Stephanie van Hoppe

104 Chapter 5 homogeneous and clear solution. Ponatinib was administered orally at a dose of 10 mg/ kg (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 pona t i n i b To minimize variation in absorption, mice were fasted for two hours prior to oral administration of ponatinib, 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-hour experiment, tail vein sampling took place at 0.5, 1, 2, 4 and 8 h after oral administration. For the 2-hour experiment, tail vein sampling took place at 0.25, 0.5 and 1 h after oral administration. At 24 or 2 h, respectively, mice were sacrificed by cervical dislocation and a set of organs was rapidly removed, weighed and subsequently frozen as whole organ at -30˚C. Prior to analysis, organs were allowed to thaw 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. Blood samples were immediately centrifuged at 2,100 g for 6 min at 4˚C, and plasma was collected and stored at -30°C until analysis. Ponatinib concentrations in brain tissue were corrected for the presence of plasma in the vascular space (1.4%) [34]. D r ug ana l y s i s Ponatinib and N-desmethyl ponatinib (DMP) concentrations in culture medium, plasma and tissue homogenates were analyzed with a previously reported liquid- chromatography tandem mass spectrometric (LC-MS/MS) assay, using deuterated internal standards [28]. Briefly, plasma and tissue homogenate samples were pre-treated using liquid-liquid extraction with tert- butylmethylether, evaporated and reconstituted before separation by reversed-phase liquid chromatography under alkaline conditions. The calibration curves of the linear assay ranged from 5-5,000 ng/ml for ponatinib and 1-1,000 ng/ml for DMP. Culture medium samples were pre-treated using protein precipitation with acetonitrile and diluted before chromatographic injection. Ponatinib was quantified in the range 5-5,000 ng/ml. S t a t i s t i c s and pha rma c o k i ne t i c c a l c u l a t i on s The unpaired two-tailed Student’s t-test was used to determine significance in the transepithelial transport assays. The area under the curve (AUC) of the plasma concentration-time curve was calculated using the trapezoidal rule, without extrapolating to infinity. Individual concentration-time data were used to determine the peak plasma concentration (C max ) and the time to reach C max (T max ). Relative organ accumulation (Porgan) was calculated by dividing organ concentrations (Corgan) at