Investigation of the effect of active efflux at the blood–brain barrier on the distribution of nonsteroidal aromatase inhibitors in the central nervous system  Mari Miyajima, Hiroyuki Kusuhara, Kayo Takahashi, Tadayuki Takashima, Takamitsu Hosoya, Yasuyoshi Watanabe, Yuichi Sugiyama  Journal of Pharmaceutical Sciences  Volume 102, Issue 9, Pages 3309-3319 (September 2013) DOI: 10.1002/jps.23600 Copyright © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 1 Chemical structures of the nonsteroidal aromatase inhibitors used in this study. Journal of Pharmaceutical Sciences 2013 102, 3309-3319DOI: (10.1002/jps.23600) Copyright © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 2 Comparison of brain-to-plasma concentration ratio of aromatase inhibitors in mice. Anastrozole (0.4 μmol/kg) or vorozole (1.2 μmol/kg) was administered in male ICR mice by intravenous bolus, and letrozole was administered by intravenous infusion at a rate of 0.8 μmol/h/kg. The plasma concentration was measured at 1, 30, 60, and 90min (a), and brain concentration at 90min (b). The brain-to-plasma concentration ratio of aromatase inhibitors was calculated at 90min (c). Each point and bar represent the mean ± SE (N = 4–5). Journal of Pharmaceutical Sciences 2013 102, 3309-3319DOI: (10.1002/jps.23600) Copyright © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 3 Time profiles of changes in brain interstitial fluid concentration and unbound plasma concentration of anastrozole after intravenous bolus administration. Microdialysis probes were inserted in the cerebral cortex of ICR male mice (1.1 mm lateral, 1.1 mm anterior, 3 mm depth). One day after surgery, anastrozole was administered by intravenous bolus (24 μmol/kg), and KRP buffer was perfused through the probe at 1 μL/min. The plasma concentration and dialysate concentration were measured. The unbound plasma concentration (Cu,p) was calculated by multiplying the unbound fraction in plasma by the total plasma concentration. Antipyrine was used as a reference compound. The brain interstitial fluid concentration of anastrozole (CISF) was extrapolated from the dialysate concentration using in vitro and in vivo probe recovery of antipyrine, as described in Materials and Methods. Each point represents the mean ± SE (N = 3). Journal of Pharmaceutical Sciences 2013 102, 3309-3319DOI: (10.1002/jps.23600) Copyright © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 4 In situ uptake clearance of aromatase inhibitors by mouse brain. Initial uptake clearance (CLuptake) of aromatase inhibitors in the mouse cerebral cortex was examined for 1min by brain perfusion using diazepam as a marker for perfusion rate. Each bar represents the mean ± SE (N = 4). Journal of Pharmaceutical Sciences 2013 102, 3309-3319DOI: (10.1002/jps.23600) Copyright © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 5 Transcellular transport of aromatase inhibitors by human P-gp- and BCRP-overexpressing MDCK II cells. Mock-vector-transfected cells (open symbols) and P-gp- or BCRP-overexpressing MDCK II cells (closed symbols) were cultured as a monolayer on porous filter. Apical-to-basal transport (AtoB, squares) and basal-to-apical transport (BtoA, circles) of anastrozole, letrozole, and vorozole were measured along with time (a and c). The flux ratio of mock and P-gp–or BCRP–MDCK II cells was obtained as the ratio of transcellular transport (basal-to-apical transport relative to apical-to-basal transport). The ratio of the flux ratios of P-gp- and BCRP-overexpressing cells to mock cells is termed “CFR” (b and d). Each symbol and bar represents the mean ± SE (N = 3). Statistical significance was examined by one-way analysis of variance followed by Tukey's test. *p < 0.05, **p < 0.01 Mock-vector-transfected cells versus P-gp- or BCRP-overexpressing MDCK II cells; †p < 0.05, ††p < 0.01 AtoB versus BtoA in P-gp- or BCRP-overexpressing MDCK II cells; ‡p < 0.05, ‡‡p < 0.01 AtoB versus BtoA in Mock-vector-transfected cells. Journal of Pharmaceutical Sciences 2013 102, 3309-3319DOI: (10.1002/jps.23600) Copyright © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions

Figure 6 Brain-to-plasma ratios of aromatase inhibitors in wild-type and Mdr1a/b/Bcrp(−/−) mice. Anastrozole (0.4 μmol/kg) or vorozole (1.2 μmol/kg) was administered by intravenous bolus in male wild-type (open symbols and bars) and Mdr1a/b/Bcrp(−/−) (closed symbols and bars) mice. Plasma concentrations at designated time (a), and brain concentration at 1.5h (b) were measured. Brain-to-plasma concentration ratio of aromatase inhibitors was calculated at 1.5h (c). Each point and bar represents the mean ± SE (N = 4). **p < 0.01 wild-type mice versus Mdr1a/b/Bcrp(−/−) mouse. Journal of Pharmaceutical Sciences 2013 102, 3309-3319DOI: (10.1002/jps.23600) Copyright © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association Terms and Conditions