RT Journal Article
SR Electronic
T1 Novel Cytochrome P450 Reaction Phenotyping for Low-Clearance Compounds Using the Hepatocyte Relay Method
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 460
OP 465
DO 10.1124/dmd.115.067876
VO 44
IS 3
A1 Xin Yang
A1 Karen Atkinson
A1 Li Di
YR 2016
UL http://dmd.aspetjournals.org/content/44/3/460.abstract
AB A novel cytochrome P450 (P450) reaction phenotyping method for low-clearance compounds has been developed for eight P450 enzymes (CYP1A2, 2B6, 2D6, 2C8, 2C9, 2C19, 3A, and 3A4) and pan-cytochrome using the hepatocyte relay approach. Selective mechanism-based inhibitors were used to inactivate the individual P450 enzymes during preincubation, and inactivators were removed from the incubation before adding substrates to minimize reversible inhibition and maximize inhibitor specificity. The inhibitors were quite selective for specific P450 isoforms using the following inhibitor concentrations and preincubation times: furafylline (1 µM, 15 minutes) for CYP1A2, phencyclidine (20 µM, 15 minutes) for 2B6, paroxetine (1.8 µM, 15 minutes) for CYP2D6, gemfibrozil glucuronide (100 µM, 30 minutes) for 2C8, tienilic acid (15 µM, 30 minutes) for 2C9, esomeprazole (8 µM, 15 minutes) for 2C19, troleandomycin (25 µM, 15 minutes) for 3A4/5, CYP3cide (2 µM, 15 minutes) for 3A4, and 1-aminobenzotriazole (1 mM, 30 minutes) supplemented with tienilic acid (15 µM, 30 minutes) for pan-cytochrome. 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 the 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.