title: Use of a benzyloxy-substituted lactone cyclooxygenase-2 inhibitor as a selective fluorescent probe for CYP3A activity in primary cultured rat and human hepatocytes.
abstract: The benzyloxy-substituted lactone cyclooxygenase-2 inhibitor DFB [3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-(methylsulfonyl)phenyl]furan-2(5H)-one] is metabolized in human and rat liver microsomal incubations and hepatocytes to a fluorescent metabolite, DFH [3-hydroxy-5,5-dimethyl-4-[4-(methylsulfonyl)phenyl]furan-2(5H)-one]. This process is CYP3A-mediated in both species, as demonstrated by incubations with recombinant CYP3A enzymes and experiments with inhibitory antibodies. Measurement of DFH fluorescence can be used as a rapid readout of CYP3A activity following microsomal or cultured hepatocyte incubations. In rat and human hepatocytes treated with prototypical inducers, the formation of DFH was linear for the first 30 min, with no secondary metabolism of DFH, such as phase II glucuronidation, observed at early time points. Using a panel of four prototypical inducers (phenobarbital, dexamethasone, phenytoin, and rifampicin), the correlation between testosterone 6beta-hydroxylation in cultured human hepatocytes and CYP3A enzyme level in cell lysate was confirmed. DFB debenzylation was then shown to correlate well with testosterone 6beta-hydroxylation in hepatocytes treated with these four inducers. Primary cultured rat and human hepatocyte induction assays were optimized for 24- and 96-well plates, respectively. Controls were established to evaluate whether test compounds demonstrate time-dependent CYP3A inhibition to avoid false negative results. Thus, the use of DFB, a fluorogenic CYP3A-selective probe substrate, affords a fast, efficient, and robust assay for the measurement of CYP3A induction in both rat and human cultured primary hepatocytes.
authors.affiliation: Merck Frosst Centre for Therapeutic Research, P.O. Box 1005, Pointe-Claire-Dorval, Quebec H9R 4P8, Canada. deborah_nicoll-griffith@merck.com
authors.forename: Deborah A
authors.initials: DA
authors.lastname: Nicoll-Griffith
authors.forename: Nathalie
authors.initials: N
authors.lastname: Chauret
authors.forename: Robert
authors.initials: R
authors.lastname: Houle
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Day
authors.forename: Michelle
authors.initials: M
authors.lastname: D'Antoni
authors.forename: José M
authors.initials: JM
authors.lastname: Silva
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015153:Blotting, Western; D002469:Cell Separation; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology*; D051544:Cytochrome P-450 CYP3A; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D005464:Fluorobenzenes / Q000494:pharmacology*; D005663:Furans / Q000494:pharmacology*; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D006900:Hydroxylation; D008565:Membrane Proteins; D008862:Microsomes, Liver; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013739:Testosterone / Q000378:metabolism
chemical_list: C421259:3,4-difluorobenzyloxy-5,5-dimethyl-4-(4-methylsulfonylphenyl)-(5H)-furan-2-one; D003470:Culture Media; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D005464:Fluorobenzenes; D005663:Furans; D008565:Membrane Proteins; D013739:Testosterone; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D010089:Oxidoreductases, N-Demethylating