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Research ArticleArticle

Role of a Potent Inhibitory Monoclonal Antibody to Cytochrome P-450 3A4 in Assessment of Human Drug Metabolism

Qin Mei, Cuyue Tang, Carol Assang, Yuh Lin, Donald Slaughter, A. David Rodrigues, Thomas A. Baillie, Thomas H. Rushmore and Magang Shou
Journal of Pharmacology and Experimental Therapeutics November 1999, 291 (2) 749-759;
Qin Mei
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Cuyue Tang
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Carol Assang
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Yuh Lin
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Donald Slaughter
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A. David Rodrigues
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Thomas A. Baillie
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Thomas H. Rushmore
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Magang Shou
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Abstract

Cytochrome P-450 (CYP) 3A4 is an inordinately important CYP enzyme that catalyzes the metabolism of a vast array of clinically used drugs. Microsomal proteins of Spodoptera frugiperda (Sf21) insect cells infected with recombinant baculoviruses encoding CYP3A4 cDNA were used to immunize mice and to develop a monoclonal antibody (mAb3A4a) specific to CYP3A4 through the use of hybridoma technology. The mAb is both a potent inhibitor and a strong binder of CYP3A4. One and 5 μl (0.5 and 2.5 μM IgG2a) of the mAb mouse ascites in 1-ml incubation containing 20 pmol of CYP3A4 strongly inhibited the testosterone 6β-hydroxylation by 95 and 99%, respectively, and, to a lesser extent, cross-inhibited CYP3A5 and CYP3A7 activity. mAb3A4a exhibited no cross-reactivity with any of the other recombinant human CYP isoforms (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1) in the course of CYP reaction phenotyping and Western immunoblot analyses. The potency of mAb-induced inhibition is insensitive to substrate concentration in human liver microsomes. Therefore, mAb3A4awas used to assess the quantitative role of CYP3A4/5 to the metabolism of testosterone and diazepam in five human liver microsomes. The results showed that CYP3A4 and CYP3A5 contribute >95% to both testosterone 6β-hydroxylation and diazepam 3-hydroxylation and 52 to 73% to diazepam N-demethylation, respectively. In addition, mAb3A4a significantly inhibited testosterone 6β-hydroxylase activity in rhesus monkey liver microsomes to a degree equal to that observed with CYP3A4 in human liver microsomes. By comparison, no inhibition of testosterone 6β-hydroxylase activity was observed in the presence of dog, rat, and mouse liver microsomes. The selectivity of ketoconazole, a chemical inhibitor of CYP3A4, was probed with mAb3A4a and was shown to be highly concentration dependent in the diazepam N-demethylation by human liver microsomes. The results demonstrate that inhibitory and immunoblotting mAb3A4a can offer a precise and useful tool for quantitative identification of CYP3A4/5 in the metabolism of drugs in clinical use and drugs in development.

Footnotes

  • Send reprint requests to: Dr. Qin Mei, Department of Drug Metabolism, Merck Research Laboratories, WP75A-203, West Point, PA 19486. E-mail: qin_mei{at}merck.com

  • Abbreviations:
    CYP
    cytochrome P-450
    OR
    cytochrome P-450 oxidoreductase
    mAb
    monoclonal antibody
    DDC
    diethyldithiocarbamate
    DZ
    diazepam
    TMZ
    temazepam
    NDZ
    nordiazepam
    B[a]P t-4,5-diol,benzo[a]pyrene trans-4,5-dihydrodiol
    BA t-5,6-diol, benz[a]anthracenetrans-5,6-dihydrodiol
    m.o.i.
    multiplicity of infection
    KPi
    potassium phosphate buffer
    ELISA
    enzyme-linked immunosorbent assay
    IgG (H + L)
    IgG (H chain + L chain)
    • Received May 18, 1999.
    • Accepted July 25, 1999.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 291 (2)
Journal of Pharmacology and Experimental Therapeutics
Vol. 291, Issue 2
1 Nov 1999
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Research ArticleArticle

Role of a Potent Inhibitory Monoclonal Antibody to Cytochrome P-450 3A4 in Assessment of Human Drug Metabolism

Qin Mei, Cuyue Tang, Carol Assang, Yuh Lin, Donald Slaughter, A. David Rodrigues, Thomas A. Baillie, Thomas H. Rushmore and Magang Shou
Journal of Pharmacology and Experimental Therapeutics November 1, 1999, 291 (2) 749-759;

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Research ArticleArticle

Role of a Potent Inhibitory Monoclonal Antibody to Cytochrome P-450 3A4 in Assessment of Human Drug Metabolism

Qin Mei, Cuyue Tang, Carol Assang, Yuh Lin, Donald Slaughter, A. David Rodrigues, Thomas A. Baillie, Thomas H. Rushmore and Magang Shou
Journal of Pharmacology and Experimental Therapeutics November 1, 1999, 291 (2) 749-759;
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