Stephanie van Hoppe

49 Brain accumulation of osimertinib and its active metabolite is restricted by ABCB1 & ABCG2 S t a t i s t i c a l and pha rma c o k i ne t i c c a l c u l a t i on s The area under the curve (AUC) of the plasma concentration-time data was calculated using the trapezoidal rule, without extrapolating to infinity using GraphPad Prism software 7.0e (GraphPad Software Inc., La Jolla, CA, USA). The maximum drug concentration in plasma (Cmax) and the time to reach maximum drug concentration in plasma (Tmax) were determined directly from individual concentration-time data. Tissue accumulation of osimertinib was calculated by determining the osimertinib tissue concentration relative to its plasma AUC from 0 - 24 h or 0 - 1.5 h. Average tissue to plasma ratios were calculated from individual mouse data. Statistical differences between individual groups were assessed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc multiple comparisons using GraphPad Prism. When variances were not homogeneously distributed, data were log-transformed before statistical tests were applied. A P value of < 0.05 was considered statistically significant. Data are presented as mean ± SD, with each experimental group containing 5-6 mice. R E S U LT S O s ime r t i n i b i s mode s t l y t r an s po r t ed by hABCB1 and mAb c g2 i n v i t r o We first studied the transport of osimertinib (2 µM) in vitro by measuring translocation through polarized monolayers of MDCK-II cell lines transduced with human (h)ABCB1, hABCG2 or mouse (m)Abcg2 cDNA. As shown in Figure 1A, in the parental line, both apically and basolaterally directed translocation of osimertinib were identical (efflux transport ratio r = 1.0). This r was somewhat, but not significantly, decreased when adding the ABCB1 inhibitor zosuquidar (Figure 1B). In hABCB1-overexpressing MDCK-II cells, osimertinib was modestly transported in the apical direction ( r = 1.8, Figure 1C), and this transport was completely blockedby zosuquidar ( r = 1.0, Figure 1D).To suppress any possible endogenous canine ABCB1 transport activity, zosuquidar was added in subsequent experiments with MDCK-II cells overexpressing hABCG2 and mAbcg2. No significant active transport of osimertinib by hABCG2 was observed, and accordingly, addition of the hABCG2/mAbcg2 inhibitor Ko143 had little effect on overall translocation (Figure 1E and 1F). In contrast, in mAbcg2-overexpressing cells, clear apically directed transport of osimertinib was observed ( r = 2.3), and this transport was completely abrogated by the addition of Ko143 (Figure 1G and 1H). Osimertinib thus appears to be modestly transported by hABCB1 and more efficiently by mAbcg2, but not detectably by hABCG2 in vitro . It is worth noting, however, that the effective dimeric transporter level per cell is about 5.5-fold lower inMDCKII-hABCG2 cells than inMDCKII-hABCB1 cells [43]. This comparatively lowexpression level might render a low level of osimertinib transport by hABCG2 undetectable.