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Rapid CommunicationShort Communication

[O-ETHYL 14C]PHENACETINO-DEETHYLASE ACTIVITY IN HUMAN LIVER MICROSOMES

A. David Rodrigues, Bruce W. Surber, Ye Yao, Shekman L. Wong and Ellen M. Roberts
Drug Metabolism and Disposition September 1997, 25 (9) 1097-1100;
A. David Rodrigues
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Bruce W. Surber
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Ye Yao
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Shekman L. Wong
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Ellen M. Roberts
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Abstract

The activity of human liver microsomal cytochrome P450 1A2 (CYP1A2) is readily estimated by following the O-deethylation of [O-ethyl 14C]phenacetin (PODase). The basis of the assay is the quantitative measurement of [14C]acetaldehyde, remaining in the supernatant of assay incubates, after extraction of unmetabolized [O-ethyl 14C]phenacetin with charcoal. In the presence of native human liver microsomes (Km = 54 ± 27 μM;Vmax = 14 ± 2.3 nmol/hr/mg; mean ± SD; N = 3 different livers) and human B-lymphoblastoid cell microsomes containing cDNA-expressed CYP1A2 (Km = 46 μM;Vmax = 55 nmol/hr/nmol CYP), PODase activity conformed to monophasic Michaelis-Menten kinetics. Furthermore, PODase activity in a panel of microsomes prepared from a series of human livers was significantly correlated (r= 0.91; p < 0.001; N = 11) with CYP1A2-selective 7-ethoxyresorufin O-deethylase activity, and was markedly inhibited (≥ 92%) by furafylline (FURA, IC50 = 0.4 μM) and 7,8-benzoflavone (ANF, IC50 = 0.1 μM), two well known CYP1A2 inhibitors. Inhibitors selective for other forms of CYP (e.g. CYP3A, CYP2C, CYP2D6, CYP2E1) elicited a marginal effect (≤ 17% inhibition) at relatively high concentrations (≥ 10·Ki ). It is concluded that the inhibition of human liver microsomal CYP1A2 activity can be readily determined by using a charcoal-based radiometric method employing [O-ethyl 14C]phenacetin as substrate.

Footnotes

  • Send reprint requests to: A. David Rodrigues, Ph.D., Drug Metabolism I, Merck Research Laboratories, Sumneytown Pike, P. O. Box 4, WP26-A 2044, West Point, PA 19486-0004.

  • ↵2 Nominal molar ratio of NADPH-CYP reductase (∼15 pmol/mg) to CYP1A2 (133 pmol CYP/mg) is 1:9.

  • ↵3 At a final phenacetin concentration of 100 μM, PODase activity varied from 2.2-21.2 nmol/hr/mg (8.4 ± 5.6 nmol/hr/mg; mean ± SD; N = 11 livers).

  • ↵4 No PODase activity was detected when [O-ethyl 14C]phenacetin (40 μM; ∼ Km) was incubated with human B-lymphoblastoid microsomes (Gentest Corp.) containing cDNA-expressed CYP2A6 (A. D. Rodrigues, unpublished results).

  • Abbreviations used are::
    CYP
    cytochrome P450
    PODase
    [O-ethyl 14C]phenacetinO-deethylase
    ANF
    7, 8-benzoflavone
    FURA
    furafylline
    COUM
    coumarin
    IC50
    concentration of drug required to inhibit activity by 50%
    Ki
    apparent inhibition constant (dissociation constant of the enzyme-inhibitor, or EI, complex)
    Km
    apparent Michaelis constant
    Vmax
    apparent maximal initial reaction velocity
    • Received February 24, 1997.
    • Accepted May 1, 1997.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition
Vol. 25, Issue 9
1 Sep 1997
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Rapid CommunicationShort Communication

[O-ETHYL 14C]PHENACETINO-DEETHYLASE ACTIVITY IN HUMAN LIVER MICROSOMES

A. David Rodrigues, Bruce W. Surber, Ye Yao, Shekman L. Wong and Ellen M. Roberts
Drug Metabolism and Disposition September 1, 1997, 25 (9) 1097-1100;

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[O-ETHYL 14C]PHENACETINO-DEETHYLASE ACTIVITY IN HUMAN LIVER MICROSOMES

A. David Rodrigues, Bruce W. Surber, Ye Yao, Shekman L. Wong and Ellen M. Roberts
Drug Metabolism and Disposition September 1, 1997, 25 (9) 1097-1100;
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