Stephanie van Hoppe

103 Brain accumulation of ponatinib and its active metabolite is limited by ABCB1 and ABCG2 100 U/ml penicillin, 100 µg/ml streptomycin (complete medium), and used as described previously [29]. Transepithelial transport assays were performed in complete medium, but without antibiotics, in triplicate on 12-well microporous polycarbonate membrane filters (3.0-µm pore size, Transwell 3402, Corning Inc., Lowell, MA) as previously described [29]. In short, cells were allowed to grow 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 in 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 5 µM ponatinib alone or in combination with the appropriate inhibitors. At 4 and 8 hours, 50 µl samples were collected from the acceptor compartment and stored at -30˚C until analysis. TEER was re-checked at the end of the transport experiment to confirm continuing intactness of the monolayer. The amount of transported drugwas 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 transport at a defined time point. Continual testing of transport of a range of other drugs (such as ceritinib and afatinib) and other compounds confirmed proper activity of hABCB1, mAbcg2 and hABCG2 in the MDCKII cell lines used. An ima l s Female wild-type, Abcb1a/1b -/- [30], Abcg2 -/- [31], Abcb1a/1b;Abcg2 -/- [32] and Cyp3a -/- mice [33], all of a >99% FVB genetic background, were used. Mice between 9 and 14 weeks of age were used in groups of 3-5 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 Ponatinib stock solution was prepared in DMSO at 20 mg/ml and stock aliquots were frozen at -80˚C. All working solutions were prepared freshly on the day of the experiment by allowing an aliquot to thaw and subsequent dilution of the aliquot 20-fold in 25 mM sodium citrate buffer in water (pH 2.75) resulting in a 1 mg/ml solution. This solution was immediately protected from light and vigorously shaken for 1 minute to obtain a