title: Compound profiling for P-glycoprotein at the blood-brain barrier using a microplate screening system.
abstract: OBJECTIVE
  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 Mrpl 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, INF 366 69120 Heidelberg, Germany.
authors.forename: Björn
authors.initials: B
authors.lastname: Bauer
authors.forename: David S
authors.initials: DS
authors.lastname: Miller
authors.forename: Gert
authors.initials: G
authors.lastname: Fricker
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000818:Animals; D001812:Blood-Brain Barrier / Q000378:metabolism*; D001921:Brain / Q000098:blood supply; D002196:Capillaries / Q000166:cytology / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D005452:Fluoresceins; D005456:Fluorescent Dyes; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D010599:Pharmacokinetics; D013552:Swine
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005452:Fluoresceins; D005456:Fluorescent Dyes; D004364:Pharmaceutical Preparations; C085925:calcein AM