CYP Inhibition IC50 In Human Liver Microsomes Using LC-MS/MS

This assay is used to determine the potency with which a test compound inhibits cytochrome P450 (CYP) enzymes

Catalog number EA413

Required from Customer

Deliverables

Substrate

Assay System

Assay Conditions

Assay QC

Notes

  1. This assay is often preceded by EA406 and/or EA407 to identify CYP inhibitors.
  2. The results from this assay can be used in assay EA410, ki Determination In Human Liver Microsomes.
  3. For known irreversible inhibitors, the reaction is initiated by the addition of substrate after preincubating the microsomes with test compound and NADPH.
  4. The IC50 is equal to the ki of a test compound only if the compound is a non-competitive inhibitor of the CYP. For all other modes of reversible inhibition, the ki value must be determined beyond a relatively simple IC50 determination in order to predict the effect of interaction of an inhibitor with a substrate of the target CYP.
CYP Specific Substrates and Metabolites 
CYP Substrate Metabolite Formed
1A2 Phenacetin Acetaminophen
2A6 Coumarin Umbelliferone
2B6 Bupropion Hydroxybupropion
2C8 Paclitaxel 6α -Hydroxypaclitaxel
2C9 Diclofenac 4’-Hydroxydiclofenac
2C19 S-mephenytoin 4’-Hydroxymephenytoin
2D6 Bufuralol 1’-Hydroxybufuralol
2E1 Chlorzoxazone 6-Hydroxychlorzoxazone
3A Testosterone 6β-Hydroxytestosterone
3A Nifedipine Oxidized nifedipine
Testosterone or Nifedipine may be added to or substituted for Midazolam
   
CYP Specific Inhibitors used as Positive Controls
CYP Inhibitor
1A2 Fluvoxamine
2A6 Tranylcypromine
2B6 N-(α-Methylbenzyl)-1-aminobenzotriazole
2C8 Quercetin
2C9 Sulfaphenazole
2C19 Ticlopidine
2D6 Quinidine
2E1 4-Methylpyrazole
3A Ketoconazole
 

Options

  1. The customer must specify: • the concentration(s) of test compound • either the standard or custom report format
  2. The customer can request: • an alternative microsomal protein concentration • additional replicates • alternative substrates, which we may need to validate in terms of Km, time course and analytical method • alternative positive control inhibitors