In vivo stable 211At-labeled prostate-specific membrane antigen-targeted tracer using a neopentyl glycol structure.

BACKGROUND: Prostate cancer is a common cancer among men worldwide that has a very poor prognosis, especially when it progresses to metastatic castration-resistant prostate cancer (mCRPC). Therefore, novel therapeutic agents for mCRPC are urgently required. Because prostate-specific membrane antigen (PSMA) is overexpressed in mCRPC, targeted alpha therapy (TAT) for PSMA is a promising treatment for mCRPC. Astatine-211 (211At) is a versatile α-emitting radionuclide that can be produced using a cyclotron. Therefore, 211At-labeled PSMA compounds could be useful for TAT; however, 211At-labeled compounds are unstable against deastatination in vivo. In this study, to develop in vivo stable 211At-labeled PSMA derivatives, we designed and synthesized 211At-labeled PSMA derivatives using a neopentyl glycol (NpG) structure that can stably retain 211At in vivo. We also evaluated their biodistribution in normal and tumor-bearing mice. RESULTS: We designed and synthesized 211At-labeled PSMA derivatives containing two glutamic acid (Glu) linkers between the NpG structure and asymmetric urea (NpG-L-PSMA ((L-Glu)2 linker used) and NpG-D-PSMA ((D-Glu)2 linker used)). First, we evaluated the characteristics of 125I-labeled NpG derivatives because 125I was readily available. [125I]I-NpG-L-PSMA and [125I]I-NpG-D-PSMA showed low accumulation in the stomach and thyroid, indicating their high in vivo stability against deiodination. [125I]I-NpG-L-PSMA was excreted in urine as hydrophilic radiometabolites in addition to the intact form. Meanwhile, [125I]I-NpG-D-PSMA was excreted in urine in an intact form. In both cases, no radioactivity was observed in the free iodine fraction. [125I]I-NpG-D-PSMA showed higher tumor accumulation than [125I]I-NpG-L-PSMA. We then developed 211At-labeled PSMA using the NpG-D-PSMA structure. [211At]At-NpG-D-PSMA showed low accumulation in the stomach and thyroid in normal mice, indicating its high stability against deastatination in vivo. Moreover, [211At]At-NpG-D-PSMA showed high accumulation in tumor similar to that of [125I]I-NpG-D-PSMA. CONCLUSIONS: [211At]At-NpG-D-PSMA showed high in vivo stability against deastatination and high tumor accumulation. [211At]At-NpG-D-PSMA should be considered as a potential new TAT for mCRPC.

Functional intestinal monolayers from organoids derived from human iPS cells for drug discovery research.

BACKGROUND: Human induced pluripotent stem (iPS) cell-derived enterocyte-like cells (ELCs) are expected to be useful for evaluating the intestinal absorption and metabolism of orally administered drugs. However, it is difficult to generate large amounts of ELCs with high quality because they cannot proliferate and be passaged. METHODS: To solve the issue above, we have established intestinal organoids from ELCs generated using our protocol. Furthermore, monolayers were produced from the organoids. We evaluated the usefulness of the monolayers by comparing their functions with those of the original ELCs and the organoids. RESULTS: We established organoids from ELCs (ELC-org) that could be passaged and maintained for more than a year. When ELC-org were dissociated into single cells and seeded on cell culture inserts (ELC-org-mono), they formed a tight monolayer in 3 days. Both ELC-org and ELC-org-mono were composed exclusively of epithelial cells. Gene expressions of many drug-metabolizing enzymes and drug transporters in ELC-org-mono were enhanced, as compared with those in ELC-org, to a level comparable to those in adult human small intestine. The CYP3A4 activity level in ELC-org-mono was comparable or higher than that in primary cryopreserved human small intestinal cells. ELC-org-mono had the efflux activities of P-gp and BCRP. Importantly, ELC-org-mono maintained high intestinal functions without any negative effects even after long-term culture (for more than a year) or cryopreservation. RNA-seq analysis showed that ELC-org-mono were more mature as intestinal epithelial cells than ELCs or ELC-org. CONCLUSIONS: We have successfully improved the function and convenience of ELCs by utilizing organoid technology.

