| Assay Method Information | |
| | Inhibitory Effect of Compound of the Present Invention on Enzymatic Activity of CYP2C9 and CYP2D6 |
| Description: | I. Experimental Materials and Instruments1. Human liver microsome (Corning 452117)2. NADPH (Solarbio 705Y021)3. Positive substrates diclofenac (Sigma SLBV3438) and dextromethorphan (TRC 3-EDO-175-1)4. Positive inhibitors sulfaphenazole (D. Ehrenstorfer GmbH 109012) and quinidine (TCI WEODL-RE)5. AB Sciex Triple Quad 5500 liquid chromatography-mass spectrometry systemII. Procedures1. Preparation of 100 mM phosphate-buffered saline (PBS): 7.098 g Na2HPO4 was weighed. 500 mL pure water was added. The mixture was dissolved by sonication to give solution A. 3.400 g KH2PO4 was weighed. 250 mL pure water was added. The mixture was dissolved by sonication to give solution B. The solution A was placed in a stirrer, and the solution B was slowly added until the pH reached 7.4, so that the 100 mM PBS buffer was prepared.2. A 10 mM NADPH solution was prepared with a 100 mM PBS buffer. A 10 mM stock solution of the compound of the present invention was diluted with DMSO to give a compound working solution at a concentration of 200×(6000, 2000, 600, 200, 60, 20, and 0 μM). The positive inhibitor stock solution was diluted with DMSO to give a positive inhibitor working solution at a concentration of 200×(sulfaphenazole, 1000, 300, 100, 30, 10, 3, and 0 μM; quinidine, 100, 30, 10, 3, 1, 0.3, and 0 μM). Substrate working solutions (120 μM diclofenac and 400 μM dextromethorphan) at a concentration of 200× were 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 well, and placed in a 37° C. water bath for pre-incubation for 15 min. 1 μL of sulfaphenazole or quinidine working solution was added to the positive control group instead of the compound working solution. At the same time, 10 mM NADPH solution was placed together in the 37° C. water bath for pre-incubation for 15 min. After 15 min, 20 μL of NADPH was added to each well to initiate the reaction. The mixture was incubated at 37° C. for 5 min (CYP2C9) or 20 min (CYP2D6). All incubated samples were in duplicate. After incubation for the corresponding period of time, 400 μL of icy methanol containing internal standard was added to all samples to stop the reaction. The mixture was vortexed, mixed well, and centrifuged for 40 min at 4° C. at 3220 g. 100 μL of the supernatant was transferred to a feeding plate after the centrifugation was completed, and 100 μL ultrapure water was added. The mixture was well mixed for LC-MS/MS analysis. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |