Chise Tateno

Near Completely Humanized Liver in Mice Shows Human-Type Metabolic Responses to Drugs

Human hepatocytes were transplanted into urokinase-type plasminogen activator-transgenic SCID mice (uPA/SCID mice), which are immunodeficient and undergo liver failure. The transplanted cells were characterized in terms of their in vivo growth potential and functions. The human hepatocytes progressively repopulated the murine host liver. However, the recipients died when the replacement index (RI) of the human hepatocytes exceeded 50%. The hosts (chimeric mice) survived at RI >50% when treated with a drug that has anti-human complement factor activity, and these mice developed livers with RI values as high as 96%. In total, 36 chimeric mice were generated, and the rate of successful engraftment was as high as 92%. The yield of chimeric mice with RI >70% was 32%. The human hepatocytes in the murine host liver expressed mRNAs for a variety of human cytochrome P450 (hCYP) subtypes, in a manner that was similar to the donor liver. The mRNAs for hCYP3A4 and hCYP1A1/2 were induced in the liver in a CYP type-specific manner when the mice were treated with rifampicin and 3-methylcholanthrene, respectively. These results indicate that human hepatocytes that propagate in mice retain their normal pharmacological responses. We conclude that the chimeric mouse developed in the present study is a useful model for assessing the functions and pharmacological responses of human hepatocytes.

Infection of human hepatocyte chimeric mouse with genetically engineered hepatitis B virus

Studies of hepatitis B virus (HBV) mutants have been hampered by the lack of a small animal model with long‐term infection of cloned HBV. Using a mouse model in which liver cells were highly replaced with human hepatocytes that survived over a long time with mature human hepatocyte function, we performed transmission experiments of HBV. Human serum containing HBV and the virus produced in HepG2 cell lines that transiently or stably transfected with 1.4 genome length HBV DNA were inoculated. Genetically modified e‐antigen–negative mutant strain also was produced and inoculated into the mouse model. A high‐level (≈1010 copies/mL) viremia was observed in mice inoculated with HBV‐positive human serum samples. The level of viremia tended to be high in mice with a continuously high human hepatocyte replacement index. High levels and long‐lasting viremia also were observed in mice injected with the in vitro generated HBV. The viremia continued up to 22 weeks until death or killing. Passage experiments showed that the serum of these mice contained infectious HBV. Genetically engineered hepatitis B e antigen–negative mutant clone also was shown to be infectious. Lamivudine effectively reduced the level of viremia in these infected mice. In conclusion, this mouse model of HBV infection is a useful tool for the study of HBV virology and evaluation of anti‐HBV drugs. Our results indicate that HBeAg is dispensable for active viral production and transmission. (HEPATOLOGY 2005;42:1046–1054.)

Single lymphocyte analysis with a microwell array chip.

Following genomics and proteomics, cytomics, a novel method of looking at life, has emerged for analyzing large populations of cells on a single‐cell basis with multiple parameters in a quantitative manner. We have developed a highly integrated live‐cell microarray system for analyzing the cellular responses of individual cells using a microwell array chip that has 234,000 microwells each of which is just large enough to fit a single cell. Compared with flow cytometry and microscope‐based methods, our system can analyze the history of the cellular responses of a large number of cells. We have successfully applied the system to analyze human antigen‐specific B‐cells and produced human monoclonal antibodies (MoAb) against hepatitis B virus surface antigen. We have also constructed a mouse system to assess hepatitis B virus‐neutralization activity and have demonstrated the neutralization activity of our antibodies. Our technology should expand the horizons of cell analysis as well as enable generation of human MoAb for antibody‐based therapeutics and diagnosis for infectious diseases such as hepatitis viruses.

EXPRESSION OF HUMAN CYTOCHROMES P450 IN CHIMERIC MICE WITH HUMANIZED LIVER

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.

Expression of human phase II enzymes in chimeric mice with humanized liver.

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.

