Metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin by human hepatic CYP isoforms: evidence for selectivity towards CYP3A4

Citation
Ab. Renwick et al., Metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin by human hepatic CYP isoforms: evidence for selectivity towards CYP3A4, XENOBIOTICA, 31(4), 2001, pp. 187-204
Citations number
37
Categorie Soggetti
Pharmacology & Toxicology
Journal title
XENOBIOTICA
ISSN journal
00498254 → ACNP
Volume
31
Issue
4
Year of publication
2001
Pages
187 - 204
Database
ISI
SICI code
0049-8254(200104)31:4<187:MO2>2.0.ZU;2-0
Abstract
1. The metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethyl coumarin (BFBFC) to 7-hydroxy-4-trifluoromethylcourmarin (HFC) was studied in human liver microsomes and in cDNA-expressed human liver CYP isoforms. F or purpose of comparsion, some limited studies were also performed with 7-b enzyloxyquinoline (7BQ). 2. Initial interactive docking studies with a homology model of human CYP3A 4 indicated that BFBFC was likely to be a selective substrate for CYP3A4 wi th a relatively high binding affinity, due to the presence of several key h ydrogen bonds with active site amino acid residues. 3. Kinetic analysis of NADPH-dependent BFBFC metabolism to HFC in three pre paration of pooled human liver microsomes revealed mean (+/- SEM) K-m and V -max = 4.6 +/-0.3 muM 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 substr ate concentration of 10 muM (i.e. around twice K-m). Good correlations (r(2 ) = 0.736-0.904) were observed between BFBFC metabolism and markers of CYP3 A isoforms. 5. While 10 mum BFBFC was metabolized to HFC by cDNA-expressed CYP3A4, litt le or no metabolism was observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6 , CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1. 6. The metabolism of 10 muM BFBFC in human liver microsomes was markedly in hibited by 5-50 muM troleandomycin and 0.2-5 muM ketoconazole, but stimulat ed by 0.2-10 muM alpha -naphthoflavone. The metabolism of 10 muM BFBFC in h uman 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 K-m and V-max = 70 muM and 3.39 n mol/min/mg protein, respectively. 8. While 80 muM 7BQ was metabolized to 7HQ by cDNA-expressed CYP3A4, only l ow rates of metabolism were observed with cDNA-expressed CYP1A2, CYP2A6, CY P2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 9. In summary, by correlation analysis, the use of cDNA-expressed CYP isofo rms, chemical inhibition and inhibitory antibodies, BFBFC metabolism in hum an liver microsomes appears to be primarily catalysed by CYP3A4, BFBFC may be useful fluorescent probed substrate for human hepatic CYP3A4, but compar ed with 7BQ has only a low rate of metabolism in human liver microsomes.