Received for publication August 31, 2005.
Revised March 14, 2006.
Accepted for publication March 15, 2006.

Comparison of Drug Efflux Transport Kinetics in Various Blood-Brain Barrier Models

Abstract

The present study quantitatively compared the drug efflux transport kinetics of BCECF-AM, and its fluorescent metabolite, BCECF, in various blood-brain barrier (BBB) models. BCECF-AM was exposed to freshly isolated bovine brain microvessels (BBM), primary cultured BBMEC, and MDCK-MDR1 cells for 30 minutes in the presence or absence of the P-gp inhibitor, GF120918 (N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide). P-gp transport kinetics were determined indirectly by calculating the difference in BCECF accumulation when P-gp was functional and completely inhibited by GF120918 (3.2 µM). MRP transport kinetics were determined by measuring the amount of BCECF transported out of the cell over time. For P-gp-related transport, Km values for BCECF-AM were approximately the same in all three models (around 2 µM), while the Vmax was 4-fold greater in the BBM than in the BBMEC or MDCKII-MDR1 cells. For MRP-related transport, Km values for BCECF varied widely among the three BBB models with a rank order of MDCKII-MDR1 < BBMEC < BBM. Like P-gp, the Vmax of BCECF for MRP-related transport was overwhelmingly higher in the BBM compared to the cultured cells. As differences in the expression of P-gp, MRP5 and MRP6 were observed in the various BBB models using RT-PCR techniques, the disparity in transport kinetics between the BBB models may be linked to variations in the amount or type of drug efflux transporters expressed in each model. The present study introduces a method of quantitatively evaluating drug efflux transport kinetics in the BBB.

Key words: ABC transporters, blood-brain barrier, kinetics, MRP, multi-drug resistance, p-glycoprotein