The cell therapy products (CTPs) derived from pluripotent stem cells (PSCs) have potentials to remain the PSCs and the residual PSCs are naturally tumorigenic, giving rise to teratoma. The droplet digital PCR (ddPCR) assay is known as in vitro assay to detect residual undifferentiated PSCs. The MEASURE project has developed ddPCR assay using LIN28 as a marker of undifferentiated PSCs and the reported detection sensitivity (0.001%) of the ddPCR assay for hiPSC was confirmed at multisite. This result indicates that ddPCR assay is rapid and robust assay for detection of residual undifferentiated PSCs in CTPs.

The highly efficient culture (HEC) assay is known as in vitro assay to detect residual undifferentiated pluripotent stem cells (PSCs) which possess tumorigenic potential. The MEASURE multisite experiments confirmed reported detection sensitivity (0.001%) under various conditions of hiPSC cell lines and culture medium/coating substrate. In addition, improved detection sensitivity (0.00002%) was achieved by utilizing magnetic activated cell sorting. These results indicate that HEC assay is highly sensitive and robust assay for detection of residual undifferentiated PSCs.

The Cellular immortality testing (CIT) was selected as an in vitro test to detect malignant transformed cells, and multicenter validation was performed. In the method HeLa cell was used as a malignant transformed cell, and human mesenchymal stem cell was used as a normal cell. Bulk growth speed was analyzed according to the previous report (Kono et al, Biologicals, 2017). As a result, we have confirmed that contamination of 10-4% HeLa cell could be detected with good reproducibility. Currently, similar examination is carried out with the cells of different properties.

The Digital Soft Agar Colony Formation (DSACF) assay is known as an assay for detecting transformed cells. In order to control the risk of false negatives, we improved method, which tried to judge the contamination with transformed cells within required test wells which were statistically estimated considering risk factor 5% from the colony formation efficiency. All facilities could detect colonies derived from 0.0001% HeLa spiked in hMSC. It is considered that DSACF using new methodology enables us to evaluate more quantitatively the risk of contamination with transformed cells.

The biodistribution evaluation of cell therapy products is performed to clarify the distribution site and to predict efficacy and safety. The MEASURE multisite experiments confirmed the qPCR quantitation and the biodistribution of human mesenchymal stem cells (hMSC) after intravenous (iv) administration to mice. The qPCR method using the cell-based calibration curve showed good results. The biodistribution of hMSC after iv administration to mice observed the similar tissue distribution profiles in all facilities, with the lungs showing the highest cell concentration among the examined tissues.

The 21st century has seen remarkable advances in computational power and biotechnology. These tools are transforming the way we predict the potential of chemicals to adversely affect human health. The sequencing of the genome supported the development of methods for evaluating global gene expression in tissues or cells, providing an unprecedented opportunity to investigate the initial effects of toxicants on cellular processes. These methods can be used in medium-to-high throughput, resulting in large data sets, which can be used alone or with other big data sets to gain insight into the toxicity of chemicals that have not previously been tested in animal models. Because human-derived cells are used in these assays, the results may be more relevant for predicting toxicity in humans. We are developing a large data set of gene expression signatures on toxicants that is compatible with other large data sets developed for drugs and gene manipulations (LINCS) and can be used for discovering which chemicals have similar biological activity using a statistical process called connectivity mapping (CMAP). We have also compiled a large data set of all toxicology studies in the public domain (either published or submitted to regulatory agencies) that is searchable by chemical structure. These tools provide a basis for read-across from tested chemicals to untested ones. The big data set of toxicology information can be used to identify close structural analogs, and the gene expression data confirms that the analogs have similar biological activity.

Climate change and shifts in economic activity markedly increase risks from catastrophic chemical contamination events resulting from climate-related or man-made emergencies. The complexities of hazardous chemical exposures, potential adverse health impacts, and the need to rapidly and comprehensively evaluate environmental mixtures call for novel approach methods to disasters. Disaster research is possible when a team of scientists from biomedical, epidemiological, data and engineering disciplines come together to design comprehensive solutions for complex exposure- and hazard-related challenges. New approach methods for rapid exposure and hazard characterization involve a number of sensitive analytical and toxicological methods that generate large amounts of information that can be classified as “big data.” However, toxicologists and risk assessors are not usually trained in the analysis of “big data” and need to collaborate with the data scientists and statisticians. This presentation will describe the efforts of the Superfund Research Center at Texas A&M University that is developing exposure and hazard assessment tools that can be used by the first responders, impacted communities, and the government agencies. We created a number of computational and statistical methods for analysis and integration of “big data” in environmental health. In addition, we work closely with industry and government decision-makers to determine what types of “big data” are most useful and how best to communicate large amounts of information to support environmental health decisions.

