| Assay Method Information | |
| | CYP inhibitio |
| Description: | CYP inhibition by test compounds in human liver microsomes (HLM) for seven major CYP450 isoforms CYP1A2, CYP2C9, CYP2D6, CYP2B6, CYP2C8, CYP2C19 and CYP3A4 were assessed. Reactions were performed by incubating a test compound at concentrations of 0.02, 0.070, 0.21, 0.62, 1.85, 5.56, 16.67 and 50 μM in <1% DMSO with HLM (0.2 mg/mL for CYP1A2 and 0.03 mg/ml for CYP3A4, 0.2 mg/mL for CYP2C19, CYP2D6, and CYP2C9) in 0.1M phosphate buffer, 1 mM NADPH and selective probe substrates of individual isoforms at 37° C. 50 μM phenacetin, 2 μM midazolam, 5 μM diclofenac, 50 μM mephenytoin, 80 μM bupropion, and 5 μM dextromethorphan were used as probe substrates of CYP1A2, CYP3A4, CYP2C9, CYP2C19, CYP2B6, and CYP2D6, respectively. The incubation times were 20 min for CYP 1A1, CYP2D6, CYP2C9, CYP2B6, and CYP2C8; 40 min for CYP2C19; and 10 min for CYP 3A4. Following incubations, the reactions were terminated with acetonitrile containing internal standard. The samples were centrifuged and the supernatants were analyzed for the formation of metabolites (1-hydroxymidazolam (CYP3A4), 4-hydroxydiclofenac (CYP2C9), 4-hydroxymephenytoin (CYP2C19) and dextrorphan (CYP2D6), hydroxybupropion (CYP2B6), acetaminophen (CYP1A2), desethylamodiaquine (CYP2C8)) by LC/MS/MS. Selective inhibitors for all isoforms were screened alongside as positive controls. A decrease in the formation of metabolites compared to vehicle control (100%) was used to estimate % inhibition, and the IC50 was estimated from concentration-response curves. |
| Affinity data for this assay | |
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