Establishment of human intestinal organoids derived from commercially available cryopreserved intestinal epithelium and evaluation for pharmacokinetic study.

Human intestinal organoids (HIOs) have been reported to exert their functions in a way that mimics living organs, and HIOs-derived monolayers are expected to be applied to in vitro intestinal pharmacokinetic studies. However, HIOs are established from human tissue, which raises issues of availability and ethics. In the present study, to solve these problems, we have established intestinal organoids using commercially available cryopreserved human intestinal epithelial cells (C-IOs), and compared their functions with biopsy-derived human intestinal organoids (B-IOs) from a pharmacokinetic point of view. Both C-IOs and B-IOs reproduced the morphological features of the intestinal tract and were shown to be composed of epithelial cells. Monolayers generated from C-IOs and B-IOs (C-IO-2D, B-IO-2D, respectively) structurally mimic the small intestine. The C-IOs showed gene expression levels comparable to those of the B-IOs, which were close to those of adult human small intestine. Importantly, the C-IOs-2D showed levels of pharmacokinetics-related protein expression and activity-including cytochrome P450 3A4 (CYP3A4) and carboxylesterase 2 (CES2) enzymatic activities and P-glycoprotein (P-gp) transporter activities -similar to those of B-IOs-2D. This study addresses the difficulties associated with B-IOs and provides fundamental characteristics for the application of C-IOs in pharmacokinetic studies.

Novel conditionally replicating adenovirus-mediated efficient detection of circulating tumor cells in lung cancer patients.

Circulating tumor cells (CTCs) are present in the blood of cancer patients from the early stage of cancer development, and their presence has been correlated with patient prognosis and treatment responses. Accordingly, CTCs have been attracting attention as a novel biomarker for early detection of cancer and monitoring of treatment responses. However, since patients typically have only a few CTCs per milliliter of blood, development of an accurate and highly sensitive CTC detection method is crucial. We previously developed a CTC detection method using a novel conditionally replicating adenovirus (Ad) that expresses green fluorescence protein (GFP) in a tumor cell-specific manner by expressing the E1 gene using a tumor-specific human telomerase reverse transcriptase (hTERT) promoter (rAdF35-142T-GFP). CTCs were efficiently detected using rAdF35-142T-GFP, but GFP expression levels in the CTCs and production efficiencies of rAdF35-142T-GFP were relatively low. In this study, in order to overcome these problems, we developed four types of novel GFP-expressing conditionally replicating Ads and examined their ability to visualize CTCs in the blood samples of lung cancer patients. Among the four types of novel recombinant Ads, the novel conditionally replicating Ad containing the 2A peptide and the GFP gene downstream of the E1A gene and the adenovirus death protein (ADP) gene in the E3 region (rAdF35-E1-2A-GFP-ADP) mediated the highest number of GFP-positive cells in the human cultured tumor cell lines. Titers of rAdF35-E1-2A-GFP-ADP were significantly higher (about 4-fold) than those of rAdF35-142T-GFP. rAdF35-E1-2A-GFP-ADP and rAdF35-142T-GFP efficiently detected CTCs in the blood of lung cancer patients at similar levels. GFP+/CD45- cells (CTCs) were found in 10 of 17 patients (58.8%) for both types of recombinant Ads.

Ensemble Machine Learning Approaches Based on Molecular Descriptors and Graph Convolutional Networks for Predicting the Efflux Activities of MDR1 and BCRP Transporters.

