Time Dependent Inhibition, LC-MS/MS Detection (IC50 Shift)
Assay System:
- Human liver microsomes, pooled from 50 donors.
- Inhibition potential against CYP isoforms (IC50) measured after pre-incubation with and without NADPH in the presence of different concentrations of test compound by monitoring activity of individual CYP enzymes using isoform-specific probe substrates with LC-MS/MS detection.
- Time-dependent inhibition (IC50 shift) is determined by comparing IC50 values determined after pre-incubation with and without NADPH.
Assay Conditions:
| Test Compound Concentration | Eight concentrations ranging from 0.05 µM to 100 µM |
| Microsome Concentration | 0.1 mg/mL |
| Percent Organic Solvent in Incubation | 2% acetonitrile |
| CYP Isoforms Available for Testing | CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP 3A4 |
| Pre-Incubation Time | 30 minutes |
| Incubation Time | 10-20 minutes (depending on CYP isoform) |
| pH | 7.4 |
| Temperature | 37 °C |
| Analysis | LC-MS/MS |
| Number of Repeats | 2 |
Deliverable:
- IC50 of test compound and positive control compound both with and without NADPH pre-incubation.
- IC50 shift (IC50 -NADPH/ IC50 +NADPH)
Test Compound Requirement: 1-2 mg dry powder or 100-200 µL of 10 mM acetonitrile stock solution.
| CYP Isoform | Probe Substrate | Monitored Metabolite | Positive Control Inhibitor |
|---|---|---|---|
| 1A2 | Phenacetin | Acetaminophen | Furafylline |
| 2B6 | Bupropion | OH Bupropion | Ticlopidine |
| 2C8 | Amodiaquine | Desethylamodiaquine | Gemfibrozil Glucuronide |
| 2C9 | Tolbutamide | 4-OH Tolbutamide | Tienilic Acid |
| 2C19 | S-Mephenytoin | 4’-OH Mephenytoin | (S)-Fluoxetine |
| 2D6 | Dextromethorphan | Dextrorphan | Paroxetine |
| 3A4 | Testosterone | 6β-OH Testosterone | Mibefradil |
| 3A4 | Midazolam | 1’-OH Midazolam | Mibefradil |