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CYP IC50 in Human Liver Microsomes – LC-MS/MS
This assay is used to determine the IC50 for reversible inhibition of individual cytochrome P450 (CYP) enzymes by a test compound.
Required from Customer
- A minimum of 300 μL of test compound at 100 mM in DMSO, or 10 mg of powder
- Molecular mass (exact mass) of test compound and salt form
- MSDS or handling and storage information, e.g., light-sensitive, store at -20°C, etc.
- Percent of control activity of each CYP isoform in the presence of each concentration of test compound incubated under reversible conditions
- Percent inhibition of each CYP isoform by one concentration of a positive control inhibitor run in parallel under reversible conditions
- Microsomal lot certification for IC50 of each CYP-specific positive control inhibitor under reversible conditions
- IC50 of test compound for each CYP isoform
- Seven concentrations of test compound dissolved in aqueous buffer, acetonitrile (≤1% final), methanol (≤1% final), or DMSO (≤0.1% final)
- Pooled, mixed-gender, human liver microsomes from a minimum 10 donors with NADPH added in high molar excess
- Concentration of each individual CYP-specific probe substrate is near the Km for the enzyme
- LC-MS/MS is used to determine the peak area response ratio (peak area corresponding to a specific metabolite of each CYP-specific probe substrate divided by that of an analytical internal standard) without running a standard curve
- The standard protein concentration is 0.25 mg/mL
- Run the assay with a single replicate (N=1 incubation)
- Include a maximum activity control, seven concentrations of test compound, and a single, high concentration of a positive control inhibitor
- Initiate reaction by adding NADPH
- Sample reaction mixture at a single time point (typically between 10 and 30 minutes)
- >50% inhibition by each positive control run in parallel
- The results from this assay are sent to the customer in the ExpressPlus report format, which may include graphical representations of data and comparison with historical data for reference compounds.
- IC50 values are estimated by fitting the experimental data (percent of control activity remaining at each concentration of test compound) to a sigmoidal model and non-linear regression analysis.
|Individual CYP Substrates and Positive Control Inhibitors|
|CYP Isoform||Probe Substrate||Metabolite||Positive Control Inhibitor|