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 μM 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 μM 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.
Footnotes
- Abbreviations:
- Pgp
- P-glycoprotein
- CYP3A4
- cytochrome P450 3A4
- di-OH vit D3
- 1α, 25-di-hydroxyvitamin D3
- CsA
- cyclosporin A
- HBSS
- Hanks' balanced-salt solution
- MES
- 2-(N-morpholino)ethanesulfonic acid
- mAb
- monclonal antibody
- VBL
- vinblastine sulfate
- calcein AM
- calcein acetoxymethyl ester
- MRP
- multidrug resistance protein
- LC/MS
- liquid chromatography/mass spectrometry
- LC/MS/MS
- liquid chromatography/tandem mass spectrometry
- A-to-B
- apical-to-basolateral
- B-to-A
- basolateral-to-apical
- Received December 29, 2000.
- Accepted March 30, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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