A living organism is a self-contained system driven by a variety of internal signals governed by receptors to interact with the environment. Although signals are measurable by omics technologies, the entire system is still too complex to decipher completely. One way to approach the system through accumulated measurements is to apply the approach for the “Big Data”.

Since 2003, we have been developing the Percellome databases consisting of mouse transcriptome data exposed to various chemicals measured by Affymetrix GeneChip expression arrays. Crucial points to obtain high-quality, reliable data are managing circadian rhythm of the mice, and the applying the “Percellome” normalization method (BMC Genomics. 2006 Mar 29;7:64.). At present, the Percellome database has comprehensive transcriptome data of liver and other organs on 151 chemicals by oral or inhalation route of exposure. Prediction of toxicity and mining of new mechanistic hypothesis of toxicity can be performed through a comparative analysis of “gene lists” made of significantly altered genes by the chemical administration.

Those new hypotheses are often verified by literature-based databases. Applying AI to such process is one of our approaches to integrate AI methods to our project. Another approach includes introduction of AI image recognition technology to verify the biological significance of the gene expression data. Any AI improvements will be reflected in the Percellome open database service soon. The goal is to evaluate or predicte the toxicity of new chemicals from short and small-scale animal bioassays.

The Tohoku Medical Megabank Project (TMM) has been launched to accomplish creative reconstruction in the aftermath of the Great East Japan Earthquake and ensuing tsunami on March 11, 2011. TMM aims to establish an integrated biobank on the basis of two prospective large-scale cohort studies; a population-based cohort study, which has recruited approximately 84,000 participants, and a birth and three-generation cohort study, which has recruited approximately 73,000 participants including newborns and their parents, siblings, grandparents. Two prospective cohorts of TMM show in combination a highly unique and strategical design. The bio-specimen and personal health records collected through these cohorts are stored in the integrated biobank, which storage both bio-specimens and genome-omics big data generated in-house. TMM will share both information data and samples with the research community to facilitate biomedical research, including toxicology, and development of personalized health care. TMM believes that constructing the integrated biobank by way of large-scale genome cohort studies will be effective in establishing the personalized health care and medicine, and in developing future toxicology.

[References]

1. http://www.megabank.tohoku.ac.jp

Recently cryo-electron Microscopy (cryoEM) are drawing attention as new technology, as it is likely to provide structure information on large and flexible proteins such as channels, transporters, or complexes. Through revealing structure information of those proteins, it is expected to improve efficiency to reduce off-target liability by SBDD approach.

As toxicity related proteins or pharmacokinetics related proteins are common among pharmas, we are collaborating to obtain structure information on non-competitive proteins. In this symposium, I would like to introduce our attempts and discuss future opportunities for cryoEM technology.

Approximately 80% of all sensory input is received via the eyes, so suffering from chronic retinal diseases that lead to blindness causes a significant decrease in the quality of life (QOL). And because retinal diseases are most common among the elderly, developing pathological analyses and treatments for retinal diseases has become an urgent issue in super-aging countries such as Japan. Although evaluation of drug candidates against retinal diseases has been done on animal models, serious concerns arise regarding the ethics and costs in addition to the limitations of translating data from animal models to clinical settings. In vitro cell culture models, an alternative to animal models, enable researchers to investigate specific molecules-of-interest and simply recapitulate complex and chronic conditions. Here, an organ-on-a-chip that mimics retinal diseases using microfluidic devices will be presented.

Steatohepatitis, marked by inflammation and fibrosis, is a common liver disease that lacks effective drug therapies. Recently we have developed a reproducible method to generate human pluripotent stem cell-derived multi-cellular liver organoids that recapitulate key pathological features of steatohepatitis. Interestingly, measurement with atomic force microscopy revealed that organoid stiffness reflects the fibrosis severity. In this presentation, we will discuss a new human organoid strategy combined with stiffness screening for facilitating drug discovery and drug safety research.

