October 3, 2018

Assay System: 

  • Human liver microsomes, pooled from 50 donors.
  • Inhibition potential against CYP isoforms (IC50) measured after pre-incubation with and without NADPH in the presence of different concentrations of test compound by monitoring activity of individual CYP enzymes using isoform-specific probe substrates with LC-MS/MS detection.
  • Time-dependent inhibition (IC50 shift) is determined by comparing IC50 values determined after pre-incubation with and without NADPH.

Assay Conditions: 

Test Compound ConcentrationEight concentrations ranging from 0.05 µM to 100 µM
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 Time30 minutes
Incubation Time10-20 minutes (depending on CYP isoform)
pH7.4
Temperature37 °C
AnalysisLC-MS/MS
Number of Repeats2

Deliverable:

  • IC50 of test compound and positive control compound both with and without NADPH pre-incubation.
  • IC50 shift (IC50 -NADPH/ IC50 +NADPH)

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

CYP IsoformProbe SubstrateMonitored MetabolitePositive Control Inhibitor
1A2PhenacetinAcetaminophenFurafylline
2B6BupropionOH BupropionTiclopidine
2C8AmodiaquineDesethylamodiaquineGemfibrozil Glucuronide
2C9Tolbutamide4-OH TolbutamideTienilic Acid
2C19S-Mephenytoin4’-OH Mephenytoin(S)-Fluoxetine
2D6DextromethorphanDextrorphanParoxetine
3A4Testosterone6β-OH TestosteroneMibefradil
3A4Midazolam1’-OH MidazolamMibefradil

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