Netherlands. Electronic address:

BLU-554 is a potent, highly selective oral FGFR4 inhibitor. A bioanalytical assay for quantification of BLU-554 in mouse plasma and six tissue homogenates (brain, kidney, liver, lung, small intestine, and spleen) was developed and validated using liquid chromatography with tandem mass spectrometric detection and with erlotinib as internal standard. After protein precipitation with acetonitrile in a 96-well format and separation on an XBridge® Peptide BEH C18 column by gradient elution using 0.2% (v/v) ammonium hydroxide (in water) and methanol, analytes were ionized by positive electrospray and monitored in the selected reaction monitoring mode by triple quadrupole mass spectrometry. The assay was validated in a 1-1000 ng/ml concentration range using calibration in mouse plasma. Precisions (intra-day and inter-day) were in the range 2.8-10.1% and accuracies were in between 88.5 and 96.6% for all levels in all matrices. The assay was successfully applied for a pilot pharmacokinetic and tissue distribution study in wild-type mice.
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http://dx.doi.org/10.1016/j.jchromb.2019.02.017DOI Listing
March 2019

Oral coadministration of elacridar and ritonavir enhances brain accumulation and oral availability of the novel ALK/ROS1 inhibitor lorlatinib.

Eur J Pharm Biopharm 2019 Mar 17;136:120-130. Epub 2019 Jan 17.

The Netherlands Cancer Institute, Division of Pharmacology, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands. Electronic address:

Lorlatinib, a novel generation oral anaplastic lymphoma kinase (ALK) and ROS1 inhibitor with high membrane and blood-brain barrier permeability, recently received accelerated approval for treatment of ALK-rearranged non-small-cell lung cancer (NSCLC), and its further clinical development is ongoing. We previously found that the efflux transporter P-glycoprotein (MDR1/ABCB1) restricts lorlatinib brain accumulation and that the drug-metabolizing enzyme cytochrome P450-3A (CYP3A) limits its oral availability. Using genetically modified mouse models, we investigated the impact of targeted pharmacological inhibitors on lorlatinib pharmacokinetics and bioavailability. Upon oral administration of lorlatinib, the plasma AUC in CYP3A4-humanized mice was ∼1.8-fold lower than in wild-type and Cyp3a mice. Oral coadministration of the CYP3A inhibitor ritonavir caused reversion to the AUC levels seen in wild-type and Cyp3a mice, without altering the relative tissue distribution of lorlatinib. Moreover, simultaneous pharmacological inhibition of P-glycoprotein and CYP3A4 with oral elacridar and ritonavir in CYP3A4-humanized mice profoundly increased lorlatinib brain concentrations, but not its oral availability or other relative tissue distribution. Oral lorlatinib pharmacokinetics was not significantly affected by absence of the multispecific Oatp1a/1b drug uptake transporters. The absolute oral bioavailability of lorlatinib over 8 h in wild-type, Cyp3a, and CYP3A4-humanized mice was 81.6%, 72.9%, and 58.5%, respectively. Lorlatinib thus has good oral bioavailability, which is markedly restricted by human CYP3A4 but not by mouse Cyp3a. Pharmacological inhibition of CYP3A4 reversed these effects, and simultaneous P-gp inhibition with elacridar boosted absolute brain levels of lorlatinib by 16-fold without obvious toxicity. These insights may help to optimize the clinical application of lorlatinib.
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http://dx.doi.org/10.1016/j.ejpb.2019.01.016DOI Listing
March 2019

P-glycoprotein (MDR1/ABCB1) and Breast Cancer Resistance Protein (BCRP/ABCG2) limit brain accumulation of the FLT3 inhibitor quizartinib in mice.

Int J Pharm 2019 Feb 12;556:172-180. Epub 2018 Dec 12.

The Netherlands Cancer Institute, Division of Pharmacology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Electronic address:

Quizartinib, a second-generation FLT3 inhibitor, is in clinical development for the treatment of acute myeloid leukemia. We studied its pharmacokinetic interactions with the multidrug efflux transporters ABCB1 and ABCG2 and the multidrug metabolizing enzyme CYP3A, using in vitro transport assays and knockout and transgenic mouse models. Quizartinib was transported by human ABCB1 in vitro, and by mouse (m)Abcb1 and mAbcg2 in vivo. Upon oral administration, the brain accumulation of quizartinib was 6-fold decreased by mAbcb1 and 2-fold by mAbcg2 (together: 12-fold). Unexpectedly, the absence of mAbcb1 resulted in a ∼2-fold lower plasma exposure in Abcb1a/1b and Abcb1a/1b;Abcg2 mice, suggesting that loss of mAbcb1 causes compensatory alterations in alternative quizartinib elimination or uptake systems. mAbcb1 and mAbcg2 themselves did not appear to restrict quizartinib oral availability. Oral and intravenous pharmacokinetics of quizartinib were not substantially altered between wild-type, Cyp3a knockout and CYP3A4-humanized mice. All three strains showed relatively high (33-51%) oral bioavailability. If this also applies in humans, this would suggest a limited risk of CYP3A-related inter-individual variation in exposure for this drug. Our results provide a possible rationale for using pharmacological ABCB1/ABCG2 inhibitors together with quizartinib when treating malignant lesions situated in part or in whole behind the blood-brain barrier.
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http://dx.doi.org/10.1016/j.ijpharm.2018.12.014DOI Listing
February 2019

