ABSTRACT:Recently, a chimeric mouse line in which the liver could be replaced by more than 80% with human hepatocytes was established in Japan. Because the chimeric mouse produces human albumin (hAlb), replacement by human hepatocytes could be estimated by the hAlb concentration in the blood of chimeric mice. In this study, we investigated human major cytochrome P450 (P450) in the livers of chimeric mice by mRNA, protein, and enzyme activity using real-time polymerase chain reaction, Western blot analysis, and high-performance liquid chromatography, respectively. Chimeric mice with humanized liver generated using hepatocytes from a Japanese and white donor were used. Human P450 mRNAs were expressed in the liver of chimeric mice, and major human P450 proteins such as CYP1A2, CYP2C9, and CYP3A4 were detected. The expression of P450 mRNA and protein was correlated with the hAlb concentration in the blood. The enzyme activities such as diclofenac 4-hydroxylase activity, dexamethasone 6-hydroxylase activity, and coumarin 7-hydroxylase activity, activities that are specific to human P450 but not to murine P450, were increased in a hAlb concentration-dependent manner. The chimeric mice with nearly 90% replacement by human hepatocytes demonstrated almost the same protein contents of human P450s and drug-metabolizing enzyme activity as those of the donor. It was confirmed that genomic DNA from the livers of the chimeric mice and that from the liver of the donor exhibited the same genotype. In conclusion, the chimeric mice exhibited a similarly efficient capacity of drug metabolism as humans, suggesting that they could be a useful animal model for drug development.

ABSTRACT:We clarified that major human cytochrome P450 (P450) enzymes were expressed in a chimeric mouse line established recently in Japan, in which the liver could be replaced by more than 80% with human hepatocytes. In this study, we investigated major human phase II enzymes such as UDP-glucuronosyltransferase (UGT), sulfotransferase (SULT), N-acetyltransferase (NAT), and glutathione S-transferase (GST) in the livers of chimeric mice by mRNA, protein, and enzyme activity using reverse transcription-polymerase chain reaction, Western blot analysis, and high-performance liquid chromatography, respectively. Human UGT, SULT, NAT, and GST mRNA were expressed in the liver of the chimeric mice, and UGT2B7, SULT1E1, SULT2A1, and GSTA1 proteins could be detected. The expression of mRNA and protein was correlated with the human albumin (hAlb) concentration in mouse blood, the replacement of which by human hepatocytes could be estimated by the hAlb concentration in the blood of the chimeric mice, because the chimeric mice produce human albumin. The enzyme activities, such as morphine 6-glucuronosyltransferase activity and estrone 3-sulfotransferase activity, activities that are specific to humans but not to mice, were increased in a hAlb concentration-dependent manner. The chimeric mice with humanized liver with nearly 90% replacement by human hepatocytes demonstrated almost the same protein contents of human phase II enzymes and enzyme activities as those of the donor. In conclusion, the chimeric mice exhibited an efficient capacity of drug conjugation similar to that in humans. These chimeric mice expressed human phase II enzymes as well as P450s, suggesting that they could be a useful animal model in drug development.Drug metabolism, drug interactions involving drug-metabolizing enzymes, and genetic polymorphisms of such enzymes have been well studied. To clarify the mechanism of the changed pharmacokinetics in studies of drug interactions, we usually focus on cytochrome P450 (P450) enzymes. However, a major metabolic pathway of a compound with polar functional groups is sometimes a conjugation reaction. The contribution of phase II conjugation to the clearance of a drug was estimated to be approximately 30% or higher (Bjornsson et al., 2003). Major hepatic phase II enzymes in humans are UDP-glucuronosyltransferase (UGT), sulfotransferase (SULT), N-acetyltransferase (NAT), and glutathione S-transferase (GST). There have been few reports on drug interactions that focused on phase II enzymes. Recently, several advances in the understanding of the inhibition and induction of such phase II enzymes have been made, especially concerning UGT. The effects of many drugs on glucuronidation in humans were clarified and listed by Kiang et al. (2005). Moreover, genetic polymorphisms of UGT, NAT, SULT, and GST have been demonstrated and the allelic frequency and effects of such mutations toward enzyme activities have become clearer. The UGT1A1*28 allele showed reduced UGT1A1 enzyme activity and was suggested to be a significant ri...

