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Molecular Pharmacology

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Research ArticleArticle

Characterization of Ritonavir-Mediated Inactivation of Cytochrome P450 3A4

Brooke M. Rock, Shawna M. Hengel, Dan A. Rock, Larry C. Wienkers and Kent L. Kunze
Molecular Pharmacology December 2014, 86 (6) 665-674; DOI: https://doi.org/10.1124/mol.114.094862
Brooke M. Rock
Department of Pharmacokinetics and Drug Metabolism, Amgen, Inc., Seattle, Washington (B.M.R., D.A.R., L.C.W.); and Department of Medicinal Chemistry, University of Washington, Seattle, Washington (S.M.H.,1 K.L.K.)
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Shawna M. Hengel
Department of Pharmacokinetics and Drug Metabolism, Amgen, Inc., Seattle, Washington (B.M.R., D.A.R., L.C.W.); and Department of Medicinal Chemistry, University of Washington, Seattle, Washington (S.M.H.,1 K.L.K.)
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Dan A. Rock
Department of Pharmacokinetics and Drug Metabolism, Amgen, Inc., Seattle, Washington (B.M.R., D.A.R., L.C.W.); and Department of Medicinal Chemistry, University of Washington, Seattle, Washington (S.M.H.,1 K.L.K.)
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Larry C. Wienkers
Department of Pharmacokinetics and Drug Metabolism, Amgen, Inc., Seattle, Washington (B.M.R., D.A.R., L.C.W.); and Department of Medicinal Chemistry, University of Washington, Seattle, Washington (S.M.H.,1 K.L.K.)
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Kent L. Kunze
Department of Pharmacokinetics and Drug Metabolism, Amgen, Inc., Seattle, Washington (B.M.R., D.A.R., L.C.W.); and Department of Medicinal Chemistry, University of Washington, Seattle, Washington (S.M.H.,1 K.L.K.)
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Abstract

Ritonavir is a human immunodeficiency virus (HIV) protease inhibitor and an inhibitor of cytochrome P450 3A4, the major human hepatic drug-metabolizing enzyme. Given the potent inhibition of CYP3A4 by ritonavir, subtherapeutic doses of ritonavir are used to increase plasma concentrations of other HIV drugs oxidized by CYP3A4, thereby extending their clinical efficacy. However, the mechanism of inhibition of CYP3A4 by ritonavir remains unclear. To date, data suggests multiple types of inhibition by ritonavir, including mechanism-based inactivation by metabolic-intermediate complex formation, competitive inhibition, irreversible type II coordination to the heme iron, and more recently heme destruction. The results presented here demonstrate that inhibition of CYP3A4 by ritonavir occurs by CYP3A4-mediated activation and subsequent formation of a covalent bond to the apoprotein. Incubations of [3H]ritonavir with reconstituted CYP3A4 and human liver microsomes resulted in a covalent binding stoichiometry equal to 0.93 ± 0.04 moles of ritonavir bound per mole of inactivated CYP3A4. The metabolism of [3H]ritonavir by CYP3A4 leads to the formation of a covalent adduct specifically to CYP3A4, confirmed by radiometric liquid chromatography–trace and whole-protein mass spectrometry. Tryptic digestion of the CYP3A4-[3H]ritonavir incubations exhibited an adducted peptide (255-RMKESRLEDTQKHR-268) associated with a radiochromatic peak and a mass consistent with ritonavir plus 16 Da, in agreement with the whole-protein mass spectrometry. Additionally, nucleophilic trapping agents and scavengers of free oxygen species did not prevent inactivation of CYP3A4 by ritonavir. In conclusion, ritonavir exhibited potent time-dependent inactivation of CYP3A, with the mechanism of inactivation occurring though a covalent bond to Lys257 of the CYP3A4 apoprotein.

Footnotes

    • Received July 22, 2014.
    • Accepted October 1, 2014.
  • ↵1 Current affiliation: Bioanalytical Development, Seattle Genetics, Bothell, WA 98011.

  • dx.doi.org/10.1124/mol.114.094862.

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

  • Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 86 (6)
Molecular Pharmacology
Vol. 86, Issue 6
1 Dec 2014
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Research ArticleArticle

Inactivation of CYP3A4 by Ritonavir

Brooke M. Rock, Shawna M. Hengel, Dan A. Rock, Larry C. Wienkers and Kent L. Kunze
Molecular Pharmacology December 1, 2014, 86 (6) 665-674; DOI: https://doi.org/10.1124/mol.114.094862

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Research ArticleArticle

Inactivation of CYP3A4 by Ritonavir

Brooke M. Rock, Shawna M. Hengel, Dan A. Rock, Larry C. Wienkers and Kent L. Kunze
Molecular Pharmacology December 1, 2014, 86 (6) 665-674; DOI: https://doi.org/10.1124/mol.114.094862
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