Stephanie van Hoppe

72 Chapter 4 HER2, JAK3, HCK and ITK [8, 10-13]. Given the high ABCB1 and ABCG2 expression in the BBB, these transporters could potentially limit brain accumulation of ibrutinib, which might reduce the therapeutic efficacy against CNS lesions including brain (micro-)metastases. However, the current FDA and EMA documentation for this drug states that in vitro studies suggest that ABCB1 and ABCG2 do not transport ibrutinib, although they might be inhibited by ibrutinib at clinical doses [6]. A recent study further reported that ibrutinib can stimulate hABCB1- mediated ATPase activity [14]. It was therefore of interest to study these interactions in more detail. The absolute oral bioavailability of ibrutinib in patients is low (around 3-8%), and this is likely due in part to extensive first-pass metabolism, primarily by cytochrome-P450 (CYP) 3A. Its main metabolite, dihydrodiol-ibrutinib (DiOH, PCI-45227, Supplemental Figure 1), has an inhibitory activity towards BTK approximately 10-15 times lower than that of the parent compound [6]. Ibrutinib-DiOH, which is thus modestly pharmacodynamically active, is formed through epoxidation and subsequent oxidation of the reactive acrylamide group [15]. Ibrutinib is mainly excreted via feces after phase I and II biotransformation. In humans, CYP3A4 is also the predominant CYP isoenzyme involved in the biotransformation of a range of other TKIs such as imatinib, nilotinib, bosutinib, dasatinib and ponatinib [16-20]. In the present study we investigated whether ibrutinib is a transported substrate of ABCB1 and ABCG2 in vitro and in vivo , and how this might affect the oral plasma pharmacokinetics and tissue distribution, including brain penetration, of ibrutinib and ibrutinib-DiOH in appropriate knockout mouse models. Furthermore, the substantial metabolism of ibrutinib by CYP3A [6] means that induction or inhibition of this enzyme complex may dramatically influence ibrutinib exposure. We therefore also studied the in vivo tissue-specific influence of CYP3A on the oral systemic availability and tissue exposure of ibrutinib and ibrutinib-DiOH in Cyp3a knockout and humanized transgenic mouse models. MAT E R I A L S AND ME T HOD S Chemi c a l s Ibrutinib (>99%) was purchased from Alsachim (Illkirch Graffenstaden, France), zosuquidar was obtained from Sequoia Research Products (Pangbourne, UK) and Ko143 was from Tocris Bioscience (Bristol, UK). Isoflurane was purchased from Pharmachemie (Haarlem, The Netherlands), heparin (5000 IU ml -1 ) was from Leo Pharma (Breda, The Netherlands), and Bovine Serum Albumin (BSA) Fraction V from Roche (Mannheim,