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Research ArticleArticle

AUTOMATED SCREENING WITH CONFIRMATION OF MECHANISM-BASED INACTIVATION OF CYP3A4, CYP2C9, CYP2C19, CYP2D6, AND CYP1A2 IN POOLED HUMAN LIVER MICROSOMES

Heng-Keang Lim, Nicholas Duczak Jr., Linda Brougham, Michael Elliot, Krupa Patel and Kelvin Chan
Drug Metabolism and Disposition August 2005, 33 (8) 1211-1219; DOI: https://doi.org/10.1124/dmd.104.003475
Heng-Keang Lim
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Nicholas Duczak Jr.
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Linda Brougham
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Michael Elliot
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Krupa Patel
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Kelvin Chan
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Abstract

A strategy is proposed to profile compounds for mechanism-based inactivation of CYP3A4, CYP2C19, CYP2C9, CYP2D6, and CYP1A2 based on an apparent partition ratio screen. Potent positives from the screen are confirmed by time- and concentration-dependent inactivation assays. Quasi-irreversible inhibitions are then differentiated from irreversible inactivations by oxidation with potassium ferricyanide and/or dialysis. The three-step screening procedure has been validated with acceptable accuracy and precision for detection and confirmation of mechanism-based inactivators in drug discovery. We report here the apparent partition ratios for 19 mechanism-based inactivators and four quasi-irreversible inhibitors obtained under the same experimental conditions. The apparent partition ratio screen was automated to provide throughput for determining structure-mechanism-based inactivation relationships. Information about reversibility can be used to assess potential toxicity mediated by covalent adducts, as well as the potential for pharmacokinetic drug-drug interactions. Direct comparison of known mechanism-based inactivators and quasi-irreversible inhibitors, based on our screening of apparent partition ratios, has identified ritonavir, mibefradil, and azamulin as highly effective mechanism-based inactivators; e.g., 1 mol of CYP3A4 was inactivated on turnover of about 2 mol of compound. Other mechanism-based inactivators we identified include bergamottin (CYP1A2 besides previously reported CYP3A4), troglitazone (CYP3A4), rosiglitazone (CYP3A4), and pioglitazone (CYP3A4). Comparison of the apparent partition ratios and inactivation clearance data for the three glitazones suggests that the chromane moiety on troglitazone contributes to its greater potency for mechanism-based inactivation.

Footnotes

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

  • doi:10.1124/dmd.104.003475.

  • ABBREVIATIONS: DDI, drug-drug interaction; P450, cytochrome P450; HLM, human liver microsome; MBI, mechanism-based inactivator; LC/MS/MS, liquid chromatography/tandem mass spectrometry; APR, apparent partition ratio; EMTPP, 1-[(2-ethyl-4-methyl-1H-imidazol-5-yl)methyl]-4-[4-(trifluoromethyl)-2-pyridinyl]piperazine; HPLC, high-performance liquid chromatography; MI, metabolite-intermediate.

  • ↵ Embedded Image The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.

    • Received December 23, 2004.
    • Accepted April 27, 2005.
  • The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 33 (8)
Drug Metabolism and Disposition
Vol. 33, Issue 8
1 Aug 2005
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AUTOMATED SCREENING WITH CONFIRMATION OF MECHANISM-BASED INACTIVATION OF CYP3A4, CYP2C9, CYP2C19, CYP2D6, AND CYP1A2 IN POOLED HUMAN LIVER MICROSOMES
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Research ArticleArticle

AUTOMATED SCREENING WITH CONFIRMATION OF MECHANISM-BASED INACTIVATION OF CYP3A4, CYP2C9, CYP2C19, CYP2D6, AND CYP1A2 IN POOLED HUMAN LIVER MICROSOMES

Heng-Keang Lim, Nicholas Duczak, Linda Brougham, Michael Elliot, Krupa Patel and Kelvin Chan
Drug Metabolism and Disposition August 1, 2005, 33 (8) 1211-1219; DOI: https://doi.org/10.1124/dmd.104.003475

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Research ArticleArticle

AUTOMATED SCREENING WITH CONFIRMATION OF MECHANISM-BASED INACTIVATION OF CYP3A4, CYP2C9, CYP2C19, CYP2D6, AND CYP1A2 IN POOLED HUMAN LIVER MICROSOMES

Heng-Keang Lim, Nicholas Duczak, Linda Brougham, Michael Elliot, Krupa Patel and Kelvin Chan
Drug Metabolism and Disposition August 1, 2005, 33 (8) 1211-1219; DOI: https://doi.org/10.1124/dmd.104.003475
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