| Assay Method Information | |
| | CYP2C9, CYP2D6 and CYP3A4 Enzymatic Activity Assay |
| Description: | 1. Preparation of 100 mM phosphate buffered saline (PBS): 7.098 g of Na2HPO4 was weighed, 500 mL of pure water was added and subjected to ultrasonic dissolution to obtain solution A. 3.400 g of KH2PO4 was weighed, 250 mL of pure water was added and subjected to ultrasonic dissolution to obtain solution B. Solution A was placed on a stirrer and solution B was slowly added until the pH value reached 7.4 to prepare 100 mM PBS buffer.2. Preparation of 10 mM NADPH solution with 100 mM PBS buffer. The 10 mM stock solution of the compound of the present disclosure was diluted with DMSO to obtain a 200× concentration of compound working solution (6000, 2000, 600, 200, 60, 20, 0 μM). The stock solution of positive inhibitors was diluted with DMSO to obtain a 200× concentration of positive inhibitor working solution (sulfaphenazole, 1000, 300, 100, 30, 10, 3, 0 μM; quinidine/ketoconazole, 100, 30, 10, 3, 1, 0.3, 0 μM). A 200× concentration of substrate working solution (120 μM diclofenac, 400 μM dextromethorphan, and 200 μM midazolam) was prepared with water, acetonitrile, or acetonitrile/methanol.3. 2 μL of 20 mg/ml liver microsome solution, 1 μL of substrate working solution, 1 μL of compound working solution and 176 μL of PBS buffer were taken, mixed uniformly, and pre-incubated in a 37° C. water bath for 15 min. To the positive control group was added 1 μL of sulfaphenazole, quinidine or ketoconazole working solution instead of compound working solution. A 10 mM NADPH solution was also pre-incubated in the 37° C. water bath for 15 min. After 15 min, 20 μL of NADPH was taken and added to each well to initiate the reaction, and the reaction was incubated at 37° C. for 5 min (CYP2C9), 20 min (CYP2D6) or 5 min (CYP3A4). Double samples were set for all incubation samples. After the corresponding time of incubation, 400 μL of glacial methanol containing internal standard was added to all samples to stop the reaction. The mixture was mixed uniformly by vortex and centrifuged at 3220 g for 40 min at 4° C. After the completion of the centrifugation, 100 μL of the supernatant was transferred to a loading plate, and 100 μL of ultrapure water was added and mixed uniformly for LC-MS/MS analysis. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |