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Research ArticleArticle

SUBSTRATE SPECIFICITY AND KINETIC PROPERTIES OF SEVEN HETEROLOGOUSLY EXPRESSED DOG CYTOCHROMES P450

Magang Shou, Ryan Norcross, Grit Sandig, Ping Lu, Yinghe Li, Yuh Lin, Qin Mei, A. David Rodrigues and Thomas H. Rushmore
Drug Metabolism and Disposition September 2003, 31 (9) 1161-1169; DOI: https://doi.org/10.1124/dmd.31.9.1161
Magang Shou
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Ryan Norcross
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Grit Sandig
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Ping Lu
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Yinghe Li
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Yuh Lin
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Qin Mei
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A. David Rodrigues
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Thomas H. Rushmore
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Abstract

Seven dog cytochromes P450 (P450s) were heterologously expressed in baculovirus-Sf21 insect cells. Of all enzymes examined, CYP1A1 exhibited high 7-ethoxyresorufin O-deethylase activity (low Km enzyme, 1 μM). CYP2B11 and CYP3A12 effectively catalyzed the N1-demethylation and C3-hydroxylation of diazepam (and its derivatives), whereas CYP3A12 and CYP2D15 catalyzed exclusively the N- and O-demethylation, respectively, of dextromethorphan. However, no saturation velocity curves for the N-demethylation of dextromethorphan (up to 500 μM) were achieved, suggesting a high Km for CYP3A12. In contrast to CYP3A12, the CYP2D15-dependent O-demethylation of dextromethorphan was a low Km process (Km = 0.7 μM), similar to that in dog liver microsomes (Km = 2.3 μM). CYP2D15 was also capable of metabolizing bufuralol (1′-hydroxylation), with a Km of 3.9 μM, consistent with that obtained with dog liver microsomes. CYP3A12 was shown to primarily oxidize testosterone at 16α-, 2α/2β-, and 6β-positions. Selectivity of CYP3A12 was observed toward testosterone 6β-(Km = 83 μM) and 2α/2β-hydroxylations (Km = 154 μM). However, the 16α-hydroxylation of testosterone was catalyzed by CYP2C21 also (Km = 6.4 μM for CYP2C21). Therefore, the 6β- and 16α-hydroxylation of testosterone can potentially be employed as markers of CYP3A12 and CYP2C21 (at low concentration), respectively. CYP2C21 was also capable of catalyzing diclofenac 4′-hydroxylation, although some activity was detected with CYP2B11. Surprisingly, none of the P450s selectively metabolized (S)-mephenytoin 4′-hydroxylation. The results described herein are a first step toward the systematic evaluation of a panel of dog P450s and the development of dog P450 isoenzyme-selective marker substrates, as well as providing useful information on prediction and extrapolation of the results from in vitro to in vivo and from dog to human.

Footnotes

  • ↵1 Abbreviations used are: P450, cytochrome P450; OR, cytochrome P450 oxidoreductase; DZ, diazepam; TMZ, temazepam; NDZ, nordazepam; OX, oxazepam; PCR, polymerase chain reaction; SFM, serum-free medium; Sf21, Spodoptera frugiperda insect cells; MOI, multiplicity of infection; HPLC, high-performance liquid chromatography; LC/MS-MS, liquid chromatography-tandem mass spectrometry; DLM, dog liver microsome; HLM, human liver microsome.

    • Received April 2, 2003.
    • Accepted May 15, 2003.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 31 (9)
Drug Metabolism and Disposition
Vol. 31, Issue 9
1 Sep 2003
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Research ArticleArticle

SUBSTRATE SPECIFICITY AND KINETIC PROPERTIES OF SEVEN HETEROLOGOUSLY EXPRESSED DOG CYTOCHROMES P450

Magang Shou, Ryan Norcross, Grit Sandig, Ping Lu, Yinghe Li, Yuh Lin, Qin Mei, A. David Rodrigues and Thomas H. Rushmore
Drug Metabolism and Disposition September 1, 2003, 31 (9) 1161-1169; DOI: https://doi.org/10.1124/dmd.31.9.1161

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Research ArticleArticle

SUBSTRATE SPECIFICITY AND KINETIC PROPERTIES OF SEVEN HETEROLOGOUSLY EXPRESSED DOG CYTOCHROMES P450

Magang Shou, Ryan Norcross, Grit Sandig, Ping Lu, Yinghe Li, Yuh Lin, Qin Mei, A. David Rodrigues and Thomas H. Rushmore
Drug Metabolism and Disposition September 1, 2003, 31 (9) 1161-1169; DOI: https://doi.org/10.1124/dmd.31.9.1161
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