title: P-glycoprotein-mediated efflux of indinavir metabolites in Caco-2 cells expressing cytochrome P450 3A4.
abstract: The role of P-glycoprotein in secretion of indinavir metabolites produced by CYP3A4 was evaluated in Caco-2 cells expressing CYP3A4. Metabolism of indinavir by CYP3A4 expressing Caco-2 cells grown on filters resulted in the formation of N-dealkylation products (M5 and M6) and hydroxylation of indinavir, which were preferentially secreted into the apical compartment. Apical secretion of the metabolites was inhibited by cyclosporin A (CsA) with all three classes of metabolites showing similar sensitivity to CsA, suggesting that they are all secreted by the same pathway. M6 stimulated P-glycoprotein (Pgp)-ATPase activity in a concentration-dependent manner. This stimulation was inhibited by the Pgp-specific monoclonal antibody C219. A method was developed to specifically inhibit Pgp using the monoclonal antibody UIC2 to determine whether Pgp efflux accounts for a significant proportion of the apical secretion of indinavir metabolites. UIC2 recognizes an extracellular transient conformational epitope that is stabilized by some Pgp substrates or by ATP depletion. Incubation of Caco-2 cells with UIC2 in the presence of 1 microM CsA resulted in 50 to 80% inhibition of Pgp-mediated vinblastine efflux, with no significant inhibition observed by UIC2 or CsA alone. Inhibition of Pgp in CYP3A4-expressing Caco-2 cells by UIC2 and 1 microM CsA resulted in a significant decrease in the apical secretion of M6, M5, and OH-indinavir and an increase in the amount of the metabolites secreted in the basolateral compartment and retained in the cytosol. These results are consistent with a role of Pgp in elimination of CYP3A4-generated metabolites and indicate that even relatively polar metabolites may be secreted from the cell by Pgp.
authors.affiliation: Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania, USA. jerome_hochman@merck.com
authors.forename: J H
authors.initials: JH
authors.lastname: Hochman
authors.forename: M
authors.initials: M
authors.lastname: Chiba
authors.forename: M
authors.initials: M
authors.lastname: Yamazaki
authors.forename: C
authors.initials: C
authors.lastname: Tang
authors.forename: J H
authors.initials: JH
authors.lastname: Lin
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000502:physiology*; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases; D018938:Caco-2 Cells / Q000187:drug effects / Q000378:metabolism*; D016572:Cyclosporine / Q000494:pharmacology; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017320:HIV Protease Inhibitors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D019469:Indinavir / Q000737:chemistry / Q000378:metabolism*; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D017320:HIV Protease Inhibitors; D019469:Indinavir; D016572:Cyclosporine; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating