Directional transport of FLZ across physiological (A) and PD pathological (B) BBB models.

Transport was measured in the absence or presence of P-gp inhibitor zosuquidar or BCRP inhibitor FTC with time up to 2.5 h. The apical-to-basolateral (A–B) and basolateral-to-apical (B–A) transepithelial flux of various concentrations of FLZ (1, 5, 10 µM) was assessed in physiological (Aa) and pathological (Ba) BBB models, respectively. Papp B–A transport was significantly higher than those for A–B transport at each FLZ concentration (Ac and Bc), indicating the presence of an efflux pump to remove FLZ from within cell membranes. Higher efflux ratio and lower BBB penetration of FLZ were showed under pathology model (Ad and Bd). To examine the contribution of P-gp and BCRP to FLZ transport, permeability of FLZ at initial concentrations of 10 µM in the absence and presence of 5 µM zosuquidar to block P-gp or 10 µM FTC to inhibit BCRP (Ab and Bb) were measured. Only the P-gp inhibitor zosuquidar effectively inhibited efflux of FLZ across the two BBB models, and the black arrows indicate the direction of change in FLZ transport caused by addition of zosuquidar (Ac and Bc). The magnitude of P-gp or BCRP-mediated efflux was estimated by the efflux ratio (ER), defined as the ratio of Papp B–A to the Papp A–B. Upon specific blocking of P-gp using zosuquidar resulted in significantly lower efflux ratio of FLZ in either physiological (Ad) or pathological (Bd) BBB models. However, the efflux ratio between BCRP inhibitor and inhibitor-free group were not significantly different (Ad and Bd). All data are expressed as the means ± SD (n = 3). *P<0.05, significantly different from each corresponding control.