Hepatocyte Intrinsic Innate Antiviral Immunity against Hepatitis Delta Virus Infection: The Voices of Bona Fide Human Hepatocytes.

The pathogenesis of viral infection is attributed to two folds: intrinsic cell death pathway activation due to the viral cytopathic effect, and immune-mediated extrinsic cellular injuries. The immune system, encompassing both innate and adaptive immunity, therefore acts as a double-edged sword in viral infection. Insufficient potency permits pathogens to establish lifelong persistent infection and its consequences, while excessive activation leads to organ damage beyond its mission to control viral pathogens. The innate immune response serves as the front line of defense against viral infection, which is triggered through the recognition of viral products, referred to as pathogen-associated molecular patterns (PAMPs), by host cell pattern recognition receptors (PRRs). The PRRs-PAMPs interaction results in the induction of interferon-stimulated genes (ISGs) in infected cells, as well as the secretion of interferons (IFNs), to establish a tissue-wide antiviral state in an autocrine and paracrine manner. Cumulative evidence suggests significant variability in the expression patterns of PRRs, the induction potency of ISGs and IFNs, and the IFN response across different cell types and species. Hence, in our understanding of viral hepatitis pathogenesis, insights gained through hepatoma cell lines or murine-based experimental systems are uncertain in precisely recapitulating the innate antiviral response of genuine human hepatocytes. Accordingly, this review article aims to extract and summarize evidence made possible with bona fide human hepatocytes-based study tools, along with their clinical relevance and implications, as well as to identify the remaining gaps in knowledge for future investigations.

A mechanistic biomarker investigation of fialuridine hepatotoxicity using the chimeric TK-NOG Hu-liver mouse model and in vitro micropatterned hepatocyte cocultures.

Fialuridine (FIAU) is a nucleoside-based drug that caused liver failure and deaths in a human clinical trial that were not predicted by nonclinical safety studies. A recent report concluded that a TK-NOG humanized liver (hu-liver) mouse model detected human-specific FIAU liver toxicity, and broader use of that model could improve drug safety testing. We further evaluated this model at similar dose levels to assess FIAU sensitivity and potential mechanistic biomarkers. Although we were unable to reproduce the marked acute liver toxicity with two separate studies (including one with a "sensitized" donor), we identified molecular biomarkers reflecting the early stages of FIAU mitochondrial toxicity, which were not seen with its stereoisomer (FIRU). Dose dependent FIAU-induced changes in hu-liver mice included more pronounced reductions in mitochondrial to nuclear DNA (mtDNA/nucDNA) ratios in human hepatocytes compared to mouse hepatocytes and kidneys of the same animals. FIAU treatment also triggered a p53 transcriptional response and opposing changes in transcripts of nuclear- and mitochondrial-encoded mitochondrial proteins. The time dependent accumulation of FIAU into mtDNA is consistent with the ≥9-week latency of liver toxicity observed for FIAU in the clinic. Similar changes were observed in an in vitro micro-patterned hepatocyte coculture system. In addition, FIAU-dependent mtDNA/nucDNA ratio and transcriptional alterations, especially reductions in mitochondrially encoded transcripts, were seen in livers of non-engrafted TK-NOG and CD-1 mice dosed for a shorter period. Conclusion: These mechanistic biomarker findings can be leveraged in an in vitro model and in a more routine preclinical model (CD-1 mice) to identify nucleosides with such a FIAU-like mitochondrial toxicity mechanistic liability potential. Further optimization of the TK-NOG hu-liver mouse model is necessary before broader adoption for drug safety testing.

Comprehensive gene profiling of the metabolic landscape of humanized livers in mice.

