Stephanie van Hoppe

24 Chapter 2 information mentions that afatinib is a transported substrate and inhibitor of ABCB1 and ABCG2 [5, 16, 17], but the limited documentation provided does not allow an assessment of the extent of these processes. In case these efflux transporters can efficiently transport afatinib, this might potentially lead to a decreased accumulation of this drug in target tumors expressing these transporters. Moreover, brain metastases are likely to occur with a subset of cancers, the frequency being highest in lung cancer relative to other common epithelial malignancies [18]. Given the high ABCB1 and ABCG2 expression in the BBB, these transporters could potentially limit afatinib brain accumulation, which might lead to reduced therapeutic efficiency against NSCLC brain metastases. We therefore aimed in this study to investigate whether and to what extent afatinib is transported by one or both of these transporters, and how this might affect oral plasma pharmacokinetics and brain penetration of the drug in Abcb1a/1b and Abcg2 mouse models. MAT E R I A L S AND ME T HOD S Chemi c a l s Afatinib (>99%) was obtained fromAlsachim(Illkirch, France). Zosuquidarwas purchased from Sequoia Research Products (Pangbourne, UK) and Ko143 was obtained fromTocris Bioscience (Bristol, UK). All chemicals used in the chromatographic afatinib assay were described before [19]. Tr an s po r t a s s ay s Polarized Madin-Darby Canine Kidney (MDCKII) cell lines and subclones transduced with either human (h)ABCB1, (h)ABCG2 [20], or mouse (m)Abcg2 cDNA were cultured and used in the transepithelial transport assays as described previously [21]. Transport assays were performed using 12-well Transwell® 3402 plates, 3.0 µm pore size (Corning Inc., USA) in DMEM with 10% fetal bovine serum (FBS). The parental cells and subclones were seeded at a density of 2.5 × 10 5 cells per well and cultured for 3 days to allow formation of an intact monolayer. Membrane tightness was assessed by measurement of transepithelial electrical resistance (TEER) using reference values that were previously established and well correlated to <1% transepithelial [ 14 C]-inulin leakage per hour [21]. The transepithelial transport experiment was started by pre-incubating the cells with or without the relevant inhibitors during 1 h, thereafter (t = 0) medium in the donor compartments was replaced with complete medium containing 2 µM afatinib alone or in combination with the appropriate inhibitors. In the inhibition experiments, 5 μM zosuquidar (ABCB1 inhibitor) and/or 5 μM Ko143 (ABCG2/Abcg2 inhibitor) were added