Publications by authors named "Rolf W Sparidans"

112 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

Bioanalysis of erlotinib, its O-demethylated metabolites OSI-413 and OSI-420, and other metabolites by liquid chromatography-tandem mass spectrometry with additional ion mobility identification.

J Chromatogr B Analyt Technol Biomed Life Sci 2021 Mar 18;1166:122554. Epub 2021 Jan 18.

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

Erlotinib is a first-generation epithelial growth factor receptor inhibitor used in the treatment of non-small cellular lung cancers. Our previously published method on a Thermo TSQ Quantum Ultra triple quadrupole mass spectrometer for the quantitation of erlotinib, OSI-420, and OSI-413 and some other kinase inhibitors was transferred to a more sensitive Sciex QTRAP5500 system. Both methods showed comparable performance in the previous range (5-5000 and 1-1000 ng/mL for erlotinib and OSI-420) with comparable accuracies and precisions (98.9-106.2 vs 98.7.0-104.0, and 3.7-13.4 vs 4.6-13.2), and a high level of agreement between the methods (R = 0.9984 and 0.9951) for the quality control samples. The new system however was also capable of quantifying lower concentrations of both erlotinib and OSI-420 (0.5 and 0.1 ng/mL) with sufficient accuracy and precision. Along with the increased sensitivity we included the semi-quantitative determination of additional erlotinib metabolites M2, M3, M5, M6, M7, M8, M9, M10, M11, M12, M16 (hydroxy-erlotinib), M17, M18, M19, M20, M21 in a 0.1-1000 ng/mL range to the method. With a simple crash, dilute, and shoot sample preparation with acetonitrile and a 4.5 min analytical run time the method outperformed most other published methods in speed and simplicity and was suitable for TDM. Further, enhancement of the understanding of the pharmacokinetics of erlotinib and its metabolites was demonstrated.
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http://dx.doi.org/10.1016/j.jchromb.2021.122554DOI Listing
March 2021

Chromatographic bioanalytical assays for targeted covalent kinase inhibitors and their metabolites.

J Chromatogr B Analyt Technol Biomed Life Sci 2021 Jan 30;1162:122466. Epub 2020 Nov 30.

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

Deriving from targeted kinase inhibitors (TKIs), targeted covalent kinase inhibitors (TCKIs) are a new class of TKIs that are covalently bound to their target residue of kinase receptors. Currently, there are many new TCKIs under clinical development besides afatinib, ibrutinib, osimertinib, neratinib, acalabrutinib, dacomitinib, and zanubrutinib that are already approved by the FDA. Subsequently, there is an increasing demand for bioanalytical methods to qualitatively and quantitively investigate those compounds, leading to a number of papers reporting the development, validation, and use of bioanalytical methods for TCKIs. Most publications describe the technological set up of analytical methods that allow quantification of TCKIs in various biomatrices such as plasma, cerebrospinal fluid, urine, tissue, and liver microsomes. In addition, the identification of metabolites and biotransformation pathways of new TCKIs has gained more interest in recent years. We provide an overview of bioanalytical methods of this new class of TCKIs. The included issues are sample pretreatment, chromatographic separation, detection, and method validation. In the scope of bioanalysis of TCKIs, protein precipitation is mostly applied to treat the biological matrices sample. Liquid chromatographic in reversed-phase mode (RPLC) and mass detection with triple quadrupole (QqQ) are the most often utilized separation and quantitative detection modes, respectively. There may be a possibility of increased use of the high-resolution mass spectrometry (HRMS) for qualitative investigation purposes in the future. We also found that US FDA and EMA guidelines are the most common guidelines employed as validation framework for the bioanalytical methods of TCKIs.
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http://dx.doi.org/10.1016/j.jchromb.2020.122466DOI Listing
January 2021

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

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

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

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

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

P-glycoprotein (MDR1/ABCB1) restricts brain accumulation and cytochrome P450-3A (CYP3A) limits oral availability of the novel ALK/ROS1 inhibitor lorlatinib.

Int J Cancer 2018 10 10;143(8):2029-2038. Epub 2018 Jul 10.

