Stephanie van Hoppe

26 Chapter 2 D r ug ana l y s i s Afatinib concentrations in culture medium, plasma and tissues were determined with a previously reported liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for afatinib, with a calibration curve ranging from 0.5 to 500 ng/ml for plasma [19] and tissue homogenates and from 1.5 to 1500 ng/ml for culture medium. Tissue concentrations were calculated correcting for the individual tissue weights, resulting in ng afatinib per gram tissue. S t a t i s t i c a l ana l y s i 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 statistical significance for transepithelial transport. In pharmacokinetic experiments, the area under the curve (AUC) of the plasma concentration-time plot was calculated using the trapezoidal rule, without extrapolating to infinity. The maximum drug concentration in plasma (C max ) and the time to reach maximum drug concentration in plasma (T max ) were determined directly from individual concentration−time data. The elimination rate constant was calculated with the Microsoft Excel plug in PKsolver [25] with the following formula: (ln(y1) - ln(y2)) / (t2 - t1). Relative organ accumulation (P organ ) was calculated by dividing organ concentrations (C organ ) at either t = 2 h or t = 24 h by the area under the plasma concentration-time curve from 0–2 h (AUC 0-2h ) or 0-24 h (AUC 0-24h ), respectively. One- way analysis of variance (ANOVA) was used to determine significance of differences between groups, after which post-hoc tests with Tukey correction were performed for comparison between individual groups. When variances were not homogeneous, data were log-transformed before statistical tests were applied. Differences were considered statistically significant when P < 0.05. Data are presented as means ± SD. R E S U LT S A f a t i n i b i s t r an s po r t ed by hABCB1 , hABCG2 and mAb c g2 Wefirst studiedthe transport of afatinib invitro bymeasuringafatinib (2µM) translocation through polarized monolayers of MDCKII cell lines transduced with human (h)ABCB1, hABCG2 or mouse (m)Abcg2 cDNA. As shown in Fig. 1A, we observed low apically directed afatinib transport in the parental cell line (transport ratio r = 1.3). This was somewhat decreased (to an r of 0.8) when adding the ABCB1 inhibitor zosuquidar (Fig. 1B), suggesting that this background transport could be mediated by the low amount of endogenous canine ABCB1 present in the MDCKII cells [26]. In MDCKII cells transduced with hABCB1, we observed extensive apically directed transport with r = 7.6, which was completely blocked by zosuquidar ( r = 0.9, Fig. 1C, D). To suppress any endogenous