Multidrug resistance (MDR1) and breast cancer resistance protein (BCRP) play important roles in drug absorption and distribution. Computational prediction of substrates for both transporters can help reduce time in drug discovery. This study aimed to predict the efflux activity of MDR1 and BCRP using multiple machine learning approaches with molecular descriptors and graph convolutional networks (GCNs). In vitro efflux activity was determined using MDR1- and BCRP-expressing cells. Predictive performance was assessed using an in-house dataset with a chronological split and an external dataset. CatBoost and support vector regression showed the best predictive performance for MDR1 and BCRP efflux activities, respectively, of the 25 descriptor-based machine learning methods based on the coefficient of determination (R2). The single-task GCN showed a slightly lower performance than descriptor-based prediction in the in-house dataset. In both approaches, the percentage of compounds predicted within twofold of the observed values in the external dataset was lower than that in the in-house dataset. Multi-task GCN did not show any improvements, whereas multimodal GCN increased the predictive performance of BCRP efflux activity compared with single-task GCN. Furthermore, the ensemble approach of descriptor-based machine learning and GCN achieved the highest predictive performance with R2 values of 0.706 and 0.587 in MDR1 and BCRP, respectively, in time-split test sets. This result suggests that two different approaches to represent molecular structures complement each other in terms of molecular characteristics. Our study demonstrated that predictive models using advanced machine learning approaches are beneficial for identifying potential substrate liability of both MDR1 and BCRP.

Effects of sleep restriction on food intake and appetite under free-living conditions: A randomized crossover trial.

To investigate changes in subjective psychological factors and dietary intake during sleep restriction, we carried out a randomized crossover trial with a 3-day sleep restriction condition (SR; 5 h of sleep) and control sleep condition (CS; 8 h of sleep). Days 3 and 4 involved free-living and laboratory (in the morning) conditions, respectively. Subjective psychological factors (hunger, appetite, desire for sweets and fatty foods, sleepiness, and fatigue) were assessed using a 0.0-10.0 cm visual analog scale (VAS) every hour throughout the day on day 3, and at 8:00 a.m. on day 4. Dietary intake on day 3 was assessed on the basis of the food purchased and eaten. Fasting blood samples were collected at 8:00 a.m. on day 4. Dietary intake during the ad libitum breakfast was assessed on day 4. The participants were 13 women and 11 men (mean age, 21.4 ± 1.0 years; mean body mass index, 19.8 ± 1.7 kg/m2). The areas under the curve 0-16 h after waking for hunger, desire for fatty foods, sleepiness, and fatigue were higher in the SR than CS on day 3 (P < 0.05). Energy and carbohydrate intakes from snacks (daytime and nighttime) on day 3 were higher in the SR than CS (P < 0.05) but total dietary intake on day 3 was not different between the conditions (P > 0.05). The 2-arachidonoylglycerol level was different between the conditions (P < 0.05), but was not associated with sweet taste preference, dietary intake, or the active ghrelin level on day 4 (P > 0.05). In conclusion, ratings for subjective psychological factors and energy and carbohydrate intakes from snacks increased in association with sleep restriction under free-living conditions.

Regional Transcriptomics and Proteomics of Pharmacokinetics-Related Genes in Human Intestine.

The intestine is an organ responsible for the absorption and metabolism of orally administered drugs. To predict pharmacokinetics behavior in the small intestine, it is necessary to examine the human intestinal expression profiles of the genes related to drug absorption, distribution, metabolism, and excretion (ADME). In this study, to obtain more accurate expression profiles in various regions of the human intestine, biopsy samples were collected from endoscopically noninflamed mucosa of the duodenum, jejunum, ileum, colon, and rectum from Japanese including Crohn's disease or ulcerative colitis patients, and both RNA-seq and quantitative proteomics analyses were performed. We also analyzed the expression of drug-metabolizing enzymes (cytochromes P450 (CYPs) and non-CYP enzymes), drug transporters, and nuclear receptors. Overall, the mRNA expression levels of these ADME-related genes correlated highly with the protein expression levels. The characteristics of the expression of ADME-related genes differed significantly between the small and large intestines, including the expression levels of CYP enzymes, which were higher and lower in the small and large intestines, respectively. Most CYPs were expressed dominantly in the small intestine, especially the jejunum, but were rarely expressed in the large intestine. On the other hand, non-CYP enzymes were expressed in the large intestine but at lower expression levels than in the small intestine. Moreover, the expression levels of drug metabolizing enzyme genes differed even between the proximal and distal small intestine. Transporters were expressed most highly in the ileum. The data in the present study will enhance understanding of the intestinal ADME of drug candidates and would be useful for drug discovery research.

Comparison of human biopsy-derived and human iPS cell-derived intestinal organoids established from a single individual.

