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).

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.

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

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.

Brain accumulation of the EML4-ALK inhibitor ceritinib is restricted by P-glycoprotein (P-GP/ABCB1) and breast cancer resistance protein (BCRP/ABCG2).

We aimed to clarify the roles of the multidrug transporters ABCB1 and ABCG2 in oral availability and brain accumulation of ceritinib, an oral anaplastic lymphoma kinase (ALK) inhibitor used to treat metastatic non-small cell lung cancer (NSCLC) after progression on crizotinib. Importantly, NSCLC is prone to form brain metastases. Transport of ceritinib by human (h) ABCB1 or hABCG2 or mouse (m) Abcg2 was assessed in vitro. To study the single and combined roles of Abcb1a/1b and Abcg2 in ceritinib disposition in vivo, we used appropriate knockout mouse strains. Ceritinib was very efficiently transported by hABCB1, and efficiently by hABCG2 and mAbcg2 in vitro, and transport was specifically inhibited by the ABCB1 inhibitor zosuquidar and ABCG2 inhibitor Ko143, respectively. Absorption and 24-h oral availability were not significantly affected by the absence of Abcb1 and/or Abcg2, but the brain concentrations were greatly increased (>38-fold) in Abcb1a/1b(-/-) mice at 3 and 24h after oral administration of 20mg/kg ceritinib. The brain concentrations increased another ∼ 3-fold (to >90-fold) in Abcb1a/1b;Abcg2(-/-) mice, indicating that there was a significant additional effect of Abcg2-mediated transport of ceritinib as well in vivo. Overall, brain accumulation, but not the 24-h oral availability of ceritinib were profoundly restricted by Abcb1a/1b and Abcg2, with Abcb1a/1b being the dominant efflux protein. Our data suggest that coadministration of ceritinib with a dual ABCB1 and ABCG2 inhibitor may improve treatment of brain (micro) metastases positioned behind a functionally intact blood-brain barrier, and possibly also of tumors resistant to ceritinib due to ABCB1 or ABCG2 overexpression.

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.

Brain and Testis Accumulation of Regorafenib is Restricted by Breast Cancer Resistance Protein (BCRP/ABCG2) and P-glycoprotein (P-GP/ABCB1).

PURPOSE: Regorafenib is a novel multikinase inhibitor, currently approved for the treatment of metastasized colorectal cancer and advanced gastrointestinal stromal tumors. We investigated whether regorafenib is a substrate for the multidrug efflux transporters ABCG2 and ABCB1 and whether oral availability, brain and testis accumulation of regorafenib and its active metabolites are influenced by these transporters. METHODS: We used in vitro transport assays to assess human (h)ABCB1- or hABCG2- or murine (m)Abcg2-mediated active transport at high and low concentrations of regorafenib. To study the single and combined roles of Abcg2 and Abcb1a/1b in oral regorafenib disposition and the impact of Cyp3a-mediated metabolism, we used appropriate knockout mouse strains. RESULTS: Regorafenib was transported well by mAbcg2 and hABCG2 and modestly by hABCB1 in vitro. Abcg2 and to a lesser extent Abcb1a/1b limited brain and testis accumulation of regorafenib and metabolite M2 (brain only) in mice. Regorafenib oral availability was not increased in Abcg2(-/-);Abcb1a/1b(-/-) mice. Up till 2 h, metabolite M5 was undetectable in plasma and organs. CONCLUSIONS: Brain and testis accumulation of regorafenib and brain accumulation of metabolite M2 are restricted by Abcg2 and Abcb1a/1b. Inhibition of these transporters may be of clinical relevance for patients with brain (micro)metastases positioned behind an intact blood-brain barrier.