Human Cytochrome P450: Metabolism of Testosterone by CYP3A4 and Inhibition by Ketoconazole

Khawja A. Usmani1, Jun Tang1

1 North Carolina State University, Raleigh, North Carolina
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.13
DOI:  10.1002/0471140856.tx0413s20
Online Posting Date:  June, 2004
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This unit describes methods for measuring CYP3A4 activity using testosterone as a specific substrate, and for measuring CYP3A4 inhibition using ketoconazole as a selective inhibitor of testosterone oxidation. CYP3A4 is one of the most important and most abundant drug‐metabolizing CYP isoforms in human liver microsomes (∼40% of total CYP), and it has the broadest substrate specificity. It is important to determine whether CYP3A4 is involved in its metabolism.

Keywords: P450; CYP3A4; testosterone; ketoconazole; inhibition

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Table of Contents

  • Basic Protocol 1: Testosterone Oxidation by CYP3A4
  • Basic Protocol 2: Inhibition of CYP3A4‐Mediated Testosterone 6β‐Hydroxylase by Ketoconazole
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Testosterone Oxidation by CYP3A4

  • 0.1 M potassium phosphate buffer, pH 7.4, with 5 mM MgCl 2 (see recipe)
  • 100 mM testosterone stock (Steraloids, Inc.; dissolve 28.841 mg testosterone in 1 ml methanol; store up to 3 months at −20°C)
  • NADPH regenerating system (see recipe)
  • 1 pmol/µl CYP3A4 (BD Bioscience)
  • Methanol, ice‐cold
  • Testosterone metabolites in methanol for standard curve (see recipe and Table 4.13.1)
  • 1‐ml HPLC vials
  • HPLC system and reagents (Table 4.13.2)
    Table 4.3.1   Materials   HPLC Retention Times for Testosterone and Hydroxylated Testosterone Metabolites a   HPLC Retention Times for Testosterone and Hydroxylated Testosterone Metabolites

    Common name Chemical name Retention time (min)
    6α‐Hydroxytestosterone 4‐Androsten‐6α,17β‐diol‐3‐one 14.38
    15β‐Hydroxytestosterone 4‐Androsten‐15β,17β‐diol‐3‐one 15.11
    6β‐Hydroxytestosterone 4‐Androsten‐6β,17β‐diol‐3‐one 16.25
    11‐Ketotestosterone 4‐Androsten‐17β‐ol‐3,11‐dione 18.24
    16β‐Hydroxytestosterone 4‐Androsten‐16β,17β‐diol‐3‐one 19.34
    11β‐Hydroxyandrostenedione 4‐Androsten‐11β‐ol‐3,17‐dione 19.68
    2α‐Hydroxytestosterone 4‐Androsten‐2α,17β‐diol‐3‐one 20.68
    2β‐Hydroxytestosterone 4‐Androsten‐2β,17β‐diol‐3‐one 21.55
    Androstenedione 4‐Androsten‐3,17‐dione 24.92
    4‐Hydroxyandrostenedione 4‐Androsten‐4‐ol‐3,17‐dione 27.20
    Testosterone 4‐Androsten‐17β‐ol‐3‐one 28.90

     aTestosterone and its metabolites are purchased from Steraloids, Inc.

Basic Protocol 2: Inhibition of CYP3A4‐Mediated Testosterone 6β‐Hydroxylase by Ketoconazole

  • 1 pmol/µl CYP3A4 (BD Bioscience) or liver microsomes (unit 4.3; 20 mg protein/ml)
  • 0.1 M potassium phosphate buffer, pH 7.4, with 5 mM MgCl 2 (see recipe)
  • 25 mM testosterone (Steraloids, Inc.) in methanol (store up to 3 months at −20°C)
  • 0.5 mM ketoconazole stock (Sigma) in DMSO (store up to 3 months at −20°C)
  • NADPH regenerating system (see recipe)
  • Methanol, ice‐cold
  • 6β‐hydroxytestosterone in methanol for standard curve (see recipe for testosterone metabolites)
  • 1‐ml HPLC vials
  • HPLC system and reagents (Table 4.13.2)
  • Additional reagents and equipment for measurement of testosterone metabolites by HPLC (see protocol 1)
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Literature Cited

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Key References
   Madan et al., 2001. See above.
  Describes in detail the methods for inhibition and reaction phenotyping studies.
   Rodrigues, 1999. See above.
  Describes in details the method for reaction phenotyping study.
Internet Resources
  FDA, 1997. Guidance for Industry: Drug metabolism/drug interaction studies in the drug development process: Studies in vitro. U.S. Food and Drug Administration, Rockville, Md.
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