BACKGROUND & AIMS: The human liver transcriptome is complex and highly dynamic, e.g. one gene may produce multiple distinct transcripts, each with distinct posttranscriptional modifications. Direct knowledge of transcriptome dynamics, however, is largely obscured by the inaccessibility of the human liver to treatments and the insufficient annotation of the human liver transcriptome at transcript and RNA modification levels. METHODS: We generated mice that carry humanized livers of identical genetic background and subjected them to representative metabolic treatments. We then analyzed the humanized livers with nanopore single-molecule direct RNA sequencing to determine the expression level, m6A modification and poly(A) tail length of all RNA transcript isoforms. Our system allows for the de novo annotation of human liver transcriptomes to reflect metabolic responses and for the study of transcriptome dynamics in parallel. RESULTS: Our analysis uncovered a vast number of novel genes and transcripts. Our transcript-level analysis of human liver transcriptomes also identified a multitude of regulated metabolic pathways that were otherwise invisible using conventional short-read RNA sequencing. We revealed for the first time the dynamic changes in m6A and poly(A) tail length of human liver transcripts, many of which are transcribed from key metabolic genes. Furthermore, we performed comparative analyses of gene regulation between humans and mice, and between two individuals using the liver-specific humanized mice, revealing that transcriptome dynamics are highly species- and genetic background-dependent. CONCLUSION: Our work revealed a complex metabolic response landscape of the human liver transcriptome and provides a novel resource to understand transcriptome dynamics of the human liver in response to physiologically relevant metabolic stimuli (https://caolab.shinyapps.io/human_hepatocyte_landscape/). IMPACT AND IMPLICATIONS: Direct knowledge of the human liver transcriptome is currently very limited, hindering the overall understanding of human liver pathophysiology. We combined a liver-specific humanized mouse model and long-read direct RNA sequencing technology to establish a de novo annotation of the human liver transcriptome and identified a multitude of regulated metabolic pathways that were otherwise invisible using conventional technologies. The extensive regulatory information on human genes we provided could enable basic scientists to infer the pathological relevance of their genes of interest and physician scientists to better pinpoint the changes in metabolic networks underlying a specific pathophysiology.

SGX523 causes renal toxicity through aldehyde oxidase-mediated less-soluble metabolite formation in chimeric mice with humanized livers.

SGX523 is a c-Met tyrosine kinase inhibitor that failed in clinical trials because of renal toxicity caused by crystal deposits in renal tubules. SGX523 is metabolized by aldehyde oxidase (AOX) in a species-dependent manner to the considerably less soluble 2-quinolinone-SGX523, which is likely involved in the clinically observed obstructive nephropathy. This study investigated the metabolism and renal toxicity of SGX523 in chimeric mice with humanized livers (humanized-liver mice). The 2-quinolinone-SGX523 formation activity was higher in humanized-liver mouse and human hepatocytes than in mouse hepatocytes. Additionally, this activity in the liver cytosolic fraction from humanized-liver mice was inhibited by the AOX inhibitors raloxifene and hydralazine. After oral SGX523 administration, higher maximum concentrations, larger areas under the plasma concentration versus time curves, and higher urinary concentrations of 2-quinolinone-SGX523 were observed in humanized-liver mice than in non-humanized mice. Serum creatinine and blood urea nitrogen levels were elevated in humanized-liver mice following repeated oral SGX523 administration. The accumulation of amorphous material in the tubules and infiltration of inflammatory cells around tubules were observed in the kidneys of humanized-liver mice after repeated oral SGX523 administration. These findings demonstrate that humanized-liver mice are useful for understanding the metabolism and toxicity of SGX523.

Humanized mouse liver reveals endothelial control of essential hepatic metabolic functions.

Hepatocytes, the major metabolic hub of the body, execute functions that are human-specific, altered in human disease, and currently thought to be regulated through endocrine and cell-autonomous mechanisms. Here, we show that key metabolic functions of human hepatocytes are controlled by non-parenchymal cells (NPCs) in their microenvironment. We developed mice bearing human hepatic tissue composed of human hepatocytes and NPCs, including human immune, endothelial, and stellate cells. Humanized livers reproduce human liver architecture, perform vital human-specific metabolic/homeostatic processes, and model human pathologies, including fibrosis and non-alcoholic fatty liver disease (NAFLD). Leveraging species mismatch and lipidomics, we demonstrate that human NPCs control metabolic functions of human hepatocytes in a paracrine manner. Mechanistically, we uncover a species-specific interaction whereby WNT2 secreted by sinusoidal endothelial cells controls cholesterol uptake and bile acid conjugation in hepatocytes through receptor FZD5. These results reveal the essential microenvironmental regulation of hepatic metabolism and its human-specific aspects.

