Stephanie van Hoppe

48 Chapter 3 by the amount of basolaterally directed transport at the 8 h time point. An ima l s Male wild-type (WT) FVB, Abcb1a/1b -/- [39], Abcg2 -/- [40], Abcb1a/1b;Abcg2 -/- [41], Cyp3a -/- and Cyp3aXAV [42] mice of identical genetic background (>99% FVB) were used. Groups of 5-6 mice per strain, aged between 9 to 14 weeks, were used. Animals were kept in a temperature-controlled environment with a 12 h light/dark cycle and received a standard diet (Transbreed, SDS Diets, Technilab-BMI, Someren, The Netherlands) and acidified water ad libitum . Mice 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 Osimertinib was first dissolved in dimethyl sulfoxide (DMSO) at a concentration of 25 mg/mL and further diluted with a mixture of polysorbate 80: ethanol (1:1 v/v), and 5 % w/v glucose water to yield a 1 mg/mL working solution. Final concentrations (v/v) of DMSO, polysorbate 80, ethanol, and glucose water were therefore 4%, 2.5%, 2.5% and 91%, respectively. Osimertinib was administered orally at a dose of 10 mg/kg body weight (10 µL/g). P l a sma and t i s s ue pha rma c o k i ne t i c s o f o s ime r t i n i b To minimize variation in absorption, mice were fasted for about 3 h prior to the oral administration of osimertinib using a blunt-ended needle. Fifty µL blood samples were drawn from the tail vein using heparin-coated capillaries (Sarstedt, Germany). At the last time point, mice were anesthetized using isoflurane inhalation, and blood was collected via cardiac puncture. For the 24 h experiment, tail vein sampling took place at 0.5, 1, 2, 4 and 8 h after oral administration; for the 1.5 h experiment, tail vein sampling took place at 5, 10, 15, 30 and 60 min after oral administration. At the end point, mice were sacrificed by cervical dislocation and a set of organs was rapidly removed, weighed, and subsequently frozen as whole organs at -30 °C. Organs were allowed to thaw on ice and homogenized in appropriate volumes of 4% (w/v) BSA in water using a FastPrep-24 device (MP Biomedicals, SA, California, USA). Homogenates were stored at -30 °C until analysis. Blood samples were immediately centrifuged at 9000 × g for 6 min at 4 °C, and plasma was collected and stored at -30 °C until analysis. D r ug ana l y s i s Osimertinib concentrations in culture medium, plasma, and tissue homogenates were analyzedwith a previously reported liquid-chromatography tandemmass spectrometric (LC-MS/MS) [37].