Assessment of drug-drug interactions caused by metabolism-dependent cytochrome P450 inhibition

Chem Biol Interact. 2012 Jun 25;198(1-3):49-56. doi: 10.1016/j.cbi.2012.05.007. Epub 2012 May 28.


This study was designed to develop methods for detecting metabolism-dependent reversible, quasi-irreversible, and irreversible cytochrome P450 (CYP) inhibition using pooled human liver microsomes and a liquid chromatography/tandem mass spectrometry (LC-MS/MS) system. Metabolism-dependent inhibition (MDI) was identified based on IC(50) shifts after pre-incubation of the tested compounds with NADPH. To distinguish reversible MDI from mechanism-based inhibition (MBI), R-fluoxetine and ticlopidine were used as positive inhibitors for reversible MDI and MBI of CYP2C19, respectively. R-fluoxetine and ticlopidine inhibited CYP2C19 activity, as determined using S-mephenytoin as a substrate, and caused 8.7- and 2.3-fold IC(50) shifts, respectively, after pre-incubation. Inhibition of CYP2C19 by R-fluoxetine, but not ticlopidine, was markedly reversed by ultracentrifugation, and two or three ultracentrifugations were not more effective than one, indicating that ultracentrifugation only once may be sufficient to reverse the reversible MDI. To distinguish between quasi-irreversible and irreversible inhibition, diltiazem and mifepristone were used as quasi-irreversible and irreversible inhibitors of CYP3A4, respectively, and CYP3A4 activity was measured using midazolam and testosterone as substrates. After pre-incubation, CYP3A4 IC(50) shifts caused by diltiazem and mifepristone were greater than 2.5- and 3.7-fold, respectively. Incubation with 2mM potassium ferricyanide for 10min reversed the MDI of CYP3A4 by diltiazem, but not mifepristone. Increases in potassium ferricyanide concentration and incubation time reduced the recovery of CYP3A4 activity. The established methods were confirmed using three CYP3A4 inhibitors including diltiazem, mifepristone and amiodarone (a reversible metabolism-dependent inhibitor). We consider these methods to be useful tools for discriminating between reversible MDI and MBI.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromatography, Liquid
  • Cytochrome P-450 CYP3A
  • Cytochrome P-450 CYP3A Inhibitors*
  • Drug Interactions
  • Enzyme Inhibitors / pharmacology*
  • Humans
  • Inhibitory Concentration 50
  • Microsomes, Liver / drug effects
  • Microsomes, Liver / enzymology
  • Spectrometry, Mass, Electrospray Ionization
  • Tandem Mass Spectrometry
  • Ultracentrifugation


  • Cytochrome P-450 CYP3A Inhibitors
  • Enzyme Inhibitors
  • Cytochrome P-450 CYP3A
  • CYP3A4 protein, human