HepaSH cells: Experimental human hepatocytes with lesser inter-individual variation and more sustainable availability than primary human hepatocytes.

Primary human hepatocytes (PHHs) have been commonly used as the gold standard in many drug metabolism studies, regardless of having large inter-individual variation. These inter-individual variations in PHHs arise primarily from genetic polymorphisms, as well as from donor health conditions and storage conditions prior to cell processing. To equalize the effects of the latter two factors, PHHs were transplanted to quality-controlled mice providing human hepatocyte proliferation niches, and engrafted livers were generated. Cells that were harvested from engrafted livers, call this as experimental human hepatocytes (EHH; termed HepaSH cells), were stably and reproducibly produced from 1014 chimeric mice produced by using 17 different PHHs. Expression levels of acute phase reactant (APR) genes as indicators of a systemic reaction to the environmental/inflammatory insults of liver donors varied widely among PHHs. In contrast to PHHs, the expression of APR genes in HepaSH cells was found to converge within a narrower range than in donor PHHs. Further, large individual differences in the expression levels of drug metabolism-related genes (28 genes) observed in PHHs were greatly reduced among HepaSH cells produced in a unified in vivo environment, and none deviated from the range of gene expression levels in the PHHs. The HepaSH cells displayed a similar level of drug-metabolizing enzyme activity and gene expression as the average PHHs but retained their characteristics for drug-metabolizing enzyme gene polymorphisms. Furthermore, long-term 2D culture was possible and HBV infection was confirmed. These results suggest that the stably and reproducibly providable HepaSH cells with lesser inter-individual differences in drug-metabolizing properties, may have a potential to substitution for PHH as practical standardized human hepatocytes in drug discovery research.

Drug transporter expression and activity in cryopreserved human hepatocytes isolated from chimeric TK-NOG mice with humanized livers.

Chimeric mice with humanized liver are thought to represent a sustainable source of isolated human hepatocytes for in vitro studying detoxification of drugs in humans. Because drug transporters are now recognized as key-actors of the hepatic detoxifying process, the present study was designed to characterize mRNA expression and activity of main hepatic drug transporters in cryopreserved human hepatocytes isolated from chimeric TK-NOG mice and termed HepaSH cells. Such cells after thawing were shown to exhibit a profile of hepatic solute carrier (SLC) and ATP-binding cassette (ABC) drug transporter mRNA levels well correlated to those found in cryopreserved primary human hepatocytes or human livers. HepaSH cells used either as suspensions or as 24 h-cultures additionally displayed notable activities of uptake SLCs, including organic anion transporting polypeptides (OATPs), organic anion transporter 2 (OAT2) or sodium-taurocholate co-transporting polypeptide (NTCP). SLC transporter mRNA expression, as well as SLC activities, nevertheless fell in HepaSH cells cultured for 120 h, which may reflect a partial dedifferentiation of these cells with time in culture in the conventional monolayer culture conditions used in the study. These data therefore support the use of cryopreserved HepaSH cells as either suspensions or short-term cultures for drug transport studies.

Roles of human cytochrome P450 3A4/5 in dexamethasone 6ß-hydroxylation mediated by liver microsomes and humanized liver in chimeric mice metabolically suppressed with azamulin.

