A novel testosterone 6 beta-hydroxylase activity assay for the study of CYP3A-mediated metabolism, inhibition, and induction in vitro

J Pharmacol Toxicol Methods. 2001 Sep-Oct;46(2):117-23. doi: 10.1016/s1056-8719(02)00168-5.


Introduction: In order to examine CYP3A-mediated metabolism in vitro, a unique analytical assay was developed to detect the formation of 63-hydroxytestosterone (63-OHT). This assay has been determined to be useful for the study of both inhibition- and induction-related drug-drug interactions in vitro and involves simple incubation and sample handling procedures.

Methods: A primary and three backup sets of analytical conditions were developed to detect interference between a test compound and either 6beta-OHT or the internal standard.

Results: The primary set of conditions was validated with a three-batch validation, and the remaining sets of conditions were validated with one-batch validations, all in human liver microsomes. The primary assay was also validated with a single batch for CYP3A induction studies in primary human hepatocytes. Enzyme kinetic parameters of 6beta-OHT formation (Km, Vmax) were determined to be reproducible in human liver microsomes.

Discussion: Utility of the assay in inhibition studies and induction studies, respectively, was confirmed with the test compounds ketoconazole and rifampicin. In addition, superiority to existing methods was demonstrated in three areas: ease of sample preparation, short run times, and low detection limits.

MeSH terms

  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 Enzyme Inhibitors
  • Cytochrome P-450 Enzyme System / analysis*
  • Cytochrome P-450 Enzyme System / metabolism
  • Enzyme Activation / drug effects
  • Enzyme Inhibitors / pharmacology
  • Hepatocytes / drug effects
  • Hepatocytes / enzymology
  • Humans
  • Microsomes, Liver / drug effects
  • Microsomes, Liver / enzymology
  • Steroid Hydroxylases / analysis*
  • Steroid Hydroxylases / antagonists & inhibitors
  • Steroid Hydroxylases / metabolism


  • Cytochrome P-450 Enzyme Inhibitors
  • Enzyme Inhibitors
  • Cytochrome P-450 Enzyme System
  • Steroid Hydroxylases
  • CYP3A protein, human
  • Cytochrome P-450 CYP3A
  • steroid hormone 6-beta-hydroxylase