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

Björn Bauer, David S. Miller, Gert Fricker

Research output: Contribution to journalArticlepeer-review

45 Scopus citations


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 languageEnglish
Pages (from-to)1170-1176
Number of pages7
JournalPharmaceutical Research
Issue number8
StatePublished - 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).


  • 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)


Dive into the research topics of 'Compound profiling for P-glycoprotein at the blood-brain barrier using a microplate screening system'. Together they form a unique fingerprint.

Cite this