One Health is an integrated, unifying approach that aims to sustainably balance and optimize the health of people, animals, and ecosystems. One Health is recognized by public health and veterinary medical experts with the advance of research on zoonotic diseases and antimicrobial resistance. Meanwhile, environmental toxicologists have not been aware of the term “One Health” until recently, but the establishment of platforms for investigating environmental contaminants (ECs) from One Health perspective will provide an opportunity to integrate knowledge on human, animal, and ecosystem health and accelerate activities to monitor, evaluate, and conserve the health of all species on the planet.

　We have engaged in research on the effects and risks of ECs on environmental and non-model organisms. In the research, we have shown that in vitro systems constructed using cDNA clones of intracellular receptors (AHR, PPAR, ER, etc.) from these organisms can be a tool to predict the selectivity and sensitivity to ECs and to assess their risk. Furthermore, in silico homology models of these receptor proteins and docking models with ECs often supported the in vitro results, suggesting the potential of in silico high-throughput ligand screening. Recently, we have worked to establish a method for risk assessment of ECs in an in vitro experimental system using fibroblasts isolated from environmental organisms and using neurons induced from fibroblasts. We also attempt to evaluate the effects of ECs on wild populations by analyzing their transcriptome and proteome.

　In this presentation, we will present these results and discuss the challenges and prospects for promoting One Health for ECs.

　The causative agent of Minamata disease was methylmercury in contaminated waste-water produced during acetaldehyde manufacturing process at Chisso Co. Ltd. Adult patients with Minamata disease displayed main neurological symptoms reflecting specific regions of the brain damaged by methylmercury. In addition, a high incidence of cerebral palsy was observed between 1955 and 1959, when the pollution was most severe; this was attributed to fetal-type Minamata disease. The babies were normal during pregnancy and delivery. However, the mothers noticed severe developmental retardation of the mental and motor functions in infants after early childhood. The photograph of a mother taking a bath with a fetal-type patient, taken by Eugene Smith, in the film "Minamata," aired last year, is symbolic in showing the characteristics of fetal-type Minamata disease. A mother said she might be healthy because the baby had absorbed toxins from her body.

　When I started working at the National Institute for Minamata Disease, the World Health Organization published the Environmental Health Criteria 101 Methylmercury (1990), in a report which summarized the health risks of methylmercury. They recommended studying the effects of methylmercury on fetuses. I then decided to understand the effects of methylmercury on fetuses to protect them from methylmercury toxicity.

　The topics of my presentation are as follows:

1.Background of Minamata disease

2.Methylmercury transfer from mothers to infants via the placenta and breast milk

3.Methylmercury pollution in the Minamata area revealed by the concentrations of methylmercury in preserved umbilical cords

4.Decreased sex ratio of boys in Minamata

5.Distribution of neuronal degeneration in the brain at various stages of development

6.Prevention of methylmercury toxicity with selenium

7.Risks and benefits of fish consumption in pregnant women

8.Increased selenium concentration in Minamata disease

The basic proposition of safety assessment in medicinal drug is "not to guarantee the safety of drugs, but to present the toxic effects, and to evaluate and manage risks in humans". In its implementation, it is necessary to understand that safety assessments (medicinal and toxic effects) are on the same platform of life-science based on multi-disciplinary sciences. In drug research and development, it is required to present an accurate information of the efficacy and toxicity, the mechanism of action, extrapolation to humans, risk evaluation (degree, tolerability, mitigation, avoidance), and risk management about appropriate application to patients.

In a practical manner, safety evaluation begins with understanding the pharmacological effects on disease-etiology, and follows by identifying the inducing toxicities. The basic concept of elucidating the toxicological action mechanism is to consider the two-sided thinking of MOA (mode of action) in efficacy and AOP (adverse outcome pathway) in toxicity. Presentation of toxicological action-mechanism leads to set the related biomarkers, and plays an important role in presenting the direction of risk management when applied to humans. In fact, it contributes for decision-making (Go or No-Go) at the timing of lead / candidate compound selection in the process of drug development. And also, it provides meaningful information for resolving the issue of clinical development.

In reality, it is undeniable that drug safety assessment would be developed based on regulatory scientific thinking from the aspect of approval application as a common practice. However, we must not forget that regulatory science is to be based on the foundation of life science, and that life science thinking is indispensable in future new medical field.

Anthropocene is proposed as a term for contemporary geologic time scale. It can be recognized as an era in which the earth has been shrunken relative to rapidly growing human activities. In this talk, I would like to discuss about new challenges for toxicology (in its broad sense) in Anthropocene, taking another relatively new concept of Planetary Health. Planetary Health refers to two health, i.e., health for earth and ecosystems on it, and health for human and its civilization, identifying and quantifying the way in which both health are dependent with each other and seeks sustainable relationship for two health.

The shrunken earth is important for toxicology in two respects. First, it allows chemicals released into environment may diffuse or be diluted but not instantly extinguish and stay for some time. Then, their environmental distribution/fate in various temporal/spatial scales in relation to the distribution of living organisms in ecosystems are crucial in determining the toxicological importance of chemicals. Second, not only human but also plants and animals are living on the shrunken earth, exerting their impact on human species through various type of ecosystem services. Of course, toxicological effects on these living organisms matter by hampering their health and lives. At the same time, the effects may impact on human health and society through the deterioration of the ecosystem services. Both of these issues would require toxicologist, while acknowledging experimental research, to utilize mathematical models to describe environmental chemistry and ecosystems and to do data collection in the real field monitoring. Thus, toxicology in the Anthropocene will be very busy but very attractive field as it has been.

The pandemic of the new coronavirus has transformed the world and has had a profound impact on science, medicine, government, and even diplomacy and economics. In particular, the year 2020 will go down in history as the year of two revolutions in vaccine development research. The first is the emergence of a new vaccine called mRNA, and the second is a revolution in the way vaccines are tested in clinical trials.

Never before have vaccines become a "personal matter" for people around the world as they have this time, and new trends are emerging in the fields of basic and clinical research involving not only infectious diseases and immunology, but also toxicology, which is expected to promote the fusion of different fields. On the other hand, looking around the world, there are many countries where people avoid vaccines or vaccinations have not progressed, and Japan is expected to contribute to global health coverage by providing safer and more effective vaccines to the world. In this lecture, we hope to discuss "100 Days Mission to Respond to Future Pandemic Threats" and new developments in post-corona vaccine development research.

We will all use pharmaceuticals at some stage in our life. During manufacturing and following their use, these substances can be released to the natural environment. As pharmaceuticals are biologically active substances, in recent years there has been increasing interest from scientists and the general public over the potential impacts of these molecules on aquatic and terrestrial organisms and on humans that consume contaminated drinking water and food items. To understand the impacts of pharmaceuticals in the environment it is essential to understand the concentrations of these molecules in the environment. Over the past two years, the University of York have been co-ordinating the Global Pharmaceutical Monitoring project which has quantified levels of pharmaceutical pollution at 1000 locations across 105 countries. Highest levels of pollution are seen in rivers in Africa and Asia receiving inputs from manufacturing plants, raw sewage disposal or trash dumping. Comparison of the data with available ecotoxicological effects data suggest that, at the most contaminated sites, the growth and reproduction of fish and invertebrate populations will be impaired and the levels of antimicrobial resistant bacteria will be enhanced. Solutions are therefore urgently needed to this problem in order to protect the health of ecosystems and human populations across the globe.

