title: Metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin by human hepatic CYP isoforms: evidence for selectivity towards CYP3A4.
abstract: 1. The metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (BFBFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomes and in cDNA-expressed human liver CYP isoforms. For purposes of comparison, some limited studies were also performed with 7-benzyloxyquinoline (7BQ). 2. Initial interactive docking studies with a homology model of human CYP3A4 indicated that BFBFC was likely to be a selective substrate for CYP3A4 with a relatively high binding affinity, due to the presence of several key hydrogen bonds with active site amino acid residues. 3. Kinetic analysis of NADPH-dependent BFBFC metabolism to HFC in three preparations of pooled human liver microsomes revealed mean (+/- TSEM) Km and Vmax = 4.6 +/- 0.3 microM and 20.0 +/- 3.8 pmol/min/mg protein, respectively. 4. The metabolism of BFBFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing a BFBFC substrate concentration of lO microM (i.e. around twice Km). Good correlations (r2 = 0.736-0.904) were observed between BFBFC metabolism and markers of CYP3A isoforms. 5. While 10O microM BFBFC was metabolized to HFC by cDNA-expressed CYP3A4, little or no metabolism was observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 6. The metabolism of 10 microM BFBFC in human liver microsomes was markedly inhibited by 5-50 microM troleandomycin and 0.2-5 microM ketoconazole, but stimulated by 0.2-10 microM alpha-naphthoflavone. The metabolism of 10 microM BFBFC in human liver microsomes was also markedly inhibited by an antibody to CYP3A4. 7. Kinetic analysis of NADPH-dependent 7BQ metabolism to 7-hydroxyquinoline (7HQ) in human liver microsomes revealed Km and Vmax = 70 microM and 3.39 nmol/min/mg protein, respectively. 8. While 80 microM 7BQ was metabolized to 7HQ by cDNA-expressed CYP3A4, only low rates of metabolism were observed with cDNA-expressed CYPIA2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 9. In summary, by correlation analysis, the use of cDNA-expressed CYP isoforms, chemical inhibition and inhibitory antibodies, BFBFC metabolism in human liver microsomes appears to be primarily catalysed by CYP3A4. BFBFC may be a useful fluorescent probe substrate for human hepatic CYP3A4, but compared with 7BQ has only a low rate of metabolism in human liver microsomes.
authors.affiliation: TNO BIBRA International Ltd, Carshalton, Surrey, UK.
authors.forename: A B
authors.initials: AB
authors.lastname: Renwick
authors.forename: D F
authors.initials: DF
authors.lastname: Lewis
authors.forename: S
authors.initials: S
authors.lastname: Fulford
authors.forename: D
authors.initials: D
authors.lastname: Surry
authors.forename: B
authors.initials: B
authors.lastname: Williams
authors.forename: P D
authors.initials: PD
authors.lastname: Worboys
authors.forename: X
authors.initials: X
authors.lastname: Cai
authors.forename: R W
authors.initials: RW
authors.lastname: Wang
authors.forename: R J
authors.initials: RJ
authors.lastname: Price
authors.forename: B G
authors.initials: BG
authors.lastname: Lake
authors.forename: D C
authors.initials: DC
authors.lastname: Evans
mesh_terms: D003374:Coumarins / Q000737:chemistry* / Q000378:metabolism* / Q000493:pharmacokinetics; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008956:Models, Chemical; D008958:Models, Molecular; D010641:Phenotype; D011485:Protein Binding; D020033:Protein Isoforms; D011804:Quinolines / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: C444101:2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin; C419147:7-benzyloxy-4-trifluoromethylcoumarin; D003374:Coumarins; D018076:DNA, Complementary; D020033:Protein Isoforms; D011804:Quinolines; C065956:7-benzyloxyquinoline; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human