Stephanie van Hoppe

73 ABCB1 restricts brain penetration of the BTK inhibitor ibrutinib while CYP3A limits its oral bioavailability Germany). All chemicals used in the chromatographic ibrutinib assay were described before [21]. 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, murine (m)Abcg2 or hABCG2 cDNA were cultured and used as described previously [22]. Transepithelial transport assays were performed in triplicate on 12-well microporous polycarbonate membrane filters (3.0-µm pore size, Transwell 3402, Corning Inc., Lowell, MA) as described [22]. In short, cells were allowed to grow to an intact monolayer in 3 days, which was monitored with transepithelial electrical resistance (TEER) measurements. On the third day, cells were pre-incubated with the relevant inhibitors for 1 hour, 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 Abcg2 and MDCK-II ABCG2 cell lines, we added 5 µM zosuquidar (ABCB1 inhibitor) to the culture medium throughout the experiment. The experiment was initiated by replacing the incubation medium from the donor compartment with freshly prepared medium containing 2 µM ibrutinib alone or in combination with the appropriate inhibitors. At 1, 2, 4 and 8 h, 50-µl samples were collected from the acceptor compartment and stored at -30˚C until analysis. The amount of transported drug was calculated after correction for volume loss due to sampling at each time point. Active transport was expressed by the transport ratio ( r ), which is defined as the amount of apically directed transport divided by the amount of basolaterally directed translocation at a defined time point. An ima l s Female wild-type (WT), Abcb1a/1b -/- [23], Abcg2 -/- [24], Abcb1a/1b;Abcg2 -/- [25], and Cyp3a -/- , hCyp3aXA, hCyp3aXV and hCyp3aXAV mice [26], all of a >99% FVB strain background, were used. Mice between 9 and 14 weeks of age were used in groups of 5-6 mice per strain. The mice were kept in a temperature-controlled environment with a 12 h light/dark cycle and received a standard diet (AM-II, Hope Farms, Woerden, The Netherlands) and acidified water ad libitum . Animals were housed and handled according to institutional guidelines in compliance with Dutch and EU legislation. D r ug s o l u t i on s Ibrutinib was dissolved at a concentration of 24.4 mg/ml in DMSO, polysorbate 80 and ethanol (final solvent ratios 1:20:20 (v/v/v)). It was then further diluted with 5% (w/v) glucose in water, to obtain a 1 mg/ml ibrutinib solution in water containing 0.1% DMSO, 2% (v/v) polysorbate 80, 2% (v/v) ethanol, and 4.795% (w/v) glucose. Ibrutinib was