One of the educational goals for pathology is to understand "Defining theetiology and pathogenesis of the disease." On the other hand, understandingthe rationality of pharmaceutical treatments based on the etiology or thepathogenesis has been considered beyond pathology education, despite the recentrapid progress in understanding the diseases at the molecular level.

As molecular pathological diagnosis provides evidence for determining the use oftherapeutic agents that target abnormal molecules, so far as we know,understanding its efficacy must require knowledge to connect pharmacologicaleffects with changes at the molecular cell level. Furthermore, it is notuncommon to point out the presence or absence of adverse drug reactions inclinical practices, clinic-pathological conference, and even in autopsy, wheretherapeutic effects and pathologic reaction may share the molecular interaction.Therefore, medical education must expand the chance to know the pathophysiologytriggered by therapeutic drug use. For these reasons, this paper willdemonstrate the necessity of pharmacology and pathology collaboration in medicaleducation and would like to propose and discuss a pharmacological pathology forunderstanding the rational selections and appropriate effect evaluation of thetherapeutic drug.

In the research and development of pharmaceuticals, the efficacy and safety areevaluated in non-clinical trials using animals or commercialized human frozencells before actually administering them to humans in clinical trials. However,compared to the use of fresh human blood or cells, it often does not reflect invivo functions, and extrapolation to humans is not necessarily high. In otherwords, by using fresh human blood, it is possible to accurately evaluate thedrug response in cell function, and pharmacodynamic (PD) assay can lead totranslational research to clinical trials.

In this presentation, I will explain the use of fresh blood by showingexamples.

Human-derived experimental materials are valuable in drug discovery research. Inparticular, patient-derived samples such as blood samples and iPS cells arethought to have a genetic predisposition to cause disease and, at least in part,some disease characteristics and are expected to be applied for elucidating theetiology and drug discovery research. In this session, examples of utilizationof human-derived samples in preclinical neuroscience research in our companieswill be introduced.

The prediction of drug bioavailability is one of the essential issues in the development of orally-administered drugs. Drug-induced intestinal abnormality is one of the most frequently-observed side effects and sometimes limits the clinical dose of anti-cancer drugs. However, their prediction at the preclinical stage is still a challenge due to the large species differences and the lack of an appropriate in vitro experimental system that mimics the functions of intestinal epithelial cells (e.g. Caco-2 cells). Therefore, we introduced the differentiated absorptive epithelial cells derived from intestinal stem cells at crypt region in humans/animals for the prediction of intestinal absorption and toxicity of drugs.

Advantages of this experimental system are that it is possible to use the unified protocol for the culture of intestinal stem cells and their differentiated ones regardless of any animal species and that it is also expected to reproduce the region-specific intestinal functions with our in vitro system since previous papers suggested that differentiated cells maintained the gene expression pattern at the crypt collection site. The expression and function of multiple transporters and metabolic enzymes in our differentiated intestinal cells were more similar to those in Caco-2 cells, which have been routinely utilized for characterization of drug intestinal absorption as a gold standard in the drug development. Furthermore, the intestinal availabilities of multiple CYP3A substrates were successfully predicted from their transcellular transport clearances. These evidences suggested that it is possible to predict the whole picture of intestinal absorption including its dynamic regulation by metabolic enzymes and transporters. Moreover, we demonstrated that PCFT and ASBT are limitedly expressed and uptake of methotrexate and taurocholate was only observed in the differentiated cells prepared from crypts at upper jejunum and terminal ileum, respectively, suggesting that region-specific function of detoxification system can also be well reproduced in this system.

Regarding the evaluation of the risk of drug-induced intestinal toxicity, we confirmed that the extent of the decrease in cellular ATP levels of spheroids by various EGFR tyrosine kinase inhibitors was well matched with clinical incidence of severe diarrhea and such toxicity was related to their inhibition potency against EGFR tyrosine kinase and mode of inhibition. We hope our in vitro system is expected to be widely used for the reproduction of various events at the intestinal tract such as drug intestinal absorption and drug toxicity.