Quantitative bioanalytical assay for the tropomyosin receptor kinase inhibitor larotrectinib in mouse plasma and tissue homogenates using liquid chromatography-tandem mass spectrometry.

J Chromatogr B Analyt Technol Biomed Life Sci 2018 Dec 30;1102-1103:167-172. Epub 2018 Oct 30.

Utrecht University, Faculty of Science, Department of Pharmaceutical Sciences, Division of Pharmacoepidemiology & Clinical Pharmacology, Universiteitsweg 99, 3584 CG Utrecht, the Netherlands; The Netherlands Cancer Institute, Department of Clinical Pharmacology, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands; MC Slotervaart, Department of Pharmacy & Pharmacology, Louwesweg 6, 1066 EC Amsterdam, the Netherlands. Electronic address:

Larotrectinib is a promising tyrosine kinase inhibitor for solid tumors harboring tropomyosin receptor kinase gene fusions. A bioanalytical assay was developed for this drug in small volume samples using a 96-well format to efficiently support multiple mouse studies. The assay was completely validated for mouse plasma and partially for homogenates of eight different tissues: brain, heart, kidneys, liver, lungs, small intestine, spleen, and testes. Proteins in 10-μl samples were precipitated using acetonitrile containing momelotinib as internal standard. Chromatographic separation of analyte and internal standard from endogenous interferences was performed on an ethylene bridged octadecyl silica column using 0.1% (v/v) formic acid (in water) and methanol for gradient elution. Electrospray ionization and selected reaction monitoring on a triple quadrupole mass spectrometer were used for detection. In the range 1-2000 ng/ml the drug could be quantified in all 9 matrices with precisions (within-day and between-day) in the range 2.7-11.1% and accuracies in the range 87.4-101.4%. Compounds were sufficiently stable under all investigated conditions except for kidney homogenate. A pilot pharmacokinetic and tissue distribution study in mice demonstrated the applicability of the new presented assay for larotrectinib.
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http://dx.doi.org/10.1016/j.jchromb.2018.10.023DOI Listing
December 2018

P-glycoprotein and breast cancer resistance protein restrict brigatinib brain accumulation and toxicity, and, alongside CYP3A, limit its oral availability.

Pharmacol Res 2018 11 22;137:47-55. Epub 2018 Sep 22.

The Netherlands Cancer Institute, Division of Pharmacology, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands. Electronic address:

Brigatinib is an FDA-approved oral anaplastic lymphoma kinase (ALK) inhibitor for treatment of metastatic non-small cell lung cancer (NSCLC). Using genetically modified mouse models, we investigated the roles of the multidrug efflux transporters ABCB1 and ABCG2, and the multispecific drug-metabolizing enzyme CYP3 A in plasma pharmacokinetics and tissue distribution of brigatinib. In vitro, brigatinib was exceptionally well transported by human ABCB1 and mouse Abcg2, and efficiently by human ABCG2. Following oral brigatinib administration (10 mg/kg), brain accumulation was dramatically increased in Abcb1a/1b (19.3-fold) and Abcb1a/1b;Abcg2(41.8-fold), but not in single Abcg2 mice compared to wild-type mice. Brigatinib testis accumulation showed qualitatively similar behavior. mAbcb1a/1b and mAbcg2 together restricted systemic exposure of brigatinib: with both systems absent oral availability increased 1.9-fold. Coadministration of elacridar, an ABCB1/ABCG2 inhibitor, caused a pronounced increase (36-fold) in brain-to-plasma ratios of brigatinib, approaching the levels seen in Abcb1a/1b;Abcg2 mice. Unexpectedly, lethal toxicity of oral brigatinib was observed in mice with genetic knockout or pharmacological inhibition of mAbcb1a/1b and mAbcg2, indicating a pronounced protective role for these transporters. In Cyp3a mice, brigatinib plasma exposure increased 1.3-fold, and was subsequently 1.8-fold reduced by transgenic overexpression of human CYP3 A4 in liver and intestine. The relative tissue distribution of brigatinib, however, remained unaltered. ABCB1 and ABCG2 thus limit brain accumulation, toxicity, and systemic exposure of brigatinib, whereas CYP3 A also markedly restricts its oral availability. Unexpected toxicities should therefore be carefully monitored when brigatinib is coadministered with ABCB1/ABCG2 inhibitors in patients. Collectively, these insights may support the clinical application of brigatinib.
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http://dx.doi.org/10.1016/j.phrs.2018.09.020DOI Listing
November 2018

P-Glycoprotein (MDR1/ABCB1) Restricts Brain Penetration of the Bruton's Tyrosine Kinase Inhibitor Ibrutinib, While Cytochrome P450-3A (CYP3A) Limits Its Oral Bioavailability.

