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Research ArticleArticle

Metabolite Formation Kinetics and Intrinsic Clearance of Phenacetin, Tolbutamide, Alprazolam, and Midazolam in Adenoviral Cytochrome P450-Transfected HepG2 Cells and Comparison with Hepatocytes and In Vivo

M. Teresa Donato, David Hallifax, Laura Picazo, José V. Castell, J. Brian Houston, M. José Gomez-Lechón and Agustin Lahoz
Drug Metabolism and Disposition September 2010, 38 (9) 1449-1455; DOI: https://doi.org/10.1124/dmd.110.033605
M. Teresa Donato
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David Hallifax
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Laura Picazo
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José V. Castell
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J. Brian Houston
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M. José Gomez-Lechón
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Agustin Lahoz
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Abstract

Cryopreserved human hepatocytes and other in vitro systems often underpredict in vivo intrinsic clearance (CLint). The aim of this study was to explore the potential utility of HepG2 cells transduced with adenovirus vectors expressing a single cytochrome P450 enzyme (Ad-CYP1A2, Ad-CYP2C9, or Ad-CYP3A4) for metabolic clearance predictions. The kinetics of metabolite formation from phenacetin, tolbutamide, and alprazolam and midazolam, selected as substrates probes for CYP1A2, CYP2C9, and CYP3A4, respectively, were characterized in this in vitro system. The magnitude of the Km or S50 values observed in Ad-P450 cells was similar to those found in the literature for other human liver-derived systems. For each substrate, CLint (or CLmax), values from Ad-P450 systems were scaled to human hepatocytes in primary culture using the relative activity factor (RAF) approach. Scaled Ad-P450 CLint values were approximately 3- to 6-fold higher (for phenacetin O-deethylation, tolbutamide 4-hydroxylation, and alprazolam 4-hydroxyaltion) or lower (midazolam 1′-hydroxylation) than those reported for human cryopreserved hepatocytes in suspension. Comparison with the in vivo data reveals that Ad-P450 cells provide a favorable prediction of CLint for the substrates studied (in a range of 20–200% in vivo observed CLint). This is an improvement compared with the consistent underpredictions (<10–50% in in vivo observed CLint) found in cryopreserved hepatocyte studies with the same substrates. These results suggest that the Ad-P450 cell is a promising in vitro system for clearance predictions of P450-metabolized drugs.

Footnotes

  • This work was supported in part by the Fundacion Valenciana para el Desarrollo de Modelos Alternativos ala Experimentación Animal and the European Commission [Grants LSHB-CT-2004-512051, LSSB-CT-2005-037499]; and the Spanish Ministry of Science and Innovation/Instituto de Salud Carlos III [Miguel Server Contract CP08/00125] (to A.L.).

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

    doi:10.1124/dmd.110.033605.

  • ABBREVIATIONS:

    HLM
    human liver microsomes
    P450
    cytochrome P450
    Ad-P450
    adenovirus expressing P450
    PCR
    polymerase chain reaction
    CMV
    cytomegalovirus
    MOI
    multiplicity of infection
    GFP
    green fluorescence protein
    RAF
    relative activity factor.

  • Received March 30, 2010.
  • Accepted May 25, 2010.
  • Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 38 (9)
Drug Metabolism and Disposition
Vol. 38, Issue 9
1 Sep 2010
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Research ArticleArticle

Metabolite Formation Kinetics and Intrinsic Clearance of Phenacetin, Tolbutamide, Alprazolam, and Midazolam in Adenoviral Cytochrome P450-Transfected HepG2 Cells and Comparison with Hepatocytes and In Vivo

M. Teresa Donato, David Hallifax, Laura Picazo, José V. Castell, J. Brian Houston, M. José Gomez-Lechón and Agustin Lahoz
Drug Metabolism and Disposition September 1, 2010, 38 (9) 1449-1455; DOI: https://doi.org/10.1124/dmd.110.033605

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Research ArticleArticle

Metabolite Formation Kinetics and Intrinsic Clearance of Phenacetin, Tolbutamide, Alprazolam, and Midazolam in Adenoviral Cytochrome P450-Transfected HepG2 Cells and Comparison with Hepatocytes and In Vivo

M. Teresa Donato, David Hallifax, Laura Picazo, José V. Castell, J. Brian Houston, M. José Gomez-Lechón and Agustin Lahoz
Drug Metabolism and Disposition September 1, 2010, 38 (9) 1449-1455; DOI: https://doi.org/10.1124/dmd.110.033605
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