Selvi Durmus

Oral availability and brain penetration of the B-RAFV600E inhibitor vemurafenib can be enhanced by the P-GLYCOprotein (ABCB1) and breast cancer resistance protein (ABCG2) inhibitor elacridar.

Vemurafenib (PLX4032) is a novel tyrosine kinase inhibitor that has clinical efficacy against metastatic melanoma harboring a BRAF(V600E) mutation. We aimed to establish whether oral availability and brain penetration of vemurafenib could be restricted by the multidrug efflux transporters P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2), as these might limit therapeutic efficacy, especially against brain metastases. In vitro, vemurafenib was efficiently transported by both human ABCB1 and ABCG2, and very efficiently by mouse Abcg2, but not by mouse Abcc2. Upon oral administration of vemurafenib (5 mg/kg), Abcb1a/1b(-/-) mice had a 1.6-fold increased, Abcg2(-/-) mice a 2.3-fold increased, and Abcb1a/1b(-/-);Abcg2(-/-) mice a 6.6-fold increased plasma AUC, respectively, compared to wild-type (WT) mice, indicating a marked and additive role of these transporters in limiting vemurafenib oral availability. Brain-to-plasma ratios of vemurafenib (oral, 25 mg/kg) were not increased in Abcg2(-/-) mice, only 1.7-fold in Abcb1a/1b(-/-) mice, but 21.4-fold in Abcb1a/1b(-/-);Abcg2(-/-) mice, indicating pronounced overlapping functions of these transporters in reducing vemurafenib brain accumulation. Oral coadministration of the dual ABCB1 and ABCG2 inhibitor elacridar almost completely eliminated the roles of Abcb1 and Abcg2 in restricting oral availability and brain accumulation of vemurafenib. As predicted by previously described pharmacokinetic modeling, halving the amount of active efflux transport at the WT blood-brain barrier by testing heterozygous Abcb1a/1b(+/-);Abcg2(+/-) mice had little impact on vemurafenib brain accumulation. Our data suggest that elacridar coadministration may be considered to improve the therapeutic efficacy of vemurafenib, especially for brain metastases located behind a functional blood-brain barrier.

Apical ABC Transporters and Cancer Chemotherapeutic Drug Disposition

ATP-binding cassette (ABC) transporters are transmembrane efflux transporters that mediate cellular extrusion of a broad range of substrates ranging from amino acids, lipids, and ions to xenobiotics including many anticancer drugs. ABCB1 (P-GP) and ABCG2 (BCRP) are the most extensively studied apical ABC drug efflux transporters. They are highly expressed in apical membranes of many pharmacokinetically relevant tissues such as epithelial cells of the small intestine and endothelial cells of the blood capillaries in brain and testis, and in the placental maternal-fetal barrier. In these tissues, they have a protective function as they efflux their substrates back to the intestinal lumen or blood and thus restrict the intestinal uptake and tissue disposition of many compounds. This presents a major challenge for the use of many (anticancer) drugs, as most currently used anticancer drugs are substrates of these transporters. Herein, we review the latest findings on the role of apical ABC transporters in the disposition of anticancer drugs. We discuss that many new, rationally designed anticancer drugs are substrates of these transporters and that their oral availability and/or brain disposition are affected by this interaction. We also summarize studies that investigate the improvement of oral availability and brain disposition of many cytotoxic (e.g., taxanes) and rationally designed (e.g., tyrosine kinase inhibitor) anticancer drugs, using chemical inhibitors of these transporters. These findings provide a better understanding of the importance of apical ABC transporters in chemotherapy and may therefore advance translation of promising preclinical insights and approaches to clinical studies.

P-glycoprotein (MDR1/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) restrict brain accumulation of the JAK1/2 inhibitor, CYT387.

CYT387 is an orally bioavailable, small molecule inhibitor of Janus family of tyrosine kinases (JAK) 1 and 2. It is currently undergoing Phase I/II clinical trials for the treatment of myelofibrosis and myeloproliferative neoplasms. We aimed to establish whether the multidrug efflux transporters P-glycoprotein (P-gp; MDR1; ABCB1) and breast cancer resistance protein (BCRP;ABCG2) restrict oral availability and brain penetration of CYT387. In vitro, CYT387 was efficiently transported by both human MDR1 and BCRP, and very efficiently by mouse Bcrp1 and its transport could be inhibited by specific MDR1 inhibitor, zosuquidar and/or specific BCRP inhibitor, Ko143. CYT387 (10 mg/kg) was orally administered to wild-type (WT), Bcrp1(-/-), Mdr1a/1b(-/-) and Bcrp1;Mdr1a/1b(-/-) mice and plasma and brain concentrations were analyzed. Over 8h, systemic exposure of CYT387 was similar between all the strains, indicating that these transporters do not substantially limit oral availability of CYT387. Despite the similar systemic exposure, brain accumulation of CYT387 was increased 10.5- and 56-fold in the Bcrp1;Mdr1a/1b(-/-) mice compared to the WT strain at 2 and 8h after CYT387 administration, respectively. In single Bcrp1(-/-) mice, brain accumulation of CYT387 was more substantially increased than in Mdr1a/1b(-/-) mice, suggesting that CYT387 is a slightly better substrate of Bcrp1 than of Mdr1a at the blood-brain barrier. These results indicate a marked and additive role of Bcrp1 and Mdr1a/1b in restricting brain penetration of CYT387, potentially limiting efficacy of this compound against brain (micro) metastases positioned behind a functional blood-brain barrier.

