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Research ArticleArticle

Broad Application of CYP3A4 Liquid Chromatography-Mass Spectrometry Protein Quantification in Hepatocyte Cytochrome P450 Induction Assays Identifies Nonuniformity in mRNA and Protein Induction Responses

John Paul Savaryn, Jun Sun, Junli Ma, Gary J. Jenkins and David M. Stresser
Drug Metabolism and Disposition February 2022, 50 (2) 105-113; DOI: https://doi.org/10.1124/dmd.121.000638
John Paul Savaryn
AbbVie Inc., DMPK-BA, North Chicago, Illinois
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Jun Sun
AbbVie Inc., DMPK-BA, North Chicago, Illinois
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Junli Ma
AbbVie Inc., DMPK-BA, North Chicago, Illinois
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Gary J. Jenkins
AbbVie Inc., DMPK-BA, North Chicago, Illinois
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David M. Stresser
AbbVie Inc., DMPK-BA, North Chicago, Illinois
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Abstract

Screening for cytochrome P450 (CYP) induction potential is routine in drug development. Induction results in a net increase in CYP protein and is assessed typically by measuring indirect endpoints, i.e., enzyme activity and mRNA in vitro. Recent methodological advancements have made CYP protein quantification by liquid chromatography-mass spectrometry in vitro induction studies more accessible and amenable to routine testing. In this study, we evaluated CYP3A4 concentration dependence of induction response for 11 compounds (rifampin, rifabutin, carbamazepine, efavirenz, nitrendipine, flumazenil, pioglitazone, rosiglitazone, troglitazone, pazopanib, and ticagrelor) in plated hepatocytes from two or three donors incorporating in the assessment all three endpoints. In addition, the time-dependence of the induction was examined over 1, 2, or 3 days of treatment. For most compounds, mRNA, enzyme activity, and protein endpoints exhibited similarity in induction responses. Pazopanib and ticagrelor were notable exceptions as neither protein nor enzyme activity were induced despite mRNA induction of a magnitude similar to efavirenz, pioglitazone, or rosiglitazone, which clearly induced in all three endpoints. Static modeling of clinical induction responses supported a role for protein as a predictive endpoint. These data highlight the value of including CYP protein quantification as an induction assay endpoint to provide a more comprehensive assessment of induction liability.

SIGNIFICANCE STATEMENT Direct, liquid chromatography-mass spectrometry (LC-MS)-based quantification of cytochrome P450 (CYP) protein is a desirable induction assay endpoint; however such application has been limited due to inefficient workflows. Here, we incorporate recent advancements in protein quantitation methods to efficiently quantify CYP3A4 protein in in vitro induction assays with 11 compounds in up to 3 donors. The data indicate induction responses from mRNA do not always align with those of protein suggesting assessment of induction liability is more complex than thought previously.

Footnotes

    • Received August 12, 2021.
    • Accepted November 30, 2021.
  • AbbVie sponsored and funded the study; contributed to the design; participated in the collection, analysis, and interpretation of data, and in writing, reviewing, and approval of the final publication.

  • All authors are or were employees of AbbVie at the time the work was conducted and may own AbbVie stock.

  • ↵1 J.P.S. and J.S. contributed equally to this work.

  • https://dx.doi.org/10.1124/dmd.120.000638.

  • ↵Embedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

  • Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 50 (2)
Drug Metabolism and Disposition
Vol. 50, Issue 2
1 Feb 2022
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Research ArticleArticle

Broad Profiling of the Three CYP3A4 Induction Endpoints

John Paul Savaryn, Jun Sun, Junli Ma, Gary J. Jenkins and David M. Stresser
Drug Metabolism and Disposition February 1, 2022, 50 (2) 105-113; DOI: https://doi.org/10.1124/dmd.121.000638

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Research ArticleArticle

Broad Profiling of the Three CYP3A4 Induction Endpoints

John Paul Savaryn, Jun Sun, Junli Ma, Gary J. Jenkins and David M. Stresser
Drug Metabolism and Disposition February 1, 2022, 50 (2) 105-113; DOI: https://doi.org/10.1124/dmd.121.000638
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