In Vivo and In Vitro Induction of Cytochrome P450 3A4 by Thalidomide in Humanized-Liver Mice and Experimental Human Hepatocyte HepaSH cells.

Autoinduction of cytochrome P450 (P450) 3A4-mediated metabolism of thalidomide was investigated in humanized-liver mice and human hepatocyte-derived HepaSH cells. The mean plasma ratios of 5-hydroxythalidomide and glutathione adducts to thalidomide were significantly induced (3.5- and 6.0-fold, respectively) by thalidomide treatment daily at 1000 mg/kg for 3 days and measured at 2 h after the fourth administration (on day 4). 5-Hydroxythalidomide was metabolically activated by P450 3A4 in HepaSH cells pretreated with 300 and 1000 µM thalidomide, and 5,6-dihydroxythalidomide was detected. Significant induction of P450 3A4 mRNA expression (4.1-fold) in the livers of thalidomide-treated mice occurred. Thalidomide exerts a variety of actions through multiple mechanisms following bioactivation by induced human P450 3A enzymes.

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.

Chronic Toxoplasma infection affects gene expression of drug-metabolizing enzymes in mouse liver.

Toxoplasma gondii is an intracellular protozoan parasite causing toxoplasmosis, an infectious disease affecting warm-blooded vertebrates worldwide. Many drug-metabolizing enzymes are located in the liver, a major organ of drug metabolism, and their function can be affected by pathogen infection.Using next-generation sequencing (RNA-seq) and quantitative polymerase chain reaction (qPCR), changes in the hepatic expressions of drug-metabolizing enzymes were analysed in mice chronically infected with T. gondii. The analysis found that, among drug-metabolizing enzymes, 22 genes were upregulated and 28 genes were downregulated (≥1.5-fold); of these 5 and 17 genes, respectively, were cytochromes P450 (Cyp or P450).Subsequent qPCR analysis showed that six P450 genes were upregulated significantly (≥1.5-fold, p < 0.05), namely, Cyp1b1, Cyp2c29, Cyp2c65, Cyp2d9, Cyp2d12, and Cyp3a59, whereas nine P450 genes were downregulated significantly (≥1.5-fold, p < 0.05), namely, Cyp2c38, Cyp2c39, Cyp2c44, Cyp2c69, Cyp2d40, Cyp2e1, Cyp3a11, Cyp3a41, and Cyp3a44.Moreover, metabolic assays in infected mouse liver using typical P450 substrates revealed that midazolam 1'-hydroxylation and testosterone 2-hydroxylation activities decreased significantly (≥1.5-fold, p < 0.05), whereas testosterone 16-hydroxylation activity increased significantly (≥1.5-fold, p < 0.05).Chronic Toxoplasma infection affects drug metabolism, at least partly, by altering the gene expressions of drug-metabolizing enzymes, including P450s.

Cytochrome P450 1A2 and 2C enzymes autoinduced by omeprazole in dog hepatocytes and human HepaRG and HepaSH cells are involved in omeprazole 5-hydroxylation and sulfoxidation.

The induction assay for the cytochromes P450 (P450s) is an important tool in drug discovery and development. The inductions of dog P450 1A2 and 3A12 by omeprazole and rifampicin were functionally characterised in dog hepatocytes and were compared with induction in human HepaRG and HepaSH cells.P450 1A2-dependent ethoxyresorufin O-deethylation was induced by R,S-omeprazole and P450 3 A-dependent midazolam 1'-hydroxylation was induced by rifampicin, and both reactions were significantly enhanced in cultured dog hepatocytes and human HepaRG and HepaSH cells.Recombinant dog P450 1A2 exhibited activities towards R- and S-omeprazole 5-hydroxylation with low Km values of 23-28 µM, whereas dog P450 2C21 and 3A12 efficiently mediated S-omeprazole 5-hydroxylation and sulfoxidation, respectively, with high Vmax values of 12-17 min-1.Although omeprazole 5-hydroxylation by human P450 2C19 (and sulfoxidation by P450 3A4) in human HepaSH cells were slightly (∼2-fold) induced by R,S-omeprazole, dog P450 1A2 was autoinduced by omeprazole in dog hepatocytes and showed enhanced R-omeprazole 5-hydroxylation activity (∼5-fold).These results indicate that omeprazole can be an autoinducer of P450 1A2 in hepatocytes, and this enzyme was found to be involved in omeprazole 5-hydroxylation and sulfoxidation in dog hepatocytes and human HepaRG and HepaSH cells.