The liver is susceptible to foreign substances such as drugs and pathogens. Various in vitro hepatocyte models have been used to elucidate the pathophysiology of liver disease caused by exposure to foreign substances. However, the clinical predictability of drug evaluation tests using hepatocytes cultured in two-dimensional and static conditions is not enough high. Therefore, it is expected that the liver-on-a-chip developed using MPS technology that can reproduce three-dimensional and fluidic conditions will be used for pharmaceutical research. To establish a highly functional and practical liver-on-a-chip, technologies for manufacturing microfluidic devices and generating liver cells are indispensable. We mainly use the devices using polydimethylsiloxane (PDMS), but there is concern that PDMS is easy to absorb drugs. We found that the logD value of a drug can be used to predict the level of drug adsorption into the PDMS devices. Recently, we have developed a device using a low adsorption material, and have confirmed that it is possible to perform pharmaceutical research with almost no risk of drug adsorption by using this device. We have developed a liver-on-a-chip with vascular and bile duct structures by seeding liver cells such as hepatocytes, bile duct epithelial cells, and vascular endothelial cells in the device developed above. Using this liver-on-a-chip, not only drug toxicity and metabolism testing can be performed, but also the excretion of bile acid components and drugs into the bile duct channel can be evaluated. We are now conducting research on the pathophysiology of liver diseases caused by infection with pathogens such as SARS-CoV-2 using this liver-on-a-chip.

It is necessary to precisely assess intestinal and hepatic first-pass effects in pharmacokinetic prediction of oral drugs, because bioavailability is a very important factor to exhibit drug efficacy and toxicity. In the pharmacokinetic studies of liver, cryopreserved human primary hepatocytes are the most used. On the other hand, human colon adenocarcinoma cell line Caco-2 are frequently used for a prediction of drug absorption in the small intestine. However, in Caco-2 cells, the expression levels of transporters and drug metabolizing enzymes are different from human intestine. We have developed the differentiation methods of human iPS cells (hiPSC) into pharmacokinetically functional small intestinal epithelial cells using small molecular compounds. Microphysiological system (MPS) has been recently developed, and utilization of the chip in the drug development is attracted attention as advanced research field of human in vitro models. We also attempted to develop MPS that can predict the first-pass effects in vitro. The probe substrates for cytochrome P450 enzymes were added in the small intestinal compartment. We sequentially collected solution from the part of corresponding to portal vein blood and hepatic vein blood, and detected substrates and its metabolites. We are expecting that hiPSC-derived cells and MPS will be used in drug evaluation systems.

Successful implementation of microphysiological systems (MPS) into drug discovery requires close communication between model developers and end-users to connect emerging technologies and context of use to build better systems (1). We have evaluated not only development devices but also commercial devices in the AMED project. Each of these devices has its own characteristics, and it was considered desirable to make the best use of those characteristics. For that purpose, the user needs to understand each device and select the device that suits each purpose.

In this study, we collaboratively evaluated a new MPS as a tool for detecting hepatotoxicity after repeated exposure to chemicals. In addition, we explored the advantages of non-invasive monitoring, such as by optical coherence tomography (OCT), in the long-term culture in the MPS.

We studied a new MPS device, consisting of a circulating small intestine-liver two-organ connection device developed at the Matsunaga Laboratory, Graduate School of Pharmaceutical Sciences, Nagoya City University (2). Human primary hepatocytes were seeded onto micro-patterned culture surfaces coated with mouse 3T3 feeder cells to form liver spheroids. The liver spheroids were exposed to acetaminophen (APAP) for 7 days, and the cytotoxicity of APAP was evaluated by ATP assay. Albumin, and APAP and its metabolites in the culture media were measured. Cell morphology was recorded by a phase-contrast microscopy and OCT. OCT data underwent image analysis.

After APAP treatment, the appearance of the spheroids changed to a black-coloration. APAP concentration-dependent ATP reduction was observed, suggesting that this liver MPS is strongly able to detect hepatotoxicity after repeated exposure to potential toxicants. Image analysis of OCT data succeeded in counting spheroid numbers. In addition, APAP concentration-dependent decreases in volume, height, and surface area were observed. Albumin also showed an APAP-dependent reduction in culture media.

This liver MPS is potentially applicable to the assessment of hepatotoxicity of chemicals after repeated exposure. OCT is useful for non-invasive monitoring of cellular morphology by image analysis.

[Reference] (1) Biol. Pharm. Bull., 43 375-383(2020).

(2) http://www.scetra.or.jp/business/

[Funding] This research was supported in part by the AMED-MPS project

MPS can generally be defined as "a culture device that can mimic the blood flow in vitro and elucidate its mechanical effects on cells as well as its effects of mass transfer of nutrients, metabolites, or drugs in the culture devices." MPS, which can mimic the cellular environment of living organisms, is expected to be a culture technology that will solve the unmet needs of pharmaceutical companies for in vitro evaluation in new drug development. For the development of a new test system, acceptance of the defined test method in industries (industrial implementation) is considered as the first step toward regulatory acceptance. There is currently a great deal of discussion regarding the specific issues that must be addressed in order for MPS to be incorporated into the drug discovery process. In Europe and the U.S., forums have been created for stakeholders to gather, such as at the MPS World Summit. In this presentation, based on the discussions in the AMED-MPS project in which the speaker has participated until FY2021, the speaker presents the concept of "technical requirements for industrial implementation of MPS" based on unmet needs for drug discovery, and discusses issues of MPS development from the viewpoint of regulatory science.

Organ-on-a-Chip (OOC) technology is increasingly able to provide a comprehensive view of physiological phenomena in healthy and diseased states. In particular, this technology is being applied primarily for preclinical testing as an alternative to animal testing. Furthermore, in order to efficiently create drugs, it is important to comprehensively evaluate drug efficacy as well as toxicity and drug metabolism from the early stages of drug discovery. In the early stages of drug discovery, high-throughput screening (HTS) is necessary to evaluate a wide variety of compounds at once using a small amount of compounds. However, the complexity and precision of OOC makes automated culture difficult and has not yet been put to practical use. In addition, the complex three-dimensional structure of cultured tissues poses a challenge to the development of assay technologies that can obtain sufficient data to quantify and interpret physiological phenomena with high efficiency.

Here, we introduce a case study of screening related to kidney and blood vessels using MIMETAS Organ-on-a-chip, which is designed to be applied to automated culture and analysis devices, and an attempt to quantify the behavior of cells in the immune system, and discuss how OOC can be used for HTS in the future. Challenges and prospects for its use will be presented.

In target safety assessment that identifies toxicological concerns for a pharmacological target, information on the phenotype of animals with a modified gene encoding the target is very useful. However, in the case that on-target toxicity to an inhibitor/antagonist is predicted using information on knockout animals, it should be noted that there are two different loss-of-function modes. Generally, the phenotype observed in knockout animals is expressed upon the irreversible and constitutive deletion of the gene encoding the target from the embryonic development stage. On the other hand, the on-target-based phenotype by the inhibitor/antagonist is expressed by reversible and time/concentration-dependent binding between the target and the inhibitor/antagonist during a specific period after birth. It is suggested that this difference in the loss-of-function mode may lead to a difference in adaptive responses, which may ultimately lead to a difference in phenotype. Therefore, we do not judge the validity of a pharmacological target only by the phenotype observed in genetically modified animals, but always judge it based on the results of toxicity tests using an inhibitor/antagonist with appropriate pharmacological activity.

Genetically modified animals and gene-knockdown cultured cells are also useful in determining whether the observed toxicity is based on on-target or off-target effects. It is expected that sharing and discussing our application of gene-modified animals or cultured cells in our presentation would support efficient new drug development in the future.

RORγt antagonist can inhibit the differentiation and function of IL-17-producing cells such as Th17, Tc17 andγδT cells, and thus are expected to be a therapeutic agent for IL-17-mediated autoimmune disorders including psoriasis. However, RORγt is also essential in the regulation of thymopoiesis. RORγt controls the apoptosis of CD4+CD8+ double-positive thymocytes, and mice deficient in Rorc have been shown to develop thymic lymphoma at high incidence. In the present study, we examined whether RORγt antagonist could be a potential therapeutic agent for psoriasis without the risk of on-target side effects. First, we developed doxycycline-inducible Rorc knockdown mice and analyzed them after administration of doxycycline from 10 weeks of age. As a result, we observed thymic T cell abnormalities and lymphoma development similar to those in congenital knockout mice, indicating that RORγt inhibition in adulthood also have side effect concerns. Next, we investigated the effect of systemic administration of RORγt antagonist with good bioavailability. Oral administration of RORγt inhibitor to psoriasis model mice for two weeks inhibited the development of psoriatic skin inflammation, but at the same time, caused decreased expression of anti-apoptotic molecules and abnormal T cell differentiation in the thymus. In contrast, our novel RORγt antagonist, S18-000003, markedly inhibited psoriasis-like symptoms after 2 weeks of topical application, with almost no effect on thymic T cells. Our study showed that the development of topical RORγt antagonists with high retention in the skin and low blood transfer could lead to potent psoriasis treatments with low risk of side effects.

