EA431

Time Dependent CYP Inhibition – IC50 Shift

This assay is used to identify time-dependent inhibition of individual cytochrome P450 (CYP) enzymes by a test compound using the IC50 shift approach.

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 and positive control vs. each CYP isoform 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 in duplicate (N=2 separate incubations)
  • 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 controls 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.
Individual CYP Substrates and Positive Control Inhibitors 
CYP Isoform Probe Substrate Metabolite Positive Control Inhibitor
1A2 Phenacetin Acetaminophen Furafylline
2A6 Coumarin 7-OH coumarin 8-Methoxypsoralen
2B6 Bupropion Hydroxybupropion Thio-TEPA
2C8 Amodiaquine Desethylamodiaquine Gemfibrozil Glucuronide
2C9 Diclofenac 4’-OH diclofenac Tienilic Acid
2C19 S-mephenytoin 4’-OH mephenytoin Ticlopidine
2D6 Bufuralol 1’-OH bufuralol Paroxetine
2E1 Chlorzoxazone 6-OH chlorzoxazone Diethyldithiocarbamate
3A4 Testosterone 6β-OH testosterone Troleandomycin
3A4 Midazolam 1’-OH midazolam Troleandomycin

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