An issue in drug discovery research at pharmaceutical companies is the decline in the probability of market launch, and there is a need to improve the POC acquisition rate by further improving clinical predictability.For this purpose, we need to change the mindset from animal to human at the drug discovery stage, that is, to shift from an animal-based evaluation system to an evaluation system that utilizes human clinical samples, to deepen our understanding of human pathophysiology, and to make efficient of non-clinical research by utilizing human clinical data and samples.In particular, with the aim of improving the efficiency of non-clinical research by utilizing human clinical data/samples, we have established a human sample utilization support team, then we are collecting information on organizations (biobank, academia, supplier, CRO) that can provide various human samples and accumulating know-how on obtaining human samples. In addition, we have built a one-stop support system for surveying human samples to meet research needs, negotiating, and contracting with the organizations, applying to ethical committees, importing samples, and dealing with customs clearance. As results, our researchers can obtain high-quality human samples to meet research needs rapidly/easily. By introducing this system, the number of the research projects that implement target validation, pharmacological evaluation, BM identification, and patient stratification, etc. using human samples from the early stage of drug discovery, has increased, and then, the evidence obtained by using human samples contribute to create drug candidates with high prediction accuracy of clinical effect.In addition, for the streamlining non-clinical research as described above, fresh and/or chronological patient samples are required in many cases. The current situation and challenges in conducting experiments using prospective and fresh human samples are as follows: i) The number of overseas CROs which can obtain patient samples and conduct the experiments with high quality, is not so many, ii) It takes long time to initiate collaborative researches with domestic or overseas academia for contracts, ethical committees, also our researchers have to stay the academia until patient samples are obtained, iii) It is difficult to conduct collaborative research to share outcome/IP with collaborators due to open innovation to acquire good drug candidates. In order to solve these problems, our team is working to strengthen cooperation with the preferred biobanks where fresh and prospective samples can be obtained. As an example, we obtained fresh patient samples from Tsukuba Human Tissue Biobank Center in Tsukuba Univ, which is located near our labs, under the material transfer agreements. Therefore, we can conduct many non-clinical research using fresh samples in our own labs. We will introduce our activities while showing the support flow that was actually constructed.

Parkinson's disease is one of the neurodegenerative disorders, it is pathologically characterized by loss of dopaminergic neurons and accumulation of α-synuclein. The treatment of diseases can be broadly classified into causal and symptomatic therapies. All the drugs currently on the market for Parkinson's disease are used for symptomatic treatments. Levodopa, a dopamine precursor, is the mainstay of treatment for Parkinson's disease to correct the malfunction of basal ganglia circuits caused by dopamine deficiency in the brain. In addition, dopamine agonists, anticholinergics, COMT inhibitors, MAO-B inhibitors and other drugs have been marketed. In clinical trials, the efficacy of these medications was mainly evaluated using Unified Parkinson's Disease Rating Scale and symptom diaries which were somewhat subjective methods. Evaluation using medical devices has been attempted, but is not yet common. With regard to the disease-modifying drugs, anti-α-synuclein antibodies, GLP-1 agonists, and kinase inhibitors have been examined in clinical trials. However, no drug has been obviously demonstrated to inhibit the progression of Parkinson's disease to date. It is more difficult to demonstrate clinical efficacy of disease-modifying drugs than symptomatic drugs, because there is no useful biomarker to quantify the degree of neuronal degeneration in clinical practice. In addition, the difficulty of using placebos for long periods in a clinical trial also makes proper assessment difficult.