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

Lorlatinib (PF-06463922) is a promising oral anaplastic lymphoma kinase (ALK) and ROS1 inhibitor currently in Phase III clinical trials for treatment of non-small-cell lung cancer (NSCLC) containing an ALK rearrangement. With therapy-resistant brain metastases a major concern in NSCLC, lorlatinib was designed to have high membrane and blood-brain barrier permeability. We investigated the roles of the multidrug efflux transporters ABCB1 and ABCG2, and the multispecific drug-metabolizing enzyme CYP3A in plasma pharmacokinetics and tissue distribution of lorlatinib using genetically modified mouse strains. In vitro, human ABCB1 and mouse Abcg2 modestly transported lorlatinib. Following oral lorlatinib administration (at 10 mg/kg), brain accumulation of lorlatinib, while relatively high in wild-type mice, was still fourfold increased in Abcb1a/1b and Abcb1a/1b;Abcg2 mice, but not in single Abcg2 mice. Lorlatinib plasma levels were not altered. Oral coadministration of the ABCB1/ABCG2 inhibitor elacridar increased the brain accumulation of lorlatinib in wild-type mice fourfold, that is, to the same level as in Abcb1a/1b;Abcg2 mice, without altering plasma exposure. Similar results were obtained for lorlatinib testis accumulation. In Cyp3a mice, the plasma exposure of lorlatinib was increased 1.3-fold, but was then twofold reduced upon transgenic overexpression of human CYP3A4 in liver and intestine, whereas relative tissue distribution of lorlatinib remained unaltered. Our data indicate that lorlatinib brain accumulation is substantially limited by P-glycoprotein/ABCB1 in the blood-brain barrier, but this can be effectively reversed by elacridar coadministration. Moreover, oral availability of lorlatinib is markedly restricted by CYP3A4 activity. These insights may be used in optimizing the therapeutic application of lorlatinib.
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http://dx.doi.org/10.1002/ijc.31582DOI Listing
October 2018

Bioanalytical assay for the quantification of the ALK inhibitor lorlatinib in mouse plasma using liquid chromatography-tandem mass spectrometry.

J Chromatogr B Analyt Technol Biomed Life Sci 2018 Apr 9;1083:204-208. Epub 2018 Mar 9.

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:

A bio-analytical assay for the first third generation ALK inhibitor lorlatinib in mouse plasma was developed and validated. Ten-μl plasma samples were prepared by adding rucaparib as the internal standard and precipitation of the plasma proteins. For LC-MS/MS analysis, compounds were eluted at 0.5 mL/min and separated on a 3-μm particle-size, polar embedded octadecyl silica column by gradient elution using 0.1% of formic acid (in water) and methanol. Compounds were monitored with positive electrospray ionization using a triple quadrupole mass spectrometer in selected reaction monitoring mode. The assay was fully validated in the 2-2000 ng/mL calibration range. Within-day (8.0-11.6%) and between-day (10.0-15.0%) precisions and accuracies (99.0-113.3%) were within acceptable range. Plasma samples were deemed stable for 6 h at ambient temperature, during three freeze-thaw cycles and for 2 months at -30 °C. Finally, the new assay was applied successfully to pilot pharmacokinetic studies in male and female wild-type mice.
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http://dx.doi.org/10.1016/j.jchromb.2018.03.014DOI Listing
April 2018

Quantification of cystine in human renal proximal tubule cells using liquid chromatography-tandem mass spectrometry.

Biomed Chromatogr 2018 Aug 15;32(8):e4238. Epub 2018 Apr 15.

Faculty of Science, Department of Pharmaceutical Sciences, Division of Pharmacology, Utrecht University, Utrecht, The Netherlands.

Nephropathic cystinosis is characterized by abnormal intralysosomal accumulation of cystine throughout the body, causing irreversible damage to various organs, particularly the kidneys. Cysteamine, the currently available treatment, can reduce lysosomal cystine and postpone disease progression. However, cysteamine poses serious side effects and does not address all of the symptoms of cystinosis. To screen for new treatment options, a rapid and reliable high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed to quantify cystine in conditionally immortalized human proximal tubular epithelial cells (ciPTEC). The ciPTEC were treated with N-ethylmaleimide, lysed and deproteinized with 15% (w/v) sulfosalicylic acid. Subsequently, cystine was measured using deuterium-labeled cystine-D4, as the internal standard. The assay developed demonstrated linearity to at least 20 μmol/L with a good precision. Accuracies were between 97.3 and 102.9% for both cell extracts and whole cell samples. Cystine was sufficiently stable under all relevant analytical conditions. The assay was successfully applied to determine cystine levels in both healthy and cystinotic ciPTEC. Control cells showed clearly distinguishable cystine levels compared with cystinotic cells treated with or without cysteamine. The method developed provides a fast and reliable quantification of cystine, and is applicable to screen for potential drugs that could reverse cystinotic symptoms in human kidney cells.
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http://dx.doi.org/10.1002/bmc.4238DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055858PMC
August 2018

