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) Km and Vmax of 8.3 +/- 1.3 microM 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 microM (i.e. about two and six times Km respectively). With 20 microM 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 microM 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 beta-lymphoblastoid cell microsomes containing cDNA-expressed CYP isoforms, 20 microM 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 Km with the CYP1A2 preparation, but a higher Vmax with the CYP3A4 preparation. 5. The metabolism of 20 microM BFC in human liver microsomes was inhibited to 37-48% of control by 5-100 microM of the mechanism-based CYP1A2 inhibitor furafylline and to 64-69% of control by 5-100 microM of the mechanism-based CYP3A4 inhibitor troleandomycin. While some inhibition of BFC metabolism was observed in the presence of 100 and 200 microM diethyldithiocarbamate, the addition of 2-50 microM sulphaphenazole, 50-500 microm S-mephenytoin and 2-50 microM quinidine had little effect. 6. The metabolism of 20 microM 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.

In Vitro Evaluation of Metabolic Drug–Drug Interactions: Concepts and Practice

“…It is important to use substrate concentrations similar to expected plasma concentrations. An artifactually high concentration would cause the substrate to be metabolized similarly by high and low affinity enzyme pathways (Renwick et al, 2004). Using liver microsomes with physiologically relevant substrate concentrations should provide the best results.…”

Fluorescence-Based Assays for Screening Nine Cytochrome P450 (P450) Activities in Intact Cells Expressing Individual Human P450 Enzymes

“…Different O-alkyl derivatives of resorufin (Burke et al, 1985), fluorescein (Stresser et al, 2000), 7-hydroxycoumarins (White, 1988;Ekins et al, 1997;Venhorst et al, 2000;Bapiro et al, 2001;Chauret et al, 2001), 6-hydroxyquinolines (Stresser et al, 2000), and 4-methylsulfonylphenyl furanones (Chauret et al, 1999) have been proposed as useful probes for this purpose. Some fluorimetric probes have been reported to be selective for individual P450 activities (Nerurkar et al, 1993;Chauret et al, 1999Chauret et al, , 2001Renwick et al, 2000), but most of them are not selective. Nonselective substrates are not appropriate for in vitro models showing several P450s such as human liver microsomes or hepatocytes.…”

A microtiter‐plate‐based cytochrome P450 3A activity assay in fish cell lines

“…We first wanted to know whether this human CYP3A substrate is also metabolized by CYP3A enzymes of different fish. For this reason, we adapted the protocol from Mensah-Osman et al [29] to evaluate whether the human-specific CYP3A substrate BFC [30][31][32] can also be catalyzed through fish CYP3A enzymes into the fluorescent product, as in carp [24]. As shown in Figure 1, fish CYP3A enzymes indeed can catalyze BFC.…”