Micromachines such as sensors contribute to high-performance for smartphones, which are necessary for our daily lives. Sensors and actuators made with microfabrication technology that developed LSI integrated circuit technology called MEMS have become indispensable for the development of various information devices. MEMS have been developed from the technology of microfabrication of Si which is a material for LSI. MEMS have been applied to microfluidics by developing micro grooves for microchannels and microactuators for valve and pump in early stage. Microfluidics based on MEMS has been used in biochemical field. Bio MEMS, which is called μTAS or LOC, have developed through successful fusion of MEMS and life sciences. It has become possible to culture cells and tissues on the biochemical analysis chips, which are important for toxicology science. Furthermore, OoC and BoC technologies may allow organs and living body on a chip. This presentation plans to introduce several examples among our research activities on biomedical applications of MEMS: Analysis technologies of biopsied samples including blood and muscle tissues, openable artificial intestinal micromachine, microfingers for manipulation of cellular aggregate.

Liquid biopsy (LB) such as circulating tumor cells, circulating tumor DNA, and exosomes places great expectations on clinical application for solid cancer patietns.

Plasma EGFR mutation test was approved as companion diagnostics for 3rd generation of EGFR-tyrosine kinase inhibitors (TKIs). Technological innovation using digital PCR and ultra-sensitive next-generation sequencers drives the clinical application of liquid biopsy.

Copy number gain and methylation of circulating tumor DNA as well as gene mutation could be analyzed using LB. The purposes of LB are prediction of the effects of molecular targeted drugs and immune checkpoint inhibitors, early detection of cancer, and proof of concept of the drugs. LB-based minimal residual disease (MDR) monitoring is gathering worldwide attention as a field of clinical implementation. In this presentation, we report on clinical applications of LB for precision oncology, including our own studies.

We have devolved nanobiodevices, quantum technology, and AI for biomedical applications and healthcare, including single cancer cell diagnosis for cancer metastasis, circulating tumour cell (CTC) detection by microfluidic devices, nanopillar devices for ultrafast analysis of genomic DNA and microRNA, nanopore devices for single DNA sequencing, nanowire devices for exosome analysis, single-molecular epigenetic analysis, AI-powered nano-IoT sensors, quantum switching intra vital imaging of iPS cells and stem cells, and quantum technology-based cancer theranostics.

The key technologies for realizing diagnosis of systemic diseases using saliva samples are biomarkers and biosensors. Chemical salivary test (salivary test) has overwhelming advantages over blood test in that it is intrinsically safe and test samples can be self-collected any time. However, a diagnostic methodology for systemic diseases cannot be established on the basis of these advantages alone. It is considered that the practical application of salivary test depends on the successful demonstration that either the diagnostic accuracy of the test is significantly higher than that of conventional techniques, or it can provide additional new information to aid medical treatments. To be specific, this involves the use of new biomarkers. Although rapid, low-cost and high sensitivity analytical techniques using saliva sampling have improved tremendously in recent years, the suggested applications of such techniques are still limited to viral infections and human stress tests. However, in the near future, salivary sensors will be practically applied to various systemic diseases as point-of-care testing (POCT). As a topic, cancer-related activation of cytokine networks are central aspects of tumor development. One of the goal of my study is to examine the possibility of salivary cytokines for the screening of cancer. This review focuses on the current status and future prospects of diagnostic methodologies using saliva samples for detection of diseases. The detection of new applications for salivary sensors is discussed.

Pretreatment of biological samples with very low concentration and small volume is an important procedures for LC-MS analysis. Solid phase extraction (SPE) is widely used for pretreatment of cannabis in biological samples. However, SPE methods involve time consuming steps, unsuitable for small sample volume and expensive preparatory equipment. Therefore, recent investigations have focused on pretreatment of cannabis to adapt for small sample volume, rapid treatment time, and the low cost. In this study, we reports a novel SEP spin column for extraction of drugs from biological samples.

In recent years, the term of “modality” has been used frequently in the pharmaceutical industry. It is considered that “modality” means drug discovery and therapeutic measures by innovative and new methods and mechanisms which are different from conventional ones. In the 20 century, small molecule drugs which chemically synthesized consist mostly of pharmaceutical products, and safety evaluation of these drugs was evaluable with corresponding guidelines for toxicity studies and testing methods, etc. Biological drugs such as antibodies and protein drugs have been gradually developed, making it difficult to handle conventional toxicity studies, and for this reason, new ways of thinking and evaluation have been discussed.

