title: Functional assessment of multiple P-glycoprotein (P-gp) probe substrates: influence of cell line and modulator concentration on P-gp activity.
abstract: Compounds known to modulate P-glycoprotein (P-gp) activity were evaluated in cell monolayers expressing P-gp for their effects on the secretory transport of P-gp substrates paclitaxel, vinblastine, and digoxin. Paclitaxel has been proposed to selectively interact with a binding site on P-gp that is distinct from the vinblastine and digoxin-binding site. Using Madin-Darby canine kidney (MDCK)-multidrug resistance-1 (MDR1), MDCK-wild-type (WT), and Caco-2 cell monolayers, the basal-to-apical (BL-AP) apparent permeability (Papp) of [3H]paclitaxel, [3H]vinblastine, and [3H]digoxin in the presence of various concentrations of a series of structurally diverse P-gp substrates and modulators of P-gp function were determined. MDCK-WT cell monolayers demonstrated active secretory transport of all P-gp substrate probes, although the sensitivity to inhibition by verapamil was lower than that demonstrated in MDCK-MDR1 cell monolayers. When evaluated as competitive inhibitors, several known P-gp substrates had no effect or only a slight modulatory effect on the BL-AP Papp of all probe substrates in MDCK-MDR1 cells. The secretory transport of P-gp substrates in MDCK-WT cells was more sensitive to inhibition by known P-gp modulators compared with MDCK-MDR1 cells. Low concentrations of ketoconazole (1-3 microM) activated the BL-AP Papp of [3H]vinblastine and [3H]digoxin in MDCK-MDR1 cells but not in MDCK-WT or Caco-2 cells. Determination of secretory transport in P-gp expressing cell monolayers, such as MDCK-MDR1 and Caco-2, may be complicated by substrate cooperativity and allosteric binding, which may result in the activation of P-gp. In addition, expression of other efflux transporters in these cell lines introduces additional complexity in distinguishing which transporter is responsible for substrate recognition and transport.
authors.affiliation: Drug Metabolism & Pharmacokinetics, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Rd., P. O. Box 368, Ridgefield, CT 06877-0368, USA. mtaub@rdg.boehringeringelheim.com
authors.forename: Mitchell E
authors.initials: ME
authors.lastname: Taub
authors.forename: Lalitha
authors.initials: L
authors.lastname: Podila
authors.forename: Diane
authors.initials: D
authors.lastname: Ely
authors.forename: Iliana
authors.initials: I
authors.lastname: Almeida
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D018938:Caco-2 Cells; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D016764:Cell Polarity; D003078:Colchicine / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D004077:Digoxin / Q000378:metabolism*; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D018432:Drug Resistance, Multiple; D006801:Humans; D006854:Hydrocortisone / Q000494:pharmacology; D017239:Paclitaxel / Q000378:metabolism*; D011339:Probenecid / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014747:Vinblastine / Q000378:metabolism*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D014747:Vinblastine; D004077:Digoxin; D003907:Dexamethasone; D017239:Paclitaxel; D011339:Probenecid; D003078:Colchicine; D006854:Hydrocortisone