Cocktail-substrate assay system for mechanism-based inhibition of CYP2C9, CYP2D6, and CYP3A using human liver microsomes at an early stage of drug development

Xenobiotica. 2009 Jun;39(6):415-22. doi: 10.1080/00498250902822204.


We established a mechanism-based inhibition cocktail-substrate assay system using human liver microsomes and drug-probe substrates that enabled simultaneous estimation of the inactivation of main cytochrome P450 (CYP) enzymes, CYP2C9, CYP2D6, and CYP3A, in drug metabolism. The inactivation kinetic parameters of typical mechanism-based inhibitors, tienilic acid, paroxetine, and erythromycin, for each enzyme in the cocktail-substrate assay were almost in agreement with the values obtained in the single-substrate assay. Using this system, we confirmed that multiple CYP inactivation caused by mechanism-based inhibitors such as isoniazid and amiodarone could be detected simultaneously. Mechanism-based inhibition potency can be estimated by the determination of the observed inactivation rate constants (k(obs)) at a single concentration of test compounds because the k(obs) of eleven CYP3A inactivators at 10 microM in the assay system nearly corresponded to k(inact)/K(I) values, an indicator of a compound's propensity to alter the activity of a CYP in vivo (R(2) = 0.97). Therefore, this cocktail-substrate assay is considered to be a powerful tool for evaluating mechanism-based inhibition at an early stage of drug development.

MeSH terms

  • Amiodarone / analogs & derivatives
  • Amiodarone / pharmacology
  • Aryl Hydrocarbon Hydroxylases / antagonists & inhibitors*
  • Biological Assay / methods*
  • Cytochrome P-450 CYP2C9
  • Cytochrome P-450 CYP2D6 Inhibitors*
  • Cytochrome P-450 CYP3A Inhibitors*
  • Dose-Response Relationship, Drug
  • Drug Discovery / methods*
  • Enzyme Activation / drug effects
  • Humans
  • Isoniazid / pharmacology
  • Kinetics
  • Microsomes, Liver / drug effects
  • Microsomes, Liver / enzymology*
  • Reference Standards
  • Substrate Specificity / drug effects
  • Time Factors


  • Cytochrome P-450 CYP2D6 Inhibitors
  • Cytochrome P-450 CYP3A Inhibitors
  • CYP2C9 protein, human
  • Cytochrome P-450 CYP2C9
  • Aryl Hydrocarbon Hydroxylases
  • desethylamiodarone
  • Amiodarone
  • Isoniazid