Genetically modified mice are essential experimental animals in medical and life science research, but their production required stable technology and a relatively long time. In particular, in order to produce knockout mice and knock-in mice that require mouse genome modification, homologous gene recombination using embryonic stem cells (ES cells) and chimeric mice using ES cells are required. It took about 1 to 2 years to produce the product. However, by establishing the CRISPR / Cas9 system reported in 2013, it became possible to generate genetically modified mice in fertilized eggs without using ES cells. At our center, we are engaged in the contract production of genetically modified mice using fertilized eggs, and we have already produced and supplied many genetically modified mice. In this talk, I would like to introduce the actual production of humanized mice using the CRISPR / Cas9 system and fertilized mouse eggs, examples of their applications, and points to note.

Genetically modified mice have been used as animal models to recapitulate the pathology of human diseases for long time and been used for testing the efficacy of drugs. However, there are several cases that mice are not suitable for the recapitulation of human diseases.

Nonhuman primates (NHPs) are considered one of the most valuable animal models, because NHPs are closer to humans in organ size and anatomical structure, and therefore have higher potential to recapitulate human diseases, while difficult genetic manipulation is a major issue for creation of the disease models. So far, we have established techniques to create transgenic and genome editing cynomolgus monkeys. By using these techniques, we have explored an intractable human disease, Autosomal dominant polycystic kidney disease (ADPKD) with CRISPR/Cas9 technique, and demonstrated that homozygous disruption of PKD1 allele, a causative gene for ADPKD can result in the massive renal cyst development. We also have developed a technique to modify specific allele and generated PKD1 heterozygotes selectively. Importantly, the PKD1 heterozygotes exhibited renal cysts during fetal stages, showing the recapitulation of some of features of human ADPKD pathology.

We had long done genotoxicity tests on biologics since the first biotherapeutic was approved by FDA in 1982. Ames test on biologics was waived by ICH S6 guideline in 2000. Despite the scientific understanding that Ames test or non-cross reactive rodent tests have no meaning, both industry and regulatory experts sticked to the traditional standard test battery for fear of taking a new step forward without accumulated evidences. The traditional test battery could not detect lethal toxicity, which resulted in the tragedy of TGN1412. We learned a lesson from the history of biologics that scientifically appropriate test battery is important to support human safety with a new modality. Recently, various new modality therapeutics have been emerged. An AAV vector gene therapeutic, BMN307, induced tumors in 6/7 mice. The traditional genotoxicity test battery could not predict cancer risk of this compound. There might be an excuse that insertion of a DNA fragment into random site has a limited impact whereas insertion into a specific site provides high risk. However, many of known genotoxic carcinogens caused mutations at random sites. Here, we discuss how we can make scientifically appropriate assessment for genotoxic potential of middle-sized peptides and gene/nucleotide therapeutics.

Contamination of valsartan drug substance with N-nitrosodimethylamine (NDMA), a well-known strong genotoxic carcinogen, was reported in 2018. Since then, regulatory authorities have issued notification to perform a risk assessment of contamination of nitrosamines as impurities in drug substance synthesis and drug product manufacturing processes for all marketed drugs. In Japan, a notification for risk assessment of nitrosamine impurity contamination was issued on October 8, 2021.

Once a risk of contamination is identified, an appropriate control strategy is required to reduce the amount of nitrosamines to a level at which there is negligible carcinogenic concern or to zero. To reduce the contamination level, the M7 concept requires control to less than or equal to 1 in 105 carcinogenic risk and the EMA has provided a default value of less than or equal to 18 ng/day for compounds without robust carcinogenicity data. This default value is set on the basis of carcinogenicity data such as NDEA, which is the most potent carcinogenic among all nitrosamines. However, there is a report suggesting that not all nitrosamines are strongly carcinogenic. While the ICHM7 Guideline provides an assessment of mutagenicity using QSAR, nitroso compounds have been handled as "impurities that should not be contaminated" in the Cohort of Concern. Therefore, they are a class of compounds for which QSAR has not been optimized and carcinogenic risk cannot be estimated appropriately. Therefore, the Agency and industry are discussing what information is needed and how to proceed with the evaluation of mutagenicity and carcinogenicity risks of this compound class.

In this presentation, the presenter introduces the background and the risk assessment method of nitrosamine impurities and the discussion points between the regulatory authorities and the industry.

Evaluation of genotoxicity is essential in the development of chemical substances such as pharmaceuticals. Currently available in vitro and in vivo genotoxicity studies use phenotypes associated with mutations in target genes, therefore there has long been concern about bias in the results of such studies. On the other hand, mutation analysis using next-generation sequencers is attracting attention as a non-biased method. In particular, a mutation signature, which is classified according to the surrounding sequences, and is known to reflect environmental exposure. In addition, a difference in mutation frequency between the transcribed and non-transcribed strands of the mutation signature is sometimes observed, which is called strand bias. This phenomenon is believed to be caused by a transcription-coupled repair mechanism, and DNA adducts are thought to contribute to the induction of mutations at these sites. In other words, these DNA adducts are considered to be important for mutagenesis, i.e. "driver adducts. Therefore, screening and identification of driver adducts associated with mutation signatures will also enable us to obtain the Adverse Outcome Pathway of chemical-induced genotoxicity, which is expected to more accurately assess genotoxicity. In this symposium, I will introduce studies using multidisciplinary approaches such as genomic analysis by next-generation sequencers and comprehensive analysis of DNA adducts, and discuss the prospects of applying these methods to genotoxicity assessment of chemical substances.

The nature of genome editing is the DNA double-strand break at the target site. Although the "off-target toxicity" due to sequence homology has been the focus of attention as the toxicity of genome editing, from the viewpoint of genotoxicity, the safety of DNA double-strand breaks themselves, i.e., "on-target toxicity" is important.

As in the case of radiation, DNA double-strand breaks are likely to cause chromosomal aberrations, and it was reported last year that chromosomal aberrations such as micronuclei induced by genome editing with CRISPR-Cas9 can cause Chromothripsis. This has attracted attention as an "on-target toxicity" of genome editing because it causes massive genomic instability by single event and can be a direct cause of cancer and genetic disease. We are attempting to artificially synthesize fusion genes (chromosomal translocations) between the genes that have been cut by genome editing at different locations. The resulted chromosomal aberrations have been examined by micronucleus induction and chromosome painting analysis.

To create translocation fusion genes between the ALK gene on chromosome 2 and the MET or SMO genes on chromosome 7 in HEK293T cells, we designed sgRNA expression vectors that cut specific sites in each gene and transfected them together with Cas9 expression vectors.

Chromosome painting analysis of ALK/MET and ALK/SMO fusions immediately after genome editing showed that the target chromosomal translocation was observed in a few percent of the cells, and more frequently, the chromosomal breaks and fragments were observed. On the other hand, we also examined micronucleus induction and observed a large number of micronuclei, which turned out to be an artifact derived from the vector plasmid we used. Therefore, we are now examining micronucleus induction using a method of transfection of Cas9 protein and sgRNA complexes without using a plasmid.

In the future, we plan to investigate the possibility that these abnormalities result into chromothripsis.

Inheritance of the genome stably and correctly is one of the most fundamental life activities of all living things. When DNA is damaged for some reason, cells arrest the cell cycle until it is repaired. If DNA damages are not repaired correctly, it will be fixed in the DNA as a mutation and the wrong genetic information will be passed on to the next generation. Besides the external factors such as ultraviolet rays, it is also known that DNA damages are provoked by some metabolites produced during intracellular energy metabolism that show genetic toxicity. For example, methylglyoxal (MG), which is generated from an intermediate of triosephosphate isomerase reaction, which is one of glycolytic enzymes, forms an adduct with a guanine residue in DNA.