Pharmacokinetic Models Scaled-up from Humanized-liver Mouse Data Can Account for Drug Monitoring Results of Atomoxetine and Its 4-Hydroxylated and N-Demethylated Metabolites i n Pediatric Patients Genotyped for Cytochrome P450 2D6.

Atomoxetine is a cytochrome P450 (P450) 2D6 probe substrate and an approved medicine for attention-deficit/hyperactivity disorder. In this humanized-liver mouse study, interactions between atomoxetine and the P450 2D6 probe drug paroxetine were observed. Human physiologically based pharmacokinetic (PBPK) models were established by scaling up humanized-liver mouse data obtained in the absence or presence of paroxetine. These models could explain the drug monitoring results of atomoxetine and its primary 4-hydroxylated and N-demethylated metabolites in Japanese children aged 8-14 years and could be used to help establish the correct dosage and for the evaluation of clinical outcomes. The results of simple PBPK models (using input parameters that reflected the subjects' small body size and normal or reduced P450 2D6-dependent clearance) were in general agreement with one-point measured plasma concentrations of atomoxetine and its 4-hydroxylated and N-demethylated metabolites in 13 pediatric participants. Unexpectedly high hepatic exposure, possibly in intermediate-metabolizer patients harboring CYP2D6*10 or 2D6*36 alleles, might in part explain the adverse effects of atomoxetine prescribed alone recorded in a Japanese adverse-event database. The steady-state, one-point drug monitoring data from the participants indicated extensive biotransformation of atomoxetine to 4-hydroxyatomoxetine under individually prescribed doses of atomoxetine. These results also suggest that a relatively narrow range of 4-hydroxyatomoxetine and N-desmethylatomoxetine concentration ratios in spot urine and/or plasma samples from pediatric patients could be a simple semiquantitative determinant factor for P450 2D6 intermediate metabolizers, compared with the wide range of concentrations of the two primary metabolites and substrate in extensive metabolizers. Significance Statement Validated simple pharmacokinetic models are able to predict steady-state plasma concentrations of the approved medicine atomoxetine and its primary metabolites in the majority of pediatric patients. The package insert advises careful dose escalation, especially for poor metabolizers; however, no simple way exists to determine P450 2D6 phenotypes. A relatively narrow range ratio of 4-hydroxyatomoxetine and N-desmethylatomoxetine in spot urine/plasma samples could be a simple semi-quantitative determinant factor for P450 2D6 intermediate metabolizers to optimize or confirm the correct dosage.

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.

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.

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.

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.

Forward and reverse dosimetry for aniline and 2,6-dimethylaniline in humans extrapolated from humanized-liver mouse data using simplified physiologically based pharmacokinetic models.

Although human urinary aniline and 2,6-dimethylaniline were unexpectedly detected in biomonitoring data, little is known about the daily intake doses of aniline and 2,6-dimethylaniline in the living environment or their relation to tolerable daily intake (TDI) values in humans. In the current study, to evaluate the daily oral intake of aniline and 2,6-dimethylaniline in humans, forward and reverse dosimetry was carried out using simplified in silico physiologically based pharmacokinetic (PBPK) modeling established using in vivo experimental pharmacokinetic data. These data were from humanized-liver mice after single oral doses of 100 mg/kg aniline (previously determined) and 116 mg/kg 2,6-dimethylanine (currently investigated). The in vivo elimination rates of 2,6-dimethylaniline from plasma in humanized-liver mice were generally slow compared with those of aniline. Faster in vitro metabolic elimination rates of aniline mediated by liver 9000 × g supernatant fractions from rats than those from humans may suggest the existence of higher first-pass effects in rats than in humanized-liver mice. In silico aniline and 2,6-dimethylaniline concentration curves in human urine after virtual oral administrations were estimated by human PBPK models created with data from humanized-liver mice. Reverse dosimetry analysis using human PBPK models estimated the daily intake of aniline, based on reported human urinary concentrations in biomonitoring data, to be roughly similar to the aniline TDI level. These results suggest that forward and reverse dosimetry using simplified human PBPK models founded on data from humanized-liver mice can be used to evaluate possible higher than expected exposures of aniline and 2,6-dimethylaniline in humans.

