Stephanie van Hoppe

47 Brain accumulation of osimertinib and its active metabolite is restricted by ABCB1 & ABCG2 MAT E R I A L S AND ME T HOD S Chemi c a l s OsimertinibandzosuquidarwerepurchasedfromSequoiaResearchProducts(Pangbourne, U.K.), and Ko143was obtained fromTocris Bioscience (Bristol, U.K.). GlutaMAX TM Dulbecco’s Modified Eagle Medium (DMEM) and Dulbecco’s phosphate buffered saline (PBS) were purchased from Gibco® by Life Technologies TM (The Netherlands). Glucose water 5% w/v was acquired from B. Braun Medical Supplies, Inc. (Melsungen, Germany). Bovine serum albumin (BSA) Fraction V was obtained from Roche Diagnostics GmbH (Mannheim, Germany). Isoflurane was purchased from Pharmachemie (Haarlem, The Netherlands), heparin (5000 IUml –1 ) was from Leo Pharma (Breda, The Netherlands). All other chemicals were acquired from Sigma-Aldrich (Steinheim, Germany) unless stated otherwise. All chemicals used in the chromatographic osimertinib assay were described before [37]. Tr an s po r t a s s ay s Polarized Madin-Darby canine kidney (MDCK-II) cell lines transduced with either human (h)ABCB1, hABCG2 or murine (m)Abcg2 cDNA were used and cultured as described previously [38]. Transepithelial transport assays were performed in triplicate on 12-well microporous polycarbonate membrane filters (3.0 µmpore size, Transwell 3402, Corning Inc., Lowell, MA), as described [38]. In brief, cells were allowed to grow to an intact monolayer over 3 days, which was monitored with transepithelial electrical resistance (TEER; Millipore Corporation, USA) measurements. For all cell lines TEERs had to be above 80 Ohm.cm2 before the start of the transport experiment, and should not have decreased when re-measured after 8 h at the end of the experiment. On the third day, cells were washed with phosphate-buffered saline and pre-incubated with fresh DMEM medium including 10% fetal bovine serum and the relevant transport inhibitors for 1 h, where 5 µM zosuquidar (ABCB1 inhibitor) and/or 5 µM Ko143 (ABCG2/Abcg2 inhibitor) were added to both apical and basolateral compartments. To inhibit endogenous canine ABCB1 when testing the MDCK-II-mAbcg2 and MDCK-II-hABCG2 cell lines, we added 5 μM zosuquidar (ABCB1 inhibitor) to the culture medium throughout the experiment. The transport experiment was initiated by replacing the pre-incubation medium from the donor compartment (either basolateral or apical) with freshly prepared medium containing 2 µM osimertinib alone or in combination with the appropriate inhibitors. Plates were kept at 37°C in 5% CO2 and 50 μl aliquots were taken from the acceptor compartment at 1, 2, 4 and 8 h and stored at -30°C until analysis. The total amount of drug transported to the acceptor compartment was calculated after correction for volume loss due to sampling at each time point. Active transport was expressed by the relative transport ratio (r), defined as the amount of apically directed transport divided