In vivo disposition of doxorubicin is affected by mouse Oatp1a/1b and human OATP1A/1B transporters

Organic anion‐transporting polypeptides (OATPs) are important drug uptake transporters, mediating distribution of substrates to several pharmacokinetically relevant organs. Doxorubicin is a widely used anti‐cancer drug extensively studied for its interactions with various drug transporters, but not OATPs. Here, we investigated the role of OATP1A/1B proteins in the distribution of doxorubicin. In vitro, we observed ∼2‐fold increased doxorubicin uptake in HEK293 cells overexpressing human OATP1A2, but not OATP1B1 or OATP1B3. In mice, absence of Oatp1a/1b transporters led to up to 2‐fold higher doxorubicin plasma concentrations and 1.3‐fold higher plasma AUC. Conversely, liver AUC and liver‐to‐plasma ratios of Oatp1a/1b−/− mice were 1.4‐fold and up to 4.1‐fold lower than in wild‐type mice, respectively. Decreased doxorubicin levels in the small intestinal content reflected those in the liver, indicating a reduced biliary excretion of doxorubicin in Oatp1a/1b−/− mice. These results demonstrate important control of doxorubicin plasma clearance and hepatic uptake by mouse Oatp1a/1b transporters. This is unexpected, as the fairly hydrophobic weak base doxorubicin is an atypical OATP1A/1B substrate. Interestingly, transgenic liver‐specific expression of human OATP1A2, OATP1B1 or OATP1B3 could partially rescue the increased doxorubicin plasma levels of Oatp1a/1b−/− mice. Hepatic uptake and bile‐derived intestinal excretion of doxorubicin were completely reverted to wild‐type levels by OATP1A2, and partially by OATP1B1 and OATP1B3. Thus, doxorubicin is transported by hepatocyte‐expressed OATP1A2, −1B1 and −1B3 in vivo, illustrating an unexpectedly wide substrate specificity. These findings have possible implications for the uptake, disposition, therapy response and toxicity of doxorubicin, also in human tumors and tissues expressing these transporters.

Liquid chromatography–tandem mass spectrometric assay for the mutated BRAF inhibitor vemurafenib in human and mouse plasma

A bioanalytical assay for the mutated BRAF inhibitor vemurafenib was developed and validated. For the quantitative assay, human plasma samples were pre-treated using protein precipitation with water-acetonitrile (1/3, v/v) containing sorafenib as internal standard. The extract was directly injected into the chromatographic system. This system consisted of a sub-2 μm particle, trifunctional bonded octadecyl silica column with isocratic elution using 0.01% (v/v) of formic acid in a mixture of water and methanol. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 0.1-100 μg/ml calibration range. Within day precisions were 1.6-3.2%, between day precisions 2.7% and 8.2% and accuracies were between 99% and 106% for the whole calibration range. The drug was stable under all relevant conditions. Finally, the assay was successfully used to assess drug levels in a pharmacokinetic mouse study.

P-glycoprotein, CYP3A and plasma carboxylesterase determine brain and blood disposition of the mTOR inhibitor everolimus (Afinitor) in mice

Purpose: To clarify the role of ABCB1, ABCG2, and CYP3A in blood and brain exposure of everolimus using knockout mouse models. Experimental Design: We used wild-type, Abcb1a/1b−/−, Abcg2−/−, Abcb1a/1b;Abcg2−/−, and Cyp3a−/− mice to study everolimus oral bioavailability and brain accumulation. Results: Following everolimus administration, brain concentrations and brain-to-liver ratios were substantially increased in Abcb1a/1b−/−and Abcb1a/1b;Abcg2−/−, but not Abcg2−/−mice. The fraction of everolimus located in the plasma compartment was highly increased in all knockout strains. In vitro, everolimus was rapidly degraded in wild-type but not knockout plasma. Carboxylesterase 1c (Ces1c), a plasma carboxylesterase gene, was highly upregulated (∼80-fold) in the liver of knockout mice relative to wild-type mice, and plasma Ces1c likely protected everolimus from degradation by binding and stabilizing it. This binding was prevented by preincubation with the carboxylesterase inhibitor BNPP. In vivo knockdown experiments confirmed the involvement of Ces1c in everolimus stabilization. Everolimus also markedly inhibited the hydrolysis of irinotecan and p-nitrophenyl acetate by mouse plasma carboxylesterase and recombinant human CES2, respectively. After correcting for carboxylesterase binding, Cyp3a−/−, but not Abcb1a/1b−/−, Abcg2−/−, or Abcb1a/1b;Abcg2−/−mice, displayed highly (>5-fold) increased oral availability of everolimus. Conclusions: Brain accumulation of everolimus was restricted by Abcb1, but not Abcg2, suggesting the use of coadministered ABCB1 inhibitors to improve brain tumor treatment. Cyp3a, but not Abcb1a/1b, restricted everolimus oral availability, underscoring drug–drug interaction risks via CYP3A. Upregulated Ces1c likely mediated the tight binding and stabilization of everolimus, causing higher plasma retention in knockout strains. This Ces upregulation might confound other pharmacologic studies. Clin Cancer Res; 20(12); 3133–45. ©2014 AACR.