Newly identified tree shrew cytochrome P450 2B6 (CYP2B6) and pig CYP2B6b are functional drug-metabolising enzymes.

Tree shrews have high phylogenetic affinity to humans and are used in various fields of biomedical research, especially hepatitis virus infection; however, cytochromes P450 (P450s or CYPs) have not been investigated in this species.In this study, tree shrew CYP2B6 and pig CYP2B6b were newly identified and had amino acid sequences highly identical (80% and 78%, respectively) to human CYP2B6, containing sequence motifs characteristic of P450s.Phylogenetic analysis revealed that novel tree shrew CYP2B6 was more closely related to known human CYP2B6 than dog, pig, or rat CYP2Bs are.Among the tissue types analysed, tree shrew CYP2B6 mRNA was preferentially expressed in liver and lung, whereas pig CYP2B6b mRNA was preferentially expressed in jejunum and lung.Tree shrew CYP2B6 and pig CYP2B6b proteins heterologously expressed in Escherichia coli metabolised human CYP2B6 substrates efavirenz, ethoxycoumarin, propofol, and testosterone, suggesting that these novel CYP2Bs are functional drug-metabolizing enzymes in liver and/or lung.

Humanized liver TK-NOG mice with functional deletion of hepatic murine cytochrome P450s as a model for studying human drug metabolism.

Chimeric TK-NOG mice with a humanized liver (normal Hu-liver) are a unique animal model for predicting drug metabolism in humans. However, residual mouse hepatocytes occasionally prevent the precise evaluation of human drug metabolism. Herein, we developed a novel humanized liver TK-NOG mouse with a conditional knockout of liver-specific cytochrome P450 oxidoreductase (POR cKO Hu-liver). Immunohistochemical analysis revealed only a few POR-expressing cells around the portal vein in POR cKO mouse livers. NADPH-cytochrome c reductase and cytochrome P450 (P450)-mediated drug oxidation activity in liver microsomes from POR cKO mice was negligible. After the intravenous administration of S-warfarin, high circulating and urinary levels of S-7-hydroxywarfarin (a major human metabolite) were observed in POR cKO Hu-liver mice. Notably, the circulating and urinary levels of S-4'-hydroxywarfarin (a major warfarin metabolite in mice) were much lower in POR cKO Hu-liver mice than in normal Hu-liver mice. POR cKO Hu-liver mice with minimal interference from mouse hepatic P450 oxidation activity are a valuable model for predicting human drug metabolism.

Selection of the candidate compound at an early stage of new drug development: retrospective pharmacokinetic and metabolic evaluations of valsartan using common marmosets.

Valsartan is an antihypertensive drug that was developed using common marmosets (Callithrix jacchus) in pivotal toxicity studies as a non-rodent species. The aim of the present study was to investigate the utility of marmosets in the candidate selection of this drug from a pharmacokinetic and metabolic viewpoint.Valsartan, as well as three other angiotensin II type-I receptor blockers, assumed as competitive candidates, were administered to common marmosets. Human pharmacokinetic parameters predicted by single-species allometric scaling and Wajima superposition suggested that valsartan may exhibit promising pharmacokinetic properties in humans.In vitro metabolic studies of valsartan using isolated rat, dog, marmoset, cynomolgus monkey, and human hepatocytes revealed that the marmoset was the most relevant animal species to humans presenting with the most abundant human metabolite, 4-hydroxyvalsartan. Oral administration of an elevated dose of valsartan to a common marmoset demonstrated that the level of 4-hydroxyvalsartan in the plasma was comparable to that in clinical practice and suggested that safety of the human metabolite might have been confirmed in the toxicity studies using common marmosets.These results suggest that common marmosets, the small, non-human primates, had been a suitable species for the development of valsartan.

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.

Polymorphic cytochromes P450 in non-human primates.

