Stephanie van Hoppe

84 Chapter 4 on reducing oral ibrutinib plasma levels by metabolizing it to ibrutinib-DiOH. Detailed analysis of changes in the tissue levels of ibrutinib and ibrutinib-DiOH was hampered by the high interindividual variation at this early time point, but the ibrutinib-DiOH to ibrutinib ratios in all tested tissues (liver, kidney, spleen) generally reflected those seen in plasma (Supplemental Figure 9A-E), indicating a relatively rapid equilibration of both compounds between these tissues and plasma. D I S C U S S I ON Our results show that ibrutinib is modestly transported in vitro by human ABCB1 and mouse Abcg2, but not detectably by human ABCG2. In vivo in mouse models, mAbcb1 andmAbcg2 do not appear to restrict the oral bioavailability of ibrutinib, although under some circumstances mAbcb1 deficiency may indirectly reduce ibrutinib availability (see below). However, the brain distribution of ibrutinib is markedly restricted by mAbcb1 in the BBB, but not substantially by mAbcg2, even in the absence of mAbcb1 activity. The brain distribution of ibrutinib-DiOH, the primary active metabolite of ibrutinib, is also strongly limited by mAbcb1 activity. In contrast, the distribution of ibrutinib and ibrutinib-DiOH to other major tissues such as liver, kidney, and spleen is not markedly affected by mAbcb1 and/or mAbcg2 activity, with the possible exception of a small effect of Abcb1a/1b deficiency in ibrutinib spleen distribution. No ibrutinib toxicity was observed in the Abcb1a/1b;Abcg2-deficient mice. We further found that mouse Cyp3a deficiency caused a profound increase in ibrutinib plasma levels, apparently due to reduced conversion of ibrutinib to ibrutinib-DiOH. Overexpression of human CYP3A4 in either liver or intestine of Cyp3a -/- mice markedly reduced ibrutinib oral bioavailability, and to roughly similar extents. The concomitant ibrutinib-DiOH levels observed in these strains qualitatively reflected the predicted changes in conversion of ibrutinib to ibrutinib-DiOH by Cyp3a and CYP3A4. Our invitro transportdatacontrastwiththeFDAandEMAdocumentationon ibrutinib, stating that in vitro studies suggest that ABCB1 and ABCG2 do not transport ibrutinib [6, 29]. Instead, we found clear transport of ibrutinib by hABCB1 and mAbcg2, with efflux ratios (2.33, 1.93) well above the often-used cut-off value of 1.5, and fully inhibitable with specific ABCB1 and ABCG2 inhibitors. We attribute this apparent discrepancy to the relative sensitivity of the transepithelial transport assays applied by us. The in vivo significance of these findings is further supported by the marked effect of mAbcb1 deficiency on brain distribution of ibrutinib (Figure 3, Table 1). mAbcg2, instead, did not appear to have a marked impact in restricting brain accumulation of ibrutinib. It is possible that ABCB1 in humans will also limit brain distribution of ibrutinib, which could