Mol Pharm 2018 11 10;15(11):5124-5134. Epub 2018 Oct 10.

Division of Pharmacology , The Netherlands Cancer Institute , 1066 CX Amsterdam , The Netherlands.

Ibrutinib (Imbruvica), an oral tyrosine kinase inhibitor (TKI) approved for treatment of B-cell malignancies, irreversibly inhibits the Bruton's tyrosine kinase (BTK). Its abundant metabolite, dihydrodiol-ibrutinib (ibrutinib-DiOH), which is primarily formed by CYP3A, has a 10-fold reduced BTK inhibitory activity. Using in vitro transport assays and genetically modified mouse models, we investigated whether the multidrug efflux transporters ABCB1 and ABCG2 and the multidrug-metabolizing CYP3A enzyme family can affect the oral bioavailability and tissue disposition of ibrutinib and ibrutinib-DiOH. In vitro, ibrutinib was transported moderately by human ABCB1 and mouse Abcg2 but not detectably by human ABCG2. In mice, Abcb1 markedly restricted the brain penetration of ibrutinib and ibrutinib-DiOH, either alone or in combination with Abcg2, resulting in 4.5- and 5.9-fold increases in ibrutinib brain-to-plasma ratios in Abcb1a/1b and Abcb1a/1b;Abcg2 mice relative to wild-type mice. Abcb1 and/or Abcg2 did not obviously restrict ibrutinib oral bioavailability, but Cyp3a deficiency increased the ibrutinib plasma AUC by 9.7-fold compared to wild-type mice. This increase was mostly reversed (5.1-fold reduction) by transgenic human CYP3A4 overexpression, with roughly equal contributions of intestinal and hepatic CYP3A4 metabolism. Our results suggest that pharmacological inhibition of ABCB1 during ibrutinib therapy might benefit patients with malignancies or (micro)metastases positioned behind an intact blood-brain barrier, or with substantial expression of this transporter in the malignant cells. Moreover, given the strong in vivo impact of CYP3A, inhibitors or inducers of this enzyme family will likely strongly affect ibrutinib oral bioavailability and, thus, its therapeutic efficacy, as well as its toxicity risks.
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http://dx.doi.org/10.1021/acs.molpharmaceut.8b00702DOI Listing
November 2018

Bioanalytical liquid chromatography-tandem mass spectrometric assay for the quantification of the ALK inhibitors alectinib, brigatinib and lorlatinib in plasma and mouse tissue homogenates.

J Pharm Biomed Anal 2018 Nov 19;161:136-143. Epub 2018 Aug 19.

Utrecht University, Faculty of Science, Department of Pharmaceutical Sciences, Division of Pharmacoepidemiology & Clinical Pharmacology, Universiteitsweg 99, 3584 CG, Utrecht, The Netherlands; The Netherlands Cancer Institute, Department of Clinical Pharmacology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands; MC Slotervaart, Department of Pharmacy & Pharmacology, Louwesweg 6, 1066 EC Amsterdam, The Netherlands. Electronic address:

Several second and third generation ALK inhibitors have been introduced in recent years. A bioanalytical assay for simultaneous quantification of alectinib, brigatinib, and lorlatinib was developed and validated for human plasma. The method was also partially validated for diluted mouse plasma and tissue homogenates of brain, liver, kidney, and spleen. Samples (40 μl) were pretreated in a 96-well plate by protein precipitation with acetonitrile containing the internal standard [H]-alectinib. After chromatographic separation on an ethylene bridged octadecyl silica column by gradient elution at 600 μl/min using 1% (v/v) formic acid (in water) and acetonitrile, compounds were ionized by a turbo electrospray and monitored by selected reaction monitoring on a triple quadrupole mass spectrometer. Validation was performed in a 2-2000 ng/ml concentration range for alectinib and lorlatinib and a 4-4000 ng/ml range for brigatinib. Precisions (within-day and between-day) were in the range 2.2-15.0% and accuracies were in between 87.2 and 110.2% for all matrices and levels. Compounds were sufficiently stable under most investigated conditions. Results of a pilot pharmacokinetic and tissue distribution study for brigatinib in mice are reported. Finally, successful incurred samples reanalysis of tissue homogenate samples containing brigatinib and lorlatinib is presented. Lorlatinib homogenate samples were also successfully reanalyzed using a second independent assay (cross-validation).
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http://dx.doi.org/10.1016/j.jpba.2018.08.038DOI Listing
November 2018