Cynomolgus macaques (Macaca fascicularis, an Old World monkey) are widely used in drug development because of their genetic and physiological similarities to humans, and this trend has continued with the use of common marmosets (Callithrix jacchus, a New World monkey). Information on the major drug-metabolizing cytochrome P450 (CYP, P450) enzymes of these primate species indicates that multiple forms of their P450 enzymes have generally similar substrate selectivities to those of human P450 enzymes; however, some differences in isoform, activity, and substrate specificity account for limited species differences in drug oxidative metabolism. This review provides information on the P450 enzymes of cynomolgus macaques and marmosets, including cDNA, tissue expression, substrate specificity, and genetic variants, along with age differences and induction. Typical examples of important P450s to be considered in drug metabolism studies include cynomolgus CYP2C19, which is expressed abundantly in liver and metabolizes numerous drugs. Moreover, genetic variants of cynomolgus CYP2C19 affect the individual pharmacokinetic data of drugs such as R-warfarin. These findings provide a foundation for understanding each P450 enzyme and the individual pharmacokinetic and toxicological results in cynomolgus macaques and marmosets as preclinical models. In addition, the effects of induction on some drug clearances mediated by P450 enzymes are also described. In summary, this review describes genetic and acquired individual differences in cynomolgus and marmoset P450 enzymes involved in drug oxidation that may be associated with pharmacological and/or toxicological effects.

Contribution of Humanized Liver Chimeric Mice to the Study of Human Hepatic Drug Transporters: State of the Art and Perspectives.

Chimeric mice with humanized livers constitute an attractive emergent experimental model for investigating human metabolism and disposition of drugs. The present review was designed to summarize key findings about the use of this model for studying human hepatic drug transporters, which are now recognized as important players in pharmacokinetics and consequently have to be considered from a regulatory perspective during pharmaceutical drug development. The reviewed data indicate that chimeric mice with humanized livers have been successfully used for analysing the implications of human hepatic drug transporters for drug hepatobiliary elimination, drug-drug interactions and drug-induced cholestasis. Such transporter studies have been performed in vivo with chimeric mice and/or in vitro with human hepatocytes isolated from humanized liver and used either in suspension or in culture. The residual presence of mouse hepatocytes and the potential morphological/histological alterations of the humanized liver, as well as its immunodeficient mouse environment, have, however, to be considered when using chimeric mice with humanized livers for transporter studies. Finally, if the proof of concept of applying chimeric mice with humanized livers to hepatic drug transport is established, more experimental data on this topic, including from standardization approaches, are likely required to completely and accurately demonstrate the robustness, convenience and added value of this chimeric mouse model for drug transporter studies.

Molecular and Functional Characterization of N-Acetyltransferases in Common Marmosets and Pigs.

Arylamine N-acetyltransferases (NATs) are drug-metabolizing enzymes that are essential for the metabolism of endogenous substrates and xenobiotics. The molecular characteristics of NATs have been extensively investigated in humans but remain to be investigated in common marmosets and pigs, animal species that are often used in drug metabolism studies. In this study, marmoset NAT1 and pig NAT1 cDNAs were isolated from liver samples and were characterized by molecular analyses and drug-metabolism assays. These NAT genes were intronless and formed gene clusters with one other NAT gene in the genome, just as human NAT genes do. Marmoset NAT1 and pig NAT1 amino acid sequences showed high sequence identities (94% and 85%, respectively) to human NAT1. Phylogenetic analysis indicated that marmoset NAT1 and pig NAT1 were more closely clustered with human NATs than with rat or mouse NATs. Marmoset NAT1 and pig NAT1 mRNAs were expressed in all the tissue types analyzed, with the expression levels being highest in the small intestine. Metabolic assays using recombinant proteins found that marmoset NAT1 and pig NAT1 metabolized human NAT substrates p-aminobenzoic acid, 2-aminofluorene, sulfamethazine, and isoniazid. Marmoset NAT1 and pig NAT1 substantially acetylated p-aminobenzoic acid and 2-aminofluorene relevant human NAT1, but their activities were lower toward sulfamethazine and isoniazid than those of the relevant human NAT2. Therefore, marmoset and pig NATs are functional enzymes with molecular similarities to human NAT1, but their substrate specificities, while similar to human NAT1, differ somewhat from human NAT2. SIGNIFICANCE STATEMENT: Marmoset N-acetyltransferase NAT1 and pig NAT1 were identified and showed high sequence identities to human NAT1. These NAT mRNAs were expressed in various tissues. Marmoset and pig NAT1s acetylated typical human NAT substrates, although their substrate specificities differed somewhat from human NAT2. Marmoset NAT1 and pig NAT1 have similarities with human NAT1 in terms of molecular and enzymatic characteristics.