E-store > ... > Time Dependent Inhibition – IC50 Shift

EA450
Time Dependent 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 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
- 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 |