We are studying the physiological function of MG using the budding yeast (Saccharomyces cerevisiae) as a model organism. It has been reported that high concentrations of MG gave lethal effect on all organisms including yeast. We found that sublethal concentrations of MG inhibited nuclear division and transformed the nuclear morphology from spheres to a flattened “jellybeans-like shape” in yeast. The nucleus of eukaryotes is usually spherical. Unlike higher eukaryotes, budding yeast performs closed mitosis in which the nuclear envelope does not disappear during nuclear division. Therefore, the nuclei of anaphase, where nuclear division occurs, are stretched along the growth axis between mother cells and daughter cells, but are basically spherical in other cell cycles. We found that treatment of yeast with MG induced the phosphorylation of Tyr19 of the cyclin-dependent kinase Cdc28 (G2/M arrest), and the nucleus resides in the mother cell near the bud neck (between mother and daughter cells). Furthermore, the nuclear morphology changed from a globate shape to one with a central depression aligned with the mother-bud axis, which we refer as a “jellybean-like shape” of the nucleus, following treatment with MG, and the nucleus remains in the mother cell even though the daughter cell is large enough to accept the nucleus. In this symposium, I would like to introduce the machinery involved in the MG-induced inhibition of nuclear division and predicted mechanism underlying.

The PMDA Regulatory Science Center was established in Apr 2018 as one of the key components of the government’s Healthcare Policy (partially amended on 09 Apr 2021). The Regulatory Science Center focuses on the collection and analysis of electronic data not only from clinical trials, but also for nonclinical studies (https://www.pmda.go.jp/review-services/drug-reviews/about-reviews/p-drugs/0003.html). As of February 2022, submission of nonclinical data in Japan is not required to be in SEND format, a non-clinical CDISC standard. However, PMDA may consider utilizing both clinical and non-clinical CDISC standard data in an integrated manner in the future since it may help to improve the quality of dossier review, implementation of safety measures, and creation of new guidelines.

At this symposium, we plan to present how clinical, as well as nonclinical CDISC standard datacan be utilized to efficiently develop safer new drugs. This presentation will focus on nonclinical aspects, and will cover the utilization of SEND from the following perspectives: regulatory review at the time of clinical introduction, approval for manufacturing and marketing, integrated analysis with clinical data, contribution to 3Rs by optimization of non-clinical studies (i.e., reduction of control group, etc.), utilization of accumulated data, and impact on the future of nonclinical trial guidelines.

[in Japanese]

In Japan, the submission of electronic clinical study data with new drug application began on October 1, 2016. As the transitional period was ended on March 31, 2021, currently subject-level data of clinical trials and analyses that are required by Pharmaceuticals and Medical Devices Agency (PMDA) should be submitted with the new drug application. Those data are used for the new drug review in the PMDA.

In drug development, various proposals and actual applications have been made to actively utilize available study data and related external information obtained at various development stages, leading to more efficient and faster new drug development. In considering such active utilization of data, standardized clinical trial data is considered to be particularly important. The PMDA also considers obtaining useful information in accordance with the accumulation of electronic study data that are submitted to the PMDA and their use in the new drug review process.

In this presentation, we will discuss the preparation and consideration of electronic data submission until the initiation of the data submission, the current status of data submission and utilization of the data, and future utilization of the data for our review of new drug applications and consultation for new drug development.

Data generated in accordance with the non-clinical CDISC standard format (SEND data) are able to be accumulated and utilized for historical data or statistical processing, and so on. In order to consolidate SEND data, it is necessary to create a database (DB) to store such data first. To design the DB, the proper understanding of the structure of such data (domain/variable) is prerequisite. However, the design of the domain set that corresponds to each DB and the keys between the domains that make up this set should be investigated in parallel.

One option to solve this dilemma is to simplify preparing the data storage, query, and interface construction using a relational DB (RDB) application. As a part of the AMED research group activities, we have started an investigation of the key of domains using SEND data (XPT file set) provided by sponsors on a voluntary basis as a trial.

We have prepared a simplified DB to accumulate such SEND data. This DB is designed without specific functions, which enables us to modify it in the future. During the provisional implementation stage, we recognized the need to 1) set up the structure necessary to accumulate data continuously, and 2) solve the inconsistency of substance names and other entries among different XPT file sets. We are in discussion on the future development plans of the DB using this provisional DB. In this presentation, we will present the recent progresses and future plans on the development of the database.

Since the available scientific evidence on the causal relationship between chloromethylisothiazolinone and methylisothiazolinone(CMIT/MIT) exposure and lung injury is limited, the victims exposed to CMIT/MIT exposure remains unresolved.

Two studies have performed and compared responses of the mouse lung to inhaled and intratracheally instilled the CMIT/MIT.

Intratracheal instillation of CMIT/MIT induced increase in the number of eosinophils and neutrophils, and concentrations of T helper 2(Th2) cytokine in BALF. Moreover, CMIT/MIT induced increase the epithelial cytokine expression like IL-25, IL-33 and TSLP in lung tissue. Histopathological analysis revealed increased eosinophilic inflammation, mucous cell hyperplasia, and fibrosis following CMIT/MIT instillation.

Inhalation of CMIT/MIT induced an increase in the number of inflammatory cells and the concentrations of Th2 cytokines in BALF. Epithelial cytokine expression were also increased. However, inhalation of CMIT/MIT did not induce significant histopathological injuries. We suggests that the initial stage of Th2-mediated lung injury was induced in current exposure condition.

Although our data are insufficient to reflect the entire responses induced by CMIT/MIT, this is the first study to demonstrate the association of inhalation exposure of CMIT/MIT with Th2-mediated lung inflammation in an animal model.

Further studies on chronic inhalation exposure are warranted to evaluate the CMIT/MIT–altered histological lung injuries.

This work was supported by grant from the National Institute of Environment Research (NIER-2021-04-03-001).

Polyhexamethylene guanidine phosphate (PHMG-p) was used as a disinfectant for the prevention of microorganism growth in humidifiers, without recognizing that a change of exposure route might cause significant health effects. Epidemiological and toxicological studies indicated that PHMG-p is strongly associated with interstitial lung diseases. Herein, we have outlined the cellular and molecular mechanisms underlying pulmonary fibrosis induced by PHMG-p. The leakage of zwitterionic liposomes, induced by PHMG-p, was more extensive than that of negative liposomes, indicating that PHMG-p adsorption onto lipid head groups via electrostatic interaction cannot fully explain the induced lipid membrane damage. Using PHMG-p-FITC conjugate, we identified that PHMG-p is rapidly located in the endoplasmic reticulum (ER) and causes ER-stress-mediated apoptosis. PHMG-p triggered G1/S arrest and apoptosis through p53 pathway in lung epithelial cells. We assumed that G1/S arrest induced by PHMG-p may precondition cells undergoing EMT, contributing to the expansion of fibroblasts. Epithelial cells at other phases may result in apoptosis. PHMG-p activated the Akt/β-catenin and Notch signaling pathways, resulting in increased ZEB2 expression. The interplay between these pathways induced EMT, resulting in PHMG-p-induced lung fibrogenesis.

Hazard assessment for manufactured nanomaterials: Along with the development of nanotechnology, variety of nanomaterials have been designed and manufactured. However, the hazard of nanomaterials has not been studied fully. Inhalation study is considered a golden standard for hazard assessment of nanomaterials, but alternative method of intratracheal instillation is usually used in academia because of the limitation of facilities.

Safe by design: To develop safer nanomaterials, knowledge how the design of nanomaterials can reduce the hazard of nanomaterials is needed. We compared bare silica nanoparticles and amino or carboxyl-functionalized silica nanoparticles for pulmonary toxicity in mice. The study shows amino- or carboxyl-functionalization of silica nanoparticles reduces the inflammation in lung and toxicity to macrophage, thus providing useful information for development of safer nanomaterials.

