EA427

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.

Deliverables

  • 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

Substrate

  • Seven concentrations of test compound dissolved in aqueous buffer, acetonitrile (≤1% final), methanol (≤1% final), or DMSO (≤0.1% final)

Assay System

  • 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

Assay Conditions

  • 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)

Assay QC

  • >50% inhibition by each positive control run in parallel

Notes

  1. 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.
  2. 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
1A2 Phenacetin Acetaminophen α-Naphthoflavone
2A6 Coumarin 7-OH coumarin Tranylcypromine
2B6 Bupropion Bupropion Thio-TEPA
2C8 Amodiaquine Desethylamodiaquine Montelukast
2C9 Diclofenac 4’-OH diclofenac Sulfaphenazole
2C19 S-mephenytoin 4’-OH mephenytoin (+)-N-3-benzylnirvanol
2D6 Bufuralol 1’-OH bufuralol Quinidine
2E1 Chlorzoxazone 6-OH chlorzoxazone 4-Methylpyrazole
3A4 Testosterone 6β-OH testosterone Ketoconazole
Midazolam 1’-OH midazolam

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