The urinary metabolic ratio of 6ß-hydroxydexamethasone to dexamethasone reportedly acts as a noninvasive marker for human cytochrome P450 (P450) 3A4/5, which is induced by rifampicin in humanized-liver mice. In the current study, the pharmacokinetics of dexamethasone in humanized-liver mice after intravenous administration (10 mg/kg) were investigated using azamulin (a time-dependent P450 3A4/5 inhibitor). After intravenous dexamethasone administration, significant differences were observed in the time-dependent plasma and 24-h urinary concentrations of 6ß-hydroxydexamethasone between untreated humanized-liver mice and humanized-liver mice treated with azamulin (daily oral doses of 15 mg/kg for 3 days). The mean ratios of 6ß-hydroxydexamethasone to dexamethasone for the maximum concentrations, the areas under the plasma concentration-versus-time curves, and urinary concentrations were significantly lower in the azamulin-treated group (59%, 58%, and 41% of the untreated values, respectively). 6ß-Hydroxydexamethasone formation was suppressed by 93% by replacing control human liver microsomes with P450 3A4/5-inactivated liver microsomes. These results suggest that the oxidation of dexamethasone in humans is mediated mainly by P450 3A4/5 (which is suppressed by azamulin), and that humanized-liver mice orally treated with azamulin may constitute an in vivo model for metabolically inactivated P450 3A4/5 in human hepatocytes transplanted into chimeric mice.

o-Toluidine metabolism and effects in the urinary bladder of humanized-liver mice.

Occupational exposure to aromatic amines is one of the most important risk factors for urinary bladder cancer. When considering the carcinogenesis of aromatic amines, metabolism of aromatic amines in the liver is an important factor. In the present study, we administered ortho-toluidine (OTD) in the diet to mice for 4 weeks. We used NOG-TKm30 mice (control) and humanized-liver mice, established via human hepatocyte transplantation, to compare differences in OTD-induced expression of metabolic enzymes in human and mouse liver cells. We also investigated OTD-urinary metabolites and proliferative effects on the urinary bladder epithelium. RNA and immunohistochemical analyses revealed that expression of N-acetyltransferases mRNA in the liver tended to be lower than that of the P450 enzymes, and that OTD administration had little effect on N-acetyltransferase mRNA expression levels. However, expression of CYP3A4 was increased in the livers of humanized-liver mice, and expression of Cyp2c29 (human CYP2C9/19) was increased in the livers of NOG-TKm30 mice. OTD metabolites in the urine and cell proliferation activities in the bladder urothelium of NOG-TKm30 and humanized-liver mice were similar. However, the concentration of OTD in the urine of NOG-TKm30 mice was markedly higher than in the urine of humanized-liver mice. These data demonstrate differences in hepatic metabolic enzyme expression induced by OTD in human and mouse liver cells, and consequent differences in the metabolism of OTD by human and mouse liver cells. This type of difference could have a profound impact on the carcinogenicity of compounds that are metabolized by the liver, and consequently, would be important in the extrapolation of data from animals to humans.

Characterization of a Bioengineered AAV3B Capsid Variant with Enhanced Hepatocyte Tropism and Immune Evasion.

Capsid engineering of adeno-associated virus (AAV) can surmount current limitations to gene therapy such as broad tissue tropism, low transduction efficiency, or pre-existing neutralizing antibodies (NAb) that restrict patient eligibility. We previously generated an AAV3B combinatorial capsid library by integrating rational design and directed evolution with the aim of improving hepatotropism. A potential isolate, AAV3B-DE5, gained a selective proliferative advantage over five rounds of iterative selection in hepatocyte spheroid cultures. In this study, we reanalyzed our original dataset derived from the AAV3B combinatorial library and isolated variants from earlier (one to three) rounds of selection, with the assumption that variants with faster replication kinetics are not necessarily the most efficient transducers. We identified a potential candidate, AAV3B-V04, which demonstrated significantly enhanced transduction in mouse-passaged primary human hepatocytes as well as in humanized liver chimeric mice, compared to the parental AAV3B or the previously described isolate, AAV3B-DE5. Interestingly, the AAV3B-V04 capsid variant exhibited significantly reduced seroreactivity to pooled or individual human serum samples. Forty-four percent of serum samples with pre-existing NAbs to AAV3B had 5- to 20-fold lower reciprocal NAb titers to AAV3B-V04. AAV3B-V04 has only nine amino acid substitutions, clustered in variable region IV compared to AAV3B, indicating the importance of the loops at the top of the three-fold protrusions in determining both transduction efficiency and immunogenicity. This study highlights the effectiveness of rational design combined with targeted selection for enhanced AAV transduction via molecular evolution approaches. Our findings support the concept of limiting selection rounds to isolate the best transducing AAV3B variant without outgrowth of faster replicating candidates. We conclude that AAV3B-V04 provides advantages such as improved human hepatocyte tropism and immune evasion and propose its utility as a superior candidate for liver gene therapy.