Role of Nrf2 in pulmonary effect of manufactured nanomaterials: Nrf2 is known to be a master regulator for anti-oxidative-stress genes. However, recent studies showed Nrf2 negatively regulate expression of proinflammatory cytokines. We compared pulmonary effect of exposure to zinc oxide nanoparticles between Nrf2 null mice and wild type mice. The result shows that deletion of Nrf2 enhances inflammatory response for exposure to zinc oxide nanoparticles in the lung of mice, suggesting inhibitory effect of Nrf2 on zinc oxide-induced pulmonary inflammation.

Systemic effect of particles: Recent epidemiological studies show association between air pollution and cognitive dysfunction. One of hazardous compositions of particulate matter is polycyclyic aromatic hydrocarbons such as benzopyrene. An epidemiological study also suggests neurobehavioral effect of benzopyrene on workers. We are studying neurobehavioral and neurotoxicological effect of benzopyrene in mice by pharyngeal aspiration method, and the result shows harmful effect of benzopyrene on the density of noradrenergic axons and behavior.

Conclusions: Information on effect of particles, not only nanoparticles but also conventional or unintentionally produced particles in humans suggests us the priority of research on manufacture nanomaterials. Not only pulmonary toxicity but also systemic effect of nanomaterials or particles should be studied further.

Both of exposure to crystalline silica and asbestos cause pneumoconiosis as well as lung cancer. However, the former is associated with autoimmune diseases like scleroderma, whereas the latter causes malignant mesothelioma. Inhaled those reach lung tissue, causing inflammation mediated by alveolar macrophages, but are also accumulated in lymph nodes, thought to contribute to a reason for related diseases. Therefore, our studies noted immunological effects of silica and asbestos and have conducted basic examinations with cell cultures and immunological analyses for peripheral blood specimens from patients. It has been clarified that both of lymphocytes exposed to silica and derived from silicosis showed activation and decreased suppressive function, whereas decreased anti-tumor immunity and increased suppressive function were seen in asbestos-exposed lymphocytes as well as those from mesothelioma. Silica exposure caused CD69⁺ activated cells and decreased Foxp3⁺ regulatory T (Treg) cells, and silicosis showed increased soluble IL-2R as activation marker and decreased Treg function in blood. In contrast, asbestos exposure caused decreased activating receptor on NK cells, Th1 function of CD4⁺ T cells and cytotoxicity of CD8⁺ T cells as well as increased Treg function, and mesothelioma also showed altered expression of marker molecules indicating decreased anti-tumor immunity. Those findings indicate that exposure to silica and asbestos not only causes inflammatory response in the bronchial alveoli, but also raise immune-functional effects of activation or suppression, leading to related autoimmune or malignant diseases respectively.

The Fish Short Term Reproduction Assay (TG229) evaluates endocrine disruption inducing reproductive dysfunction in fish (medaka, fathead minnow and zebrafish). TG229 evaluates effects on spawning, vitellogenin (vtg), gonadal histopathology and secondary sex characteristics (SSC). Four actions are detectable based on their effects: estrogenic, anti-estrogenic, androgenic, and steroidogenesis inhibition, but TG229 is not supposed to reveal specific actions. In the four actions, Adverse Outcome Pathways (AOP) has already been established, with reduced spawning and changes in vtg being key events. Therefore, the four actions can be discriminated to some extent due to the results of TG229. Estrogenic action can be identified by increased vtg in males. Anti-estrogenic action and steroidogenesis inhibition can be inferred from decreased vtg and spawning, but these are indistinguishable. Androgenic action can be identified by the appearance of male-like SSC in females. SSC are gross morphological changes that are indicator of masculinization: the formation of papillary processes on the anal fin in medaka and nuptial tubercles on head in fathead minnow. The appearance of male-like SSC is regulated by androgens and has been reported to be a more sensitive indicator of androgen substance than changes in vtg and spawning. On the other hands, zebrafish does not possess quantifiable SSC. This presentation will describe the characteristics of the indicators that should be evaluated for TG229 in terms of AOP and sensitivity.

For pesticide registration, effect assessment on environmental organisms is also required, in addition to human safety assessment. Recently, there has been increasing interest in endocrine disrupting (ED) effects of chemicals. Whether or not a chemical substance meets the ED criteria is evaluated in each humans and non-target organisms (environmental organisms). For ED evaluation, various in vitro/in vivo test methods focusing on EATS modalities (estrogen, androgen, thyroid, steroidogenesis) have been developed and adopted as OECD test guidelines as test methods for chemical hazard assessment. OECD test guideline 231, Amphibian Metamorphosis Assay (AMA) is one of the ED screening assays for environmental organisms. This assay is intended to identify test substances which may interfere with the normal function of the hypothalamic-pituitary-thyroid (HPT) axis. The HPT axis is strongly associated with amphibian metamorphosis, which enable to detect the effect of a test substance on the thyroid depending on whether the development of tadpoles exposed to the test substance is delayed (advanced) compared to normal. The test species is African clawed frog (Xenopus laevis) which can be easily bred in the laboratory and purchased as an experimental organism. In addition, this species can be easily propagated by hormone injections, making it possible to obtain large numbers of tadpoles for use in this assay. In this presentation, we introduce an amphibian metamorphosis test that applies the tadpole metamorphosis of African clawed frog.

Environmental toxicity tests for the registration of plant protection products are required when plant protection products are likely to affect bees. From the point of view of biodiversity, in Europe, future tests of the environmental toxicity of pesticides are being considered not only for honey bees, but also for bumblebees and solitary bees. In 2013, a Guidance Document on the risk assessment of plant protection products on bees (Apis mellifera, Bombus spp. and solitary bees) was issued by the European Food Safety Authority.

Honey bee tests are well established by OECD guidelines. The overview of honey bee tests could be used the potential for future testing of bumblebees and solitary bees.

In the case of honey bee tests, the first step is to conduct an acute contact and oral test (OECD 213 and 214), a 10-day oral chronic toxicity test (OECD 245) and (a single exposure larval test up to 7 days of age (OECD 237)) and a 22-day larval test (OECD 239), depending on the developmental process of the honey bee.

For plant protection products that affect honey bees in these laboratory tests, semi-field tests (EPPO 170 and OECD 75 using tunnels, and the Oomen de Ruijter test) are required.

If there is an effect in the semi-field test, the field test will be performed.

At present, the OECD guidelines for bumblebees exist only for acute contact and oral tests (246 and 247), while the 10-day oral chronic toxicity test and the tunnel test are under consideration by a working group of the International Commission for Plant-Pollinator Relationships.

For solitary bees, acute contact and oral tests are being developed by the ICPPR working group and preparations are underway for the development of OECD guidelines.

The extent of possible effects of chemicals on mollusks came into public regard with the dramatic effects of tributyltin (TBT) compounds, which have broadly been used as antifouling agents for ships. The females of the Dog Whelk (Nucella lapillus) and of at least 160 further species exposed to TBT developed male parts in addition to the female genital organs, a syndrome named ‘imposex’. With a number of approximately.

Therefore, a review paper of the OECD (2010) recommended test procedures for optimization and possible validation for partial life cycle (PLC) test P. antipodarum (freshwater, gastropod), full life cycle (FLC) with L. stagnalis (freshwater, gastropod), and Crassostrea gigas (bivalve, marine). It was also recommended to develop protocols for FLC with P. antipodarum and PLC with L. stagnalis. The OECD (2016 a, b) published a guideline for a reproduction test with L. stagnalis and P. antipodarum. Histopathology was detailed in the guidelines but stated as ‘…other endpoint (e.g., histopathology)…’ (OECD, 2016b) and was mentioned under point 3.3. and in the Annex for possible histopathology evaluation (OECD, 2016a).

The current guidelines target mainly endocrine disruptor effects. However, inflammatory, and degenerative processes or parasitic infestations can mask or mimic endocrine effects. Therefore, a histopathological examination should be performed. The cost and labor are not high.

References

OECD 242. 29 Juy 2016a.

OECD 243. 29 Juy 2016b.

OECD. ENV/JM/MONO(2010)9

Discussions on the revision of ICH S1 are being finalized after the prospective evaluation, drafting of the S1B(R1) addendum and correction based on public comments.

