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.

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.

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.

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.

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.

Highly efficient induction of primate iPS cells by combining RNA transfection and chemical compounds.

Induced pluripotent stem (iPS) cells hold great promise for regenerative medicine and the treatment of various diseases. Before proceeding to clinical trials, it is important to test the efficacy and safety of iPS cell-based treatments using experimental animals. The common marmoset is a new world monkey widely used in biomedical studies. However, efficient methods that could generate iPS cells from a variety of cells have not been established. Here, we report that marmoset cells are efficiently reprogrammed into iPS cells by combining RNA transfection and chemical compounds. Using this novel combination, we generate transgene integration-free marmoset iPS cells from a variety of cells that are difficult to reprogram using conventional RNA transfection method. Furthermore, we show this is similarly effective for human and cynomolgus monkey iPS cell generation. Thus, the addition of chemical compounds during RNA transfection greatly facilitates reprogramming and efficient generation of completely integration-free safe iPS cells in primates, particularly from difficult-to-reprogram cells.

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.

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.

Expression and inducibility of cytochrome P450s in human hepatocytes isolated from chimeric mice with humanised livers.

The evaluation of drug-mediated cytochrome P450 (P450) induction using human hepatocytes is important for predicting drug interactions. In this study, we prepared hepatocytes from chimeric mice with humanised livers (Hu-Liver mice) and evaluated the expression and inducibility of P450s in these hepatocytes. Up to 95% of the Hu-Liver cells stained positive for human leukocyte antigen and the mean viability exceeded 85% (n = 10). Monolayer-cultured Hu-Liver cells displayed a similar morphology to cultures of the corresponding human hepatocytes used as transplantation donors. The mRNA expression levels in Hu-Liver cells of 16 P450 forms belonging to P450 subfamilies 1-4 correlated well with the expression levels of the same enzymes in human hepatocytes. The variations in individual P450 mRNA levels between Hu-Liver cells and the corresponding human hepatocytes were within five-fold for 13 P450 forms. The production of 6ß-hydroxytestosterone in Hu-Liver cells was significantly increased (p < .05) following treatment with the CYP3A inducer, rifampicin. Hu-Liver cells have characteristics similar to those of human hepatocytes in terms of mRNA expression levels and the inducibility of the various P450 forms. Thus, Hu-Liver cells can potentially be used for in vitro drug-mediated induction assays of human hepatic P450s.

Hepatocytes buried in the cirrhotic livers of patients with biliary atresia proliferate and function in the livers of urokinase-type plasminogen activator-NOG mice.

The pathogenesis of biliary atresia (BA), which leads to end-stage cirrhosis in most patients, has been thought to inflame and obstruct the intrahepatic and extrahepatic bile ducts. BA is not believed to be caused by abnormalities in parenchymal hepatocytes. However, there has been no report of a detailed analysis of hepatocytes buried in the cirrhotic livers of patients with BA. Therefore, we evaluated the proliferative potential of these hepatocytes in immunodeficient, liver-injured mice [the urokinase-type plasminogen activator (uPA) transgenic NOD/Shi-scid IL2rγnull (NOG); uPA-NOG strain]. We succeeded in isolating viable hepatocytes from the livers of patients with BA who had various degrees of fibrosis. The isolated hepatocytes were intrasplenically transplanted into the livers of uPA-NOG mice. The hepatocytes of only 3 of the 9 BA patients secreted detectable amounts of human albumin in sera when they were transplanted into mice. However, human leukocyte antigen-positive hepatocyte colonies were detected in 7 of the 9 mice with hepatocyte transplants from patients with BA. We demonstrated that hepatocytes buried in the cirrhotic livers of patients with BA retained their proliferative potential. A liver that was reconstituted with hepatocytes from patients with BA was shown to be a functioning human liver with a drug-metabolizing enzyme gene expression pattern that was representative of mature human liver and biliary function, as ascertained by fluorescent dye excretion into the bile canaliculi. These results imply that removing the primary etiology via an earlier portoenterostomy may increase the quantity of functionally intact hepatocytes remaining in a cirrhotic liver and may contribute to improved outcomes.

The human hepatic cell line HepaRG as a possible cell source for the generation of humanized liver TK-NOG mice.

1. Humanized-liver mice, in which the liver has been repopulated with human hepatocytes, have been used to study aspects of human liver physiology such as drug metabolism, toxicology and hepatitis infection. However, the procurement of human hepatocytes is a major problem in producing humanized-liver mice because of the finite nature of the patient-derived resource. 2. In order to overcome this limitation, the human hepatic cell line HepaRG® were evaluated as promising donor cells for liver reconstitution in the TK-NOG mouse model. 3. We demonstrate that, in vivo, transplanted confluent culture or differentiated HepaRG® cells proliferated and differentiated toward both hepatocyte-like and biliary-like cells within the recipient liver. In contrast, proliferative HepaRG® cells could engraft TK-NOG mouse liver but could differentiate only toward biliary-like cells. The differentiation to hepatocyte-like cells was characterized by the detection of human albumin in the recipient mouse serum and was confirmed by immunohistochemical staining for human leukocyte antigen, human albumin, cytochrome P450 3A4, and multidrug resistance-associated protein 2. Biliary-like cells were characterized by positive staining for cytokeratin-19. 4. These results indicated that the differentiated HepaRG® cells are a possible cell source for generating humanized-liver mice, which are a useful model for in vivo studies of liver physiology.