Anti-GD2 Immunoliposomes for Targeted Delivery of the Survivin Inhibitor Sepantronium Bromide (YM155) to Neuroblastoma Tumor Cells.

Pharm Res 2018 Mar 7;35(4):85. Epub 2018 Mar 7.

Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands.

Purpose: Sepantronium bromide (YM155) is a hydrophilic quaternary compound that cannot be administered orally due to its low oral bioavailability; it is furthermore rapidly eliminated via the kidneys. The current study aims at improving the pharmacokinetic profile of YM155 by its formulation in immunoliposomes that can achieve its enhanced delivery into tumor tissue and facilitate uptake in neuroblastoma cancer cells.

Methods: PEGylated YM155 loaded liposomes composed of DPPC, cholesterol and DSPE-PEG were prepared via passive film-hydration and extrusion method. Targeted (i.e. immuno-)liposomes were prepared by surface functionalization with SATA modified monoclonal anti-disialoganglioside (GD2) antibodies. Liposomes were characterized based on their size, charge, antibody coupling and YM155 encapsulation efficiency, and stability. Flow cytometry analysis and confocal microscopy were performed on IMR32 and KCNR neuroblastoma cell lines. The efficacy of developed formulations were assessed by in-vitro toxicity assays. A pilot pharmacokinetic analysis was performed to assess plasma circulation and tumor accumulation profiles of the developed liposomal formulations.

Results: YM155 loaded immunoliposomes had a size of 170 nm and zeta potential of -10 mV, with an antibody coupling efficiency of 60% andYM155 encapsulation efficiency of14%. Targeted and control liposomal formulations were found to have similar YM155 release rates in a release medium containing 50% serum. An in-vitro toxicity study on KCNR cells showed less toxicity for immunoliposomes as compared to free YM155. In-vivo pharmacokinetic evaluation of YM155 liposomes showed prolonged blood circulation and significantly increased half-lives of liposomal YM155 in tumor tissue, as compared to a bolus injection of free YM155.

Conclusions: YM155 loaded immunoliposomes were successfully formulated and characterized, and initial in-vivo results show their potential for improving the circulation time and tumor accumulation of YM155.
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http://dx.doi.org/10.1007/s11095-018-2373-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5842274PMC
March 2018

P-glycoprotein (MDR1/ABCB1) and Breast Cancer Resistance Protein (BCRP/ABCG2) affect brain accumulation and intestinal disposition of encorafenib in mice.

Pharmacol Res 2018 03 14;129:414-423. Epub 2017 Nov 14.

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

Encorafenib (LGX818) is a promising BRAF inhibitor that has efficacy against metastatic melanoma. To better understand its pharmacokinetics, we studied its interactions with the multidrug efflux transporters ABCB1 and ABCG2 and the multidrug metabolizing enzyme CYP3A. In polarized MDCK-II cells, encorafenib was efficiently transported by canine and human ABCB1 and ABCG2 and by mouse Abcg2. Upon oral administration to wild-type, Abcb1a/1b, Abcg2, and Abcb1a/1b;Abcg2 mice, encorafenib was absorbed very quickly and to very high plasma levels, but without clear changes in oral availability between the strains. Upon oral or intravenous administration, encorafenib brain accumulation was markedly increased in Abcb1a/1b;Abcg2 mice and to a lesser extent in Abcb1a/1b mice. However, absolute brain concentrations and brain-to-plasma ratios remained very low in all strains, indicating intrinsically poor brain penetration of encorafenib. Upon intravenous administration, Abcb1a/1b;Abcg2 mice showed somewhat reduced plasma elimination of encorafenib compared to wild-type mice, and lower accumulation of the drug in the intestinal tract, suggesting a limited role for these transporters in intestinal elimination of the drug. In Cyp3a mice plasma levels of encorafenib were not markedly increased, suggesting a limited impact of Cyp3a on encorafenib oral availability. The low brain penetration of encorafenib might limit its efficacy against malignancies positioned behind a functional blood-brain barrier, but its oral bioavailability and distribution to other tested organs (liver, kidney, spleen, testis) was high.
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http://dx.doi.org/10.1016/j.phrs.2017.11.006DOI Listing
March 2018

Brain Accumulation of Ponatinib and Its Active Metabolite, N-Desmethyl Ponatinib, Is Limited by P-Glycoprotein (P-GP/ABCB1) and Breast Cancer Resistance Protein (BCRP/ABCG2).

