P-glycoprotein-mediated efflux of indinavir metabolites in Caco-2 cells expressing cytochrome P450 3A4

J Pharmacol Exp Ther. 2001 Jul;298(1):323-30.

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.

MeSH terms

  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / antagonists & inhibitors
  • ATP Binding Cassette Transporter, Subfamily B, Member 1 / physiology*
  • Antibodies, Monoclonal / pharmacology
  • Aryl Hydrocarbon Hydroxylases*
  • Caco-2 Cells / drug effects
  • Caco-2 Cells / metabolism*
  • Cyclosporine / pharmacology
  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 Enzyme System / metabolism*
  • Enzyme Inhibitors / pharmacology
  • HIV Protease Inhibitors / chemistry
  • HIV Protease Inhibitors / metabolism*
  • Humans
  • Indinavir / chemistry
  • Indinavir / metabolism*
  • Oxidoreductases, N-Demethylating / metabolism*

Substances

  • ATP Binding Cassette Transporter, Subfamily B, Member 1
  • Antibodies, Monoclonal
  • Enzyme Inhibitors
  • HIV Protease Inhibitors
  • Indinavir
  • Cyclosporine
  • Cytochrome P-450 Enzyme System
  • Aryl Hydrocarbon Hydroxylases
  • Cytochrome P-450 CYP3A
  • Oxidoreductases, N-Demethylating