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 provided 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.

Empirical scaling factor for predicting human pharmacokinetic profiles of disproportionate metabolites using the Css-MRTpo method and chimeric mice with humanised livers.

Predicting plasma concentration-time profiles of disproportionate metabolites in humans is crucial for evaluating metabolites according to the Safety Testing guidelines. We evaluated Css-MRTpo, an empirical method, using chimeric mice with humanised livers capable of generating human-disproportionate metabolites. Azilsartan and AZ-M2 were administered to humanised chimeric mice, and pharmacokinetic parameters were obtained. Pharmacokinetic data for DS-1971a and DS-M1 in humanised chimeric mice were obtained from the literature. The human plasma concentration-time profiles of these compounds were simulated using the Css-MRTpo method. Azilsartan, DS-1971a, and PF-04937319 produced human disproportionate metabolites, AZ-M2, DS-M1, and PF-M1, respectively. The predicted human pharmacokinetic profiles of PF-04937319 and PF-M1 were obtained from a previous study, and their outcomes were re-evaluated. Our findings revealed that the plasma concentrations of the three metabolites were unexpectedly underpredicted, whereas the three unchanged drugs were reasonably predicted. Further, the introduction of the empirical scaling factor of 3, obtained from six model compounds, improved the predictability of metabolites, suggesting the potential usefulness of the Css-MRTpo method in combination with humanised chimeric mice for predicting the pharmacokinetic profiles of disproportionate metabolites at the early stage of new drug development.

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.

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.

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.

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.

The Unique Human N10-Glucuronidated Metabolite Formation from Olanzapine in Chimeric NOG-TKm30 Mice with Humanized Livers.

Olanzapine is an antipsychotic agent with species-dependent pharmacokinetic profiles in both humans and animals. In the present study, the metabolic profiles of olanzapine in vitro and in vivo were compared in non-transplanted immunodeficient NOG-TKm30 mice and chimeric mice with humanized livers (hereafter humanized-liver mice). Hepatic microsomal fractions prepared from humanized-liver mice and humans mediated olanzapine N10-glucuronidation, whereas fractions from cynomolgus monkeys, marmosets, minipigs, dogs, rabbits, guinea pigs, rats, CD1 mice, and NOG-TKm30 mice did not. The olanzapine N10-glucuronidation activity in liver microsomes from humanized-liver mice was inhibited by hecogenin, a human UDP-glucuronosyltransferase (UGT) 1A4 inhibitor. In addition, hepatocytes from humanized-liver mice suggest that olanzapine N10-glucuronidation was a major metabolic pathway in the livers of humanized-liver mice. After a single oral dose of olanzapine (10 mg/kg body weight) to humanized-liver mice and control NOG-TKm30 mice, olanzapine N10-glucuronide isomers and olanzapine N4'-glucuronide were detected only in the plasma of humanized-liver mice. In contrast, the area under the curve for N4'-demethylolanzapine, 2-hydroxymethylolanzapine, and 7-hydroxyolanzapine glucuronide was higher in NOG-TKm30 mice than that in humanized-liver mice. The cumulative excreted amounts of olanzapine N10-glucuronide isomers were high in the urine and feces from humanized-liver mice, whereas the cumulative excreted amounts of 2-hydroxymethylolanzapine were higher in NOG-TKm30 mice than in humanized-liver mice. Thus, production of human-specific olanzapine N10-glucuronide was observed in humanized-liver mice, which was consistent with the in vitro glucuronidation data. These results suggest that humanized-liver mice are useful for studying drug oxidation and conjugation of olanzapine in humans. SIGNIFICANCE STATEMENT: Human-specific olanzapine N10-glucuronide isomers were generated in chimeric NOG-TKm30 mice with humanized livers (humanized-liver mice), and high UGT1A4-dependent N10-glucuronidation was observed in the liver microsomes from humanized-liver mice. Hence, humanized-liver mice may be a suitable model for studying UGT1A4-dependent biotransformation of drugs in humans.

Comparison of mouse and human cytochrome P450 mediated-drug metabolising activities in hepatic and extrahepatic microsomes.