With the rapid progress of science and technology including IT, medical technologies (therapeutic methods) called “New Modalities” such as nucleic acid medicines, genetic therapies, and cell therapies are making rapid progress. However, the corresponding method of assessment is not appropriate. The goal of toxicologic pathology is to elucidate and analyze toxic changes due to exposure and/or treatment. Toxicologic pathology explores pathological conditions and the nature of diseases that occur in the living system. Elucidation of toxicity will eventually be verified by experimental pathology. Pathology, which evaluates morphological changes in the final assessment, is essentially unchanged even when the method changes. A key item of pathology is the final assessment of safety taking into account the benefit/risk of the medicinal product.

Oligonucleotide therapeutics include antisense oligonucleotides, siRNA, and aptamers, and are expected as next-generation molecular targeted drugs following antibody drugs. Currently, many clinical trials are in running on various types of oligonucleotide candidates that are indicated for cancer, immune diseases, neuromuscular diseases, or viral infections. In addition, many non-clinical toxicity studies have been conducted in order to support these clinical trials, and then revealed characteristic common toxic changes due to the oligonucleotide drugs. Oligonucleotide drugs mainly exert their action by complimentary binding to target nucleic acids. Therefore, mechanisms of the pharmacologic and toxic effects are different from those of the low molecule drugs and antibody drugs. It is also known toxic changes associated with their relatively high molecular weight feature.

Toxic changes resulting from oligonucleotide drugs can be broadly divided into two groups; hybridization-dependent and hybridization independent toxicities. Hybridization-dependent toxic changes (related to complimentary binding to nucleic acid) include excessive pharmacologic effects due to bind the target RNA (on target toxicity) and unexpected effects due to bind to other than the target RNA (off target toxicity). These hybridization-dependent changes appear as unique to each oligonucleotide drug. The other hybridization-independent toxic changes (not related to complimentary binding to nucleic acid) are ocurred due to the structure or physical properties of the oligonucleotide compounds, which include drug accumulation, hepatotoxicity, nephrotoxicity, or inflammatory reactions by compliment activation or immune complex, and they are known to be common to several oligonucleotide drugs.

The oligonucleotide drugs are water-soluble polymers with a negative charge, so they have low affinity or poor penetration to the cells, and are rapidly degraded by exonucleases in the body. Therefore, so-called first generation compounds showed toxic changes mainly related to accumulation often limited to high dose. In order to improve these disadvantages, various molecular modifications have been developed, and second or third generation compounds that have better cellular penetration, greater nuclease resistance, and increased binding affinity have been synthesized. In the meantime, new toxic changes associated with nucleic acid modification are also known recently. In the toxicity assessment for the oligonucleotide drugs, it is important to be familiar with physicochemical characteristic profile in each drug in addition to the class effect toxicities.

In this symposium, we will introduce you well-recognized histopathological lesions commonly seen in the toxicity studies of oligonucleotide drugs, especially antisense oligonucleotides including our experiences and the literature review.

Along with the advance of the antibody engineering technologies, antibody therapeutics which specifically bind to extracellular molecules are now equipped with several pharmacological effector functions. Because of this progress, toxicological findings of antibody therapeutics depend on both their effector functions and target molecules, and we will share some of our cases in this session.

In addition, to obtain the data of tissue cross reactivity studies, our practical experience and issues will also be shared in the session.

The safety evaluation of human cell derived tissue-engineered medical products involves assessing the systemic and local effects, as well as evaluating the products themselves for unintended changes (transformation, tumorigenesis), and for engraftment to unintended organs (systemic distribution, ectopic tissue formation). In this presentation, I will introduce and discuss the current status of non-clinical safety evaluation of these products along with actual examples of issues encountered while conducting histopathological evaluation.

More than three years have passed since SEND became mandatory by the US FDA in December 2016. Some Japanese pharmaceutical companies, especially those who develop medicines globally, have already implemented SEND, and some have decided to wait for the conclusion of the introduction of SEND to Japan.

In this presentation, we would like to explain the current situation surrounding SEND from the viewpoint of pharmaceutical manufacturers, taking into account such situations, and to summarize the issues to be considered depending on the differences in companies’ positions.

The Standard for Exchange of NonClinical Data (SEND) - Navigating the regulatory landscape to get the information necessary to understand SEND requirements can be confusing. In this session, we will provide a very brief overview of what is SEND, explain the requirements, timelines and scope surrounding SEND dataset packages. We will spend time focusing on how the CDER FDA Technical Rejection Criteria will be implemented. We will also discuss what you need to know about SEND today and any upcoming SEND initiatives and challenges. All this information will provide a complete picture of the expectations surrounding SEND.

