CYP Inhibition IC50 In Human Liver Microsomes – LC-MS/MS
This assay is used to determine the potency with which a test compound inhibits cytochrome P450 (CYP) enzymes.
Required from Customer
- A study design—the customer must specify the values of several assay variables listed in the Options section
- The customer must specify which CYP activities are to be assayed for inhibition by test compound
- A minimum of 10 mg of powder
- Molecular mass (exact mass) of test compound and salt form
- Stability of test compound in the presence of human liver microsomes
- MSDS or handling and storage information, e.g., light-sensitive, store at -20°C, etc.
- Tables and graphs of percent activity remaining after exposure to each concentration of test compound relative to solvent control for each CYP
- When > 50% inhibition is observed, calculation of IC50 of test compound by fitting the plot of percent inhibition vs. concentration to a sigmoid model
- Positive control IC50 values and graphs for known CYP inhibitors run in parallel with test compound
- The test compound dissolved in aqueous buffer, acetonitrile, or methanol
- Pooled human liver microsomes from a minimum 10 donors with NADPH added in high molar excess
- Individual CYP-specific substrates in separate incubations
- LC/MS/MS is used to determine the concentration of a specific metabolite of each CYP-specific probe substrate
- The standard protein concentration is 0.25 mg/mL
- Run the assay in duplicate (N = 2 separate incubations)
- Include a solvent control and seven concentrations of test compound, typically with serial 3-fold dilutions from 100 μM
- Add each substrate to microsomes at a concentration around the Km of the appropriate CYP
- Initiate reaction by adding NADPH, based on the default assumption that test compound is not an irreversible inhibitor.
- Sample reaction mixture at a single time point in the linear portion of the rate vs. time curve
- Microsomal lot qualification at the time of the study with known inhibitors of each CYP
- This assay is often preceded by EA406 and/or EA407 to identify CYP inhibitors.
- The results from this assay can be used in assay EA410, ki Determination In Human Liver Microsomes.
- For known irreversible inhibitors, the reaction is initiated by the addition of substrate after preincubating the microsomes with test compound and NADPH.
- 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.
- The customer must specify: • the concentration(s) of test compound • either the standard or custom report format
- 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
|CYP Specific Substrates and Metabolites|
|Testosterone or Nifedipine may be added to or substituted for Midazolam|