Time-Dependent Cytochrome P450 Inhibition, 
Fluorescence Detection (IC50 Shift)
Assay System: 
  • Individual human recombinant CYP isoforms expressed in baculovirus-infected insect cells.
  • Inhibition potential against recombinant CYP isoforms (IC50) measured with and without pre-incubation in 
    the presence of different concentrations of test compound by monitoring activity of individual CYP enzymes using isoform-specific probe substrates with fluorescence detection.
  • Time-dependent inhibition (IC50 shift) is determined by comparing IC50 values determined with and without pre-incubation. 

Assay Conditions:

Test Compound Concentration   Eight concentrations ranging from 0.05 µM to 100µM
Percent Organic Solvent in Incubation   1% acetonitrile
CYP Isoforms Available for Testing   CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6,and CYP3A4
Pre-Incubation Time   15 minutes
Incubation Time   15-45 minutes (depending on CYP isoform)
pH   7.4
Temperature   37°C
Analysis   Fluorescence Detection
Number of Repeats   2

Deliverable:
 IC50  with pre-incubation, IC50 without pre-incubation, and IC50 shift.

Test Compound Requirement: 1-2 mg dry powder or 100-200 µL of 10 mM MeCN stock solution.

Positive Control Inhibitor
Furafylline
ThioTEPA
Phenelzine Sulfate Salt
Tienilic Acid
S-(+)-Fluoxetine
Paroxetine
Mifepristone
Probe Substrate
3-Cyano-7-ethoxycoumarin
7-Ethoxy-4-trifluoromethylcoumarin
Dibenzylfluorescein 
7-Methoxy-4-trifluoromethylcoumarin
3-Cyano-7-ethoxycoumarin
3-[2-(N,N-diethyl-N methylamino)ethyl]-7-methoxy-4-methylcoumarin
7-Benzyloxy-4-(trifluoromethyl)-coumarin

CYP Isoform
1A2
2B6
2C8
2C9
2C19
2D6
3A4