title: Determining P-glycoprotein-drug interactions: evaluation of reconstituted P-glycoprotein in a liposomal system and LLC-MDR1 polarized cell monolayers.
abstract: BACKGROUND
  P-Glycoprotein (ABCB1, MDR1) is a multidrug efflux pump that is a member of the ATP-binding cassette (ABC) superfamily. Many drugs in common clinical use are either substrates or inhibitors of this transporter. Quantitative details of P-glycoprotein inhibition by pharmaceutical agents are essential for assessment of their pharmacokinetic behavior and prevention of negative patient reactions. Cell-based systems have been widely used for determination of drug interactions with P-glycoprotein, but they suffer from several disadvantages, and results are often widely variable between laboratories. We aimed to demonstrate that a novel liposomal system employing contemporary biochemical methodologies could measure the ability of clinically used drugs to inhibit the P-glycoprotein pump. To accomplish this we compared results with those of cell-based approaches.
  
  
  METHODS
  Purified transport-competent hamster Abcb1a P-glycoprotein was reconstituted into a unilamellar liposomal system, Fluorosome-trans-pgp, whose aqueous interior contains fluorescent drug sensors. This provides a well-defined system for measuring P-glycoprotein transport inhibition by test drugs in real time using rapid fluorescence-based technology.
  
  
  RESULTS
  Inhibition of ATP-driven transport by Fluorosome-trans-pgp employed a panel of 46 representative drugs. Resulting IC50 values correlated well (r2=0.80) with Kd values for drug binding to purified P-glycoprotein. They also showed a similar trend to transport inhibition data obtained using LLC-MDR1 cell monolayers. Fluorosome-trans-pgp IC50 values were in agreement with published results of digoxin drug-drug interaction studies in humans.
  
  
  CONCLUSIONS
  This novel approach using a liposomal system and fluorescence-based technology is shown to be suitable to study whether marketed drugs and drug candidates are P-glycoprotein inhibitors. The assay is rapid, allowing a 7-point IC50 determination in <6 min, and requires minimal quantities of test drug. The method is amenable to robotics and offers a cost advantage relative to conventional cell-based assays. The well-defined nature of this assay also obviates many of the inherent complications and ambiguities of cell-based systems.
authors.affiliation: GLSynthesis Inc., One Innovation Drive, Worcester, Massachusetts 01605, USA. Donald.Melchior@glsynthesis.com
authors.forename: Donald L
authors.initials: DL
authors.lastname: Melchior
authors.forename: Frances J
authors.initials: FJ
authors.lastname: Sharom
authors.forename: Raymond
authors.initials: R
authors.lastname: Evers
authors.forename: George E
authors.initials: GE
authors.lastname: Wright
authors.forename: Joseph W K
authors.initials: JW
authors.lastname: Chu
authors.forename: Stephen E
authors.initials: SE
authors.lastname: Wright
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Chu
authors.forename: Jocelyn
authors.initials: J
authors.lastname: Yabut
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001693:Biological Transport, Active; D006224:Cricetinae; D016572:Cyclosporine / Q000737:chemistry / Q000378:metabolism; D004347:Drug Interactions; D006801:Humans; D020128:Inhibitory Concentration 50; D018374:LLC-PK1 Cells; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins; D013552:Swine
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008081:Liposomes; D004364:Pharmaceutical Preparations; D011994:Recombinant Proteins; D016572:Cyclosporine