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Research ArticleArticle

Cytochrome P450 Fluorometric Substrates: Identification of Isoform-Selective Probes for Rat CYP2D2 and Human CYP3A4.

David M. Stresser, Stephanie D. Turner, Andrew P. Blanchard, Vaughn P. Miller and Charles L. Crespi
Drug Metabolism and Disposition July 2002, 30 (7) 845-852; DOI: https://doi.org/10.1124/dmd.30.7.845
David M. Stresser
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Stephanie D. Turner
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Andrew P. Blanchard
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Vaughn P. Miller
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Charles L. Crespi
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Abstract

We have tested a panel of 29 cDNA-expressed rat and human enzymes with 9 fluorometric substrates to determine the P450 isoform selectivity in the catalysis of the substrates to fluorescent products. The substrates examined were dibenzyl fluorescein, 7-benzyloxyquinoline (BQ), 3-cyano-7-ethoxycoumarin, 3-cyano-7-methoxycoumarin, 7-methoxy-4-trifluoromethylcoumarin, 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (AMMC), 3-[2-(N,N-diethyl-N-methylamino)ethyl]-7-methoxy-4-trifluoromethylcoumarin, 7-benzyloxyresorufin, and 7-benzyloxy-4-trifluoromethylcoumarin (BFC). For most substrates, multiple cDNA-expressed cytochrome P450 isoforms were found to catalyze the formation of the fluorescent product. However, among the combinations tested, rat CYP2D2 displayed high selectivity for AMMC demethylation (a substrate selective for CYP2D6 in human liver microsomes). AMMC demethylation activity was 15-fold lower in microsomes isolated from female Dark Agouti rats, a model known to have a low abundance of CYP2D2, and apparentKM values were similar for cDNA-expressed CYP2D2 and male Sprague-Dawley liver microsomes. BFC dealkylation and BQ dealkylation were selective but not exclusive for human CYP3A4. A small role for CYP1A2 could be demonstrated. The CYP3A4 selectivity in hepatic microsomes was supported by studies using chemical and antibody inhibitors and a correlation analysis within a panel of liver microsomes from individual donors. BQ demonstrated a higher degree of selectivity for and higher rates of metabolism by CYP3A than BFC. However, per unit enzyme the fluorescent signal is lower for BQ than BFC. AMMC, BQ, and BFC should find uses as enzyme-selective probe substrates.

Footnotes

  • A portion of this work was presented at the 6th International Society for the Study of Xenobiotics meeting; 2001 Oct 7–11; Munich, Germany.

  • Abbreviations used are::
    P450
    cytochrome P450
    RLM
    rat liver microsomes
    HLM
    human liver microsomes
    SD
    Sprague-Dawley
    DA
    Dark Agouti
    DBF
    dibenzyl fluorescein
    BQ
    7-benzyloxyquinoline
    AMMC
    3-[2-(N,N-diethyl-N-methylamino) ethyl]-7-methoxy-4-methylcoumarin
    MeAMFC
    3-[2-(N,N-diethyl-N-methylamino) ethyl]-7-methoxy-4-trifluoromethylcoumarin
    BFC
    7-benzyloxy-4-trifluoromethylcoumarin
    CEC
    7-ethoxy-3-cyanocoumarin
    CMC
    7-methoxy-3-cyanocoumarin
    MFC
    7-methoxy-4-trifluoromethylcoumarin
    BzRes
    7-benzyloxyresorufin
    E/S
    enzyme-substrate
    MAB
    monoclonal antibody
    • Received November 1, 2001.
    • Accepted April 11, 2002.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 30 (7)
Drug Metabolism and Disposition
Vol. 30, Issue 7
1 Jul 2002
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Research ArticleArticle

Cytochrome P450 Fluorometric Substrates: Identification of Isoform-Selective Probes for Rat CYP2D2 and Human CYP3A4.

David M. Stresser, Stephanie D. Turner, Andrew P. Blanchard, Vaughn P. Miller and Charles L. Crespi
Drug Metabolism and Disposition July 1, 2002, 30 (7) 845-852; DOI: https://doi.org/10.1124/dmd.30.7.845

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Research ArticleArticle

Cytochrome P450 Fluorometric Substrates: Identification of Isoform-Selective Probes for Rat CYP2D2 and Human CYP3A4.

David M. Stresser, Stephanie D. Turner, Andrew P. Blanchard, Vaughn P. Miller and Charles L. Crespi
Drug Metabolism and Disposition July 1, 2002, 30 (7) 845-852; DOI: https://doi.org/10.1124/dmd.30.7.845
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