The US FDA has required SEND in 2016. AMED research group is discussing how to use SEND for a new drug approval reviewing by PMDA.

PMDA investigates toxicity reports in terms of human safety and clarifies the comprehensive toxicity profile in IND and NDA. If the interpretation of the toxicological findings are different between the PMDA and the applicant, PMDA will give inquiries the applicant and the final decision is shown in the reviewing reports. PMDA reviewers are required to have the specific skills to clarify the overall toxicity. In this presentation, we will show specific review points in toxicity studies, and will introduce a usefulness of visualizing toxic data using SEND in the reviewing.

As the creation and accumulation of data conforming to the non-clinical CDISC standard SEND has progressed, the utilization of SEND data for drug discovery has been actively discussed in addition to the purpose of regulatory submission. Through our task force activities, we realize that if SEND data are accumulated “as is”, it cannot always be used for the intended purposes.

Our group would like to confirm what “standard” is and, based on the current situation of SEND, what we should pay attention to when creating and accumulating data will lead to the development of better medicines faster.

Recently, FDA required that toxicology data be formatted and structured in the Standard Exchange for Nonclinical Data (SEND) model for IND/NDA submissions. The continued growth in the number of toxicology study datasets in SEND format will enable easier information exchange for due diligence, investigation of toxicity profile, cross study analysis and QSAR. The global consortium, “BioCelerate”, developed a SEND-based database (DataCelerate) for nonclinical toxicology data for use by members of the consortium. Shionogi analyzed data from two independent studies submitted to DataCelerate. These two studies were different compounds but designed to have the same pharmacologic target. Interestingly, the same specific target-organ toxicity was observed in both studies were conducted in a different facility. We considered the findings to be a class effect of this drug target. While the above example represents the benefit of cross-study analysis, there are also challenges. For example, animal age data in SEND datasets can be entered as day, week, month or year. The variability in this parameter makes it challenging to employ robust cross-study analysis due to the lack of a standard expression of age. Similar variability in populating variables can also be seen in SEND datasets. Searching across SEND datasets can help to understand a target toxicity profile, and predict toxicity for other compounds in the class. In the future, a large number of SEND datasets in DataCelerate should be a useful resource to predict potential drug target-related clinical adverse events.

In recent years, attempts to predict organ toxicity in vitro by using MPS (Microphysiological Systems) that mimics and reproduces the tissue structure in a living body have been widespread. In the background of these technologies, advances in engineering research such as cell culture, surface processing, and micro-engineering play an important role. On the other hand, there is an approach to extrapolate such in vitro results to clinical practice using PBPK and QSP/QST. This symposium considers future prospects for reducing the side effects of pharmaceuticals by fusing the two technologies.

In the field of drug toxicity and efficacy assays, cell monolayer and spheroid cultures have been widely used. However, it was mostly difficult to estimate human reaction by the methods because the complex three-dimensional (3D) tissue structures have not been reconstructed yet. In our body, endothelial and epithelial cells exist as a monolayer with tight junction. However, surrounding connective (stromal) tissues are composed of various extracellular matrix (ECM), mainly type I collagen, and also embedded fibroblasts, immune cells, and blood and lymphatic capillaries. The ECM is high density fibers and gives suitable mechanical property to the cells. Since mechanobiology- cell properties are influenced by the viscoelasticity of the surrounding connective tissues- is recent hot topic, novel tissue engineering is strongly desired for reconstructing high density ECM tissues.

We recently discovered novel tissue engineering, “sedimentary culture”, for in vitro reconstruction of the high density ECM tissues using type I collagen microfibers. Since this method allows us to manage cell property depending on the viscoelasticity of the surrounding tissues, it is expected as a new type of tissue engineering method.

The pharmacotoxicity and pharmacokinetics in the kidney are evaluated exclusively by animal tests because there is no effective in vitro evaluation systems. However, the dropout of drug candidates in preclinical and clinical trials due to differences in metabolic mechanisms between humans and experimental animals leads to enormous losses in drug development costs. Our research group has been developed a kidney microphysiological system (MPS) that uses microfluidic device technologies to mimic the physiological environment of glomeruli and tubules as novel in vitro systems for nephrotoxicity assay in drug discovery. In this talk, I will introduce both glomerular MPS and tubular MPS among the kidney MPS that we are developing, and its application to nephrotoxicity tests.

