| Assay Method Information | |
| | CYP1A2 Inhibition Assay |
| Description: | A stock solution of KCI838 was be prepared in DMSO at 50 mM and stored at −20° C. Serial dilutions of the stock solution were prepared in acetonitrile:DMSO (9:1) for CYP inhibition testing. The final DMSO content in the reaction mixture was equal in all solutions used within an assay, and was ≤0.2%. KCI838 was be incubated at seven increasing concentrations in with pooled human liver microsomes (Bioreclamation-IVT, Baltimore MD) in the presence of 2 mM NADPH in 100 mM potassium phosphate (pH 7.4) containing 5 mM magnesium chloride and the probe substrate, tacrine (5 uM), in a 200 μL assay (final volume). The selective CYP1A2 inhibitor α-naphthoflavone was screened alongside KCI838 as a positive control. After incubation for 10 min at 37° C. (Table 1), the reactions were terminated by addition of methanol containing internal standard for analytical quantification. The quenched samples were incubated at 4° C. for 10 min and centrifuged at 4° C. for 10 min. The supernatant was removed, and the probe substrate metabolite analyzed by LC-MS/MS. |
| Affinity data for this assay | |
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