Stephanie van Hoppe

74 Chapter 4 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 i b r u t i n i b To minimize variation in absorption, mice were fasted for 3 h prior to oral administration of ibrutinib, using a blunt-ended needle. 50-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 8-h experiment, tail vein sampling took place at 0.25, 0.5, 1, 2, and 4 h after oral administration; for the 1-h experiment tail vein sampling took place at 5, 10, 15, and 30 min after oral administration, and finally for the 20-min experiment, tail vein sampling took place at 5, 10, and 15 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. Ibrutinib concentrations in brain tissue were corrected for the amount of plasma present in the vascular space (~1.4%) [27]. D r ug ana l y s i s Ibrutinib and ibrutinib-DiOH concentrations in culture medium, plasma and tissue homogenates were analyzed with a previously reported liquid-chromatography tandemmass spectrometric (LC-MS/MS) assay, using deuterated internal standards [21]. 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 the significance of differences 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 ). Ordinary one-way analysis of variance (ANOVA) was used to determine significant differences between groups. Post-hoc Tukey’s multiple comparisons were used to compare significant differences between individual groups. When variances were not homogeneously distributed, data were log-transformed before applying statistical tests. Differences were considered statistically significant when P < 0.05. Data are presented as mean ± SD with each experimental group containing 5-6 mice.