| Assay Method Information | |
| | Inhibition Assay |
| Description: | Master solutions were prepared containing human liver microsomes (Gibco, 0.2 mg/mL) and MgCl2 (5 mM) in potassium phosphate buffer (10 mM). To aliquots (169 μL) of the microsome solution was added test compound in acetonitrile (1 μL) and DMSO (1 μL) to provide final test compound concentrations of 0, 0.005, 0.05, 0.25, 1, 5, 10, and 25 μM.NADPH (10 mM) in ultra-pure water (20 μL) was added, and this mixture was incubated at 37° C. for 30 minutes. The enzyme reaction then was initiated by the addition of enzyme substrate (dextromethorphan) dissolved in 1 μL of acetonitrile and 9 μL of ultra-pure water. The final substrate concentration was 10 μM.After 20 minutes, the incubation mixture was diluted with 3 volumes of cold methanol containing imipramine (200 nM), labetalol (200 nM), and ketoprofen (2 μM) as internal standards. Samples were centrifuged at 16,000 g for 10 minutes, then an aliquot of the supernatant (200 μL) was removed and a |
| Affinity data for this assay | |
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