Development of an in vitro drug-drug interaction assay to simultaneously monitor five cytochrome P450 isoforms and performance assessment using drug library compounds

J Pharmacol Toxicol Methods. Nov-Dec 2008;58(3):206-14. doi: 10.1016/j.vascn.2008.05.131. Epub 2008 Jun 25.

Abstract

Introduction: Inhibition of cytochrome P450 (CYP) is a principal mechanism for metabolism-based drug-drug interactions (DDIs). This article describes a robust, high-throughput CYP-mediated DDI assay using a cocktail of 5 clinically relevant probe substrates with quantification by liquid chromatography/tandem mass spectrometry (LC/MS-MS).

Methods: The assay consisted of human liver microsomes and a cocktail of probe substrates metabolized by the five major CYP isoforms (tacrine for CYP1A2, diclofenac for CYP2C9, (S)-mephenytoin for CYP2C19, dextromethorphan for CYP2D6 and midazolam for CYP3A4). The assay was fully automated in both 96- and 384-well formats.

Results: A series of experiments were conducted to define the optimal kinetic parameters and solvent concentrations, as well as, to assess potential reactant and product interference. The assay was validated against known CYP inhibitors (miconazole, sulfaphenazole, ticlopidine, quinidine, ketoconazole, itraconazole, fluoxetine) and evaluated in a screening environment by testing 9494 compounds.

Discussion: Our findings show that this assay has application in early stage drug discovery to economically, reliably and accurately assess compounds for DDIs.

MeSH terms

  • Aryl Hydrocarbon Hydroxylases / metabolism
  • Chromatography, Liquid / methods
  • Cytochrome P-450 CYP1A2 / metabolism
  • Cytochrome P-450 CYP2C9
  • Cytochrome P-450 Enzyme System / metabolism*
  • Dextromethorphan / metabolism
  • Dextromethorphan / pharmacology
  • Diclofenac / metabolism
  • Diclofenac / pharmacology
  • Drug Interactions
  • Humans
  • Isoenzymes / metabolism
  • Mass Spectrometry / methods
  • Mephenytoin / metabolism
  • Mephenytoin / pharmacology
  • Miconazole / metabolism
  • Miconazole / pharmacology
  • Microsomes, Liver / drug effects
  • Microsomes, Liver / metabolism*
  • Midazolam / metabolism
  • Midazolam / pharmacology
  • Substrate Specificity
  • Tacrine / metabolism
  • Tacrine / pharmacology
  • Testosterone / metabolism
  • Testosterone / pharmacology

Substances

  • Isoenzymes
  • Diclofenac
  • Testosterone
  • Tacrine
  • Dextromethorphan
  • Miconazole
  • Cytochrome P-450 Enzyme System
  • CYP2C9 protein, human
  • Cytochrome P-450 CYP2C9
  • Aryl Hydrocarbon Hydroxylases
  • Cytochrome P-450 CYP1A2
  • Mephenytoin
  • Midazolam