Metabolism of 7-benzyloxy-4-trifluoromethylcoumarin by human hepatic cytochrome P450 isoforms

Abstract

1. The metabolism of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomal preparations and in cDNA-expressed human cytochrome P450 (CYP) isoforms. 2. Kinetic analysis of the NADPH-dependent metabolism of BFC to HFC in four preparations of pooled human liver microsomes revealed mean (±SEM) K_m and V_max of 8.3±1.3 μM and 454±98 pmol/min/mg protein respectively. 3. The metabolism of BFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing BFC substrate concentrations of 20 and 50 μM (i.e. about two and six times K_m respectively). With 20 μM BFC the highest correlations were observed between BFC metabolism and markers of CYP1A2 (r² = 0.784-0.797) and then with CYP3A (r² = 0.434-0.547) isoforms, whereas with 50 μM BFC the highest correlations were observed between BFC metabolism and markers of CYP3A (r² = 0.679-0.837) and then with CYP1A2 (r² = 0.421-0.427) isoforms. At both BFC substrate concentrations, lower correlations were observed between BFC metabolism and enzymatic markers for CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP4A9/11. 4. Using human β-lymphoblastoid cell microsomes containing cDNA-expressed CYP isoforms, 20 μM BFC was metabolized by CYP1A2 and CYP3A4, with lower rates of metabolism being observed with CYP2C9 and CYP2C19. Kinetic studies with the CYP1A2 and CYP3A4 preparations demonstrated a lower K_m with the CYP1A2 preparation, but a higher V_max with the CYP3A4 preparation. 5. The metabolism of 20 μM BFC in human liver microsomes was inhibited to 37-48% of control by 5-100 μM of the mechanism-based CYP1A2 inhibitor furafylline and to 64-69% of control by 5-100 μM of the mechanism-based CYP3A4 inhibitor roleandomycin. While some inhibition of BFC metabolism was observed in the presence of 100 and 200 μM diethyldithiocarbamate, the addition of 2-50 μM sulphaphenazole, 50-500 μM Smephenytoin and 2-50 μM quinidine had little effect. 6. The metabolism of 20 μM BFC to HFC in human liver microsomes was also inhibited by an antibody to CYP3A4, whereas antibodies to CYP2C8}9 and CYP2D6 had no effect. 7. In summary, by correlation analysis, use of cDNA-expressed CYP isoforms, chemical inhibition and inhibitory antibodies, BFC appears metabolized by a number of CYP isoforms in human liver. BFC metabolism appears to be primarily catalysed by CYP1A2 and CYP3A4, with possibly some contribution by CYP2C9, CYP2C19 and perhaps other CYP isoforms. 8. The results also demonstrate the importance of the selection of an appropriate substrate concentration when conducting reaction phenotyping studies with human hepatic CYP isoforms.