Rodent-derived intestinal tissues or human colon cancer-derived Caco-2 cells are widely used for in vitro pharmacokinetic tests. However, both entail problems such as species differences from humans and low expression levels of specific pharmacokinetic-related factors, respectively. To solve these problems, many groups, including ours, have been focusing on human biopsy-derived intestinal organoids (b-IOs) and human iPS cell-derived intestinal organoids (i-IOs). However, no reports directly compare the two. Therefore, we established both from a single individual and conducted a comparative study. b-IOs had a shorter doubling time than i-IOs: about 59 h vs 148 h. b-IOs also had higher gene expression levels of major drug transporters and drug-metabolizing enzymes than i-IOs. To evaluate their applicability to pharmacokinetics, both organoids were two-dimensionally cultured. Although the b-IO monolayer had a lower transepithelial electrical resistance than the i-IO monolayer, it had higher gene expression levels of many drug transporters and major drug-metabolizing enzymes than the i-IO monolayer. RNA-seq analysis showed that the i-IOs monolayer had a more complex structure than the b-IOs monolayer because the former contained neuronal and vascular endothelial cells. This study provides basic information for pharmacokinetic applications of human biopsy-derived and human iPS cell-derived intestinal organoids.

Establishment of MDR1-knockout human enteroids for pharmaceutical application.

In the drug development process, it is important to assess the contributions of drug-metabolizing enzymes and/or drug transporters to the intestinal pharmacokinetics of candidate compounds. For such assessments, chemical inhibitors are often used in in vitro systems. However, this practice poses two problems: one is the low expression levels of pharmacokinetic-related genes in conventional in vitro systems, such as Caco-2 cells, and the other is the off-target and less-efficient effects of their inhibitors. Here, as a model, we have established human biopsy-derived enteroids deficient in MDR1, a key efflux transporter. The expression levels and activities of other pharmacokinetic-related genes, such as CYP3A4, in the MDR1-knockout (KO) enteroid-derived monolayers were maintained at levels as high as those in the WT enteroid-derived monolayers. The contribution of MDR1 to the cytotoxicity of vinblastine, which CYP3A4 metabolized, was accurately evaluated by using the MDR1-KO enteroid-derived monolayers. In contrast, it could not be evaluated in the WT enteroid-derived monolayers treated by verapamil, a widely used MDR1 inhibitor, due to the off-target effect of verapamil, which also inhibits CYP3A4. The combination of human enteroid-derived monolayers and genome editing technology would be a powerful tool to evaluate the contributions of specific pharmacokinetic-related molecules.

Monolayer platform using human biopsy-derived duodenal organoids for pharmaceutical research.

The human small intestine is the key organ for absorption, metabolism, and excretion of orally administered drugs. To preclinically predict these reactions in drug discovery research, a cell model that can precisely recapitulate the in vivo human intestinal monolayer is desired. In this study, we developed a monolayer platform using human biopsy-derived duodenal organoids for application to pharmacokinetic studies. The human duodenal organoid-derived monolayer was prepared by a simple method in 3-8 days. It consisted of polarized absorptive cells and had tight junctions. It showed much higher cytochrome P450 (CYP)3A4 and carboxylesterase (CES)2 activities than did the existing models (Caco-2 cells). It also showed efflux activity of P-glycoprotein (P-gp) and inducibility of CYP3A4. Finally, its gene expression profile was closer to the adult human duodenum, compared to the profile of Caco-2 cells. Based on these findings, this monolayer assay system using biopsy-derived human intestinal organoids is likely to be widely adopted.

Comparison of culture media for human intestinal organoids from the viewpoint of pharmacokinetic studies.

Human intestinal organoids are expected to be applied in pharmaceutical research. Various culture media for human intestinal organoids have been developed, but it remains unclear which media are preferable for pharmacokinetic studies. Here, we cultured human intestinal organoids with three major culture media that are already used widely around the world: the medium of Sato et al. (S-medium; reported in 2011), Fujii et al. (F-medium; 2018), and Miyoshi et al. (M-medium; 2013). The growth of human intestinal organoids cultured in S-medium was faster than that in F- or M-medium. The gene expression levels of most pharmacokinetic-related enzymes or transporters in human intestinal organoids cultured in M-medium were higher than those in S- or F-medium, and comparable to those in the adult human small intestine. The level of cytochrome P450 (CYP) 3A4 activity was also highest in human intestinal organoids cultured in M-medium. Collectively, the results underscored the importance of selection and optimization of culture medium for various applications using human intestinal organoids, including pharmacokinetic studies.

