Stephanie van Hoppe

112 Chapter 5 strains compared to WT mice, especially for Abcb1a/1b -/- mice with a significant 1.9- fold increase. However, combined transporter deficiency resulted in an 18-fold increase in brain accumulation (Figure 4C). Thus, also at 2 h, both Abcg2 and Abcb1a/1b alone could still quite effectively restrict the brain accumulation of ponatinib, so that only the combination transporter knockout demonstrated a highly increased brain penetration of ponatinib. Similar to the 24 h experiment, we observed no substantial impact of Abcb1a/1b and/or Abcg2 deficiency on the liver distribution of ponatinib (Figure 4D-F). Accordingly, plotting of the brain-to-liver ratios of ponatinib at both 2 and 24 h resulted in qualitatively similar results, indicating a strong effect of the combination knockout on relative brain accumulation of ponatinib (Supplementary Figure 2). The e f f e c t o f Cy p3a and Ab c g2 and Ab c b1a / 1b on N - de sme t hy l pona t i n i b d i s po s i t i on and b r a i n a c c umu l a t i on The ponatinib metabolite DMP is pharmacodynamically active, and can thus contribute to ponatinib treatment response. We therefore tested to what extent DMP pharmacokinetics was affected by Cyp3a and/or ABC transporter deficiency in mice. Conversion of ponatinib to DMP in humans is thought to be mediated primarily by CYP3A [1]. Indeed, the plasma AUC of ponatinib was modestly (1.4-fold) increased in Cyp3a -/- mice (Figure 2A, Table 1), but the plasma concentrations or AUC 0-24 of DMP were not significantly decreased in Cyp3a -/- mice (Figure 5A, Table 1). However, the plasma DMP/ponatinib ratio was decreased, albeit not significantly, for most time points up to 8 h in Cyp3a -/- mice (Figure 5B, inset), which is in line with somewhat reduced (Cyp3a- mediated) formation of DMP in these mice. The brain concentration and accumulation of DMP was not significantly affected by Cyp3a deficiency, and the same applies for DMP liver concentration and accumulation (Table 1, Supplementary Figure 2). Interestingly, instrikingcontrast totheplasma levelsof parental ponatinib, theplasma levels and AUC 0-24 of DMP were increased by about 3- to 4-fold in both Abcg2-deficient strains (Figure 5A and Table 1). This suggests that Abcg2 is involved in the clearance of DMP from plasma. On the other hand, Abcb1a/1b deficiency had no marked effect on DMP plasma levels (Figure 5A, Table 1). We further investigated the impact of single and combined Abcg2 and Abcb1a/1b deficiency on DMP brain levels 24 hours after oral intake of ponatinib. While the brain concentrations and accumulations in the single transporter knockout mice were not markedly different from those in WT mice, in the combined Abcb1a/1b;Abcg2 -/- mice they were highly increased (Table 1, Supplementary Figure 3A-C). Incidentally, the comparatively high DMP liver concentrations, liver-to- plasma ratios, and liver accumulations we observed in both the Abcg2-deficient strains (Supplementary Figure 3D-F) could reflect diminished elimination of DMP from the liver at this late (24 h) phase, perhaps by reduced hepatobiliary excretion of DMP. When we