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Research ArticleArticle

Selective Mechanism-Based Inactivation of CYP3A4 by CYP3cide (PF-04981517) and Its Utility as an In Vitro Tool for Delineating the Relative Roles of CYP3A4 versus CYP3A5 in the Metabolism of Drugs

Robert L. Walsky, R. Scott Obach, Ruth Hyland, Ping Kang, Sue Zhou, Michael West, Kieran F. Geoghegan, Christopher J. Helal, Gregory S. Walker, Theunis C. Goosen and Michael A. Zientek
Drug Metabolism and Disposition September 2012, 40 (9) 1686-1697; DOI: https://doi.org/10.1124/dmd.112.045302
Robert L. Walsky
Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Groton, CT (R.L.W., R.S.O., M.W., K.F.G., C.J.H., G.S.W., T.C.G.); Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., San Diego, CA (S.Z., P.K., M.A.Z.); and Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Sandwich, United Kingdom (R.H.)
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R. Scott Obach
Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Groton, CT (R.L.W., R.S.O., M.W., K.F.G., C.J.H., G.S.W., T.C.G.); Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., San Diego, CA (S.Z., P.K., M.A.Z.); and Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Sandwich, United Kingdom (R.H.)
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Ruth Hyland
Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Groton, CT (R.L.W., R.S.O., M.W., K.F.G., C.J.H., G.S.W., T.C.G.); Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., San Diego, CA (S.Z., P.K., M.A.Z.); and Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Sandwich, United Kingdom (R.H.)
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Ping Kang
Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Groton, CT (R.L.W., R.S.O., M.W., K.F.G., C.J.H., G.S.W., T.C.G.); Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., San Diego, CA (S.Z., P.K., M.A.Z.); and Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Sandwich, United Kingdom (R.H.)
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Sue Zhou
Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Groton, CT (R.L.W., R.S.O., M.W., K.F.G., C.J.H., G.S.W., T.C.G.); Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., San Diego, CA (S.Z., P.K., M.A.Z.); and Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Sandwich, United Kingdom (R.H.)
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Michael West
Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Groton, CT (R.L.W., R.S.O., M.W., K.F.G., C.J.H., G.S.W., T.C.G.); Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., San Diego, CA (S.Z., P.K., M.A.Z.); and Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Sandwich, United Kingdom (R.H.)
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Kieran F. Geoghegan
Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Groton, CT (R.L.W., R.S.O., M.W., K.F.G., C.J.H., G.S.W., T.C.G.); Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., San Diego, CA (S.Z., P.K., M.A.Z.); and Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Sandwich, United Kingdom (R.H.)
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Christopher J. Helal
Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Groton, CT (R.L.W., R.S.O., M.W., K.F.G., C.J.H., G.S.W., T.C.G.); Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., San Diego, CA (S.Z., P.K., M.A.Z.); and Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Sandwich, United Kingdom (R.H.)
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Gregory S. Walker
Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Groton, CT (R.L.W., R.S.O., M.W., K.F.G., C.J.H., G.S.W., T.C.G.); Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., San Diego, CA (S.Z., P.K., M.A.Z.); and Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Sandwich, United Kingdom (R.H.)
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Theunis C. Goosen
Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Groton, CT (R.L.W., R.S.O., M.W., K.F.G., C.J.H., G.S.W., T.C.G.); Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., San Diego, CA (S.Z., P.K., M.A.Z.); and Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Sandwich, United Kingdom (R.H.)
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Michael A. Zientek
Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Groton, CT (R.L.W., R.S.O., M.W., K.F.G., C.J.H., G.S.W., T.C.G.); Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., San Diego, CA (S.Z., P.K., M.A.Z.); and Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Pfizer, Inc., Sandwich, United Kingdom (R.H.)
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Abstract