Vinblastine treatment decreases the undifferentiated cell contamination of human iPSC-derived intestinal epithelial-like cells.

Human induced pluripotent stem cell-derived intestinal epithelial cells (hiPSC-IECs) are expected to be utilized in regenerative medicine. To perform a safe transplantation without the risk of tumor formation, residual undifferentiated hiPSCs must be removed from hiPSC-IECs. In this study, we examined whether vinblastine (a multiple drug resistance 1 [MDR1] substrate) could remove residual undifferentiated hiPSCs in hiPSC-IECs and attempted to generate hiPSC-IECs applicable to transplantation medicine. We found that the expression levels of pluripotent markers were largely decreased and those of intestinal markers were increased by vinblastine treatment. The treatment of undifferentiated hiPSCs with vinblastine significantly decreased their viability. These results suggested that undifferentiated hiPSCs can be eliminated from hiPSC-IECs by vinblastine treatment. We hypothesized that MDR1-negative cells (such as undifferentiated hiPSCs) die upon vinblastine treatment because they are unable to excrete vinblastine. As expected, the cell viability of MDR1-knockout hiPSC-IECs was significantly decreased by vinblastine treatment. Furthermore, teratomas were formed by subcutaneous transplantation of hiPSC-IECs mixed with undifferentiated hiPSCs into mice, but they were not observed when the transplanted cells were pre-treated with vinblastine. Vinblastine-treated hiPSC-IECs would be an effective cell source for safe regenerative medicine.

In Vivo Gene Expression Profile of Human Intestinal Epithelial Cells: From the Viewpoint of Drug Metabolism and Pharmacokinetics.

Orally administered drugs are absorbed and metabolized in the intestine. To accurately predict pharmacokinetics in the intestine, it is essential to understand the intestinal expression profiles of the genes related to drug absorption, distribution, metabolism, and excretion (ADME). However, in many previous studies, gene expression analysis in the intestine has been carried out using specimens from patients with cancer. In this study, to obtain more accurate gene expression profiles, biopsy samples were collected under endoscopic observation from the noninflammatory regions of 14 patients with inflammatory bowel disease, and RNA-seq analysis was performed. Gene expression analysis of drug-metabolizing enzymes (cytochromes P450), non-cytochrome P450 enzymes, nuclear receptors, drug-conjugating enzymes (UDP-glucuronosyltransferases and sulfotransferases), and apical and basolateral drug transporters was performed in biopsy samples from the duodenum, ileum, colon, and rectum. The proportions of the cytochromes P450 expressed in the ileum were 25% (CYP3A4), 19% (CYP2C18), and 14% (CYP3A5). CYP3A4 and CYP2C19 were highly expressed in the duodenum and ileum, but not in the colon and rectum. In the ileum, apical transporters such as P-gp, peptide transporter 1, breast cancer resistance protein, MRP2, and ASBT were strongly expressed, and the expression levels of P-gp and ASBT in the ileum were higher than those in other regions. In the ileum, basolateral transporters such as OSTα, OSTß, and MRP3 were strongly expressed. We succeeded in obtaining gene expression profiles of ADME-related genes in human intestinal epithelial cells in vivo. We expect that this information would be useful for accurate prediction of the pharmacokinetics of oral drugs. SIGNIFICANCE STATEMENT: To obtain gene expression profiles of ADME-related genes in human intestinal epithelial cells in vivo, biopsy samples were collected under endoscopic observation from the noninflammatory regions of 14 patients with inflammatory bowel disease, and RNA-seq analysis was performed. Gene expression profiles of drug-metabolizing enzymes (cytochromes P450), non-cytochrome P450 enzymes, nuclear receptors, drug-conjugating enzymes (UDP-glucuronosyltransferases and sulfotransferases), and apical and basolateral drug transporters in biopsy samples from the duodenum, ileum, colon, and rectum were obtained in this study.