Despite the importance of mice as a preclinical species in drug testing, their hepatic and extrahepatic drug-metabolising characteristics are poorly understood. Here, we compared the P450-dependent drug oxidation activity in tissue microsomes and distribution patterns of P450 protein/mRNA between humans and mice.The activities of midazolam 1'-/4-hydroxylation in the liver and intestine and chlorzoxazone 6-hydroxylation in the liver were similar in humans and mice. The activities of coumarin 7-hydroxylation, flurbiprofen 4'-hydroxylation, and S-mephenytoin 4'-hydroxylation in the liver were higher in humans than in mice. The activities of 7-ethoxyresorufin O-deethylation in the liver, 7-pentoxyresorufin O-depentylation in the lung/liver/intestine, bufuralol 1'-hydroxylation in the liver/intestine, propafenone 4'-hydroxylation in liver/intestine, and diazepam N-demethylation in the liver/intestine were higher in mice than in humans.CYP1A2/2E1 mRNAs were mainly expressed in the livers of humans and mice. Cyp2b9/2b10 mRNAs were abundant in the mouse lung/liver/intestine, but CYP2B6 was mainly expressed in the human liver. CYP2C/2D/3A mRNAs were expressed in the liver and intestine, with the respective proteins detected in tissue microsomes of both humans and mice.These information on P450-dependent drug-metabolising characteristics in hepatic and extrahepatic tissues is useful to understand the similarities and differences between humans and mice in drug metabolism.

Probe drug T-1032 N-oxygenation mediated by cytochrome P450 3A5 in human hepatocytes in vitro and in humanized-liver mice in vivo.

Polymorphic cytochrome P450 3A5 (CYP3A5) expression contributes to individual differences in the pharmacokinetics of probe drugs. The identification of suitable in vivo CYP3A5 probes would benefit drug metabolism and drug interaction studies using chimeric mice with humanized liver. In this study, we investigated the pharmacokinetic profiles of T-1032, which is known as an in vitro CYP3A5 probe substrate, using humanized-liver mice. Substantial N-oxygenation of T-1032 was observed in hepatocytes from humans and from humanized-liver mice. Hepatocytes from the human donor genotyped as CYP3A5∗3/∗3 (poor expressers) showed significantly lower T-1032 N-oxidation rates than those from donors harboring CYP3A5∗1. After a single oral dose of T-1032 (1.0 mg/kg) in humanized-liver mice, the plasma levels of T-1032 N-oxide were higher in five mice with CYP3A5∗1/∗7 hepatocytes than in four mice with CYP3A5∗3/∗3 hepatocytes. The maximum concentrations of T-1032 N-oxide after oral administration of T-1032 in humanized-liver mice with CYP3A5∗1/∗7 hepatocytes were twice (a significant difference) those from humanized-liver mice with CYP3A5∗3/∗3 hepatocytes. These results suggest that polymorphic CYP3A5-dependent T-1032 N-oxidation was observed in humanized liver mice in vitro and in vivo. However, the contribution of CYP3A5 genotypes may have little or only limited effects on the overall pharmacokinetic profiles of T-1032 in vivo.

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.

An improved TK-NOG mouse as a novel platform for humanized liver that overcomes limitations in both male and female animals.

We developed a novel immunodeficient NOG mouse expressing HSVtk mutant clone 30 cDNA under the control of mouse transthyretin gene enhancer/promoter (NOG-TKm30) to acquire fertility in males and high inducibility of liver injury in females. Maximum human albumin levels (approx. 15 mg/mL plasma) in both male and female NOG-TKm30 mice engrafted with human hepatocytes (humanized liver mice) were observed 8-12 weeks after transplantation. Immunohistochemical analyses revealed abundant expression of major human cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2D6, CYP2E1, and CYP3A4) in reconstituted liver with original zonal distribution. In vivo drug-drug interactions were observed in humanized liver mice as decreased area under the curve of midazolam (CYP3A4/5 substrate) and omeprazole (CYP3A4/5 and CYP2C19 substrate) after oral administration of rifampicin. Furthermore, we developed a pregnant model for evaluating prenatal exposure to drugs. The detection of thalidomide metabolites in the fetuses of pregnant humanized liver mice indicates that the novel TK model can be used for developmental toxicity studies requiring the assessment of human drug metabolism. These results suggest that the limitations of traditional TK-NOG mice can be addressed using NOG-TKm30 mice, which constitute a novel platform for humanized liver for both in vivo and in vitro studies.

Oxidative metabolism and pharmacokinetics of the EGFR inhibitor BIBX1382 in chimeric NOG-TKm30 mice transplanted with human hepatocytes.

