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Assay Method Information

Assay Name:  CYP Enzyme Inhibition Assay
Description:  Human liver microsomes (HLMs) (0.3 mg/mL in 0.1-M potassium phosphate buffer, pH 7.4) are incubated with CYP (cytochromes P450) isozyme-selective substrates (phenacetin for CYP1A2, amodiaquine for CYP2C8, diclofenac for CYP2C9, S-mephenytoin for CYP2C19, Dextromethorphan for CYP2D6, and midalozam for CYP3A4/5), and multiple concentrations of Compound (I-1) or Ko143 (0, 0.041, 0.12, 0.37, 1.11, 3.33, 10, and 30 uM) in 96-well plates. Reactions are initiated by the addition of β-NADPH (2 mM) and MgCl2 (3 mM) in 0.1-M potassium phosphate buffer, pH 7.4. Reactions are incubated for 12 minutes at 37° C., and then terminated by the addition of an equal volume of ACN containing 1-uM carbutamide (IS). The plates are refrigerated at approximately 4° C. for 15 minutes and then centrifuged in order to pellet the precipitated proteins.
Affinity data for this assay
 

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Last update November 1, 2007
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