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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

P-glycoprotein-Mediated Efflux of Indinavir Metabolites in Caco-2 Cells Expressing Cytochrome P450 3A4

Jerome H. Hochman, Masato Chiba, Masayo Yamazaki, Cuyue Tang and Jiunn H. Lin
Journal of Pharmacology and Experimental Therapeutics July 2001, 298 (1) 323-330;
Jerome H. Hochman
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Masato Chiba
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Masayo Yamazaki
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Cuyue Tang
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Jiunn H. Lin
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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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Journal of Pharmacology and Experimental Therapeutics: 298 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 298, Issue 1
1 Jul 2001
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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

P-glycoprotein-Mediated Efflux of Indinavir Metabolites in Caco-2 Cells Expressing Cytochrome P450 3A4

Jerome H. Hochman, Masato Chiba, Masayo Yamazaki, Cuyue Tang and Jiunn H. Lin
Journal of Pharmacology and Experimental Therapeutics July 1, 2001, 298 (1) 323-330;

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Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

P-glycoprotein-Mediated Efflux of Indinavir Metabolites in Caco-2 Cells Expressing Cytochrome P450 3A4

Jerome H. Hochman, Masato Chiba, Masayo Yamazaki, Cuyue Tang and Jiunn H. Lin
Journal of Pharmacology and Experimental Therapeutics July 1, 2001, 298 (1) 323-330;
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