Stephanie van Hoppe

25 ABCG2 & ABCB1 transport afatinib and restrict its oral availability and brain accumulation to both apical and basolateral compartments. Plates were kept at 37°C in 5% CO 2 during the experiment, and 50-μl aliquots were taken from the acceptor compartment at 2, 4 and 8 h for measurement of afatinib concentrations. Samples were stored at -30ºC for later LC-MS/MS measurement. Total amount of drug transported to the acceptor compartment was calculated after correction for volume loss for each sampling 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. An ima l s Female wild-type, Abcb1a/1b -/- [22], Abcg2 -/- [23] and Abcg2;Abcb1a/1b -/- mice [24], all of a >99% FVB genetic background, were used. Mice between 9 and 14 weeks of age were used in groups of five mice per strain. The mice were kept in a temperature- controlled environment with a 12 h light/12 h dark cycle and received a standard diet (Transbreed, SDS Diets, Technilab - BMI) 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 and pha rma c o k i ne t i c ex pe r imen t s Afatinib was dissolved at a concentration of 20 mg/ml in 50% (v/v) polysorbate 80, 50% (v/v) ethanol. It was then further diluted with 5% (w/v) glucose in water, to obtain a 1 mg/ml afatinib solution in water containing 2.5% (v/v) polysorbate 80, 2.5% (v/v) ethanol, and 4.75% (w/v) glucose. Afatinib was administered orally at a dose of 10 mg/ kg (10 μl/g). All working solutions were prepared freshly on the day of the experiment. To minimize variation in absorption, mice were fasted for 3 h prior to oral administration of afatinib using a blunt-ended needle. For the pharmacokinetic experiment, 50-μl blood samples were drawn from the tail vein using heparin-coated capillaries (Sarstedt, Germany) at 0.5, 1, 2, 4 and 8 h or 0.25, 0.5 and 1 h. At 24 h or 2 h, mice were anesthetized using isoflurane and blood was collected via cardiac puncture. Immediately thereafter, mice where sacrificed by cervical dislocation and brain, spleen, liver and kidney were rapidly removed, weighed and subsequently frozen as whole organ at −30°C. Prior to analysis, organs were allowed to thaw and then homogenized in appropriate volumes (1 to 3 ml) of 4% (w/v) bovine serum albumin (BSA) in water using a FastPrep®-24 homogenizer (MP-Biomedicals, NY, USA). Homogenates were stored at −30°C. Blood samples were centrifuged immediately after collection at 9000 × g for 6 min at 4°C, and plasma was collected and stored at −30°C until analysis.