Liquid chromatography–tandem mass spectrometric assay for the multikinase inhibitor regorafenib in plasma

Regorafenib has recently been approved for the treatment of colorectal cancer. A bioanalytical liquid chromatography-tandem mass spectrometric assay for this multikinase inhibitor was developed and validated in plasma. The concentration range of the assay was 25-25,000 ng/mL. Protein precipitation with acetonitrile was used as sample pre-treatment with sorafenib as internal standard. The extract was diluted with methanol (25%, v/v) and then injected onto the sub-2 µm particle, bridged ethylsilicia hybrid trifunctional bonded C18 column. Isocratic elution using 0.02% (v/v) formic acid in a methanol-water mixture was used. Compounds were monitored by a triple quadrupole mass spectrometer in the selected reaction monitoring mode after positive electrospray ionization. Double logarithmic calibration was used; within-day precisions, between-day precisions, and accuracies were 3.2-9.2, 4.1-12.3 and 94.8-103.0%, respectively. High drug stability was observed under all relevant storage conditions. The assay was used to measure drug concentrations in a pharmacokinetic study in wild-type FVB mice.

Liquid chromatography-tandem mass spectrometric assay for the JAK2 inhibitor CYT387 in plasma.

A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for the JAK2 inhibitor CYT387 was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing cediranib as internal standard. The extract was directly injected into the chromatographic system after dilution with water. This system consisted of a sub-2 μm particle, trifunctional bonded octadecyl silica column with a gradient using 0.005% (v/v) of formic acid in a mixture of water and methanol. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 0.25-1000 ng/ml calibration range. Within day precisions were 3.0-13.5%, between day precisions 5.7% and 14.5%. Accuracies were between 96% and 113% for the whole calibration range. The drug was stable under all relevant analytical conditions. Finally, the assay was successfully used to assess drug levels in mice.

P-glycoprotein, CYP3A, and Plasma Carboxylesterase Determine Brain Disposition and Oral Availability of the Novel Taxane Cabazitaxel (Jevtana) in Mice.

We aimed to clarify the roles of the multidrug-detoxifying proteins ABCB1, ABCG2, ABCC2, and CYP3A in oral availability and brain accumulation of cabazitaxel, a taxane developed for improved therapy of docetaxel-resistant prostate cancer. Cabazitaxel pharmacokinetics were studied in Abcb1a/1b, Abcg2, Abcc2, Cyp3a, and combination knockout mice. We found that human ABCB1, but not ABCG2, transported cabazitaxel in vitro. Upon oral cabazitaxel administration, total plasma levels were greatly increased due to binding to plasma carboxylesterase Ces1c, which is highly upregulated in several knockout strains. Ces1c inhibition and in vivo hepatic Ces1c knockdown reversed these effects. Correcting for Ces1c effects, Abcb1a/1b, Abcg2, and Abcc2 did not restrict cabazitaxel oral availability, whereas Abcb1a/1b, but not Abcg2, dramatically reduced cabazitaxel brain accumulation (>10-fold). Coadministration of the ABCB1 inhibitor elacridar completely reversed this brain accumulation effect. After correction for Ces1c effects, Cyp3a knockout mice demonstrated a strong (six-fold) increase in cabazitaxel oral availability, which was completely reversed by transgenic human CYP3A4 in intestine and liver. Cabazitaxel markedly inhibited mouse Ces1c, but human CES1 and CES2 only weakly. Ces1c upregulation can thus complicate preclinical cabazitaxel studies. In summary, ABCB1 limits cabazitaxel brain accumulation and therefore potentially therapeutic efficacy against (micro)metastases or primary tumors positioned wholly or partly behind a functional blood-brain barrier. This can be reversed with elacridar coadministration, and similar effects may apply to ABCB1-expressing tumors. CYP3A4 profoundly reduces the oral availability of cabazitaxel. This may potentially be greatly improved by coadministering ritonavir or other CYP3A inhibitors, suggesting the option of patient-friendly oral cabazitaxel therapy.

Liquid chromatography-tandem mass spectrometric assay for the mutated BRAF inhibitor dabrafenib in mouse plasma.

A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for the mutated BRAF inhibitor dabrafenib was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing PLX4720 as internal standard. The extract was directly injected into the reversed-phase chromatographic system after dilution with water. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 2-2000 ng/ml calibration range with r(2)=0.993±0.002 for linear regression with quadratic weighting (n=5). Within day precisions (n=6) were 3.3-5.2%, between day (3 days; n=18) precisions 4.7-8.2%. Accuracies were between 95-104% for the whole calibration range. The drug was sufficiently stable under all relevant analytical conditions. Finally, the assay was successfully used to determine drug pharmacokinetics in mice.