Flavin-Containing Monooxygenase Activity in Hepatocytes and Microsomes: In Vitro Characterization and In Vivo Scaling of Benzydamine Clearance

Abstract

ABSTRACT:Liver microsomes, and more recently cryopreserved hepatocytes, are commonly used in the in vitro characterization of the metabolism of new xenobiotics. The flavin-containing monooxygenases (FMO) are a major nonP450 oxidase present in liver microsomes and hepatocytes. Since FMO is known to be thermally labile, and this enzyme may be involved in the metabolic clearance of some drugs, we sought to more completely characterize the metabolic competency of this enzyme in cryopreserved hepatocytes and in liver microsomes preincubated under various conditions using benzydamine as an in vitro and in vivo probe. The metabolism of benzydamine to its major metabolite, the N-oxide, is mediated by FMO3 in humans. We found that the in vitro microsomal t 1/2 was 70% longer when incubations were prewarmed at 37°C in the absence of NADPH compared with prewarming in the presence of an NADPH-regenerating system, and N-oxide formation was inhibited >99%. Interestingly, the in vivo clearance predicted from these incubations and from human hepatocytes overpredicted the observed clearance of benzydamine in humans (>10.5 versus 2.4 ml/min/kg). In contrast, rat hepatocytes successfully predicted rat in vivo benzydamine clearance to within ϳ30% (>68 versus 48 ml/min/kg). Benzydamine N-oxidation in liver microsomes from all common preclinical species demonstrated heat sensitivity. This information should be considered when extrapolating metabolism data of xenobiotics from these in vitro systems.

Role of Flavin-Containing Monooxygenase in Oxidative Metabolism of Voriconazole by Human Liver Microsomes

“…Unfortunately, the lack of inhibitory antibodies and FMO-selective inhibitors does not allow us to estimate the contribution of individual FMO enzymes in the overall metabolism of voriconazole. When the kinetic parameters for voriconazole metabolism by FMO1/FMO3 (Table 1) versus CYP2C19/ CYP3A4 (Hyland et al, 2003) are scaled to a typical human liver, containing 80 (FMO3), 15 (CYP2C9), and 100 (CYP3A4) pmol of enzyme/mg of microsomal protein (Fisher et al, 2002;Koukouritaki et al, 2004;Hines 2006), it is expected that FMO would contribute to less than 2% of the voriconazole metabolic clearance by CYP3A4 and an even smaller percentage of the metabolic clearance by CYP2C19. However, the in vitro (heat inactivation) studies with human liver microsomes (Fig.…”

Metabolic Profiling of Human Long-Term Liver Models and Hepatic Clearance Predictions from In Vitro Data Using Nonlinear Mixed-Effects Modeling

“…Furthermore, substantial donor variability of the AO activity was observed by these authors (33) in primary hepatocyte suspension cultures. For FMO, loss of enzyme function due to either liver tissue preparation (34,35) or with time in plated human primary hepatocytes was found previously (36). Thus, either biological variability or in vitro conditions could explain the reduced AO and FMO activities in the long- Fig.…”

Metabolism and Disposition of a Selective α7 Nicotinic Acetylcholine Receptor Agonist in Humans

“…Sample aliquots (45 l) were removed by micropipette at 0, 5, 10, 15, 30, and 45 min after addition of 1 (or buffer), and quenched and processed as described above for LC-MS/MS analysis. FMO1 and FMO3 viability was confirmed by monitoring the NADPH-dependent conversion of the respective isozyme-selective substrates (both at 1 M) imipramine (Lemoine et al, 1990) and benzydamine (Fisher et al, 2002) to their N-oxide metabolites.…”