Establishment of hyperoxic cell culture system for predicting drug-induced liver injury: reducing accumulated lipids in hepatocytes derived from chimeric mice with humanized liver.

Drug-induced liver injury (DILI) is a major adverse reaction. Species-specific differences between humans and laboratory animals make it difficult to establish evaluation models that can accurately predict DILI in the preclinical phase. Chimeric mice with humanized liver are potential predictive models for understanding DILI. Chimeric mice generated by transplanting human hepatocytes into urokinase-type plasminogen activator/severe combined immunodeficient mice are known to develop fatty liver and show lipid accumulation in isolated hepatocytes. It is speculated that the lipids accumulated in hepatocytes may interfere with DILI assessment. It is known that normal 20% oxygen culture conditions do not meet oxygen demand because oxygen consumption rate is higher than the oxygen supply rate. Therefore, we predicted that hyperoxic cultures could induce hepatocyte function and reduce accumulated lipids. A culture of chimeric mouse hepatocytes in 40% oxygen showed reduced intracellular lipid and triglyceride levels compared to those cultured in 20% oxygen on days 7 and 10. In addition, fatty acid ß-oxidation (FAO) activity increased from day 7 under 40% oxygen conditions. On the other hand, FAO activity increased on day 10 under 20% conditions. Microarray and Ingenuity Pathway Analysis showed that lipid metabolism-related pathways were downregulated under 40% oxygen conditions for 7 days, suggesting the involvement of several mechanisms in decreasing lipid levels and increasing FAO. Furthermore, some pathways related to cellular function and maintenance were upregulated under 40% oxygen conditions for 7 days. In conclusion, chimeric mouse hepatocytes cultured under hyperoxic conditions may be useful for predicting DILI.

Long-term cell fate and functional maintenance of human hepatocyte through stepwise culture configuration.

Human hepatocyte culture system represents by far the most physiologically relevant model for our understanding of liver biology and diseases; however, its versatility has been limited due to the rapid and progressive loss of genuine characteristics, indicating the inadequacy of in vitro milieu for fate maintenance. This study, therefore, is designed to define environmental requirements necessary to sustain the homeostasis of terminally differentiated hepatocytes. Our study reveals that the supplementation of dimethyl sulfoxide (DMSO) is indispensable in mitigating fate deterioration and promoting adaptation to the in vitro environment, resulting in the restoration of tight cell-cell contact, cellular architecture, and polarity. The morphological recovery was overall accompanied by the restoration of hepatocyte marker gene expression, highlighting the interdependence between the cellular architecture and the maintenance of cell fate. However, beyond the recovery phase culture, DMSO supplementation is deemed detrimental due to the potent inhibitory effect on a multitude of hepatocyte functionalities while its withdrawal results in the loss of cell fate. In search of DMSO substitute, our screening of organic substances led to the identification of dimethyl sulfone (DMSO2), which supports the long-term maintenance of proper morphology, marker gene expression, and hepatocytic functions. Moreover, hepatocytes maintained DMSO2 exhibited clinically relevant toxicity in response to prolonged exposure to xenobiotics as well as alcohol. These observations suggest that the stepwise culture configuration consisting of the consecutive supplementation of DMSO and DMSO2 confers the microenvironment essential for the fate and functional maintenance of terminally differentiated human hepatocytes.

Evaluation of the mode of action and human relevance of liver tumors in male mice treated with epyrifenacil.

