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.
Test Compound Concentration
Six concentrations ranging around values of IC50 shift (determined from time-dependent inhibition experiments)
Percent Organic Solvent in Incubation
CYP Isoforms Available for Testing
CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4
up to 30 minutes (depending on CYP isoform)
Number of Repeats
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.