Rapid emergence of telaprevir resistant hepatitis C virus strain from wildtype clone in vivo

Telaprevir is a potent inhibitor of hepatitis C virus (HCV) NS3‐4A protease. However, the emergence of drug‐resistant strains during therapy is a serious problem, and the susceptibility of resistant strains to interferon (IFN), as well as the details of the emergence of mutant strains in vivo, is not known. We previously established an infectious model of HCV using human hepatocyte chimeric mice. Using this system we investigated the biological properties and mode of emergence of mutants by ultra‐deep sequencing technology. Chimeric mice were injected with serum samples obtained from a patient who had developed viral breakthrough during telaprevir monotherapy with strong selection for resistance mutations (A156F [92.6%]). Mice infected with the resistant strain (A156F [99.9%]) developed only low‐level viremia and the virus was successfully eliminated with interferon therapy. As observed in patients, telaprevir monotherapy in viremic mice resulted in breakthrough, with selection for mutations that confer resistance to telaprevir (e.g., a high frequency of V36A [52.2%]). Mice were injected intrahepatically with HCV genotype 1b clone KT‐9 with or without an introduced resistance mutation, A156S, in the NS3 region, and treated with telaprevir. Mice infected with the A156S strain developed lower‐level viremia compared to the wildtype strain but showed strong resistance to telaprevir treatment. Although mice injected with wildtype HCV showed a rapid decline in viremia at the beginning of therapy, a high frequency (11%) of telaprevir‐resistant NS3 V36A variants emerged 2 weeks after the start of treatment. Conclusion: Using deep sequencing technology and a genetically engineered HCV infection system, we showed that the rapid emergence of telaprevir‐resistant HCV was induced by mutation from the wildtype strain of HCV in vivo. (HEPATOLOGY 2011;).

Chimeric mice with humanized liver.

Recently, chimeric mice with humanized liver were established by transplanting human hepatocytes into an urokinase-type plasminogen activator(+/+)/severe combined immunodeficient transgenic mouse line. The replacement with human hepatocytes is more than 80-90% and is higher than any other chimeric mouse reported previously. In drug development, the liver is one of the most important organs because it is mainly involved in the pharmacokinetics of drugs and is frequently damaged by many drugs due to the accumulation of drugs and/or metabolites. The pharmacokinetics could affect the efficacy and toxicity of a drug, and thus prediction of the human pharmacokinetics is important for developing new drugs without adverse reactions and toxicity. Extrapolation from experimental animals or in vitro studies to the human in vivo pharmacokinetics is still difficult. To date, human hepatocytes and liver microsomes are recognized as better tools and are frequently used to estimate the human pharmacokinetics. We thought that chimeric mice with humanized liver could become a new tool for estimating the human toxicity and pharmacokinetics. At first, metabolism, which plays an essential role in pharmacokinetics, was investigated in the chimeric mice. In the liver of the chimeric mice, human drug metabolizing enzymes were found to be expressed and to reflect the capacities and genetic polymorphism of the donor. In an in vivo study on metabolism, human specific metabolites could be detected in the serum of the chimeric mice indicating that the chimeric mice could be used as an in vivo model to address human metabolism. These results suggested that the chimeric mice could overcome the species differences in drug metabolism and be used to evaluate drug toxicity due to genetic polymorphism. The reasons for drug interaction are often enzyme induction and inhibition. By the treatment with a typical inducer of cytochrome P450 (P450), which is the central drug-metabolizing enzyme, P450s expressed in the liver of the chimeric mice were found to possess induction potencies. After the treatment with a specific inhibitor of human P450, the area under the curve of the P450 metabolite was significantly decreased in the chimeric mice but not in the control mice. Therefore, it was indicated that the chimeric mice could be useful for assessing drug interactions in vivo. Moreover, drug excretion was determined to be humanized because cefmetazole was mainly excreted in urine both in the chimeric mice and humans but in the feces in control uPA(-/-)/SCID mice. Drug transporters expressed in the liver of the chimeric mice were also humanized. In this review, studies of the chimeric mice with humanized liver, particularly on metabolism and excretion, are summarized and the possibility of using the chimeric mice is proposed for the advanced prediction of human pharmacokinetics and toxicity.

