Required from Customer

• A minimum of 25 mg of powder
• Exact molecular mass of test compound and its salt form
• Purity of test compound
• Metabolite standard(s) and purity (if available)
• MSDS or handling and storage information (e.g., light sensitive, store at -20°C, etc.)

Deliverables

• IC₅₀ of test compound vs. each CYP isoform under time-dependent inhibition conditions
• IC₅₀ of positive control vs. a single CYP isoform (typically CYP3A) under time-dependent inhibition conditions
• Calculated IC₅₀ shift (-NADPH/ +NADPH)

Substrate

• Test compound in aqueous buffer, acetonitrile or methanol (≤1% final)

Assay System

• Pooled, mixed-gender, human liver microsomes from a minimum 10 donors
• Concentration of each individual CYP-specific probe substrate is near the Km for that enzyme
• LC-MS/MS is used to determine the peak area response ratio (peak area corresponding to metabolite of CYP-specific probe substrate) divided by that of an analytical internal standard) without running a standard curve

Assay Conditions

• The standard protein concentration is 0.25 mg/mL
• Run the assay with a single incubation (N=1) per treatment
• Include a maximum activity control, seven concentrations of test compound (typically serial dilutions from 100 µM), and seven concentrations of a positive control inhibitor
• After pre-incubating test compound with microsomes, with and without NADPH, at 37°C for 30 minutes, initiate enzyme activity assay by adding an enzyme-specific probe substrate (and NADPH, if not already present)
• Sample reaction mixture at a single time point consistent with the rate of formation of the metabolite
• Estimate IC₅₀ in the absence and presence of NADPH, calculate IC₅₀ shift (-NADPH/ +NADPH)

Assay QC

• IC₅₀ shift ≥3 for positive control run in parallel

Notes

1. IC₅₀ values are estimated by fitting the experimental data (percent of control activity remaining at each concentration of test compound or positive control) to a sigmoidal model and non-linear regression analysis.