The epidermal growth factor receptor inhibitor BIBX1382 has failed in drug development because of poor oral exposure and low bioavailability associated with its extensive metabolism by aldehyde oxidase (AOX) in humans. In this study, we investigated the metabolic profiles and pharmacokinetics of BIBX1382 in chimeric NOG-TKm30 mice with humanized liver (humanized liver mice). After intravenous and oral BIBX1382 administration, increased plasma clearance and decreased oral exposure together with high production of the predominant oxidative metabolite (M1, BIBU1476) and secondary oxidized metabolite (M2) were observed in humanized liver mice. Extensive oxidation rates of BIBX1382 were observed in hepatocytes from humanized liver mice and were suppressed by the typical human AOX1 inhibitors raloxifene and hydralazine. Liver cytosolic fractions from humans, humanized liver mice, cynomolgus monkeys, minipigs, and guinea pigs, but not fractions from dogs, rabbits, rats, and mice, displayed high BIBX1382 clearance and resulted in oxidative metabolite production. These results indicate that humanized liver mice have human-type AOX activity based on the transplanted human liver AOX1 function. Humanized liver mice can be considered an important animal model for understanding the metabolism and pharmacokinetics of AOX drug substrates.

UDP-glucuronosyltransferase 1A4-mediated N2-glucuronidation is the major metabolic pathway of lamotrigine in chimeric NOG-TKm30 mice with humanised-livers.

Lamotrigine is a phenyltriazine anticonvulsant used to treat epilepsy and bipolar disorder, with species-dependent metabolic profiles. In this study, we investigated the metabolism of lamotrigine in chimeric NOG-TKm30 mice transplanted with human hepatocytes (humanised-liver mice).Substantial lamotrigine N2-glucuronidation activities were observed in the liver microsomes from humanised-liver mice, humans, marmosets, and rabbits, compared to those from monkeys, minipigs, guinea pigs, rats, and mice. Lamotrigine N2-glucuronidation activities in the liver microsomes from humanised-liver mice were dose-dependently inhibited by hecogenin, a specific inhibitor of the human UGT1A4.The major metabolite in the hepatocytes from humanised-liver mice and humans was lamotrigine N2-glucuronide, whereas that in mouse hepatocytes was lamotrigine N2-oxide. After a single oral dose of lamotrigine (10 mg/kg), the plasma levels of N2-glucuronide, N5-glucuronide, and N2-methyl were higher in humanised-liver mice compared to that in NOG-TKm30 mice. Lamotrigine N2-glucuronide was the most abundant metabolite in the urine in humanised-liver mice, similar to that reported in humans; whereas, lamotrigine N2-oxide was predominantly excreted in the urine in NOG-TKm30 mouse.These results suggest that humanised-liver mice may be a suitable animal model for studying the UGT1A4 mediated-lamotrigine metabolism.

Establishment of novel common marmoset embryonic stem cell lines under various conditions.

Pluripotent stem cells (PSCs), embryonic stem cells (ESCs), and induced PSCs (iPSCs) are excellent tools for studying embryonic development in organisms and classified into naïve and primed states. ESC-derived germline chimera individuals can be produced by injecting naïve ESCs/iPSCs into preimplantation embryos, and conversion of primed human ESCs/iPSCs into a naïve state provides insights into epiblast cell features. Non-human ESCs/iPSCs are alternatives to human naïve ESCs/iPSCs, which elicit ethical issues. In this study, we used the common marmoset (Callithrix jacchus) as an animal model. Since 1996, 16 marmoset ESC lines have been established. Because most of these ESC lines are female and were derived >10 years ago, new ESCs, particularly male marmoset ESC lines, are needed. Here, we successfully established 17 novel marmoset ESC lines, including six male ESC lines from in vitro-fertilized (IVF) embryos and 12 ESC lines under feeder-free conditions. This report is the first to establish ESC lines using feeder-free conditions and IVF preimplantation blastocysts in marmosets, and these novel ESC lines could potentially facilitate future non-human primate ESC studies.

Methyl-hydroxylation and subsequent oxidation to produce carboxylic acid is the major metabolic pathway of tolbutamide in chimeric TK-NOG mice transplanted with human hepatocytes.

