Stephanie van Hoppe

75 ABCB1 restricts brain penetration of the BTK inhibitor ibrutinib while CYP3A limits its oral bioavailability R E S U LT S We first studied the interaction between ibrutinib and ABCB1 and ABCG2 in vitro by measuring ibrutinib (2 µM) translocation through polarized monolayers of the MDCKII parental cell line and subclones transduced with human (h)ABCB1, hABCG2 or mouse (m)Abcg2 cDNA. As shown in Figure 1A, we observed no net apically directed ibrutinib transport in the parental cell line (transport ratio r = 0.88). This was not significantly altered when the cells were treated with the ABCB1 inhibitor zosuquidar (r = 0.84, Figure 1B), suggesting that there is virtually no background transport mediated by the endogenous canine ABCB1 present in the MDCKII cells [28]. In MDCKII cells transduced with human ABCB1, we observed active apically directed transport with r = 2.33, which was almost completely blocked by zosuquidar, indicating that ibrutinib is a transport substrate for hABCB1 (Figure 1C and D). In subsequent transport experiments using MDCKII cells expressing human or mouse ABCG2, zosuquidar was included to block any background transport mediated by endogenous canine ABCB1. We observed substantial apically directed transport by mAbcg2 (r = 1.93), whereas no transport was detected for hABCG2 (r = 0.94) as shown in Figure 1E-H. Ibrutinib transport by mAbcg2 was efficiently blocked by the ABCG2 inhibitor Ko143. Ibrutinib thus appears to be transported by hABCB1 and mAbcg2, but, at this concentration, not noticeably by hABCG2. Based on these transport data, we studied the single and combined effects of Abcb1 and Abcg2 on the plasma and tissue pharmacokinetics of ibrutinib and its pharmacodynamically active metabolite, ibrutinib-DiOH, using WT, Abcb1a/1b -/- , Abcg2 -/- , and Abcb1a/1b;Abcg2 -/- mice. Because ibrutinib is taken orally by patients, we administered ibrutinib orally at a dose of 10mg/kg, roughly physiologically equivalent to the lower end of the human recommended dosages (140 mg/day). In a pilot experiment we analyzed the plasma concentrations of ibrutinib and ibrutinib-DiOH over 8 h in WT and Abcb1a/1b;Abcg2 -/- mice. In this experiment, performed in our old mouse facility, we found no significant differences in the AUC 0-8 h values between theWT and knockout mice (Supplemental Figure 2A, B; Table 1). Ibrutinib was very rapidly absorbed, with a T max occurring before 5 min, and also quite rapidly cleared. Within 15 min, ibrutinib- DiOH concentrations were substantially higher than those of the parent drug, reaching a T max around 15-30 min, and with ibrutinib-DiOH/ibrutinib ratios staying well over a factor of 5 from 30 min on to at least 3 h after administration (Supplemental Figure 3, Supplemental Table 1). A subsequent 1-h experiment including all four ABC transporter mouse strains was performed more than a year later, after transfer of these strains to our new mouse facility. This transfer included a full clean-up by hysterectomy and a complete change-over in the microflora to that of another commercial animal supplier.