ABSTRACT:The induction and inhibition of human cytochrome P450 (P450) enzymes are clinically responsible for drug interactions. Although the induction of P450s is investigated using human hepatocytes in the drug development process, there are some disadvantages, such as the decline of the enzyme activity during culture. In the present study, we examined the in vivo induction potency in chimeric mice with humanized liver, which was recently established in Japan to clarify whether this chimeric mouse model would be more suitable for human induction studies. Rifampicin and 3-methylcholanthrene (3-MC) were used in vivo as typical P450 inducers in the chimeric mice. The expression levels of human CYP3A4 mRNA and CYP3A4 protein and dexamethasone 6-hydroxylase activity, specific for human CYP3A4, were increased 8-to 22-, 3-to 10-, and 5-to 12-fold, respectively, by treatment with rifampicin. In addition, the expression levels of human CYP1A2 mRNA and CYP1A2 protein were also increased 2-to 9-and 5-fold, respectively, by treatment with 3-MC. Although other human P450s are expressed in the chimeric mice, there were few effects by the treatment of rifampicin and 3-MC on the mRNA, protein, and enzyme activity of those P450s. It was demonstrated that human P450s expressed in the chimeric mice with humanized liver were induced by rifampicin and 3-MC. This chimeric mouse model may be a useful animal model to estimate and predict the in vivo induction of P450s in humans.

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ABSTRACT:Tranilast is an oral antiallergic agent widely used in Japan. Recently, in Western populations, hyperbilirubinemia induced by tranilast was suspected during clinical trials. Tranilast has been reported to be mainly metabolized to a glucuronide and a phase I metabolite, 4-demethyltranilast (N-3). In the present study, we investigated the in vitro metabolism of tranilast in human liver and jejunum microsomes and recombinant UDP-glucuronosyltransferases (UGTs). The glucuronidation of tranilast was clarified to be mainly catalyzed by UGT1A1 in human liver and intestine. The K m values of tranilast glucuronosyltransferase activity were 51.5, 50.6, and 38.0 M in human liver microsomes, human jejunum microsomes, and recombinant UGT1A1, respectively. The V max values were 10.4, 42.9, and 19.7 pmol/min/mg protein in human liver microsomes, human jejunum microsomes, and recombinant UGT1A1, respectively. When the intrinsic clearance was calculated using the in vitro kinetic parameters, microsomal protein content, and weight of tissues, tranilast glucuronosyltransferase activity was 2.5-fold higher in liver than in intestine. Tranilast glucuronosyltransferase activity was strongly inhibited by bilirubin, a typical UGT1A1 substrate, and N-3, indicating that the phase I metabolite could affect the tranilast glucuronosyltransferase activity. In the case of N-3 formation, the K m and V max values were 37.1 M and 27.6 pmol/min/mg protein in human liver microsomes. The bilirubin glucuronosyltransferase activity was strongly inhibited by both tranilast and N-3, suggesting that tranilast-induced hyperbilirubinemia would be responsible for the inhibition by tranilast and N-3 of the bilirubin glucuronosyltransferase activity, as would the UGT1A1 genotype.Tranilast (N-(3Ј,4Ј-demethoxycinnamoyl)-anthranilic acid) is an oral antiallergic agent developed by Kissei Pharmaceutical Co. Ltd. (Nagano, Japan) and widely used in Japan for bronchial asthma, allergic rhinitis, atopic dermatitis, keloid, and hypertrophic scar. The mechanism of its efficacy is to inhibit chemical mediators from mast cells (Azuma et al., 1976) and the accumulation of collagen in granulation tissue (Isaji et al., 1987). Recently, a clinical trial regarding the prevention of restenosis after percutaneous transluminal coronary revascularization was performed in Western populations (Holmes et al., 2000). During that trial, it was found that hyperbilirubinemia might be induced by tranilast and the risk of hyperbilirubinemia was increased in individuals with Gilbert's syndrome (Danoff et al., 2004).Major metabolic pathways of tranilast have been shown to be glucuronidation, 4-demethylation (N-3), and sulfation of N-3 in the data sheet of tranilast provided by Kissei Pharmaceutical (Fig. 1). Tranilast, N-3, and N-3 sulfate were reported to be detected in human urine (Slobodzian et al., 1985). Since the urine sample was hydrolyzed by glucuronidase and/or base in that article, the formation of glucuronide could be speculated by a comparison of the chromatograms befo...