Tolbutamide is an oral anti-hyperglycaemic agent used to treat non-insulin-dependent diabetes mellitus with species-dependent metabolic profiles. In this study, we investigated tolbutamide metabolism in chimeric TK-NOG mice transplanted with human hepatocytes (humanised-liver mice).Substantial 4-hydroxytolbutamide and 4-carboxytolbutamide production was observed in hepatocytes from humanised-liver mice (Hu-Liver cells) and humans, whereas 4-carboxytolbutamide production was not detected in mouse hepatocytes. In Hu-Liver cells, 4-hydroxytolbutamide formation was inhibited by sulfaphenazole (CYP2C9 inhibitor), whereas 4-carboxytolbutamide formation was inhibited by raloxifene/ethinyloestradiol (aldehyde oxidase inhibitor) and disulfiram (aldehyde dehydrogenase inhibitor).After a single oral dose of tolbutamide (10 mg/kg), the plasma levels of 4-carboxytolbutamide and p-tolylsulfonylurea were higher in humanised-liver mice than in TK-NOG mice. Urinary excretion was the predominant route (>99% of unchanged drug and metabolites detected in excreta) of elimination in both groups. 4-Carboxytolbutamide was the most abundant metabolite in humanised-liver mouse urine, as similarly reported for humans, whereas 4-hydroxytolbutamide was predominantly excreted in TK-NOG mouse urine.These results suggest that humanised-liver mice might represent a suitable animal model for studying the successive oxidative metabolism of tolbutamide by multiple drug-metabolising enzymes. Future work is warranted to study the general nature of primary alcohol metabolism using humanised-liver mice.

Identification of human long noncoding RNAs associated with nonalcoholic fatty liver disease and metabolic homeostasis.

A growing number of long noncoding RNAs (lncRNAs) have emerged as vital metabolic regulators. However, most human lncRNAs are nonconserved and highly tissue specific, vastly limiting our ability to identify human lncRNA metabolic regulators (hLMRs). In this study, we established a pipeline to identify putative hLMRs that are metabolically sensitive, disease relevant, and population applicable. We first progressively processed multilevel human transcriptome data to select liver lncRNAs that exhibit highly dynamic expression in the general population, show differential expression in a nonalcoholic fatty liver disease (NAFLD) population, and respond to dietary intervention in a small NAFLD cohort. We then experimentally demonstrated the responsiveness of selected hepatic lncRNAs to defined metabolic milieus in a liver-specific humanized mouse model. Furthermore, by extracting a concise list of protein-coding genes that are persistently correlated with lncRNAs in general and NAFLD populations, we predicted the specific function for each hLMR. Using gain- and loss-of-function approaches in humanized mice as well as ectopic expression in conventional mice, we validated the regulatory role of one nonconserved hLMR in cholesterol metabolism by coordinating with an RNA-binding protein, PTBP1, to modulate the transcription of cholesterol synthesis genes. Our work overcame the heterogeneity intrinsic to human data to enable the efficient identification and functional definition of disease-relevant human lncRNAs in metabolic homeostasis.

Predicted values for human total clearance of a variety of typical compounds with differently humanized-liver mouse plasma data.

Prediction of human pharmacokinetics is important in the preclinical stage. Values for total clearance of compounds from plasma should be one of the most important pharmacokinetic parameters for predictions. Although several physiological and empirical methods including single-species allometry for prediction of values for human clearance of compounds using humanized-liver mice have been reported, further improvement of prediction accuracies would be still expected. To optimize these approaches, we proposed methods for unbound intrinsic clearance in virtually 100% humanized-liver mouse by incorporating unbound plasma fractions of compounds in differently humanized-liver mice. Comparisons of prediction accuracies of values for human clearance of 15 model compounds were performed among our current physiological and previously reported models and single-species allometry using humanized-liver mice. Incorporation of the actual unbound plasma fractions of compounds and correction of residual mice hepatocyte in humanized-liver mice showed comparable prediction accuracy to that by single-species allometry. After exclusion of 3 compounds with large species differences in values of clearance and unbound plasma fractions between mice and humans out of 15 compounds, prediction accuracies were improved in the methods investigated. The previously and present reported physiological methods could show the good prediction accuracy of values for clearance of drugs from plasma.

Human Aldehyde Oxidase 1-Mediated Carbazeran Oxidation in Chimeric TK-NOG Mice Transplanted with Human Hepatocytes.