Mol Pharm 2017 10 19;14(10):3258-3268. Epub 2017 Sep 19.

Division of Molecular Oncology, The Netherlands Cancer Institute , Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

Ponatinib is an oral BCR-ABL1 inhibitor for treatment of advanced leukemic diseases that carry the Philadelphia chromosome, specifically containing the T315I mutation yielding resistance to previously approved BCR-ABL1 inhibitors. Using in vitro transport assays and knockout mouse models, we investigated whether the multidrug efflux transporters ABCB1 and ABCG2 transport ponatinib and whether they, or the drug-metabolizing enzyme CYP3A, affect the oral availability and brain accumulation of ponatinib and its active N-desmethyl metabolite (DMP). In vitro, mouse Abcg2 and human ABCB1 modestly transported ponatinib. In mice, both Abcb1 and Abcg2 markedly restricted brain accumulation of ponatinib and DMP, but not ponatinib oral availability. Abcg2 deficiency increased DMP plasma levels ∼3-fold. Cyp3a deficiency increased the ponatinib plasma AUC 1.4-fold. Our results suggest that pharmacological inhibition of ABCG2 and ABCB1 during ponatinib therapy might benefit patients with brain (micro)metastases positioned behind an intact blood-brain barrier, or with substantial expression of these transporters in the malignant cells. CYP3A inhibitors might increase ponatinib oral availability, enhancing efficacy but possibly also toxicity of this drug.
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http://dx.doi.org/10.1021/acs.molpharmaceut.7b00257DOI Listing
October 2017

Liquid chromatography-tandem mass spectrometric assay for the quantitative determination of the tyrosine kinase inhibitor quizartinib in mouse plasma using salting-out liquid-liquid extraction.

J Chromatogr B Analyt Technol Biomed Life Sci 2017 Sep 23;1061-1062:300-305. Epub 2017 Jul 23.

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:

A bioanalytical assay for quizartinib -a potent, and selective FLT3 tyrosine kinase inhibitor- in mouse plasma was developed and validated. Salting-out assisted liquid-liquid extraction (SALLE), using acetonitrile and magnesium sulfate, was selected as sample pretreatment with deuterated quizartinib as internal standard. Separation was performed with reversed-phase liquid chromatography followed by detection with positive electrospray-triple quadrupole mass spectrometry in the selected reaction monitoring mode. The assay was successfully validated for mouse plasma in a 2-2000ng/ml calibration range with r=0.9958±0.0028 (n=7) for linear regression with the inverse square of the concentration as a weighting factor. The within-run precision (n=18), between-run precision and accuracy were 2.9-6.0%, 4.5-8.9% and 91.7-109.4% respectively. The drug was stable under all relevant conditions. Finally, the assay was successfully applied in a pharmacokinetic pilot study in plasma of FVB/NRj mice treated with quizartinb orally.
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http://dx.doi.org/10.1016/j.jchromb.2017.07.034DOI Listing
September 2017

Breast cancer resistance protein (BCRP/ABCG2) and P-glycoprotein (P-gp/ABCB1) transport afatinib and restrict its oral availability and brain accumulation.

Pharmacol Res 2017 Jun 10;120:43-50. Epub 2017 Mar 10.

