CYP2C9-Catalyzed Metabolism ofS-Warfarin to 7-Hydroxywarfarin in Vivo and in Vitro in Chimeric Mice with Humanized Liver

Abstract

ABSTRACT:Chimeric mice having humanized livers were constructed by transplantation of human hepatocytes. In this study, we investigated whether these mice have a capacity for drug metabolism similar to that of humans by examining hydroxylation of S-warfarin, which is predominantly metabolized to S-7-hydroxywarfarin, catalyzed by CYP2C9, in humans but not mice. The 7-hydroxylating activity of chimeric mouse liver microsomes toward S-warfarin was approximately 10-fold higher than that of control (urokinase-type plasminogen activator-transgenic severe combined immunodeficient) mice. The 7-hydroxylase activity of chimeric mouse liver microsomes was markedly inhibited by sulfaphenazole, as was that of human liver microsomes, whereas the activity of control mice was unaffected. The CYP2C isoform in chimeric mouse liver was also confirmed to be the human isoform, CYP2C9, by immunoblot analysis. In the present in vivo study, the level of S-7-hydroxywarfarin in plasma of chimeric mice was approximately 7-fold higher than that in control mice, in agreement with the in vitro data. Thus, the CYP2C isoform in chimeric mice functions in vivo and in vitro as a human isoform, CYP2C9. These results suggest that chimeric mice with humanized liver could be useful for predicting drug metabolism in humans, at least regarding CYP2C9-dependent metabolism.Chimeric mice have been constructed by transplantation of human hepatocytes into urokinase-type plasminogen activator-transgenic severe combined immunodeficient mice (Dandri et al., 2001;Mercer et al., 2001). It was suggested that these chimeric mice may be useful as an in vivo model for studies on human liver diseases and hepatotropic viruses. Prolonged infections with human hepatitis B or C virus can be maintained in these mice (Dandri et al., 2001;Mercer et al., 2001). However, the chimeric mice used in those experiments did not have a high level of replacement with human hepatocytes. In a recent study, Tateno et al. (2004) prepared chimeric mice in which the liver was almost completely repopulated with human hepatocytes. Furthermore, they reported that cytochrome P450 subtypes in liver microsomes of chimeric mice in which more than 80% of hepatocytes had been replaced with human hepatocytes were similar to those of the donor human liver . Therefore, the chimeric mice constructed by Tateno et al. (2004) appear to be an excellent in vivo model for prediction of drug metabolism and drug-drug interactions due to drug induction and inhibition of drugmetabolizing enzymes in humans. Katoh et al. (2004Katoh et al. ( , 2005a reported that cytochrome P450 (P450) isoforms and phase II enzymes in chimeric mice in which the human hepatocyte replacement rate was nearly 90% were almost the same as those in human liver, based on quantitation of enzyme proteins. They also showed that the chimeric mice are a useful animal model to estimate the inductive effect on P450 in humans (Katoh et al., 2005b). We have shown that aldehyde oxidase, a cytosolic drugmetabolizing enzyme, in chimeric mice has func...

Investigation of Drug-Drug Interactions Caused by Human Pregnane X Receptor-Mediated Induction of CYP3A4 and CYP2C Subfamilies in Chimeric Mice with a Humanized Liver

“…Although no variant sequence was detected in the CYP2C9 gene (data not shown), real-time quantitative reverse transcription-PCR analysis revealed that the mRNA expression level of CYP2C9 in the hepatocytes was relatively low compared with that in other donor hepatocytes (data not shown). In fact, the plasma elimination of WAR in this study seems to be slower than that in the previous study using PXB mice transplanted with a different lot of human hepatocytes (Inoue et al, 2008). Therefore, the main reason for the failure to detect of CYP2C9 induction in the in vivo study was probably the low hepatic expression of CYP2C9 in the PXB mice used in this study.…”

Evaluation of the pharmacokinetics, biotransformation and hepatic transporter effects of troglitazone in mice with humanized livers

“…In addition, it has been demonstrated that chimeric mice were able to generate humanspecific metabolites in vivo through primary and/or secondary metabolic reactions Inoue et al 2009;De Serres et al 2011). In agreement with these findings, in vitro studies using liver microsomes, cytosol and hepatocytes from chimeric mice have shown the formation of human selective metabolites (Aoki et al 2006;Inoue et al 2008;Kitamura et al 2008). It has been concluded that chimeric mice with humanized livers have the ability to achieve improved prediction of systemic metabolite exposure in humans when compared with conventional animal strains, providing metabolism is either completely or primarily hepatic (Kamimura et al 2010).…”

“…One approach which has the potential to address at least some of the inherent limitations of human in vitro biotransformation systems is to perform studies in vivo in "chimeric" mice with humanized livers, in which the majority of the hepatocyte population of the mouse liver has been replaced by human hepatocytes (Tateno et al 2004;Katoh and Yokoi, 2007;Okumura et al 2007;Inoue et al 2008;Inoue et al 2009;Strom et al 2010;Kamimura et al 2010;Chen et al 2011;De Serres et al 2011;Lootens et al 2011). These livers have been shown to express virtually the full complement of human Phase I and II metabolic enzymes and hepatic transporter proteins with gene-expression profiles and phenotypes similar (up to 85%) to those of the original donor liver (Tateno et al 2004;Yoshizato and Tateno, 2009;Strom et al 2010;Yamasaki et al 2010).…”

Assessment of Chimeric Mice with Humanized Liver as a Tool for Predicting Circulating Human Metabolites

“…41) In their in vitro metabolism study using liver microsomes from chimeric mice, Inoue and her colleagues 42) found that warfarin-7-hydroxylase activity increased with increasing hepatic replacement ratio with human hepatocytes, and was markedly inhibited by the CYP2C9 inhibitor sulfaphenazole. They then administered (S)-warfarin to chimeric mice and analyzed the parent compound and its major metabolite in plasma.…”