Carbazeran is a potent phosphodiesterase inhibitor with species-dependent metabolic profiles in rats, dogs, and humans. In this study, we investigated the aldehyde oxidase (AOX)-mediated oxidation of carbazeran to 4-oxo derivatives in chimeric NOD/Shi-scid IL2 receptor gamma-null mice expressing a herpes simplex virus type 1 thymidine kinase transgene with humanized livers (humanized-liver mice). Liver cytosolic fractions from humanized-liver mouse effectively catalyzed carbazeran 4-oxidation with high affinity for the substrate, similar to those of the human liver cytosolic fractions and recombinant human AOX1 protein. Furthermore, hepatocytes prepared from humanized-liver mice and humans also exhibited substantial metabolism via carbazeran 4-oxidation. After a single oral administration of carbazeran (10 mg/kg), plasma levels of 4-oxo-carbazeran, N-desethyl-4-oxo-carbazeran, and 6,7-dimethoxy-1-[4-(hydroxy)-piperidino]-4-phthalazinone (three human metabolites formed via 4-oxidation) were higher in humanized-liver mice than in the control mice. In contrast, plasma levels of O-desmethylcarbazeran (a major metabolite in dogs) in control mice were higher than those in the humanized-liver mice. Relative excreted amounts of the three 4-oxidation-derived human-specific metabolites in the urine and feces were greater for humanized-liver mice than control mice, whereas the relative excreted amounts of O-desmethylcarbazeran were predominant in the urine and feces of control mice. Thus, the production of carbazeran 4-oxo derivatives was elevated in humanized-liver mice compared with control mice, in agreement with our in vitro enzyme-mediated oxidation data. These results suggest that hepatic human AOX1 functions in humanized-liver mice at the in vivo level and that humanized-liver mice may be useful for predicting drug metabolism in humans, at least with regard to human AOX1-dependent metabolism. SIGNIFICANCE STATEMENT: We found that the production of carbazeran 4-oxo derivatives was higher in humanized-liver mice than in control mice. These results were supported by the fact that carbazeran was rapidly metabolized to 4-oxo-carbazeran in humanized-liver mouse hepatocytes expressing human aldehyde oxidase 1. These results suggest that human aldehyde oxidase 1 is functional in humanized-liver mice in vivo and that chimeric NOD/Shi-scid IL2 receptor gamma-null mice expressing a herpes simplex virus type 1 thymidine kinase transgene transplanted with human hepatocytes may be a suitable model animal for predicting aldehyde oxidase-dependent biotransformation of drugs in humans.

In vivo functional analysis of non-conserved human lncRNAs associated with cardiometabolic traits.

Unlike protein-coding genes, the majority of human long non-coding RNAs (lncRNAs) are considered non-conserved. Although lncRNAs have been shown to function in diverse pathophysiological processes in mice, it remains largely unknown whether human lncRNAs have such in vivo functions. Here, we describe an integrated pipeline to define the in vivo function of non-conserved human lncRNAs. We first identify lncRNAs with high function potential using multiple indicators derived from human genetic data related to cardiometabolic traits, then define lncRNA's function and specific target genes by integrating its correlated biological pathways in humans and co-regulated genes in a humanized mouse model. Finally, we demonstrate that the in vivo function of human-specific lncRNAs can be successfully examined in the humanized mouse model, and experimentally validate the predicted function of an obesity-associated lncRNA, LINC01018, in regulating the expression of genes in fatty acid oxidation in humanized livers through its interaction with RNA-binding protein HuR.

Metabolism of desloratadine by chimeric TK-NOG mice transplanted with human hepatocytes.

1. Desloratadine is an antiallergic drug with species-dependent metabolic profiles in mice, rats, monkeys and humans. We investigated whether humanized-liver mice could reproduce the reported human-specific in vivo metabolic profile for desloratadine in terms of the formation of 3-hydroxydesloratadine and its O-glucuronide.2. Hepatocytes prepared from humans and humanized-liver mice both preferentially catalyzed the formation of 3-hydroxydesloratadine and its O-glucuronide in vitro.3. After a single oral administration of desloratadine, plasma levels of desloratadine and its metabolites (3-hydroxydesloratadine and its O-glucuronide) in humanized-liver mice were lower and higher, respectively, than those in control mice.4. The amounts of 3-hydroxydesloratadine and its O-glucuronide excreted in humanized-liver mouse feces and urine were higher than those of the control mice, whereas 5- and 6-hydroxydesloratadine formation were predominant in the feces and urine samples from control mice. A significant correlation (r = 0.68) for the dose percentage of urinary and fecal metabolites of desloratadine was only observed between the humanized-liver mice and the reported values for humans.5. These results indicated that urinary 3-hydroxydesloratadine O-glucuronide and fecal desloratadine, 3-hydroxydesloratadine and 5-hydroxydesloratadine were the major excretion pathways of desloratadine in humanized-liver mice, which is reasonably similar to that reported for humans.