Dementia with Lewy bodies (DLB) is a form of dementia characterized by hallucinations and delusions. The concept of DLB was first reported in the world by Kosaka et al. as a disease in which Lewy bodies, which are unique neural inclusions, are found in autopsy brains, and the disease concept has been established. On the other hand, Parkinson's disease (PD), which shows characteristic motor symptoms such as tremor, muscle rigidity, and akinesia, shows Lewy body pathology due to damage to dopamine neurons in the substantia nigra, and as the disease progresses, Lewy bodies spread throughout the brain and eventually cause dementia. The clinical features of PD dementia (PDD) is quite similar to those of DLB and furthermore, the pathology of PDD is indistinguishable from that of DLB. For these reasons, it is now considered appropriate to refer to both disorders collectively as Lewy body disease. Although pharmacotherapy, mainly dopamine replacement, has shown remarkable efficacy in improving motor dysfunction in PD, it is not effective enough to treat a wide variety of non-motor symptoms, including cognitive dysfunction. It is now clear that the most important determinant of prognosis in PD is the degree of concomitant cognitive dysfunction. Recent studies have shown that the prognosis for PD is generally 3-4 years from the onset of dementia, regardless of disease duration or age of onset. It is also clear that more than 80% of PD patients will eventually develop dementia. Therefore, it is essential to establish a methodology for early detection and intervention of PD dementia. We performed the OSIT-J olfaction test in PD patients without dementia and examined its association with future onset of dementia. Results showed that about half of the patients with severe olfactory impairment (OSIT-J ≤ 4) had slightly lower cognitive function scores from the time of entry. In addition, longitudinal results showed that 10 of the 44 PD patients in the study developed new dementia during the 3-year outpatient period, all of whom had severe olfactory impairment at study entry. It was also found that brain atrophy and cerebral metabolic abnormalities were prominent in patients with severe hyposmia, even when motor impairment was mild (Brain 135:161-169, 2012). This study is the first to show that severe hyposmia is predictive of dementia in PD. Similar results have been replicated around the world. Based on these results, a randomized, double-blind, comparative study of donepezil as a biomarker for olfactory impairment in PD (DASH-PD study) was initiated as a multicenter study at 22 centers nationwide to establish an early diagnosis and treatment intervention for dementia in PD, and a 4-year follow-up period was completed. The results suggest the efficacy and safety of cholinesterase inhibitors for PD without dementia and were recently reported (eClinicalMedicine 51: 101571, 2022).

The development of therapeutic agents for neurodegenerative disorders is an urgent issue in our super-aging society. On this point, screening target patients is essential for medical treatment. Dementia and Parkinson's disease (PD) are often diagnosed after the onset, which limits the therapeutic effect after the disease has progressed. We previously demonstrated that fatty acid-binding proteins (FABPs) are critical for the pathogenesis of cognitive and motor dysfunctions, which were associated with neuroinflammation pathology. To develop quantitative biomarkers to detect pathogenesis, we measured plasma from 600 patients with mild cognition impairment (MCI), Alzheimer's disease (AD), PD, and dementia with Lewy bodies (DLB), using a high-sensitivity ELISA system. Plasma FABP3 level was increased in each disorder. Diverse disease-specific differences were observed in the plasma content of various additional biomarkers. Optimizing quantification methods resulted in unique techniques for discriminating each disease. Based on the data, we discuss our novel technique using unique biomarkers that discriminates between MCI, AD, PD, and DLB, which enables us to accelerate the quantitative diagnostics of various neurodegenerative diseases essential for early therapeutic intervention.

Foods and pharmaceuticals play important roles in public health and welfare, and ensuring that these products meet their quality assurance standards is a top priority in health and medical care. Quality assurance of products is a fundamental philosophy lies in pharmaceutical science, and it started with evaluating and assuring quality of natural medicines. 

Prior to the announcement of the report by the subcommittee of clinical pharmacy and pharmaceutical sciences in Science Council of Japan, the subcommittee conducted a questionnaire survey about the university lectures on quality assurance of foods and pharmaceuticals to all the faculty of pharmaceutical sciences (75 schools) in Japan. We received the answers from 60 schools and the result was published by Prof. Komatsu in "Yakugaku Zasshi, 141(6), 2021". In the questionnaire there were 13 essential items as contents of lecture. Of them, regulatory science, borderline of pharmaceuticals to non-pharmaceuticals, quality assurance of foods, chemistry, manufacturing and control (CMC), definition of quality, and classification of pharmaceuticals were not lectured in 37%, 20%, 25%, 48%, 20% and 20% of schools, respectively; however, the quality assurance is a basic philosophy for a pharmacist. 

Very low achievement ratio of the lectures for the above-mentioned items may be due to the lack of description about them in the "model core curriculum for pharmacy education". In this symposium I would like to introduce the report based on the questionnaire survey and describe the need of reforming pharmaceutical education.