In the prospective evaluation, the carcinogenicity assessment documents (CAD) indicating the predicted results of a 2-year rat carcinogenicity study based on "Weight of Evidence (WoE)" for small molecule pharmaceuticals under development were encouraged to submit, and compared with the views of the five regulatory agencies, along with the actual study results.

With the analysis of collected 45 CADs, more important WoEs for prediction of 2-year rat carcinogenicity outcome were extracted.

This addendum proposes possibility of the waiver of 2-year rat carcinogenicity study, when concern of carcinogenicity can be evaluated comprehensively from WoEs of the drug, the data in the same drug class and literature.

However, since the prospective evaluation is based on the results of rat studies, a carcinogenicity study in mice, either 2-year or a short-term transgenic model as specified in ICH S1B, remains a recommended component of a carcinogenicity assessment plan, even for those compounds where the integrated WoE assessment indicates a 2-year rat study would not contribute significant value.

With the revision, short-term studies using mouse transgenic models, especially rasH2-Tg mice for which background information is being accumulated, might be considered as an option.

At this symposium, for appropriate application, it is expected that information on the characteristics of the tests using rasH2-Tg mice will be shared.

The rasH2-Tg mouse was developed for use as a model for carcinogenicity studies, and its phenotype is stable during high-volume production and over successive generations. To produce rasH2-Tg mice, three strains of mice (C57BL/6J-TgrasH2, C57BL/6J, and BALB/cByJ) are maintained individually. Since the homozygous c-HRAS genotype is lethal, hemizygous transgenic mice were maintained by crossing with inbred C57BL/6J mice. After breeding, male B6-transgenic mice were mated with female BALB/cByJ mice to obtain transgenic mice. Pups that were rasH2-Tg (tg/wt) or rasH2-Wt (wt/wt) were selected by genotyping. Frozen embryos were preserved by the Central Institute for Experimental Animals (CIEA) and sent to two facilities, CLEA Japan and Taconic Biosciences, where the mice are produced. Production colonies are formed in both facilities and are supplied to customers worldwide. To prevent genetic drift, the colonies were renewed for up to 10 generation. To ensure the uniformity and maintenance of the phenotype of rasH2-Tg mice, the carcinogen susceptibilities were monitored every renewal of colony by CIEA based on a standard protocol of the short-term carcinogenicity study using the positive control compound N-methyl-N-nitrosourea (MNU). Furthermore, simple carcinogenicity monitoring targeted at the forestomach, the organ most sensitive to MNU, is performed approximately once a year. Based on the optimally designed production and monitoring systems, the quality of rasH2-Tg mice with reproducibility and stability of carcinogenicity is maintained and is supplied globally.

Carcinogenicity studies using rasH2-Tg mice may be hesitant to conduct due to lack of knowledge for background data and testing experience. In this presentation, I would like to present the pathological background and characteristic lesions to increase understanding of rasH2-Tg mice and to promote their use.

The standard 26-week carcinogenicity study using rasH2-Tg mice consists of 25 males and 25 females per group, 3 dose groups, a negative control group (NC), and a positive control group (PC) that receives a known carcinogen such as MNU or urethane to validate the test system. In our background data, the survival rate at week 26 was 98%/94% (male/female) for NC and 6%/22% for PC (MNU), and there was no significant difference in the transition of body weight from 1 to 26 weeks between NC and PC.

At necropsy, in the PC, the forestomach mass in almost all, and the skin mass, thoracic fluid, and enlargement in the thymus, lymph nodes and spleen in more than half were observed.

Histopathologically, in the NC, hemangioma or hemangiosarcoma of the spleen, adenoma or adenocarcinoma of the lungs, and squamous cell papilloma (SCP), keratoacanthoma (KA) or squamous cell carcinoma (SCC) of the skin were observed in a few. In the PC, the forestomach tumor (SCP or SCC) in almost all, malignant lymphoma of the lymphohematopoietic system in more than 3 quarters, and the skin tumor (SCP, KA or SCC) in about half were observed. SCP of the vagina and adenoma of the uterus were observed in a few cases. In my presentation, I will report these background data in detail.

The high-dose selection with 25x human exposure in 2-year rodent carcinogenicity studies specified in ICH S1C(R2) is not universally accepted for rasH2-Tg mouse carcinogenicity studies (rasH2-Tg mouse studies). Therefore, to evaluate an appropriate multiple for exposure based high-dose selection in rasH2-Tg mouse studies, we reviewed application materials and published articles of 53 drugs that have been tested in rasH2-Tg mice and 2-year rodent studies (13 drugs positive in rasH2-Tg mice; 22 drugs negative in rasH2-Tg mice and positive in 2-year rodent studies; 18 drugs negative in both models). Then, we investigated AUC margin (AM) or body surface area-adjusted dose ratios (DR) of tumorigenesis in the carcinogenicity studies to maximum recommended clinical doses.

The results showed that tumorigenic doses in rasH2-Tg mice were 0.05-5.2x DR for 6 genotoxic drugs and 0.2-47x AM/DR for 7 non-genotoxic drugs. In terms of histopathologic risk factors (HPRF, including hypertrophy, hyperplasia, altered cellular foci and preneoplastic lesions), 26 drugs yielded HPRF within 50x AM/DR. HPRF dose was 211x AM in one drug, but only a single dose level was tested. HPRF in 2 drugs that was induced only at greater than 350x AM had no relationship with tumors in the relevant rasH2-Tg mouse and 2-year rat studies. No HPRF was induced in 19 drugs. HPRF induction was unknown in 5 drugs. In conclusion, doses greater than 50x AM are not of value in rasH2-Tg mouse studies.

This presentation also includes the 2017 survey on rasH2-Tg mouse study implementation practices among JPMA member companies.

Recently, the approaches focusing individual susceptibility to drug have been taken toward drug discovery/development. The backgrounds of patients are diverse, and in fact, there are some cases in which idiosyncratic toxicity is suspected as the reason of drug development failure. Given the acceleration of aging in Japan and the diversity of patients' drugs, we could need to update drug safety strategy. Nevertheless, the conventional drug safety assessment is conducted using models such as homogenous cell lines and normal animals, and it is not enough to understand or predict the individual susceptibility to drugs in patients. As an approach for predicting individual susceptibility to drug in patients, (1) deeply understand the background of the patients, (2) create a hypothesis based on that background, and (3) extrapolate the hypothesis experimentally are applied. To promote these stepwise approaches, first, it is necessary to understand the type of disease, medical history or co-/multi-morbidity, and trend of polypharmacy in patients. Then, it is considering susceptibility to drugs in a specific patient, so that preparing an appropriate in vitro model. In particular, regarding in vitro model, recent advanced models such as iPS cell-based platforms have been versatile for some assays and are highly expected to be leveraged for predicting individual susceptibility to drugs in patients. In the presentation, I would like to discuss a concept to assess individual susceptibility to drug, including examples, and expand the future potential.

Although genome-wide association studies (GWAS) have revealed numerous genetic variants associated with complex diseases, discovering therapeutic targets and biomarkers need translation of GWAS findings into biological knowledge. We have recently proposed a novel research strategy, "Polygenicity in a dish," in which polygenic risk scores (PRS) constructed from GWAS results are analyzed in detail in human cells and organoid models. Our drug-induced liver injury (DILI) study showed that this strategy worked well. Based on the hypothesis that the DILI risk can be explained by the sum of very tiny effects of many SNPs, we constructed PRS models by collaboration with the international consortiums for DILI (iDILIC and DILIN). Clinical trial data and in vitro survival assays with primary human hepatocytes and iPS cell-derived liver organoids showed that the genetic DILI risk was associated with the DILI vulnerability of many DILI-inducible drugs. Interestingly, in vitro transcriptome analysis and survival assays demonstrated that higher genetic DILI risk was related to biological pathways such as oxidative stress responses, indicating the validity of our "Polygenicity in a dish" strategy. Besides, we have also proposed an AI model, MENTR, to predict non-coding RNA expressions (especially enhancer RNAs from an activated enhancer) from only DNA sequence patterns, inspired by the conventional sequence motif analysis. MENTR has succeeded in interpreting rare variants associated with complex diseases, such as asthma and atopic dermatitis, by linking cell-type-specific enhancer RNA expressions. In this talk, we will introduce the latest trends in utilizing statistical analysis and machine learning techniques.

