June 25, 2018
Assay System:
- Plated cryopreserved human hepatocytes from 3 individual donors.
- Induction of CYP1A2, CYP2B6 and CYP3A4 by test compound measured by comparing enzyme activity of hepatocytes treated with test compound with vehicle control.
Assay Conditions:
| Test Compound Concentration | 30 µM, 3 µM, and 0.3 µM |
| Percent Organic Solvent – Inducer Treatment | 0.1% DMSO |
| Percent Organic Solvent – Substrate Incubation | 1.5% acetonitrile |
| Inducer Treatment Time | 48 hours |
| Substrate Incubation Time | 30 minutes |
| pH | 7.4 |
| Treatment and Incubation Conditions | 37 °C, 5% CO2, and saturating humidity |
| Positive Control | Omeprazole (1A2), Phenobarbital (2B6), and Rifampicin (3A4) |
| Analysis | LC-MS/MS |
| Number of Repeats | 3 |
Deliverable: Mean fold induction of test compound and positive control compound.
Test Compound Requirement: 2-3 mg dry powder or 100-200 µL of 30 mM DMSO stock solution.
| CYP Isoform | Probe Substrate | Positive Control Inhibitor |
| 1A2 | Phenacetin | Furafylline |
| 2B6 | Bupropion | Ticlopidine HCl |
| 2C8 | Dibenzylfluorescein | Quercetin |
| 2C9 | 7-Methoxy-4-trifluoromethylcoumarin | Sulfaphenazole |
| 2C19 | 3-Cyano-7-ethoxycoumarin | Tranylcypromine |
| 2D6 | 3-[2-(N,N-diethyl-N methylamino)ethyl]-7-methoxy-4-methylcoumarin | Quinidine |
| 3A4 | 7-Benzyloxy-4-(trifluoromethyl)-coumarin | Ketoconazole |