Photoactivatable oncolytic adenovirus for optogenetic cancer therapy.

Virotherapy using oncolytic adenovirus is an effective anticancer strategy. However, the tumor selectivity of oncolytic adenoviruses is not enough high. To develop oncolytic adenovirus with a low risk of off-tumor toxicity, we constructed a photoactivatable oncolytic adenovirus (paOAd). In response to blue light irradiation, the expression of adenoviral E1 genes, which are necessary for adenoviral replication, is induced and replication of this adenovirus occurs. In vitro, efficient lysis of various human cancer cell lines was observed by paOAd infection followed by blue light irradiation. Importantly, there was no off-tumor toxicity unless the cells were irradiated by blue light. In vivo, tumor growth in a subcutaneous tumor model and a mouse model of liver cancer was significantly inhibited by paOAd infection followed by blue light irradiation. In addition, paOAd also showed a therapeutic effect on cancer stem cells. These results suggest that paOAd is useful as a safe and therapeutically effective cancer therapy.

Establishment of SLC15A1/PEPT1-Knockout Human-Induced Pluripotent Stem Cell Line for Intestinal Drug Absorption Studies.

Because many peptide and peptide-mimetic drugs are substrates of peptide transporter 1, it is important to evaluate the peptide transporter 1-mediated intestinal absorption of drug candidates in the early phase of drug development. Although intestinal cell lines treated with inhibitors of peptide transporter 1 are widely used to examine whether drug candidates are substrates for peptide transporter 1, these inhibitors are not sufficiently specific for peptide transporter 1. In this study, to generate a more precise evaluation model, we established peptide transporter 1-knockout induced pluripotent stem cells (iPSCs) by using a CRISPR-Cas9 system and differentiated the cells into intestinal epithelial-like cells. The permeability value and uptake capacity of glycylsarcosine (substrate of peptide transporter 1) in peptide transporter 1-knockout intestinal epithelial-like cells were significantly lower than those in wild-type intestinal epithelial-like cells, suggesting that peptide transporter 1 was successfully depleted in the epithelial cells. Taken together, our model can be useful in the development of peptide and peptide-mimetic drugs.

Generation of Human iPSC-Derived Intestinal Epithelial Cell Monolayers by CDX2 Transduction.

BACKGROUND & AIMS: To develop an effective and safe orally administered drug, it is important to predict its intestinal absorption rate, intestinal first-pass effect, and drug-drug interactions of orally administered drugs. However, there is no existing model to comprehensively predict the intestinal pharmacokinetics and drug-response of orally administered drugs. In this study, we attempted to generate homogenous and functional intestinal epithelial cells from human induced pluripotent stem (iPS) cells for pharmaceutical research. METHODS: We generated almost-homogenous Villin- and zonula occludens-1 (ZO1)-positive intestinal epithelial cells by caudal-related homeobox transcription factor 2 (CDX2) transduction into human iPS cell-derived intestinal progenitor cells. RESULTS: The drug absorption rates in human iPS cell-derived intestinal epithelial cell monolayers (iPS-IECM) were highly correlated with those in humans (R2=0.91). The expression levels of cytochrome P450 (CYP) 3A4, a dominant drug-metabolizing enzyme in the small intestine, in human iPS-IECM were similar to those in human small intestine in vivo. In addition, intestinal availability in human iPS-IECM (the fraction passing the gut wall: Fg=0.73) was more similar to that in the human small intestine in vivo (Fg=0.57) than to that in Caco-2 cells (Fg=0.99), a human colorectal adenocarcinoma cell line. Moreover, the drug-drug interaction and drug-food interaction could be observed by using our human iPS-IECM in the presence of an inducer and inhibitor of CYP3A4, i.e., rifampicin and grape fruit juice, respectively. CONCLUSION: Taking these results together, we succeeded in generating the human iPS-IECM that can be applied to various intestinal pharmacokinetics and drug-response tests of orally administered drugs.

Modeling of Hepatic Drug Metabolism and Responses in CYP2C19 Poor Metabolizer Using Genetically Manipulated Human iPS cells.