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

Afatinib is a highly selective, irreversible inhibitor of EGFR and HER-2. It is orally administered for the treatment of patients with EGFR mutation-positive types of metastatic NSCLC. We investigated whether afatinib is a substrate for the multidrug efflux transporters ABCB1 and ABCG2 and whether these transporters influence oral availability and brain and other tissue accumulation of afatinib. We used in vitro transport assays to assess human (h)ABCB1-, hABCG2- or murine (m)Abcg2-mediated transport of afatinib. To study the single and combined roles of Abcg2 and Abcb1a/1b in oral afatinib disposition, we used appropriate knockout mouse strains. Afatinib was transported well by hABCB1, hABCG2 and mAbcg2 in vitro. Upon oral administration of afatinib, Abcg2, Abcb1a/1b and Abcb1a/1b;Abcg2 mice displayed a 4.2-, 2.4- and 7-fold increased afatinib plasma AUC compared with wild-type mice. Abcg2-deficient strains also displayed decreased afatinib plasma clearance. At 2h, relative brain accumulation of afatinib was not significantly altered in the single knockout strains, but 23.8-fold increased in Abcb1a/1b;Abcg2 mice compared to wild-type mice. Abcg2 and Abcb1a/1b restrict oral availability and brain accumulation of afatinib. Inhibition of these transporters may therefore be of clinical importance for patients with brain (micro)metastases positioned behind an intact blood-brain barrier.
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http://dx.doi.org/10.1016/j.phrs.2017.01.035DOI Listing
June 2017

Liquid chromatography-tandem mass spectrometric assay for therapeutic drug monitoring of the B-Raf inhibitor encorafenib, the EGFR inhibitors afatinib, erlotinib and gefitinib and the O-desmethyl metabolites of erlotinib and gefitinib in human plasma.

J Chromatogr B Analyt Technol Biomed Life Sci 2016 Oct 11;1033-1034:390-398. Epub 2016 Sep 11.

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

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http://dx.doi.org/10.1016/j.jchromb.2016.09.012DOI Listing
October 2016

PI3K-mTOR Pathway Inhibition Exhibits Efficacy Against High-grade Glioma in Clinically Relevant Mouse Models.

Clin Cancer Res 2017 Mar 23;23(5):1286-1298. Epub 2016 Aug 23.

Department of Bio-Pharmacology/Mouse Cancer Clinic, The Netherlands Cancer Institute (Antoni van Leeuwenhoek Hospital), Amsterdam, the Netherlands.

The PI3K-AKT-mTOR signaling pathway is frequently activated in glioblastoma and offers several druggable targets. However, clinical efficacy of PI3K/mTOR inhibitors in glioblastoma has not yet been demonstrated. Insufficient drug delivery may limit the efficacy of PI3K/mTOR inhibitors against glioblastoma. The presence of the efflux transporters ABCB1/Abcb1 (P-glycoprotein, MDR1) and ABCG2/Abcg2 (BCRP) at the blood-brain barrier (BBB) restricts the brain penetration of many drugs. We used drug transport assays and performed pharmacokinetic/pharmacodynamic studies in wild-type and ABC-transporter knockout mice. The efficacy of PI3K-mTOR inhibition was established using orthotopic allograft and genetically engineered spontaneous glioblastoma mouse models. The mTOR inhibitors rapamycin and AZD8055 are substrates of ABCB1, whereas the dual PI3K/mTOR inhibitor NVP-BEZ235 and the PI3K inhibitor ZSTK474 are not. Moreover, ABCG2 transports NVP-BEZ235 and AZD8055, but not ZSTK474 or rapamycin. Concordantly, mice revealed increased brain penetration of rapamycin (13-fold), AZD8055 (7.7-fold), and NVP-BEZ235 (4.5-fold), but not ZSTK474 relative to WT mice. Importantly, ABC transporters limited rapamycin brain penetration to subtherapeutic levels, while the reduction in NVP-BEZ235 brain penetration did not prevent target inhibition. NVP-BEZ235 and ZSTK474 demonstrated antitumor efficacy with improved survival against U87 orthotopic gliomas, although the effect of ZSTK474 was more pronounced. Finally, ZSTK474 prolonged overall survival in Cre-LoxP conditional transgenic mice, mainly by delaying tumor onset. PI3K/mTOR inhibitors with weak affinities for ABC transporters can achieve target inhibition in brain (tumors), but have modest single-agent efficacy and combinations with (BBB penetrable) inhibitors of other activated pathways may be required. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-1276DOI Listing
March 2017

Liquid chromatography-tandem mass spectrometric assay for the T790M mutant EGFR inhibitor osimertinib (AZD9291) in human plasma.