Because pharmacology seeks to understand therapeutic effects in the whole body and in human populations down to the pharmacological effects that occur at the molecular level, pharmacology integrates basic and clinical applications. Only when the quality of both the test substance and the biological model is stable can we understand the mechanism of pharmacological action leading to a cure. Thus, quality assurance of both the test substance and the biological model is highly important to collect pharmacological data successfully with high reproducibility and reliability among different laboratories. However, the survey about the lectures on quality assurance of foods and pharmaceuticals introduced by Dr. Goda's talk revealed that education on quality assurance was lacking even though some topics are covered in pharmacology lectures. In this talk, I would like to discuss with audience how to improve education on quality assurance in pharmacology education by citing several cases where the concept of quality assessment is important in pharmacological research.

When it comes to quality assurance in a pharmaceutical company, students may think mainly of the business content of product quality control, pharmaceutical affairs, and quality assurance. In reality, there are several steps in the drug development activities of pharmaceutical companies, and each step must be dealt with from the perspective of quality assurance. For example, when conducting a pharmacological experiment using cells or animals, a study plan to be prepared before the experiment, information on the source of cells or animals used in the experiment, acquisition records and usage records of reagents to be used, storage of raw data and guarantee of accuracy of processed data after analysis, appropriate tatistical analysis method, study report containing the above information are required to guarantee the study results. For studies other than pharmacological studies, not only study plan and study report are required, but also studies are required to be conducted under the conditions stipulated by the Ordinance of the Ministry of Health, Labor and Welfare. Non-clinical toxicological studies and pharmacokinetic studies are conducted under GLP (Good Laboratory Practice), clinical studies are conducted under GCP (Good Clinical Practice), and product manufacturing control is conducted under GMP (Good Manufacturing Practice). Efforts are underway to protect patient's health and safety.

Based on the "Redefinition of Mission" published in April 2014 by the Ministry of Education, Culture, Sports, Science and Technology, the Graduate School of Pharmaceutical Sciences, Osaka University ("Grad. Sch. Pharm. Sci., Osaka Univ.") has decided to promote Regulatory Science (RS). "Grad. Sch. Pharm. Sci., Osaka Univ." was adopted by "Grant for promotion of the practical application of innovative pharmaceuticals, medical devices, and regenerative medicine products", which was supported by the Ministry of Health, Labour and Welfare (MHLW), and were in collaboration with the Pharmaceuticals and Medical Devices Agency (PMDA), the National Institute of Health Sciences, the National Institute of Biomedical Innovation, Health and Nutrition, and the National Cerebral and Cardiovascular Center, etc. This led to the conclusion of a cooperative graduate school agreement with PMDA and NIHS, and the start of full-scale research education and human resource development regarding RS. In addition, personnel exchanges with the MHLW and PMDA were conducted. Furthermore, a new laboratory, named Laboratory of Regulatory Science for Pharmaceuticals and Medical Devices, was launched in April 2018, and a "new six-year pharmacy education system" unique to "Grad. Sch. Pharm. Sci., Osaka Univ." was established from FY2019. In this lecture, I would like to talk about the current situation and future challenges of RS research education and human resource development in academia.

The acute toxicity testing is conducted as a part of chemical hazard identification. The endpoint of acute toxicity is death, which is the ultimate toxicity and the lethal dose 50% (LD₅₀) have been used because of the convenience of showing the toxicity intensity of compounds as a common index. On the other side, death is not a desirable endpoint from the viewpoint of animal welfare. Moreover, the significant shortcoming of acute toxicity study is that it is not useful for the medical treatment of human poisoning because the cause of death, target organs, etc. are not considered at all. In order to solve these issues concerning acute toxicity study, it is great way to obtain valid result with precisely measure and quantify the signs of toxicity over multiple items, and make rational criteria for determining the severity of their toxicity. This will be, however, a complicated and expensive test than a study with a mortality endpoint. For modernization of acute toxicity, we are focusing on multiple vital signs. As a part of this project, we have established a less invasive method of measuring biopotential by carbon nanotube yarn. In this presentation, we would like to new approach for assessment of acute toxicity by multiple parameters of vital signs. (Health and Labour Sciences Research Grant, Japan)

We are constructing the "Percellome Database" consisting of the transcriptomes of liver and other organs of mice exposed to different types of chemicals. Using this database we are systematically elucidating the molecular mechanism of toxicity for the development of a new toxicity prediction technology.

