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Compound Profiling for P-Glycoprotein at the Blood–Brain Barrier Using a Microplate Screening System

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Abstract

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 non-competitive 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.

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Authors and Affiliations

  1. Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, INF 366 69120, Heidelberg, Germany

    Björn Bauer & Gert Fricker

  2. National Institutes of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina

    Björn Bauer & David S. Miller

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  1. Björn Bauer
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Correspondence to Gert Fricker.

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Bauer, B., Miller, D.S. & Fricker, G. Compound Profiling for P-Glycoprotein at the Blood–Brain Barrier Using a Microplate Screening System. Pharm Res 20, 1170–1176 (2003). https://doi.org/10.1023/A:1025040712857

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