Time-Dependent Cytochrome P450 Inhibition, LC-MS/MS Detection (IC50 Shift)
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 Concentration           Eight concentrations ranging from 0.05 µM to 100 µM
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           10-20 minutes (depending on CYP isoform)
pH           7.4
Temperature           37 °C
Analysis           LC-MS/MS
Number of Repeats           2

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.

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