The database already contains comprehensive transcriptomic data on 160 chemicals. Acute toxicity of a chemical can be predicted to a certain extent by searching the similarity of the transcriptomes obtained by the single-dose exposure experiments.

In addition, we are performing the repeated-dose experiments on the selected chemicals in order to analyze the correlation between the transcriptome and the epigenome i.e. histone modification by ChIP-Seq and genomic DNA methylation by WGBS for the understanding of the molecular mechanism of the chronic toxicity.

Now, we are attempting to expand the scope of analysis with higher efficiency by introducing the artificial intelligence technologies. This approach should maximize the value of the Percellome database and optimize the utility of the epigenome data for the prediction of chronic toxicity from short-term repeated dosing toxicity studies, and for the progress of practical in silico toxicity prediction in the near future.

The success rate in the development of new drugs is generally low and dropout due to safety issues in late phase clinical trials can be a critical blow to a pharmaceutical company. In small molecule drug discovery, not only on-target toxicities but also off-target toxicities (such as genotoxicity and QT prolongation) should be considered early to prevent dropout in later phases, and related guidelines have also been established. Before the first in human dosing phase, in vivo toxicity studies are conducted to determine a safe initial dose for clinical trials, while in vivo toxicity studies at the early drug discovery stage are conducted to identify the toxicity potential of lead compounds. In addition, target safety assessments are also performed to estimate what kinds of on-target toxicity will occur in normal tissues and what kinds of non-clinical safety assessments are needed for further drug discovery. In some cases, knockout animals are used to obtain proof of concept. If critical on-target toxicity is identified, the project might be terminated depending on the indications. However, off-target toxicity may be avoided if the mechanism of toxicity is elucidated by additional experiments. In this presentation, I will introduce our early drug discovery strategy for small molecule drugs and share examples of our approach to elucidate the mechanisms of on-/off-target toxicity.

In recent years, drug development has expanded rapidly from small molecules and some biologics, such as insulin and monoclonal antibodies to new modalities including oligonucleotide, cell and gene therapies. These therapies may have the potential to transform patients' lives and cure diseases by addressing the root cause of the diseases rather than the symptoms. As technologies continues to evolve, regulatory authorities have drafted new guidance accordingly in each region. However, harmonization of guidelines for international regulatory authorities has been still limited. Although some of new modalities fall outside the scope of the ICH guidance entitled "M3(R2): Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals" and "S6: preclinical safety evaluation of biotechnology-derived pharmaceuticals", the guidance for small molecules and the conventional biologics may be useful in pre-clinical safety assessment of new modalities by considering new modalities-specific safety concerns. This presentation will provide points to consider in pre-clinical safety assessment of new modalities, focusing on differences from that of conventional modalities.

Water is by far the most abundant molecule in tissues and the common solvent for all bioactive substances including drugs. Therefore, any changes in abundance, flow and quality of water will critically affect the biological actions of substances. Despite the importance, however, dynamics of water in tissues are poorly understood, in sharp contrast to those of solutes. This situation is mainly due to the lack of appropriate analytical tool to image and characterize water dynamics in living tissues with high spatiotemporal resolution. To overcome this technical limitation, we developed a multimodal multiphoton microscopy technique with stimulated Raman scattering (SRS) imaging ability. This SRS multimodal multiphoton microscopy allows us to visualize water dynamics in brain tissues. In addition, simultaneously obtained fluorescence images enables us to characterize water dynamics together with that of fluorescent dyes (solutes), in the context of cellular structures. In this talk, I will introduce the technical background of this technique and new information obtained from our research.

The glymphatic system has been proposed as a draining system in the brain like the lymphatic system in the body. The water dynamics (inflow and outflow) should be an important key; however, the exact dynamics of intracranial water especially for extracellular and cerebrospinal fluid (CSF) are still unknown.

