Time-Dependent Cytochrome P450 Inhibition (kinact/KI)
Positive Control Inhibitor
Furafylline
Tienilic Acid
(S)-Fluoxetine
Paroxetine
Mibefradil
Mibefradil
Monitored Metabolite
Acetaminophen
4’-OH Tolbutamide
4’-OH mephenytoin
Dextrorphan
6β-OH Testosterone
1’-OH Midazolam
Probe Substrate
Phenacetin
Tolbutamide
S-mephenytoin
Dextromethorphan
Testosterone
Midazolam

CYP Isoform
1A2
2C9
2C19
2D6
3A4
3A4
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           Six 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, CYP2C9, CYP2C19, CYP2D6, and CYP3A4
Pre-Incubation Time           30 minutes
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.