Insight into the cooperation of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) at the blood-brain barrier: a case study examining sorafenib efflux clearance

Abstract

The ATP-binding cassette transporters P-glycoprotein and breast cancer resistance protein have been shown to be critical determinants limiting drug transport across the BBB into the brain. Several therapeutic agents have been shown to be substrates for these two transporters, and as a result they have limited distribution to the brain. Recently, it has been shown that these two drug transporters cooperate at the BBB and brain penetration of dual substrates increases significantly only when both are absent, e.g., in the Mdr1a/1b(-/-)Bcrp1(-/-) mice. The present study uses the brain penetration of sorafenib to investigate these findings and attempts to explain the mechanistic basis of this cooperation with a simple theory based on affinity and capacity dependent carrier-mediated transport. The brain efflux index method, combined with the organotypic brain slices, was used to determine the net contribution of P-gp and BCRP to the total clearance of sorafenib out of the brain and show that its efflux at the BBB is mediated primarily by BCRP. Sorafenib clearance out of the brain decreased 2-fold in the Bcrp1(-/-) mice and 2.5-fold in the Mdr1a/1b(-/-)Bcrp1(-/-) mice. Clearance out of brain when P-gp was absent did not change significantly compared to wild-type. We also investigated the expression of P-gp and BCRP in the genetic knockout animals and saw no differences in either P-gp or BCRP in the transporter deficient mice compared to the wild-type mice. In conclusion, this study explains the cooperation of P-gp and BCRP by analysis of the efflux clearance of sorafenib and correlating it to the "mechanisms" that determine the clearance, i.e., affinity and capacity.

Figure 1. Brain efflux index of sorafenib in FVB mice

Sorafenib was rapidly cleared out of the brain in wild-type and Mdr1a/b^-/- mice with an efflux rate constant of 0.17 ± 0.02 and 0.16 ± 03 min^-1, respectively. Sorafenib clearance out of brain decreased in the Bcrp1^-/- and Mdr1a/b^-/-Bcrp1^-/- mice with efflux rate constants of 0.1 ± 0.01 and 0.06 ± 02 min^-1, respectively. Data presented as mean ± S.D. (n = 3-5 per time point).

Figure 2. Volume of distribution of sorafenib in brain

Volume of distribution was determined by measuring uptake of [3H]-sorafenib in brain slices from wild-type, Mdr1a/b^-/-, Bcrp1^-/- and Mdr1a/b^-/-Bcrp1^-/- mice. Data presented as mean ± S.D. (n = 3-5 per time point).

Figure 3. Western blot analysis of P-gp and BCRP in isolated brain capillaries of wild-type, Mdr1a/b^-/-, Bcrp1^-/- and Mdr1a/b^-/- Bcrp1^-/- mice (pooled samples from 10 mouse brains from each genotype)

Quantification of Western blot shows no difference in expression of P-gp in the BCRP deficient mice and vice versa.