Microphysiological system (MPS) might be a promising tool for analysis of drug metabolism and toxicity in humans although limited information is available in quantitative approach for such purpose. We have introduced pressure-driven, medium circulation system for the entero-hepatic two-organ MPS recently developed in AIST (Tsukuba, Japan) into quantitative analysis of phase I- and II metabolism of several model drugs. Metabolic clearance of cytochrome P450 substrates and glucuronidation of hydroxyl metabolites in the two-organ MPS was higher than that observed in the single culture system, suggesting a certain gut-liver interaction involved in efficient drug metabolism. Plasma concentration profile of the model drug and its metabolites in humans was quantitatively simulated based on pharmacokinetic model in the MPS by incorporating several scaling factors. Thus, MPS would be effective tool for deep understanding of drug metabolism observed in multiple organs.

Arakawa H et al. Lab on a Chip 20, 537, 2020.

The National MPS project in Japan (AMED-MPS) consists of 4 research teams, central research center, and Head Quarter for establishing a close communication system among academic researchers, pharmaceutical companies as users and decision makers.

The main research program is organ model development research program focusing on cell supply and MPS model development of 4 organs: liver, gut, kidney, and BBB. In addition to the main research program, industrial programs are involved including the device manufacturing research program developing manufacturing technology for industrially produces; standardization research program developing standardization of MPS models for quality control and regulatory development in AMED-MPS. Noteworthy, senior managers and researchers in pharmacokinetics, safety fields from domestic pharmaceutical companies participate in the project as a member of decision-making body and research partners.

To bridge between the researchers and users, Central-research-center, closely collaborating with manufacturing and standardization program members, conducts researches and development to transfer newly-developed MPS technologies from model development team into practical use in users.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by MECP2 mutations. We have previously reported that MeCP2 promotes the posttranscriptional processing of particular microRNAs (miRNAs) as a component of the microprocessor Drosha complex. Among the MeCP2-regulated miRNAs, we found that expression of miR-199a rescues several impairments in MeCP2-deficient neurons, including reductions in soma size, excitatory neurotransmission and synaptic formation, which are hallmarks of RTT neuropathology. We here show that the MeCP2/miR-199a axis is participated not only in the regulation of neuronal functions but also in the fate specification of neural stem cells (NSCs). Expression of MeCP2 in NSCs promoted neuronal differentiation, while it inhibited astrocyte differentiation. Furthermore, this effect of MeCP2 was cancelled by inhibition of miR-199a. Based on the results of database search, we identified Smad1 as a target molecule of miR-199a. Smad1 is a well-known critical transcription factor downstream of bone morphogenetic protein signaling, which promotes and inhibits astrocytic and neuronal differentiation of NSCs, respectively. Consistent with these findings, we observed that Smad1 protein expression is increased in MeCP2 deficient mouse brain, in which miR-199a expression is decreased by the loss of MeCP2 expression. We further found that astrocyte differentiation of NSCs is indeed enhanced in MeCP2 deficient brain, corresponding to the fact that protein levels of typical astrocyte marker glial fibrillary acidic protein is increased in RTT patient. Since it is becoming apparent that functions of MeCP2 in glial cells are also involved in the pathogenesis of RTT, our finding implies that imbalance of neuronal and glial differentiation can be one of the causes for RTT.

Epigenetic modifications play an important role in gene transcription and are involved in numerous cellular processes. Two of the most characterized epigenetic modifications are DNA methylation and histone modification. Furthermore, the 3D-genome architecture has attracted much attention as an important epigenetic factor in gene regulation. My laboratory has been extensively analyzing their dynamic changes during early development of medaka fish using the quantitative ChIP-seq and HiC methods. In my talk, I will discuss the conservation and difference of epigenetic changes among vertebrate species and their roles in vertebrate development.

Epigenetic modifications are expected to change in fetuses and neonates and affect gene expression in the baby for long time. We collected biological samples of cases with pregnancy complications and preterm infants and found epigenomic changes in cord blood and placentas of these cases. Some changes are assumed to show "remaining immature epigenetic marks". In this symposium, we will give an overview of epigenome changes due to the environment observed in humans, including our original reports.

For safety assessment of epigenetic drugs, indirect effects associated with changes in gene expression should be considered in addition to the direct effects of DNA methylation, demethylation and histone-modification. For example, the impact on the next generation and the long-term effects such as carcinogenicity would be pointed out.