J Chromatogr B Analyt Technol Biomed Life Sci 2016 Sep 18;1031:80-85. Epub 2016 Jul 18.

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.

A method for the quantitative analysis by ultra-performance liquid chromatography-tandem mass spectrometry of the highly selective irreversible covalent inhibitor of EGFR-TK, osimertinib in human plasma was developed and validated, using pazopanib as an internal standard. The validation was performed in a range from 1 to 1000ng/ml, with the lowest level corresponding to the lower limit of quantitation. Gradient elution was performed on a 1.8μm particle trifunctional bonded C18 column by 1% (v/v) formic acid in water, and acetonitrile as mobile phase. The analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer after positive ionization with the heated electrospray interface. Within-day precisions ranged from 3.4 to 10.3%, and between-day precisions from 3.8 to 10.4%, accuracies were 95.5-102.8%. Plasma (either lithium heparin or sodium EDTA) pretreatment was performed by salting-out assisted liquid-liquid extraction using acetonitrile and magnesium sulfate. This method was used to analyze the osimertinib blood plasma levels of five adult patients with metastatic T790M mutated non-small cellular lung carcinoma for therapeutic drug monitoring purposes.
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http://dx.doi.org/10.1016/j.jchromb.2016.07.037DOI Listing
September 2016

Recent developments in the chromatographic bioanalysis of approved kinase inhibitor drugs in oncology.

J Pharm Biomed Anal 2016 Oct 21;130:244-263. Epub 2016 Jun 21.

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

In recent years (2010-present) there has been an increase in the number of publications reporting the development, validation and use of bioanalytical methods in the rapidly expanding drug class of small molecule protein kinase inhibitors. Most reports describe the technological set-up of the methods that have allowed for drug concentration measurements from various sample types. This includes plasma, dried blood-spot, and tissue-analysis. Also method development, exploration of various techniques, as well as measurement and identification of metabolites were addressed. For the bioanalysis, a variety of sample-pretreatment methods like protein-precipitation, liquid-liquid extraction, and solid-phase extraction have been employed, all varying in complexity, cleanliness and time-consumption. Chromatographic separation, nowadays, is more focused on separating components from ion-suppressive effects, since for MS/MS detection, various components do not have to be baseline separated. For detection multiple types of detectors were used, ranging from state-of-the-art high resolution, and tandem mass spectrometry with low picogram per milliliter detection limits to the classical UV-detector with several nanograms per milliliter limits. As new bioanalytical methods have arisen that do rely on chromatographic separation, for example for high-throughput analysis, these are addressed in this review as well.
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http://dx.doi.org/10.1016/j.jpba.2016.06.037DOI Listing
October 2016

Liquid chromatography-tandem mass spectrometric assay for ponatinib and N-desmethyl ponatinib in mouse plasma.

J Chromatogr B Analyt Technol Biomed Life Sci 2016 Jun 2;1023-1024:24-9. Epub 2016 May 2.

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

Ponatinib is a multi-targeted third generation BCR-ABL1 tyrosine-kinase inhibitor approved for specific types of leukemia. A bioanalytical assay for this drug and its N-desmethyl metabolite in mouse plasma was developed and validated using liquid chromatography-tandem mass spectrometric (LC-MS/MS) with liquid-liquid extraction as sample pre-treatment procedure. After extraction with tert-butyl methyl ether of both analytes with their isotopically labeled internal standards and evaporation and reconstitution of the extract, compounds were separated by reversed-phase liquid chromatography under alkaline conditions. After electrospray ionization, both compounds were quantified in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The linear assay was validated in the ranges 5-5000ng/ml for ponatinib and 1-1000ng/ml for N-desmethyl ponatinib. Within-run (n=18) and between-run (3 runs; n=18) precisions were 10% and 12% at the lower limit of quantification for the metabolite, all other precisions were ≤8% for the metabolite and ≤6% for ponatinib. Accuracies were between 92 and 108% for both compounds in the whole calibration range. The drug was sufficiently stable under most relevant analytical conditions, only ponatinib showed more than 15% hydrolytic degradation after storage for 6h and longer at ambient temperature in mouse plasma. Finally, the assay was successfully applied to determine plasma drug levels and study pharmacokinetics after oral administration of ponatinib to female FVB mice.
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http://dx.doi.org/10.1016/j.jchromb.2016.04.049DOI Listing
June 2016