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Research ArticleArticle

Midazolam Metabolism by Modified Caco-2 Monolayers: Effects of Extracellular Protein Binding

Jeannine M. Fisher, Steven A. Wrighton, Justina C. Calamia, Danny D. Shen, Kent L. Kunze and Kenneth E. Thummel
Journal of Pharmacology and Experimental Therapeutics May 1999, 289 (2) 1143-1150;
Jeannine M. Fisher
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Steven A. Wrighton
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Justina C. Calamia
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Danny D. Shen
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Kent L. Kunze
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Kenneth E. Thummel
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Abstract

It has been suggested that the binding of a drug to plasma proteins will influence the intestinal extraction efficiency when drug is delivered to the mucosal epithelium via either the gut lumen or vasculature. We evaluated this hypothesis using cytochrome P-450 (CYP)3A4-expressing Caco-2 monolayers as a model for the intestinal epithelial barrier and midazolam as a CYP3A-specific enzyme probe. The rate of 1′-hydroxylation was measured following apical or basolateral midazolam administration to monolayers incubated in the presence or absence of 4 g/dl of human serum albumin (HSA) in the basolateral compartment medium. The midazolam-free fraction in culture medium containing HSA was 3.3%. Inclusion of HSA in the basolateral medium decreased peak intracellular midazolam accumulation after an apical midazolam dose (3 μM) by 35% and reduced the 1′-hydroxymidazolam formation rate by ∼20%. Because of the accelerated diffusion of midazolam through the cell monolayer and into the basolateral compartment, there was a 61% reduction in the first-pass metabolic extraction ratio: 13.3 ± 0.12% for control versus 5.2 ± 1% with HSA. Compared with control, addition of HSA resulted in a 91% decrease in the peak intracellular midazolam level and a 86% decrease in the rate of 1′-hydroxylation after the administration of midazolam into basolateral medium. These findings suggest that, in vivo, binding of a drug to plasma proteins will impact both first-pass and systemic intestinal midazolam extraction efficiency. Furthermore, the effect will be more pronounced for a drug that is delivered to mucosal enterocytes by way of arterial blood, compared with oral drug delivery.

Footnotes

  • Send reprint requests to: Kenneth E. Thummel, Ph.D., Department of Pharmaceutics, Box 357610, University of Washington, Seattle, WA 98195-7610. E-mail:thummel{at}u.washington.edu

  • ↵1 This work was funded in part by Eli Lilly & Co. and National Institutes of Health Grant GM 32165.

  • Abbreviations:
    CYP
    cytochrome P-450
    MDZ
    midazolam
    1′-OH-MDZ
    1′-hydroxymidazolam
    1α
    25-(OH)2-D3, 1α,25-dihydroxy vitamin D3
    FBS
    fetal bovine serum
    HSA
    human serum albumin
    DMEM
    Dulbecco’s modified Eagle medium
    DM
    differentiation medium
    TEER
    transepithelial electrical resistance
    DMSO
    dimethyl sulfoxide
    ER
    extraction ratio
    • Received October 19, 1998.
    • Accepted January 16, 1999.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 289 (2)
Journal of Pharmacology and Experimental Therapeutics
Vol. 289, Issue 2
1 May 1999
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Research ArticleArticle

Midazolam Metabolism by Modified Caco-2 Monolayers: Effects of Extracellular Protein Binding

Jeannine M. Fisher, Steven A. Wrighton, Justina C. Calamia, Danny D. Shen, Kent L. Kunze and Kenneth E. Thummel
Journal of Pharmacology and Experimental Therapeutics May 1, 1999, 289 (2) 1143-1150;

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Research ArticleArticle

Midazolam Metabolism by Modified Caco-2 Monolayers: Effects of Extracellular Protein Binding

Jeannine M. Fisher, Steven A. Wrighton, Justina C. Calamia, Danny D. Shen, Kent L. Kunze and Kenneth E. Thummel
Journal of Pharmacology and Experimental Therapeutics May 1, 1999, 289 (2) 1143-1150;
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