A fluorometric screening assay for drug efflux transporter activity in the blood-brain barrier

Purpose. To examine the capability of a fluorometric assay to identify and characterize the drug efflux interactions of a broad spectrum of drug agents in an hi vitro model of the blood-brain barrier (BBB). Methods. Various concentrations of drug agent (1, 10, and 100 muM) were evaluated for their effect on the cellular accumulation of the P-glycoprotein (P-gp) probe R123 (3.2 muM), and the mixed P-gp and multidrug resistance-associated protein (MRP) probe, BCECF (1 muM), in bovine brain microvessel endothelial cell (BBMEC) monolayers. Drugs demonstrating a significant effect were further quantitated using an expanded concentration range and a nonlinear regression curve fit to determine the potency (IC50) and efficacy (Imax) of the drug for P-gp and/or MRP. Results. Several of the 36 therapeutic agents examined showed drug efflux transporter interactions in BBMEC monlayers. Melphalan and risperidone significantly enhanced the accumulation of R123 over control (1.47- and 1.82-fold, respectively) with resulting IC(50)s of 1.4 and 14.6 muM, respectively. Chlorambucil and valproic acid significantly enhanced the accumulation of BCECF compared to control monolayers (2.02- and 4.01-fold, respectively) With resulting IC50S of 146.1 and 768.5 muM, respectively. Conclusions. The current study demonstrates the feasibility of a fluorometric assay consisting of R123 and BCECF in assessing the drug efflux interactions of a variety of drugs in the BBB.