Stephanie van Hoppe

83 ABCB1 restricts brain penetration of the BTK inhibitor ibrutinib while CYP3A limits its oral bioavailability proteins are a major factor in metabolizing ibrutinib to ibrutinib-DiOH. When transgenic human CYP3A4 was expressed in both liver and intestine of Cyp3a -/- mice (Cyp3aXAV), the ibrutinib plasma concentrations decreased again by 5-fold, to less than two-fold the AUC 0-8 h in WT mice (Figure 5A, Table 2). At the same time, plasma levels of ibrutinib- DiOH in Cyp3aXAV mice increased by 4.2-fold compared to Cyp3a -/- mice, to about 28% of the ibrutinib-DiOH AUC 0-8 h seen in WT mice (Figure 5C, Supplemental Table 2. These data indicate a substantial, albeit not complete reversal of ibrutinib to ibrutinib- DiOH conversion by transgenic human CYP3A4 expression in liver and intestine. Thus, the combined endogenous mouse Cyp3a proteins have a higher overall capacity to metabolize ibrutinib to ibrutinib-DiOH than the exogenously (but orthotopically) expressed human CYP3A4. Accordingly, when we plotted the plasma ibrutinib-DiOH to ibrutinib concentration ratio over the first three hours, the high ratio observed in WT mice (rising well above 5 within 30 min) was very low in Cyp3a -/- mice (0.04 around 30 min), but only returned to a ratio of around 1 at 30 min in the Cyp3aXAV mice (Supplemental Figure 7). A small 1-h follow-up experiment in WT and Cyp3a -/- mice revealed very similar profiles (Supplemental Figure 8). To further analyze the separate and combined in vivo impact of hepatic and intestinal CYP3A4 on ibrutinib to ibrutinib-DiOH metabolism around or shortly after the T max of ibrutinib, we performed a short-term (20 min) oral pharmacokinetic experiment in WT, Cyp3a -/- , Cyp3aXA, Cyp3aXV, and Cyp3aXAV mice. As shown in Figure 5B and D, the interindividual variation in plasma levels was high, especially in WT mice, which is not uncommon this shortly after oral drug administration. Nonetheless, absence of Cyp3a led to a highly significant, 8.7-fold increase in ibrutinib plasma levels (AUC 0-0.33 h ), which was partly reversed by either hepatic CYP3A4 expression (2.3-fold reversal), or intestinal CYP3A4 expression (2.7-fold reversal). Combined hepatic and intestinal CYP3A4 expression had an additive effect, resulting in a 5.1-fold reversal of ibrutinib plasma levels (Figure 5B, Table 2). Also over this time period reversal by the transgenic human CYP3A4 was therefore extensive, but not completely back to WT levels. The changes in ibrutinib-DiOH plasma levels between the strains mirrored the changes in levels of ibrutinib metabolism, with absence of Cyp3a resulting in a 12.4-fold decrease in ibrutinib-DiOH levels (AUC 0-0.33 h ), hepatic and intestinal CYP3A4 expression causing a 4.4- and 6.5-fold reversal, respectively, and the combination expression a 7.9-fold reversal (Figure 5D, Supplemental Table 2). Plotting the plasma ibrutinib- DiOH to ibrutinib concentration ratios confirmed these separate and additive effects (Supplemental Figure 9E), including that the reversal even in the Cyp3aXAV strain was far from completely back to the WT levels. Collectively, the data indicate that in these mouse strains hepatic and intestinal CYP3A4 have a more or less comparable impact