TY - JOUR T1 - Novel Cytochrome P450 Reaction Phenotyping for Low Clearance Compounds Using Hepatocyte Relay Method JF - Drug Metabolism and Disposition JO - Drug Metab Dispos DO - 10.1124/dmd.115.067876 SP - dmd.115.067876 AU - Xin Yang AU - Karen Atkinson AU - Li Di Y1 - 2015/01/01 UR - http://dmd.aspetjournals.org/content/early/2015/12/23/dmd.115.067876.abstract N2 - A novel P450 reaction phenotyping method for low clearance compounds has been developed for eight cytochrome P450 enzymes (CYP1A2, 2B6, 2D6, 2C8, 2C9, 2C19, 3A and 3A4) and pan-CYP using the hepatocyte relay approach. Selective mechanism based inhibitors were used to inactivate the individual P450 enzymes during pre-incubation, and inhibitors were removed from the incubation prior to adding substrates to minimize reversible inhibition and maximize selectivity. With the experimental design, the inhibitors were quite selective for specific P450 isoforms using the following inhibitor concentrations and pre-incubation times: furafylline (1 μM, 15 min) for CYP1A2, phencyclidine (20 μM, 15 min) for 2B6, paroxetine (1.8 μM, 15 min) for CYP2D6, gemfibrozil glucuronide (100 μM, 30 min) for 2C8, tienilic acid (15 μM, 30 min) for 2C9, esomeprazole (8μM, 15 min) for 2C19, troleandomycin (25 μM, 15 min) for 3A4/5, CYP3cide (2 μM, 15 min) for 3A4 and 1-aminobenzotriazole (1 mM, 30 min) supplemented with tienilic acid (15 μM, 30 min) for pan-CYPs. The inhibitors were successfully applied to the hepatocyte relay method in a 48-well format for P450 reaction phenotyping of low clearance compounds. This novel method provides a new approach for determining fraction metabolized of low turnover compounds that are otherwise challenging with the traditional methods, such as chemical inhibitors with human liver microsomes and hepatocytes, or human recombinant P450 enzymes. ER -