While a fellow at the Pediatric Critical Care Medicine Department at the University of Chicago, I also had a fellowship in Clinical Pharmacology and Pharmacogenomics (PGx) in order to better understand individual differences in drug reactions. There, I participated in the 1200 Patients Project which clinically applies and studies PGx research. At the time, the University of Chicago was one of the few facilities implementing PGx, but since then the practice has steadily spread around the medical world. Currently, 53 hospitals are registered users of the Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines (US, 40; Canada, 2; Europe, 8; Australia, 1; Egypt, 1; China, 1). Undoubtedly, the practice will eventually come to Japan, but at the moment there are questions regarding how ready we are. We believe that there are many issues regarding clinical application in Japan, including the fact that within the 26 CPIC guidelines, some cannot be used by Japanese people due to the low frequency of alleles, and others have limited evidence in children. Currently, our laboratory is conducting joint research with children’s hospitals and university pediatric departments in order to realize clinical implementation of PGx in pediatric medical settings in Japan in the near future. In this symposium, I would like to introduce my own research, the latest findings of pharmacogenomics in children, and global trends around this breaking edge medicine.

Over the recent years, to select the right medicine for right patients based on the appropriate prediction of efficacy and side effect, namely “personalized medicine”, has been becoming popular. Many clinical studies have reported that gene polymorphism is one of the key factors which influence on patient’s efficacy and toxicity as disease states and lifestyles do. For nonclinical toxicity assessments in pharmaceuticals, cynomolgus monkeys, a primate genetically closed to humans, are widely used. However, inter-individual differences of toxicity are observed in monkeys more often than rodents such as mice and rats. Gene polymorphisms could be one of the reasons, but due to limitations in the number of animals, it is often difficult to analyze the relationships between toxicological sensitivity and mutations.

As macaque monkey genome have been analyzed, genetic diversity has been gradually clarified. Although extrapolation to humans is still largely unknown, the influence of each genetic variation on functions has also been reported. Given these reported data, we have been focusing on mutations of the Fcγ receptors (FcγR), which is well-known as one of the factors causing sensitivity differences to therapeutic antibodies such as trastuzumab in clinical. In our research, through genetic analysis of the FcγR in Cambodian cynomolgus monkeys, we have detected the similarity of gene polymorphism between humans and other macaques. In this session, we would like to introduce the research data and discuss individual differences of toxicological sensitivity in nonclinical studies.

TGN1412 caused the tragic cytokine release syndrome (CRS) in the clinical study. MABEL approach was recommended after TGN1412, which has successfully prevented from serious accidents with CRS in any clinical studies with antibody drugs. However, CRS has been existing as a big problem in development of antibody therapeutics. The compounds with potential risk of CRS are mostly anti-tumor drugs. MABEL approach requires very long time to reach the effective dose due to a very low starting dose where no anti-tumor effect is expected, and very small step dose escalation. Why do we need the very conservative approach instead of a much quick access to an effective dose that is strongly desired by patients? In vitro human cell assay can reveal potential risk of CRS but any non-clinical studies cannot predict severity or dose-response in human. The other reason is that widely used pre-medications and palliative treatment with steroids are often not so effective as to provide a sense of safety. In this symposium, we discuss currently ongoing efforts to improve the situation at the frontline of clinical and non-clinical development of antibody therapeutics with potential risk of CRS.

Cytokine release syndrome (CRS) is a relatively common side effect of CAR-T cell infusion therapy, presenting as mild to moderate symptoms (fever, etc.) in many patients receiving CAR-T therapy. However, in some patients, severe hypotension and dyspnea may be induced, and symptoms may develop rapidly and become severe without appropriate intervention.

On the other hand, in addition to CAR-T therapy, there have been recent developments of bispecific antibodies (BiTE, etc.) that recognize CD3 on T cells and other specific proteins (antigens) expressed on tumor cells, and CRS has been reported to occur even in such novel immune antibody therapies. Although approved for hematologic tumors at this time, there are many types and varieties, and development in solid tumors is currently progressing rapidly, making this the fastest growing area of anticancer drug development in the past few years.

Initial treatment of CRS involves symptomatic treatment of individual signs and symptoms or suppression of the excessive inflammatory response by administration of corticosteroids and other drugs. However, in more severe cases, treatment with anti-cytokine therapy is commonly used to control excessive increases in blood cytokine levels. With the increase in the diversity of drugs and the broadening of the scope of drug development, the response to CRS is becoming more important in medical practice than ever before. In solid tumors, while referring to the response to CRS in drugs for hematologic tumors as in the past, there is a need for a more individualized response to symptoms and development, taking into account inter-individual and inter-drug differences, and a practical system needs to be established based on each and every experience. In this symposium, we would like to discuss how to provide safe and effective treatment to patients, taking into account the actual situation in the medical field in Japan.

Cytokine release syndrome (CRS) is a typical adverse effect caused by an infusion of therapeutic antibodies. CRS may lead to interruption or delay in the development of new therapeutic antibodies and therefore preventing or reducing the CRS risk is an important issue. It is difficult to predict the CRS risk using animals since the expression of target molecules and FcγRs in animals are different from those in humans. Therefore, cytokine release assay using human peripheral blood mononuclear cells or whole blood is widely used for risk assessment.

In this presentation, we will introduce a case of therapeutic antibody where elevated cytokines were observed in the clinical trial, and present the nonclinical risk evaluation and investigation of underlying mechanisms conducted using human peripheral blood mononuclear cells and whole blood.

Non-clinical safety studies play a crucial role for the evaluation of safety risks in humans in the drug development. However, current non-clinical studies cannot predict safety risks sufficiently, which cause a gap known as "Death Valley" between non-clinical and clinical.

Cytokine release syndrome (CRS) is a variety of symptoms caused by cytokine release, including fever, tachypnea, headache, tachycardia, hypotension, skin rash, and hypoxia. CRS is often observed after the administration of biotechnology-derived pharmaceuticals such as antibody drugs, and more recently, after chimeric antigen receptor T cell (CAR-T) therapy and as COVID-19 infection-related symptoms. It is an important issue to avoid and prevent the risks of CRS in the drug development because severe CRS can lead a lethal condition as cytokine storm. However, it is not easy to estimate the risk of the occurrence of CRS in humans in a routine non-clinical studies because of species differences in immune system itself and binding affinity against target antigen, pharmacological activity, and sensitivity to downstream signals caused by slight differences in amino acid sequence in the target molecule. As a result, Death Valley between non-clinical and clinical has not been filled yet, but has been even widening with a muddy stream of cytokines.

Here, we present a case study of our antibody drug that showed CRS in a clinical trial along with the result of non-clinical evaluation. We would like to discuss the limitations of current prediction methods and challenges to overcome the gap based on our case study.

CD3 bispecific antibodies (CD3BiAbs) physically link CD3 on T cells to target tumor antigens, which directs T cells to kill tumors. CD3BiAbs have shown high therapeutic potency against hematologic malignancies and are also being developed to target solid tumors. In clinical settings, CD3BiAbs causes cytokine release syndrome (CRS), a difficult-to-manage side effect associated with elevated levels of circulating cytokines, which has proven be a dose-limiting toxicity (DLT) and can be life-threatening. Premedication with corticosteroid, anti-IL-6 therapy, and intra-patient dose escalation have been reported to mitigate CRS, but more is needed to successfully control CRS. In an in vivo cynomolgus monkey study, a daily ascending dosing of a surrogate CD3BiAb mitigated CRS and the tolerance to CRS remained in effect for the following weekly doses. Antibodies having ADCC also cause CRS, and CRS typically occurs with the first dose and rarely with following doses. However, the mechanisms underlying the mitigation of CRS are not fully understood. We established an in vitro model with human lymphocytes showing that the cytokine release potential was decreased by the priming treatment. Cytokine mRNA levels after the repeated treatment were lower than those after the first treatment, but CD3 signal transduction was similar. ATAC-seq analysis showed that the priming treatment altered T-cell chromatin states, suggesting that epigenetic changes played an important role in the mitigation of cytokine release. In this presentation, we will introduce these studies on the mitigation of CRS by CD3BiAbs.