Epyrifenacil (trademark name: Rapidicil®), a novel protoporphyrinogen oxidase (PPO)-inhibiting herbicide, induces hepatocellular adenomas and carcinomas in male CD-1 mice after 78 weeks treatment. The mode of action (MOA) of these mouse liver tumors and their relevance to humans was assessed based on the 2006 International Programme on Chemical Safety (IPCS) Human Relevance Framework. Epyrifenacil is not genotoxic and induced liver tumors via the postulated porphyria-mediated cytotoxicity MOA with the following key events: (#1) PPO inhibition; (#2) porphyrin accumulation; (#3) hepatocellular injury; with (#4) subsequent regenerative cell proliferation; and ultimately (#5) development of liver tumors. This article evaluates the weight of evidence for this MOA based on the modified Bradford Hill criteria. The MOA data were aligned with the dose and temporal concordance, biological plausibility, coherence, strength, consistency, and specificity for a porphyria-mediated cytotoxicity MOA while excluding other alternative MOAs. Although the postulated MOA could qualitatively potentially occur in humans, we demonstrate that it is unlikely to occur in humans because of quantitative toxicodynamic and toxicokinetic differences between mice and humans. Therefore, this MOA is considered not relevant to humans, utilizing the IPCS Human Relevance Framework; consequently, a nonlinear, threshold dose response would be appropriate for human risk assessment.

Formation of reactive metabolites of benzbromarone in humanized-liver mice.

Benzbromarone, a uricosuric drug, has the potential to cause serious hepatotoxicity. Several studies have shown the formation of reactive metabolites of benzbromarone and their association with hepatotoxicity in mice. However, it is unknown whether those reactive metabolites are generated in humans in vivo. In the present study, we firstly investigated the pharmacokinetic profiles of benzbromarone in chimeric TK-NOG mice transplanted with human hepatocytes (humanized-liver mice) and then investigated whether reactive metabolites could be generated. The area under the plasma concentration-time curve ratio of benzbromarone and its major metabolites (benzbromarone: 1'-hydroxy benzbromarone: 6-hydroxy benzbromarone) in humanized-liver mice was 1: 1.2: 0.7, which was similar to that reported in humans. In addition, glutathione conjugates and their further metabolites derived from the epoxidation of the benzofuran ring and 1',6-dihydroxylation of benzbromarone were detected in the livers, urine and plasma. Furthermore, their peak intensities in mass spectrometry showed markedly higher levels compared with those of TK-NOG mice. These results suggested that the metabolic profiles of benzbromarone in humanized-liver mice were similar to those in humans and that the reactive metabolites detected in humanized-liver mice could be generated and are associated with the benzbromarone-induced hepatotoxicity in humans.

Expression and functional activity of cytochrome P450 enzymes in human hepatocytes with sustainable reproducibility for in vitro phenotyping studies.

Primary human hepatocytes are an essential in vitro tool for evaluating drug metabolism, drug-drug interactions, and hepatotoxicity. This model is considered as the gold standard in matter of DMPK studies in both industrial and academic research. The primary human hepatocytes are used either in suspension or in monolayer, as fresh or frozen cells. However, the use of this model is limited due to the lack of availability, rapid loss of functionality, high cost as well as the variable hepatocyte plating efficiencies in culture and the limited stock of hepatocytes derived from the same origin. Chimeric TK-NOG mice with humanized livers (humanized liver mice) are an attractive platform for drug metabolism and toxicity, which were produced by transplanting human hepatocytes into immunodeficient mice with injured livers. Here, we show that, using humanized mouse liver, in vivo human hepatocyte repopulation was over ~100-fold enabling the continuous and abundant use of human hepatocytes of the same origin and improving their plateability. In our latest cell preparations, hepatocytes isolated from humanized liver mice (Hu-Liver cells) exhibited high purity (ratio of HLA-positive cells: 92±3%), good viability (75±12%), and yield (1.0×108 cells/mouse). Human hepatic drug metabolizing enzymes, transporters, and nuclear receptors genes were expressed in humanized mouse liver. Drug-metabolizing activities in Hu-Liver cells were comparable to or higher than those in primary human hepatocytes. An extensive P450-dependent human drug metabolism was observed in Hu-Liver cells. CYP1A2, CYP2B6, and CYP3A4/5 activities/mRNA in Hu-Liver cells were induced by the hepatocyte exposure to typical human P450 inducers, omeprazole, phenobarbital, and rifampicin, respectively. Finally, Human albumin secretion and CYP3A-mediated drug oxidation activity were maintained over 4-weeks. Altogether, the expression level of pharmacokinetics-related genes, enzyme activity, human-typed drug metabolism, and inducibility of P450 in Hu-Liver cells make from humanized mouse liver a relevant and robust model for in vitro preclinical studies, including drug metabolism, pharmacokinetics, and toxicology studies.

