Stephanie van Hoppe

28 Chapter 2 canine ABCB1 activity, subsequent experiments on ABCG2-mediated transport were done in the presence of zosuquidar. Both hABCG2 and mAbcg2 efficiently transported afatinib in the apical direction ( r = 5.6 and r = 23, respectively) (Fig. 1E, G). This transport was completely abrogated ( r = 0.8 and r = 1.0, respectively) by treatment with the ABCG2 inhibitor Ko143 (Fig. 1F, H). Overall, afatinib appears to be well transported by hABCB1, hABCG2 and mAbcg2. Ab c g2 and Ab c b1 e a c h r e s t r i c t t he o r a l ava i l ab i l i t y o f a f a t i n i b i n mi c e Afatinib is given orally to patients. We therefore started with a pilot experiment with oral afatinib (10mg/kg) inWTandAbcb1a/1b;Abcg2 -/- mice, analyzingplasma concentrations and tissue accumulation after 8 h. The results indicated a substantial impact of this combined transporter deficiency on oral availability and brain accumulation of afatinib, with highly increased exposure levels in the knockout strain (Supplemental Figures 1-3). We therefore studied in more detail the single and combined effects of Abcb1 and Abcg2 deficiencies on afatinib plasma pharmacokinetics and organ accumulation using WT, Abcg2 -/- , Abcb1a/1b -/- , and Abcb1a/1b;Abcg2 -/- mice. Plasma exposure of afatinib over 24 h (AUC 0-24 ) was increased by the absence of Abcg2 by 4.2-fold (P = < 0.001), by the absence of Abcb1a/1b by 2.4-fold (P < 0.01), and by the absence of both Abcg2 and Abcb1a/1b by 7-fold (P < 0.001) compared toWT mice (Fig. 2A; Table I, 24 h data). These results indicate that each of these transporters can substantially affect the intestinal uptake and/or the systemic elimination of afatinib, thus restricting its oral availability. The marked contribution of each of the individual transporters was further supported by comparison of the plasma AUC 0-24 values of the single knockout strains with the combination knockout strain (Table I, 24 h data). Figure 2. Plasma concentration-time curves of afatinib in female WT (black squares), Abcg2 -/- (blue triangles), Abcb1a/1b -/- (green triangles), and Abcg2;Abcb1a/1b -/- mice (red squares) over 24 hours ( A ) 0 25 50 75 100 125 0 50 100 150 200 250 300 350 time (minutes) Plasma concentration (ng/ml) 0 5 10 15 20 25 0 50 100 150 200 250 300 Time (hours) Plasma concentration (ng/ml) WT Abcg2;Abcb1a/1b -/- Abcb1a/1b -/- Abcg2 -/- A B *** *** *** *** * ** *** *** *** * Figure 2. Plasma concentration-time curves of afatinib in female WT (black squares), triangles), Abcb1a/1b -/- (green triangles), and Abcg2;Abcb1a/1b -/- mice (red squares) ove and 120 minutes ( B ) after oral administration of 10 mg/kg afatinib. Data are given as m rendered one-sided for improved clarity). *, P < 0.05; **, P < 0.01; ***, P < 0.001 co mice. Statistical analysis for the 24 h graph ( A ) for the time points up to 4 h yields P < 0. and 1 h, at 2 h P < 0.01 for Abcg2;Abcb1a/1b -/- compared to WT mice. 0 25 50 75 100 125 0 50 100 150 200 250 300 350 time (minutes) Plasma concentration (ng/ml) 0 5 10 15 20 25 0 50 100 150 200 250 300 Time (hours) Plasma concentration (ng/ml) WT Abcg2;Abcb1a/1b -/- Abcb1a/1b -/- Abcg2 -/- A B *** *** *** *** * ** *** *** *** * Figure 2 - Plasma concentration-time curves of afatinib in femaleWT (black squares), Abcg2 -/- (blue triangles), Abcb1a/1b -/- (green triangles), and Abcg2;Abcb1a/ b -/- mic (red squares) over 24 hours ( A ) and 120 minutes ( B ) after oral administration of 10 mg/kg afatinib. Data are given as mean ± SD (SD rendered one-sided for improved clarity). *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared toWT mice. Statistical analysis for the 24 h graph ( A ) for he time p ints up to 4 h yields P < 0.001 at 30 min and 1 h, at 2 h P < 0.01 for Abcg2;Abcb1a/1b -/- compared to WT mice.