Inhalation exposure to nanomaterials (NMs) is the most likely route to be unintentionally exposed in all life cycle of products from the manufacturing to being wasted, and greatest concern in harmfulness. With the progress of product development applying NMs, it is desired to develop an evaluation method that can rapidly and easily obtain fundamental and quantitative information necessary for preventing health damage. However, inhalation exposure study of particulate matter including NMs requires specialized equipment such as aerosol generators, particle concentration measuring equipment, and requires a great deal of time and cost. Furthermore, in the chronic inhalation exposure study of particulate matter, there has been controversy regarding, as it is called, overload; the accumulation of particles in the lungs throughout the exposure period. We have been developing an intermittent exposure method as an alternative to a chronic inhalation exposure study that is more efficient than the usual two-year continuous inhalation exposure study. We first initiated a 4-week intermittent exposure inhalation study, mimicking the lung burden of the rat study reported by Kasai et al., 2016. Male C57BL/6 mice were exposed to 53 μm mesh-filtered Mitsui MWNT-7 aerosol by Taquann system (J. Toxicol. Sci. 2013) at the mass concentrations of ca. 2.6 and 5.0 mg/m³, for 6 hours per day every 4 weeks. MMAD was ca. 500 nm. Lung burden at 6 months were ca. 6.4 and 15.2μg per animal, and at 12 months 22.3 and 45.8μg per animal, respectively. Histologically, MWNT-7-laden macrophages were found at the terminal bronchioles to alveolar region. Microgranulomas were often observed. MWNT-7 were also found in pleural cavity, local lymphnodes and, distally, in renal glomeruli. Further details will be presented. (Health and Labour Sciences Research Grant, Japan)

Although chronic toxicity of multiwalled carbon nanotube (MWCNT) has been a critical issue in the field of nanotoxicology for over a decade, there has been only one 2-year inhalation study assessing pulmonary carcinogenicity of MWNT-7 in rats by Kasai et al.. In another 2-year study, rats administered MWNT-7 by repeated intratracheal instillation at the beginning of the experimental period developed pleural mesotheliomas but not lung tumors. This differences in the 2 studies could be attributed to the difference in the temporal pattern of MWNT-7 lung burden, i.e., continuous exposure by inhalation results in increasing lung burden throughout the experimental period, while the protocol of the intratracheal instillation results in maximal the lung burden at the beginning of the study period.

We recently implemented 2 intermittent exposure studies in which we administered MWNT-7 by intratracheal instillation with 4-weeks intervals for more than 1 year, allowing lung burdens of MWNT-7 to gradually increase, similar to the inhalation study. A 2-year treatment of MWNT-7 resulted in the significant increases of the incidences of lung tumors and pleural mesotheliomas. In another experiment, a shortened MWNT-7 (pretreated by a grinder) was administered for 1 year with a subsequent 1-year non-treatment period. No lung tumors were observed; however, pleural mesotheliomas were sporadically found through the exposed groups. These suggest that the temporal pattern of the lung burden, the structure of MWCNT, and the exposure methods can affect the outcomes of carcinogenicity tests.

The development and international standardization of a test method that can efficiently and accurately evaluate the toxicity of various nanomaterials (NMs) is an urgent issue. In this study, we focused on the effects of various NMs on antigen-presenting cells (APCs), which play a fundamental role in the recognition of foreign substances, and aimed to elucidate the mechanism of toxicity and develop an in vitro test method. We evaluated various NMs using the human Cell Line Activation Test (h-CLAT), a skin sensitization test adopted by the Organisation for Economic Co-operation and Development guideline that measures activation of APCs as an indicator. All five nano silicas examined in this study were positive for h-CLAT. Some samples increased the expression of CD54 by over 20-fold that of the control. For silver nanoparticles, both CD86 and CD54 expression by THP-1 cells were enhanced as well as allergenic silver ions. The activation of THP-1 cells by silver nanoparticles was considered to be due to silver ions that leached from the nanoparticles into the culture medium. In the case of titanium dioxide NMs, CD54 expression was mildly increased. In addition, carbon nanotubes strongly enhanced CD54 expression at low concentrations. These results suggest the usefulness of the h-CLAT test as a screening test for the evaluation of NM immunotoxicity.

　In the future, we plan to elucidate the mechanism of NM-mediated THP-1 cells activation by inflammasome analysis and analyze the effect of NM inhalation on alveolar macrophages to clarify the relationship between in vivo toxicity and in vitro activation of THP-1 cells. Multivariate analysis will be used to clarify the relationship between in vivo and in vitro test results and physical properties. The collected data will be useful for the development of an in vitro immunotoxicity assay.

Particle inhalation has been causally linked to diseases including cancer, COPD, fibrosis and cardiovascular disease. We have proposed a biological mechanism of action for particle-induced cardiovascular disease causally linking particle exposure to induction of acute phase response, which is a known risk factor for atherosclerosis and cardiovascular disease.

Inhalation and pulmonary deposition of particles induces inflammation, which is proportional to the total surface area of the pulmonary-deposited particles. In mice, inflammation is accompanied by an acute phase response, which is long-lasting for insoluble particles. Acute phase protein Serum Amyloid A (SAA) is among the most differentially expressed genes in lung tissue following particle exposure.

SAA is causally implicated in atherosclerosis and both overexpression and pulmonary dosing of SAA promotes plaque formation in ApoE-/- mice. In humans, controlled exposure to metal oxides and combustion particles induce dose-dependent increases in blood levels of SAA and C-reactive protein. Blood levels of acute phase proteins SAA and C-reactive protein are risk factors for cardiovascular disease in prospective, epidemiological studies.

Nanoparticles have a higher specific surface area than larger particles with similar chemical composition, and therefore, nanoparticles are more hazardous than larger particles of similar chemical composition in relation to cardiovascular disease. This underscores cardiovascular disease as a particle-induced occupational disease.

Cancer is thought to comprise a heterogeneous population of neoplastic cells showing a complex hierarchical structure in terms of gene mutations. According to recent studies, before a cancer develops, many independent, precancerous populations of clones are presumed to be present within apparently or physiologically normal tissues. However, it is poorly understood how those clones evolve from early infancy to the end of the life span in terms of their frequency and size and how its dynamics is affected by environmental/genetic factors to contribute to cancer. In this study, we investigated clonal expansion in physiologically normal tissues and those exposed to chronic inflammation by collecting a large number of micro-scale samples from aged esophagus as well as inflamed colorectal tissues from patients with ulcerative colitis, followed by an unbiased detection of somatic mutations and copy number abnormalities (CNAs) using whole-exome sequencing (WES). We demonstrated pervasive expansion of clones in aged esophageal tissues and inflamed colorectal epithelium in UC patients. Driver genes mutated in these expanded clones showed significant overlaps to those involved in cancer development of corresponding tissues, suggesting that these expanded clones represent the origin of cancer. Nevertheless, the frequency and patterns of mutations were substantially different between normal and cancer tissues, likely reflecting discrete underlying mechanism of clonal selection between both conditions. Thus, a mutation which is positively selected in non-cancerous expansion might not promote clonal selection but even negatively affect cancer development. For example, NFKBIZ mutations are highly prevalent in UC epithelia but rarely found in both sporadic and colitis-associated cancer, suggesting negative selection of NFKBIZ-mutated cells during colorectal carcinogenesis, which was further supported by significantly attenuated colitis-induced tumor formation in Nfkbiz-deficient mice and compromised cell-competition of NFKBIZ-disrupted colorectal cancer cells. To summarize, our results disclosed discrete mechanisms of clonal selection and remodeling between aged or inflamed tissues and cancer development, which may unmask novel cancer vulnerability potentially utilized for therapeutics of colorectal cancer.