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.

Deliverables

• 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

Substrate

• The test compound dissolved in aqueous buffer, acetonitrile, or methanol

Assay System

• 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

Assay Conditions

• 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 K_m 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

Assay QC

• Microsomal lot qualification at the time of the study with known inhibitors of each CYP

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, k_i 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 k_i of a test compound only if the compound is a non-competitive inhibitor of the CYP. For all other modes of reversible inhibition, the k_i 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.

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 K_m, time course and analytical method • alternative positive control inhibitors