October 3, 2018

Assay System: 

  • Human liver microsomes (HLM), pooled from 50 donors.
  • Incubation of the test compound with HLM in the presence of NADPH with collection of samples from the incubation mixture at 6 time points.
  • The remaining activity of individual CYP enzymes measured using isoform-specific probe substrates with LC/MS/MS detection to determine the rate constant of CYP inactivation (kobs). The experiment is repeated at 6 concentrations of the test compound to measure the dependence of kobs on inactivator concentration.
  • Constants KI (equilibrium binding) and kin (irreversible CYP inactivation) for test compounds are determined from the kinetic data using a non-linear regression analysis to the four-parameter logistic model fit with XLFit 5.3 from IDBS software.

Assay Conditions: 

Test Compound ConcentrationEight concentrations ranging around values of IC50 shift (determined from time-dependent inhibition experiments)
Microsome Concentration0.1 mg/mL
Percent Organic Solvent in Incubation2% acetonitrile
CYP Isoforms Available for TestingCYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP 3A4
Pre-Incubation Time15 or 30 min
Incubation Timeup to 30 minutes (depending on CYP isoform)
Number of Repeats2

Deliverable: Values of KI (µM), kinact(min-1), and kinact/KI (mL/min/µmol) for test compounds and positive control inhibitors.

Test Compound Requirement: 1-2 mg dry powder or 100-200 µL of 10 mM acetonitrile stock solution.

CYP IsoformProbe SubstrateMonitored MetabolitePositive Control Inhibitor
2B6BupropionOH BupropionTiclopidine
2C8AmodiaquineDesethylamodiaquineGemfibrozil Glucuronide
2C9Tolbutamide4-OH TolbutamideTienilic Acid
2C19S-Mephenytoin4’-OH Mephenytoin(S)-Fluoxetine
3A4Testosterone6β-OH TestosteroneMibefradil
3A4Midazolam1’-OH MidazolamMibefradil

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