Compound profiling for p-glycoprotein at the blood-brain barrier using a microplate screening system

Purpose. The purpose of this study was to establish a fluorescent dye (calcein - acetoxymethylester; calcein- AM)-based assay to rapidly screen compounds for interactions with p-glycoprotein (p-gp) at the blood - brain barrier and to determine whether such an assay can be useful for kinetic analysis. Methods. Porcine brain capillary endothelial cells (PBCECs) were isolated and cultured in 96-well plates. Cells were incubated with calcein- AM in the absence and presence of substrates and inhibitors of ABC transporters and the extent of intracellularly appearing fluorescence was monitored with a fluorescence plate reader in a time- and a concentration-dependent manner. Results. PBCECs showed stable expression of p-gp and as a result calcein- AM was extruded by the cells. In the presence of p-gp substrates and inhibitors a significant increase of intracellular fluorescence was observed ( decreased calcein- AM efflux), the increase being well correlated with the p-gp affinity of the compounds used. Inhibitors of Mrp1 and Mrp2 did not influence fluorescence intensity. Time-dependent readouts and Michaelis-Menten kinetic analysis separated inhibitors into those showing competitive, mixed and noncompetitive inhibition of p-glycoprotein-mediated transport. Conclusion. The calcein- AM-assay based on PBCECs can be used as a rapid microplate screening system for interactions of drugs with p-glycoprotein at the blood-brain barrier and represents therefore a useful tool in the profiling of drugs. In addition, convenient kinetic assays can provide information about the mode of interaction.