Impact of miR-222-3p-mediated downregulation of arylacetamide deacetylase on drug hydrolysis and lipid accumulation.

Arylacetamide deacetylase (AADAC) is involved in drug hydrolysis and lipid metabolism. In 23 human liver samples, no significant correlation was observed between AADAC mRNA (19.7-fold variation) and protein levels (137.6-fold variation), suggesting a significant contribution of post-transcriptional regulation to AADAC expression. The present study investigated whether AADAC is regulated by microRNA in the human liver and elucidate its biological significance. Computational analysis predicted two potential miR-222-3p recognition elements in the 3'-untranslated region (UTR) of AADAC. Luciferase assay revealed that the miR-222-3p recognition element was functional in downregulating AADAC expression. In HEK293 cells transfected with an AADAC expression plasmid containing 3'-UTR, miR-222-3p overexpression decreased AADAC protein level and activity, whereas miR-222-3p inhibition increased them. Similar results were observed in human hepatoma-derived Huh-1 cells endogenously expressing AADAC and HepaSH cells that are hepatocytes from chimeric mice with humanized livers. In individual human liver samples, AADAC protein levels inversely correlated with miR-222-3p levels. Overexpression of miR-222-3p resulted in increased lipid accumulation in Huh-1 cells, which was reversed by AADAC overexpression. In contrast, miR-222-3p inhibition decreased lipid accumulation, which was reversed by AADAC knockdown. In conclusion, we found that hepatic AADAC was downregulated by miR-222-3p, resulting in decreased drug hydrolysis and increased lipid accumulation.

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.

Synthesized basement membranes direct the differentiation of mouse embryonic stem cells into pancreatic lineages.

We previously reported that embryonic stem (ES) cells cultured on M15 cells, a mesoderm-derived supportive cell line, were efficiently differentiated towards an endodermal fate, finally adopting the specific lineages of various digestive organs such as the pancreas and liver. We show here that the endoderm-inducing activity of M15 cells is in part mediated through the extracellular matrices, and that laminin alpha5 is one of the crucial components. In an attempt to establish a feeder-free ES-cell procedure for pancreatic differentiation, we used a synthesized basement membrane (sBM) substratum using an HEK293 cell line stably expressing laminin-511. On the sBM, mouse ES or induced pluripotent stem (iPS) cells sequentially differentiated into the definitive endoderm, pancreatic progenitor cells, and then insulin-expressing pancreatic beta-cells in vitro. Knockdown of ES cells with integrin beta1 (Itgb1) reduces differentiation towards pancreatic cells. Heparan sulfate proteoglycan 2 (HSPG2) knockdown and heparitinase treatment synergistically decreased the number of Pdx1-expressing cells. These findings indicate that components of the basement membrane have an important role in the differentiation of definitive endoderm lineages. This novel procedure will be useful for the study of pancreatic differentiation of ES or iPS cells and the generation of potential sources of surrogate cells for regenerative medicine.

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.

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.

Capsid Allosteric Modulators Enhance the Innate Immune Response in Hepatitis B Virus-Infected Hepatocytes During Interferon Administration.

Capsid allosteric modulators (CAMs) inhibit the encapsidation of hepatitis B virus (HBV) pregenomic RNA (pgRNA), which contains a pathogen-associated molecular pattern motif. However, the effect of CAMs on the innate immune response of HBV-infected hepatocytes remains unclear, and we examined this effect in this study. Administration of a CAM compound, BAY41-4109 (BAY41), to HBV-infected primary human hepatocytes (PHHs) did not change the total cytoplasmic pgRNA levels but significantly reduced intracapsid pgRNA levels, suggesting that BAY41 increased extracapsid pgRNA levels in the cytoplasm. BAY41 alone did not change the intracellular interferon (IFN)-stimulated gene (ISG) expression levels. However, BAY41 enhanced antiviral ISG induction by IFN-α in HBV-infected PHHs but did not change ISG induction by IFN-α in uninfected PHHs. Compared with BAY41 or IFN-α alone, coadministration of BAY41 and IFN-α significantly suppressed extracellular HBV-DNA levels. HBV-infected human liver-chimeric mice were treated with vehicle, BAY41, pegylated IFN-α (pegIFN-α), or BAY41 and pegIFN-α together. Compared with the vehicle control, pegIFN-α highly up-regulated intrahepatic ISG expression levels, but BAY41 alone did not change these levels. The combination of BAY41 and pegIFN-α further enhanced intrahepatic antiviral ISG expression, which was up-regulated by pegIFNα. The serum HBV-DNA levels in mice treated with the combination of BAY41 and pegIFN-α were the lowest observed in all the groups. Conclusion: CAMs enhance the host IFN response when combined with exogenous IFN-α, likely due to increased cytoplasmic extracapsid pgRNA.

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.