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

Björn Bauer; David S. Miller; Gert Fricker

doi:10.1023/A:1025040712857

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

Björn Bauer, David S. Miller, Gert Fricker

Pharmaceutical Sciences

Research output: Contribution to journal › Article › peer-review

45 Scopus citations

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.

Original language	English
Pages (from-to)	1170-1176
Number of pages	7
Journal	Pharmaceutical Research
Volume	20
Issue number	8
DOIs	https://doi.org/10.1023/A:1025040712857
State	Published - Aug 1 2003

Bibliographical note

Funding Information:
We would like to thank Prof. Dr. Gerhart Kurz (†), University of Freiburg, FRG, for helpful discussions concerning the kinetic assay. This work was supported by the Grant of the German Research Foundation DFG FR 1211/6-2).

Keywords

Blood-brain barrier
Calcein-AM
Compound profiling
Microplate screening system
P-glycoprotein

ASJC Scopus subject areas

Biotechnology
Molecular Medicine
Pharmacology
Pharmaceutical Science
Organic Chemistry
Pharmacology (medical)

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title = "Compound profiling for P-glycoprotein at the blood-brain barrier using a microplate screening system",

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.",

keywords = "Blood-brain barrier, Calcein-AM, Compound profiling, Microplate screening system, P-glycoprotein",

author = "Bj{\"o}rn Bauer and Miller, {David S.} and Gert Fricker",

note = "Funding Information: We would like to thank Prof. Dr. Gerhart Kurz (†), University of Freiburg, FRG, for helpful discussions concerning the kinetic assay. This work was supported by the Grant of the German Research Foundation DFG FR 1211/6-2).",

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AU - Bauer, Björn

AU - Miller, David S.

AU - Fricker, Gert

N1 - Funding Information: We would like to thank Prof. Dr. Gerhart Kurz (†), University of Freiburg, FRG, for helpful discussions concerning the kinetic assay. This work was supported by the Grant of the German Research Foundation DFG FR 1211/6-2).

PY - 2003/8/1

Y1 - 2003/8/1

N2 - 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.

AB - 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.

KW - Blood-brain barrier

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

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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