CYP3cide (PF-4981517; 1-methyl-3-[1-methyl-5-(4-methylphenyl)-1H-pyrazol-4-yl]-4-[(3S)-3-piperidin-1-ylpyrrolidin-1-yl]-1H-pyrazolo[3,4-d]pyrimidine) is a potent, efficient, and specific time-dependent inactivator of human CYP3A4. When investigating its inhibitory properties, an extreme metabolic inactivation efficiency (kinact/KI) of 3300 to 3800 ml · min−1 · μmol−1 was observed using human liver microsomes from donors of nonfunctioning CYP3A5 (CYP3A5 *3/*3). This observed efficiency equated to an apparent KI between 420 and 480 nM with a maximal inactivation rate (kinact) equal to 1.6 min−1. Similar results were achieved with testosterone, another CYP3A substrate, and other sources of the CYP3A4 enzyme. To further illustrate the abilities of CYP3cide, its partition ratio of inactivation was determined with recombinant CYP3A4. These studies produced a partition ratio approaching unity, thus underscoring the inactivation capacity of CYP3cide. When CYP3cide was tested at a concentration and preincubation time to completely inhibit CYP3A4 in a library of genotyped polymorphic CYP3A5 microsomes, the correlation of the remaining midazolam 1′-hydroxylase activity to CYP3A5 abundance was significant (R2 value equal to 0.51, p value of <0.0001). The work presented here supports these findings by fully characterizing the inhibitory properties and exploring CYP3cide's mechanism of action. To aid the researcher, multiple commercially available sources of CYP3cide were established, and a protocol was developed to quantitatively determine CYP3A4 contribution to the metabolism of an investigational compound. Through the establishment of this protocol and the evidence provided here, we believe that CYP3cide is a very useful tool for understanding the relative roles of CYP3A4 versus CYP3A5 and the impact of CYP3A5 genetic polymorphism on a compound's pharmacokinetics.

Footnotes

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

    http://dx.doi.org/10.1124/dmd.112.045302.

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

  • ABBREVIATIONS:

    P450
    cytochrome P450
    CYP3cide
    1-methyl-3-[1-methyl-5-(4-methylphenyl)-1H-pyrazol-4-yl]-4-[(3S)-3-piperidin-1-ylpyrrolidin-1-yl]-1H-pyrazolo[3,4-d]pyrimidine
    HLM
    human liver microsomes
    POR
    P450 oxidoreductase
    r
    recombinant
    amu
    atomic mass units
    KCN
    potassium cyanide
    MS
    mass spectrometry
    LC
    liquid chromatography
    HPLC
    high-performance liquid chromatography
    ESI
    electrospray ionization
    NMR
    nuclear magnetic resonance
    DMSO
    dimethyl sulfoxide.

  • Received March 6, 2012.
  • Accepted May 29, 2012.
  • Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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Drug Metabolism and Disposition: 40 (9)
Drug Metabolism and Disposition
Vol. 40, Issue 9
1 Sep 2012
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Research ArticleArticle

SELECTIVE INHIBITION OF CYP3A4 BY CYP3cide

Robert L. Walsky, R. Scott Obach, Ruth Hyland, Ping Kang, Sue Zhou, Michael West, Kieran F. Geoghegan, Christopher J. Helal, Gregory S. Walker, Theunis C. Goosen and Michael A. Zientek
Drug Metabolism and Disposition September 1, 2012, 40 (9) 1686-1697; DOI: https://doi.org/10.1124/dmd.112.045302

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Research ArticleArticle

SELECTIVE INHIBITION OF CYP3A4 BY CYP3cide

Robert L. Walsky, R. Scott Obach, Ruth Hyland, Ping Kang, Sue Zhou, Michael West, Kieran F. Geoghegan, Christopher J. Helal, Gregory S. Walker, Theunis C. Goosen and Michael A. Zientek
Drug Metabolism and Disposition September 1, 2012, 40 (9) 1686-1697; DOI: https://doi.org/10.1124/dmd.112.045302
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