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

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 (k_obs). The experiment is repeated at 6 concentrations of the test compound to measure the dependence of k_obs on inactivator concentration.
Constants K_I (equilibrium binding) and k_in (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	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 K_I (µM), k_inact(min^-1), and k_inact/K_I (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