EA426

CYP Inhibition in Human Liver Microsomes – LC-MS/MS

This assay is used to screen for reversible inhibition of individual cytochrome P450 (CYP) enzymes by a single concentration of a test compound.

Required from Customer

  • A minimum of 300 μL of test compound at 10 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 inhibition of each CYP isoform by a single concentration of test compound under reversible conditions
  • Percent inhibition of each CYP isoform by one concentration of a positive control inhibitor run in parallel under reversible conditions

Substrate

  • The test compound at a single concentration (typically 10 μM) 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=2 incubation)
  • Include a maximum activity control, a single concentration 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. Percent inhibition is calculated from the amount of metabolite formed in the presence of test compound relative to that formed in the absence of test compound, based on the peak area response ratio (PARR): % Inhibition = [(PARR without test compound – PARR with test compound) / (PARR without test compound)] x 100%.
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
3A Testosterone 6β-OH testosterone Ketoconazole
3A Midazolam 1’-OH midazolam Ketoconazole

 

ORDER ONLINE

Clear