Cytochrome P450s in chimeric mice with humanized liver.

Chimeric mice with humanized livers (humanized liver mice) are attractive experimental animal models for drug metabolism and pharmacokinetic studies. The "humanized liver" is a mature and functional liver with zonal position-specific expressions of human cytochrome P450 (P450) enzymes and a global gene expression pattern consistent with that of the mature human liver. Most P450-dependent drug oxidation activities were comparable between microsomes from livers of human and humanized liver mice based on similar expression levels of human P450 enzymes; however, some differences were observed between the two species, including considerable variations in activities of bufuralol 1'-hydroxylation and propafenone 4'-hydroxylation. Human disproportionate and/or unique metabolites of P450 substrate drugs were produced in humanized liver mice. Plasma concentration profiles of typical P450 substrate drugs in humans could be extrapolated from the corresponding data in humanized liver mice using simplified physiologically based pharmacokinetic modeling. Drug-drug interaction-mediated hepatic human CYP3A/2C induction by rifampicin (a human pregnane X receptor agonist) was observed in humanized liver mice. The major role of human CYP2C9 in in vivo diclofenac 4'-hydroxylation were determined using human CYP2C9-inactivated chimeric mice using a mechanism-based inhibitor, tienilic acid. Overall, based on the functional characteristics of hepatic human P450 enzymes, humanized liver mice are valuable experimental animals for studying metabolite profiling, pharmacokinetics, and drug interactions.

Downregulation of CYP17A1 by 20-hydroxyecdysone: plasma progesterone and its vasodilatory properties.

Aim: To investigate the effect of 20-hydroxyecdysone on steroidogenic pathway genes and plasma progesterone, and its potential impact on vascular functions. Methods: Chimeric mice with humanized liver were treated with 20-hydroxyecdysone for 3 days, and hepatic steroidogenic pathway genes and plasma progesterone were measured by transcriptomics and GC-MS/MS, respectively. Direct effects on muscle and mesenteric arterioles were assessed by myography. Results: CYP17A1 was downregulated in 20-hydroxyecdysone-treated mice compared with untreated group (p = 0.04), with an insignificant increase in plasma progesterone. Progesterone caused vasorelaxation which was blocked by 60 mM KCl, but unaffected by nitric oxide synthase inhibition. Conclusion: In the short term, 20-hydroxyecdysone mediates CYP17A1 downregulation without a significant increase in plasma progesterone, which has a vasodilatory effect involving inhibition of voltage-dependent calcium channels, and the potential to enhance 20-hydroxyecdysone vasorelaxation.

Assessment of metabolic activation of felbamate in chimeric mice with humanized liver in combination with in vitro metabolic assays.

