title: A functional assay for quantitation of the apparent affinities of ligands of P-glycoprotein in Caco-2 cells.
abstract: OBJECTIVE
  To develop a facile functional assay for quantitative determination of the apparent affinities of compounds that interact with the taxol binding site of P-glcoprotein (P-gp) in Caco-2 cell monolayers.
  
  
  METHODS
  A transport inhibition approach was taken to determine the inhibitory effects of compounds on the active transport of [3H]-taxol, a known substrate of P-gp. The apparent affinities (K(I) values) of the compounds were quantitatively determined based on the inhibitory effects of the compounds on the active transport of [3H]-taxol. Intact Caco-2 cell monolayers were utilized for transport inhibition studies. Samples were analyzed by liquid scintillation counting.
  
  
  RESULTS
  [3H]-Taxol (0.04 microM) showed polarized transport with the basolateral (BL) to apical (AP) flux rate being about 10-20 times faster than the flux rate in the AP-to-BL direction. This difference in [3H]-taxol flux could be totally abolished by inclusion of (+/-)-verapamil (0.2 mM), a known inhibitor of P-gp, in the incubation medium. However, inclusion of probenecid (1.0 mM), a known inhibitor for the multidrug resistance associated protein (MRP), did not significantly affect the transport of [3H]-taxol under the same conditions. These results suggest that P-gp, not MRP, was involved in taxol transport. Quinidine, daunorubicin, verapamil, taxol, doxorubicin, vinblastine, etoposide, and celiprolol were examined as inhibitors of the BL-to-AP transport of [3H]-taxol with resulting K(I) values of 1.5+/-0.8, 2.5+/-1.0, 3.0+/-0.3, 7.3+/-0.7, 8.5+/-2.8, 36.5+/-1.5, 276+/-69, and 313+/-112 microM, respectively. With the exception of that of quinidine, these K(I) values were comparable with literature values.
  
  
  CONCLUSIONS
  This assay allows a facile quantitation of the apparent affinities of compounds to the taxol-binding site in P-gp, however, this assay does not permit the differentiation of substrates and inhibitors. The potential of drug-drug interactions involving the taxol binding site of P-gp can be conveniently estimated using the protocol described in this paper.
authors.affiliation: Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence 66047, USA.
authors.forename: J
authors.initials: J
authors.lastname: Gao
authors.forename: O
authors.initials: O
authors.lastname: Murase
authors.forename: R L
authors.initials: RL
authors.lastname: Schowen
authors.forename: J
authors.initials: J
authors.lastname: Aubé
authors.forename: R T
authors.initials: RT
authors.lastname: Borchardt
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism*; D001692:Biological Transport; D001693:Biological Transport, Active; D018938:Caco-2 Cells; D004347:Drug Interactions; D006801:Humans; D008024:Ligands; D008962:Models, Theoretical; D017239:Paclitaxel / Q000378:metabolism*; D014316:Tritium
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000972:Antineoplastic Agents, Phytogenic; D008024:Ligands; D014316:Tritium; D017239:Paclitaxel