Assay Method Information

Assay Name:  Inhibition Assay
Description:  Solutions of each test compound were separately prepared at concentrations of 20000, 6000, 2000, 600, 200, and 60 uM by serial dilution with DMSO:MeCN (50:50 v/v). The individual test compound solutions were then diluted 20-fold with DMSO:MeCN:deionized water (5:5:180 v/v/v) to concentrations of 1000, 300, 100, 30, 10, and 3 M. Mixtures of isozyme inhibitors (sulfaphenazole, tranylcypromine, and ketoconazole as specific inhibitors of isozymes 2C9, 2C19, and 3A4, respectively) were prepared containing each inhibitor at concentrations of 6000, 2000, 600, 200, 60, 20, 6, and 2 uM by serial dilution with DMSO:CH3CN (50:50 v/v). The mixed inhibitor solutions were then diluted 20-fold with DMSO: CH3CN:deionized water (5:5:180 v/v/v) to concentrations of 300, 100, 30, 10, 3, 1, 0.3, and 0.1 uM. The percent of organic solvent attributable to the test compound or inhibitor mixture in the final reaction mixture was 2% v/v. Pooled human liver microsome suspension (20 mg/mL) was diluted with phosphate buffer to obtain a 5 mg/mL suspension.
Affinity data for this assay
 

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