Felbamate (FBM) is an antiepileptic drug that has minimal toxicity in preclinical toxicological species but has a serious idiosyncratic drug toxicity (IDT) in humans. The formation of reactive metabolites is common among most drugs associated with IDT, and 2-phenylpropenal (2-PP) is believed to be the cause of IDT by FBM. It is important to consider the species difference in susceptibility to IDT between experimental animals and humans. In the present study, we used an in vitro and in vivo model system to reveal species difference in IDT of FBM. Human cytochrome P450 (CYP) and carboxylesterase (CES) expressing microsomes were used to clarify the isozymes involved in the metabolism of FBM. The remaining amount of FBM was significantly reduced in incubation with microsomes expressing human CYP2C8, 2C9, 2E1, and CES1c isozymes. Chimeric mice with humanized liver are expected to predict IDT in humans. Therefore, metabolite profiles in chimeric mice with humanized liver were investigated after administration of FBM. Metabolites after glutathione (GSH) conjugation of 2-phenylpropenal (2-PP), which is the reactive metabolite responsible for FBM-induced IDT, were detected in chimeric mice plasma and liver homogenate. Mass spectrometry imaging (MSI) visualizes distribution of FBM and endogenous GSH, and GSH levels in human hepatocyte were decreased after administration of FBM. In this study, we identified CYP and CES isozymes involved in the metabolism of FBM and confirmed reactive metabolite formation and subsequent decrease in GSH using humanized animal model. These results would provide useful information for the susceptibility to IDT between experimental animals and humans.

Cytochrome P450-dependent drug oxidation activities and their expression levels in liver microsomes of chimeric TK-NOG mice with humanized livers.

Hepatic cytochrome P450 (P450)-dependent drug oxidation activity has not been completely characterized in chimeric TK-NOG mice with humanized livers (humanized liver mice). In this study, we examined several drug oxidation activities catalyzed by liver microsomes from humans, humanized liver mice, and TK-NOG mice using 9 P450 substrates. The catalytic activities of liver microsomes from humans and humanized liver mice showed relatively similar rates of oxidation of 7-ethoxyresorufin, coumarin, 7-pentoxyresorufin, flurbiprofen, S-mephenytoin, chlorzoxazone, and midazolam, whereas bufuralol 1'-hydroxylation and propafenone 4'-hydroxylation (rodent-specific propafenone oxidation activity) were higher in humanized liver mice than in humans. In addition, P450 protein expression levels in the humanized mouse liver were quantified using a liquid chromatography-tandem mass spectrometry-based protein quantification method. Quantification of P450 enzymes showed a 3-fold difference between human and humanized liver mouse livers, except for CYP2B6, which showed an approximately 6-fold difference. Overall, most P450-dependent drug oxidation activities were comparable between liver microsomes from human and humanized liver mice based on the similar expression levels of human P450 enzymes. However, some differences were observed between both species, including considerable differences in bufuralol 1'-hydroxylation and propafenone 4'-hydroxylation activities.

Prediction of the human pharmacokinetics of epyrifenacil and its major metabolite, S-3100-CA, by a physiologically based pharmacokinetic modeling using chimeric mice with humanized liver.

Human internal dosimetry of pesticides is essential in the risk assessment when toxicity has been confirmed in laboratory animals. While human toxicokinetics data of pesticides are hardly obtained intendedly, the use of physiologically based pharmacokinetic (PBPK) modeling has become important for predicting human internal dosimetry. Especially, when the compound exhibits complicated pharmacokinetics via active uptake, metabolism, and biliary excretion in liver, it is difficult to obtain these hepatic parameters only by the in vitro experiments. Epyrifenacil, a new herbicide, is rapidly metabolized to S-3100-CA (CA) in mammals and causes hepatotoxicity in mice. CA is eliminated from the systemic circulation by biliary excretion and metabolism in liver. Although uptake of CA by transporters is observed in mouse primary hepatocytes, significantly less of it is observed in human primary hepatocytes. In order to evaluate human internal dosimetry of CA, a precise PBPK model was developed. To obtain human hepatic parameters, i.e., hepatic elimination intrinsic clearance via biliary excretion and metabolism, we used chimeric mice with humanized liver as a model to reproduce the complicated pharmacokinetics of CA in humans. After we developed a mouse PBPK model, by replacing mouse parameters with those of humans, we calculated CA concentration in human liver. Comparing the predicted CA exposure in human liver with the measured values in mice, we demonstrated a clear interspecies difference of approximately 4 times lower Cmax and AUC in humans. This result suggested that the risk of hepatotoxicity is less in humans than in mice.