Pleiotrophin/Heparin-Binding Growth-Associated Molecule as a Mitogen of Rat Hepatocytes and Its Role in Regeneration and Development of Liver

Previously pleiotrophin (PTN) was identified among proteins secreted by Swiss 3T3 cells as a mitogen for cultured adult rat hepatocytes. The present study showed that the growth of rat hepatocytes was enhanced when cultured with rat hepatic stellate cells (HSCs). HSCs expressed PTN mRNA and secreted its protein in the co-cultures. Recombinant PTN enhanced the growth of hepatocytes in culture, suggesting that HSCs stimulate the growth of hepatocytes through the action of PTN. To know the biological role of PTN in the growth of hepatocytes in vivo, we examined the expression of PTN in four regeneration models of adult liver and embryonic liver of rat. The expression of PTN mRNA in the liver was markedly up-regulated by the treatment with D-galactosamine (GalN) or with acetylaminofluorene followed by partial hepatectomy. HSCs expressed PTN mRNA in response to GalN treatment and its protein was found on hepatocytes. The mRNA expression of N-syndecan, a PTN receptor, was up-regulated in GalN-treated hepatocytes. The mesenchymal cells in the septum transversum enclosing the embryonic liver, but not embryonic HSCs, expressed PTN mRNA. We suggest that PTN is secreted from activated adult HSCs and embryonic mesenchymal cells as a mitogen of parenchymal cells in adult and embryonic liver, respectively.

Emergence of a Novel Lamivudine-Resistant Hepatitis B Virus Variant with a Substitution Outside the YMDD Motif

ABSTRACT Lamivudine is a major drug approved for treatment of chronic hepatitis B virus (HBV) infection. Emergence of drug-resistant mutants with amino acid substitutions in the YMDD motif is a well-documented problem during long-term lamivudine therapy. Here we report a novel lamivudine-resistant strain of HBV with an intact YMDD motif, which included an amino acid substitution, rtA181T, in the reverse transcriptase (RT) domain of HBV polymerase. The substitution also induced a unique amino acid substitution (W172L) in the overlapping hepatitis B surface (HBs) protein. The YMDD mutant strains were not detected even by using the sensitive peptide nucleic acid-mediated PCR clamping method. The detected nucleotide substitution was accompanied by the emergence of an additional nucleotide substitution that induced amino acid change (S331C) in the spacer domain. The rtA181T mutant strain displayed a threefold decrease in susceptibility to lamivudine in in vitro experiments in comparison with the wild type. In vivo analysis using human hepatocyte-chimeric mice confirmed the resistance of this mutant strain to lamivudine. We developed a method to detect this novel rtA181T mutation and a previously reported rtA181T mutation with the HBs stop codon using restriction fragment length polymorphism PCR and identified one patient with the latter pattern among 40 patients with lamivudine resistance. In conclusion, although the incidence is not high, we have to be careful regarding the emergence of lamivudine-resistant mutant strains with intact YMDD motif.

Evaluation of mRNA expression of human drug-metabolizing enzymes and transporters in chimeric mouse with humanized liver

The hepatic mRNA expression of human drug-metabolizing enzymes and transporters in chimeric mise with almost-completely humanized liver (replacement index: 71–89%) was investigated. The mRNAs of 58 human phase I enzymes, 26 human phase II enzymes, 23 human transporters, and five mouse Cyps were measured in the chimeric mice with humanized liver generated using hepatocytes from a Japanese donor. The mRNA expression of 52 human phase I enzymes, which includes 20 human CYPs, 26 human phase II enzymes and 21 human transporters was ascertained in the chimeric mouse liver. Among them, the expression of the target mRNAs vital for liver function such as the metabolism and secretion of endogenous compounds appeared to be maintained. The central value for the expression ratio in all target genes in chimeric mouse liver to the donor liver was 0.46, which was lower than the substitution rate of chimeric mouse liver by donor liver. The ratio of mouse Cyp mRNA expression of chimeric mouse liver to that of control mouse liver was 0.19 or less, except for that of Cyp2b10. There were good correlations between the mRNA expression levels of human hepatic albumin gene, the values of the rate of replacement of mouse liver by human liver, and the human blood albumin concentration in the chimeric mice. The chimeric mice with humanized liver may be a useful tool for the evaluation of drug–drug interactions such as the inhibition and induction of drug-metabolizing enzymes and transporters.