In this presentation, we refer to the toxicities and adverse effects of the epigenetic drugs that have been approved in nonclinical and clinical studies and introduce the points to consider for future development of epigenetic drugs.

Recent findings reveal that extracellular vesicles (EVs), secreted from cells, are circulating in the blood.

In this study, comprehensive sequencing of EV-associated small RNAs for male C57BL/6J mice orally dosed with carbon tetrachloride in a corn oil vehiclewere carried out. As a results, several EV-associated miRNAs, including well-known hepatotoxicity biomarkers, miR-122 and miR-192, were upregulated in the blood. The liquid biopsy by using EV-associated miRNA as biomarkers would accelerate a rapid evaluation of chemical substances and drugs in Nonclinical Safety Evaluation.

Aggresome-like induced structures (ALIS) have been described as ubiquitinated protein-containing aggresomes formed in stress conditions. Recently, we have demonstrated that nuclear accumulation of ALIS acts as microdomains sensing cellular stresses and triggering oxidative stress-induced parthanatos, a newly discovered non-apoptotic cell death mediated by poly (ADP-ribose) polymerase-1 (PARP-1) as a stress-sensing protein. In this symposium, we would like to introduce the novel function of the nuclear ALIS as a sensor of oxidative stress, which may control life-and-death decisions of cells.

Electrophilic aldehydes readily react with biomolecules, such as proteins, to generate various adducts. Because of technical constraints, protein modification by aldehydes has been individually studied, and comprehensive analysis of protein modification has rarely been performed. To simultaneously detect a variety of adducts in protein samples, we established a mass spectrometry-based method for the comprehensive analysis of adducts (adductome analysis). In this presentation, we would like to introduce our studies on redox protein adductomics.

Keap1-Nrf2 system is a cytoprotective mechanism that defends against a variety of toxicants and oxidative stress. We have been examined the relationship of Keap1-Nrf2 system and detoxication in the liver using genetically modified animals. Overdose of acetaminophen (APAP), an antipyretic analgesic, leads to severer liver injury in Nrf2 knockout mice in which Nrf2-target gene products responsible for detoxication and antioxidant are downregulated. On the other hands, more than half of Nrf2 knockout mice have a congenital portosystemic shunt and suppresses APAP-induced hepatotoxicity. Unlike acetaminophen that leads to necrosis around central veins, a heme synthesis inhibitor DDC is a hepatotoxicant that causes cholangiocytes expansion around portal veins. Liver-specific Keap1 knockout mice in which Nrf2 constitutively actives significantly ameliorated DDC-induced hepatotoxicity. In addition, Nrf2 activation protects liver cancers caused by aflatoxin B1 (AFB1) that is produced by molds contaminated in foods. However, this experimental model is not suitable for mice. Therefore, we generated Nrf2 knockout rat and dosed AFB1 to the rats. We hypothesized that Nrf2 knockout rats are susceptible to liver cancers with severer DNA damage. However, Nrf2 knockout rats died of not cancers but cirrhosis with fibrosis and hepatocyte regeneration. Interestingly, Keap1 knockout rats with constitutive Nrf2 activation died at perinatal period with jaundice, lacking of intrahepatic bile ducts around portal veins. Analyses using these animal models demonstrate that Nrf2 is the master transcription factor responsible for not only detoxication but also tissue formations of the shunt and intrahepatic bile ducts at developing stage.

Accumulated evidence indicate that sulfane sulfur, which is defined as a sulfur atom with 6 valence electrons and binds covalently to other sulfur atoms, plays an essential role in protection against oxidative/electrophilic stresses because sulfane sulfur-containing molecules such as persulfide and polysulfide can be highly nucleophilic compared to the corresponding thiol group. In the symposium, I will introduce the function of sulfane sulfur in the redox regulation during oxidative/electrophilic stresses, and discuss “phase zero-reaction” of environmental electrophiles.

Reactive sulfur species (RSS) with high nucleophilicity contributes to the elimination of electrophilic stress through sulfur adduct formation of electrophiles. Nrf2 pathway, a canonical redox signaling, does not function redundantly with RSS-generating CSE pathway because CSE/Nrf2 double-KO mice and their hepatocytes were more sensitive to various electrophiles than their single-KO counterparts. Thus, RSS acts as a critical regulator for electrophilic stress. In fact, stage- and region-specific RSS content is likely to affect selective methylmercury cytotoxicity in rat brain.