Cytochrome P450 family 2 subfamily C member 19 (CYP2C19), in liver, plays important roles in terms of drug metabolism. It is known that CYP2C19 poor metabolizers (PMs) lack CYP2C19 metabolic capacity. Thus, unexpected drug-induced liver injury or decrease of drug efficacy would be caused in CYP2C19 substrate-treated CYP2C19 PMs. However, it is difficult to evaluate the safety and effectiveness of drugs and candidate compounds for CYP2C19 PMs because there is currently no model for this phenotype. Here, using human induced pluripotent stem cells (human iPS cells) and our highly efficient genome-editing and hepatocyte differentiation technologies, we generated CYP2C19-knockout human iPS cell-derived hepatocyte-like cells (CYP2C19-KO HLCs) as a novel CYP2C19 PM model for drug development and research. The gene expression levels of hepatocyte markers were similar between wild-type iPS cell-derived hepatocyte-like cells (WT HLCs) and CYP2C19-KO HLCs, suggesting that CYP2C19 deficiency did not affect the hepatic differentiation potency. We also examined CYP2C19 metabolic activity by measuring S-mephenytoin metabolites using ultra-performance liquid chromatography-tandem mass spectrometry. The CYP2C19 metabolic activity was almost eliminated by CYP2C19 knockout. Additionally, we evaluated whether clopidogrel (CYP2C19 substrate)-induced liver toxicity could be predicted using our model. Unexpectedly, there was no significant difference in cell viability between clopidogrel-treated WT HLCs and CYP2C19-KO HLCs. However, the cell viability in clopidogrel- and ketoconazole (CYP3A4 inhibitor)-treated CYP2C19-KO HLCs was significantly enhanced as compared with that in clopidogrel- and DMSO-treated CYP2C19-KO HLCs. This result suggests that CYP2C19-KO HLCs can predict clopidogrel-induced liver toxicity. We succeeded in generating CYP2C19 PM model cells using human iPS cells and genome-editing technologies for pharmaceutical research. SIGNIFICANCE STATEMENT: Although unexpected drug-induced liver injury or decrease of drug efficacy would be caused in CYP2C19 substrate-treated CYP2C19 poor metabolizers, it is difficult to evaluate the safety and effectiveness of drugs and candidate compounds for CYP2C19 poor metabolizers because there is currently no model for this phenotype. Using human iPS cells and our highly efficient genome editing and hepatocyte differentiation technologies, we generated CYP2C19-knockout human iPS cell-derived hepatocyte-like cells as a novel CYP2C19 poor metabolizer model for drug development and research.

Pharmaceutical Research for Inherited Metabolic Disorders of the Liver Using Human Induced Pluripotent Stem Cell and Genome Editing Technologies.

Orthotopic liver transplantation, rather than drug therapy, is the major curative approach for various inherited metabolic disorders of the liver. However, the scarcity of donated livers is a serious problem. To resolve this, there is an urgent need for novel drugs to treat inherited metabolic disorders of the liver. This requirement, in turn, necessitates the establishment of suitable disease models for many inherited metabolic disorders of the liver that currently lack such models for drug development. Recent studies have shown that human induced pluripotent stem (iPS) cells generated from patients with inherited metabolic disorders of the liver are an ideal cell source for models that faithfully recapitulate the pathophysiology of inherited metabolic disorders of the liver. By using patient iPS cell-derived hepatocyte-like cells, drug efficacy evaluation and drug screening can be performed. In addition, genome editing technology has enabled us to generate functionally recovered patient iPS cell-derived hepatocyte-like cells in vitro. It is also possible to identify the genetic mutations responsible for undiagnosed liver diseases using iPS cell and genome editing technologies. Finally, a combination of exhaustive analysis, iPS cells, and genome editing technologies would be a powerful approach to accelerate the identification of novel genetic mutations responsible for undiagnosed liver diseases. In this review, we will discuss the usefulness of iPS cell and genome editing technologies in the field of inherited metabolic disorders of the liver, such as alpha-1 antitrypsin deficiency and familial hypercholesterolemia.