Short Communication Aldehyde Oxidase-Catalyzed Metabolism of N-Methylnicotinamide in Vivo and in Vitro in Chimeric Mice with Humanized Liver

Aldehyde oxidase-mediated oxidation of N-methylnicotinamide to N-methyl-2-pyridine-5-carboxamide (2-PY) and N-methyl-4pyridone-5-carboxamide (4-PY) in chimeric mice constructed by transplanting human hepatocytes into urokinase-type plasminogen activator-transgenic severe combined immunodeficient mice was examined in vivo and in vitro. The activity in liver cytosol of chimeric mice with a high replacement index was approximately 4-fold higher than that in control mice. Furthermore, the oxidation products in control mice were 2-PY and 4-PY, whereas, in chimeric mice, the major product was 2-PY, as in humans. The aldehyde oxidase in chimeric mouse liver was confirmed to be of human type by immunoblotting analysis. The ratio of pyridones (2-PY/4-PY) excreted in the urine of chimeric mice was closer to that of humans than to that of control mice. Thus, the aldehyde oxidase in chimeric mice has human-type functional characteristics. Tateno et al. (2004) established chimeric mice in which the liver was almost completely repopulated with human hepatocytes. Such mice should be an excellent in vivo model for predicting drug metabolism, drug-drug interactions, drug induction, and inhibition of drug-metabolizing enzymes in humans. Katoh et al. (2004, 2005b) reported that the patterns of cytochrome P450 isoforms and phase II enzymes, such as UDP-glucuronosyltransferase, sulfotransferase, N-acetyltransferase, and glutathione S-transferase, in chimeric mice having a nearly 90% replacement rate with human hepatocytes were almost identical with those in human liver, and they used these mice to estimate the in vivo induction of cytochrome P450 enzymes in humans (Katoh et al., 2005a). Nishimura et al. (2005) reported that hepatocytes from chimeric mice with nearly completely humanized liver are useful for predictive screening of the induction potency of new drugs on drug-metabolizing enzymes in humans. We were interested in knowing whether drug metabolism by a cytosolic drugmetabolizing enzyme, aldehyde oxidase, in these mice is also similar to that in humans. Aldehyde oxidase (EC 1.2.3.1) contains flavin adenine dinucleotide, molybdenum, and iron-sulfur centers. It has been suggested to be relevant to the pathophysiology of a number of clinical disorders (Berger et al., 1995; Wright et al., 1995; Moriwaki et al., 1997). The enzyme in liver of various species catalyzes the oxidation of various aldehydes and nitrogenous heterocyclic xenobiotics, such as methotrexate and cyclophosphamide (Beedham, 1985; Kitamura et al., 2006), and also catalyzes the metabolism of physiological compounds, such as retinaldehyde (Huang and Ichikawa, 1994). However, there are marked species differences and strain differences of the enzyme activities for oxidative reaction in rats and mice (Beedham, 1985; Schofield et al., 2000; Kitamura et al., 2006; Sugihara et al., 2006). This has presented problems in preclinical studies. Many aldehyde oxidase substrates, such as methotrexate and phthalazine, show similar metabolic profiles among different species, though the level of the oxidase activity is species-dependent (Beedham, 1985; Kitamura et al., 1999, 2006). However, other substrates, such as famciclovir, 6-deoxypenciclovir, quinoxaline, 2-aminopteridine, and N-methylnicotinamide (NMN), show species differences in the metabolic profile (Beedham, 1985; Rashidi et al., 1997; Kitamura et al., 2006). We thought that NMN, which shows different metabolic profiles in mice and humans, would be the most suitable substrate for the present purpose. NMN, which is formed from nicotinamide by nicotinamide N-methyltransferase, is widely distributed in animals (Yan et al., 1997), and its metabolism to N-methyl-2-pyridone-5-carboxamide (2-PY) and N-methyl-4-pyridone-3-carboxamide (4-PY) is catalyzed by aldehyde oxidase. However, the ratio of pyridones (2PY/4-PY) formed from NMN varies among species. For example, 2-PY is mainly formed in humans, whereas 2-PY and 4-PY are equally formed in mice (Kitamura et al., 2006). In this study, we examined the characteristics of aldehyde oxidase activity in control mice and chimeric mice with a humanized liver by using NMN as a substrate. We demonstrated that oxidation of NMN in highreplacement chimeric mice showed a human-type pattern rather than a mouse-type pattern. Materials and Methods Chemicals. NMN, N -methylnicotinamide, and benzaldehyde were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), and 2-PY and 4-PY were prepared by the reported method (Sugihara et al., 2006). This work was supported by a Grant-in-Aid for Scientific Research from the Japanese Ministry of the Environment and the Japan Society for the Promotion of