Reporter gene mutation assay has been noted as an in vivo genotoxicity test considering ADME and target sites of test chemicals. However, this assay sometimes demonstrates incompatible results with those in vitro genotoxicity tests. In this presentation, further studies examined based on negative results of three known carcinogens in reporter gene mutations assays will be demonstrated. The outcomes in these studies contributed to understanding of their toxicological profiles. This implies that negative results in reporter gene mutation assays include various significance.

Gene mutation induced by environmental mutagens is an important health factor. Germline mutations are sources of genetic diversity but may cause adverse outcome such as genetic diseases. We investigated mutation frequencies (MFs) in sperm of ENU-treated male mice by reporter gene assay and de novo germline MFs in their offspring by whole exome/genome sequencing. Point mutations induced in male germ cells caused in dose-dependent increase of de novo mutations in the offspring. It is important to evaluate mutagenic effects of environmental perturbations.

Recently, large-scale genome analysis using next-generation sequencing (NGS) technology has been actively performed, and this technique can be applied to genotoxicity evaluation for chemical substances. By using whole genome / exome analyses of chemical-exposed samples, non-biased and global mutation analysis becomes possible, therefore this method is expected to complement conventional genotoxicity tests.

In this symposium, I will introduce the current status of non-biased mutation analysis using NGS, and discuss prospects for application to a novel evaluation system of chemicals.

Transgenic rodent in vivo assay is useful for assessing the risk of combined exposure of multiple chemicals in the environment. We have evaluated in vivo mutagenicity of chemical mixture contained in airborne particulate matters (PM) by using gpt delta transgenic mouse. We showed that the inhalation of diesel exhaust (DE), a model pollutant, elevated the mutation frequency in lung, and mutation spectrum of DE resembled that of 1,6-dinitropyrene. The extract of PM collected in urban area was revealed to induce G-to-T transversion at a specific position on gpt as a landmark of oxidative stress.

A risk of tumor formation due to emergence of transformed cells is one of the safety risks specific to cell therapy products. Increased genetic instability is a potential hazard for the tumor formation risk. However, there is no global consensus on the clinical safety of cells with genetic mutations. Although a couple of MHLW guidance documents raise several test methods for the evaluation of genetic instability, these tests are generally intended to be carried out to obtain reference information about the characteristics of cells rather than shipping criteria.

Any specific safety guidelines for peptide pharmaceuticals are not available so far. Since ICH S6 states “The principles outlined in this guidance may also be applicable to…chemically synthesized peptides…”, PMDA applies “case-by-case basis approach” described in ICH S6 to safety evaluation of peptide pharmaceuticals. Since there are a variety of peptide pharmaceuticals, the non-clinical safety evaluation should be assessed based on each property (e.g. biological activity, chemical structure, chemical modification).

In this presentation, our preclinical consideration for peptide pharmaceuticals will be provided.

The number of peptide drugs approved in the USA as well as Japan has increased significantly despite inherent challenges of rising cost of manufacturing, peptide instability, and patient-friendly delivery. Disparities in interpretations and application of existing International Council for Harmonization (ICH) guidance ICH M3(R2) and ICH S6 to non-natural synthetic and conjugated peptide therapeutic products will be explored. The presentation will discuss the gaps in current regulatory guidance and provide the working group’s opinion on specific case examples.

Medium-sized peptide (MP) has recently emerged as a promising new modality of drug platform. MP is composed of various amino acids other than the 20 amino acids composing human proteins, which enables numerous structural variations to achieve intended chemical property. MP is similar partially to biopharmaceuticals and partially to low molecular drugs. Therefore, the safety guidelines based on the accumulated knowledge with existing therapeutics are not directly applicable to MP. Preparation of AMED guidance document is underway and has not yet been published. Here, I’ll discuss the safety and quality issues which toxicologists are confronting under the current situation.

Peptide drugs containing non-natural/modified residues are expected to interact selectively with target proteins, to target intracellular molecules and the application of oral administration. However, there is no regulatory guidelines that specifically address the non-clinical assessment. JPMA KT4 ADME team has started to discuss about issues and perspective. In this presentation, topics “appropriate approach for pharmacokinetic study and drug interaction study”, “application of guidelines for bioanalytical method validation”, and “consideration of anti-drug antibody” will be mainly discussed.