| Assay Method Information | |
| | Assay for Inhibition of Compounds Provided in the Present Application on CYP Enzymes |
| Description: | Typical substrate metabolic responses of five major human CYP subtypes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) were evaluated using 150-donor pooled human liver microsomes (purchased from Corning, Cat. No.: 452117). The influence of different concentrations of each compound to be tested on metabolic responses of phenacetin (CYP1A2), diclofenac sodium (CYP2C9), S-mephenytoin (CYP2C19), bufuralol hydrochloride (CYP2D6), and midazolam (CYP3A4) was determined by liquid chromatography-mass spectrometry (LC/MS/MS). The reaction system (200 μL) (100 mmol/L phosphate buffer, pH 7.4, containing DMSO, acetonitrile and methanol at a volume ratio of 0.3%:0.6%:0.1%) of 30 μM phenacetin, 10 μM diclofenac sodium, 35 μM S-mephenytoin, 5 μM bufuralol hydrochloride, 3 μM midazolam, 1 mM reduced nicotinamide adenine dinucleotide phosphate (NADPH), the compound to be tested (at concentrations of 0.1, 0.3, 1, 3, 10, 30 μmol/L, respectively), or positive compound or blank control, with pooled human liver microsomes (0.2 mg/mL) was incubated at 37° C. for 5 min. Then 200 μL of acetonitrile solution containing 3% formic acid and 40 nM internal standard verapamil was added and centrifuged at 4000 rpm for 50 min. The mixture was cooled on ice for 20 min and centrifuged at 4000 rpm for 20 min to precipitate the protein. Then 200 μL of supernatant was analyzed by LC-MS/MS.The peak area was calculated from the chromatogram. |
| Affinity data for this assay | |
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