Time Dependent Inhibition, LC-MS/MS Detection (Ki/Kinact Determination)

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 Concentration Eight concentrations ranging around values of IC50 shift (determined from time-dependent inhibition experiments)
Microsome Concentration 0.1 mg/mL
Percent Organic Solvent in Incubation 2% acetonitrile
CYP Isoforms Available for Testing CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP 3A4
Pre-Incubation Time 15 or 30 min
Incubation Time up to 30 minutes (depending on CYP isoform)
pH 7.4
Temperature 37°C
Analysis LC-MS/MS
Number of Repeats 2

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 Isoform Probe Substrate Monitored Metabolite Positive Control Inhibitor
1A2 Phenacetin Acetaminophen Furafylline
2B6 Bupropion OH Bupropion Ticlopidine
2C8 Amodiaquine Desethylamodiaquine Gemfibrozil Glucuronide
2C9 Tolbutamide 4-OH Tolbutamide Tienilic Acid
2C19 S-Mephenytoin 4’-OH Mephenytoin (S)-Fluoxetine
2D6 Dextromethorphan Dextrorphan Paroxetine
3A4 Testosterone 6β-OH Testosterone Mibefradil
3A4 Midazolam 1’-OH Midazolam Mibefradil
*If you choose "Other", please use the comments section below to request your desired system.