Water molecules labeled by a stable isotope of oxygen-17 (O-17) can be visualized by MRI. When it is used as an exogenous tracer, temporal changes in tracer concentration can be elucidated. By using intravenous injection of O-17 labeled water, we found the different kinetics of CSF between subarachnoid and intraventricular spaces in humans. By using intrathecal injection, we also found the different washout of CSF between patients with idiopathic normal pressure hydrocephalus and Alzheimer's dementia.

In the animal study, different water kinetics have been noted between AQP4 knocked out and wild type (WT) mice in the water intoxication model, and between amyotrophic lateral sclerosis model and WT mice. These differences are probably due to the differences in permeability or retention of water in the brain. Further studies are now being planned to trace the outflow of water dynamics in detail in the animal study after the intrathecal injection of O-17 labeled water.

The brain is equipped with an unique water system consisting of blood-brain barrier, cerebrospinal fluid, and astrocyte AQP4. The water environment in the brain is supposed to be essential for the highly developed information processing by neurons, but the crosstalk between water and neuron is still to be elucidated. We had reported that water influx mediated by AQP4 induces ATP release and subsequent increase of extracellular adenosine, which modulates neuronal activities, as well as blood flow and immune cells. Furthermore, we are finding that the adenosine derived from astrocytes affects dopamine neurotransmission and is associated with psychiatric disorders. Depressive behavior is affected in AQP4 knockout mice, which shows altered adenosine release, adenosine receptor expression and adenosine-dependent modulations of dopamine neurotransmission. Since AQP4 localizes to the astrocytic endfeet along blood vessels, these data suggest that the water flow from the bloodstream to brain parenchyma influences neural activities. And this water flow is likely accelerated by the nutrient uptake during neuronal activities, because astrocyte active transport is the exclusive gate to the brain parenchyma. We propose water system as an emerging clue for understanding brain functions.

Alzheimer's disease (AD) is characterized by two pathological hallmarks, extracellular aggregation of Aβ as well as intracellular deposition of tau protein.

Glymphatic system has been shown to play a significant role in Aβ clearance, whose impairment has been implicated in AD development. Contrary to Aβ that is normally released from neurons, it has been long assumed that tau has no immediate interaction with extracellular clearance pathway due to its primarily cytoplasmic localization. However, recently we have discovered that tau released from neurons is also cleared from brain parenchyma to cerebrospinal fluid (CSF) by glymphatic systems and subsequently transported to deep cervical lymph nodes. Strikingly, deletion of aquaporin-4, a critical component of glymphatic system, not only elevated tau in CSF but also markedly exacerbated intracellular phosphorylated tau deposition and the associated neurodegeneration in the brains of transgenic mice expressing P301S mutant tau (Ishida, Yamada et al., J Exp Med 2022). This finding highlights the previously underappreciated, unique interplay between extracellular tau clearance and tau pathophysiology and motivates future therapies targeting modulation of extracellular tau. Along with our discoveries, this presentation will provide current knowledges on glymphatic/lymphatic systems as well as its roles on AD pathogenesis and discuss the possible future prospective.

Clioquinol was extensively used as an amebicide to treat indigestion and diarrhea in the mid-1900s. However, it was withdrawn from the market in Japan because its use was epidemiologically linked to an increase in the incidence of subacute myelo-optic neuropathy (SMON). SMON is characterized by the subacute onset of sensory and motor disturbances in the lower extremities with occasional visual impairments, which are preceded by abdominal symptoms. Although pathological studies demonstrated axonopathy of the spinal cord and optic nerves, the underlying mechanisms of clioquinol toxicity have not been elucidated in detail. We previously performed a global analysis of human neuroblastoma cells using DNA chips and demonstrated that clioquinol induced 1) DNA double-strand breaks and subsequent activation of ATM/p53 signaling; 2) the expression of VGF, the precursor of neuropeptides involved in pain reactions, by inducing c-Fos; 3) the expression of interleukin-8 by down-regulating GATA-2 and GATA-3. We also demonstrated that clioquinol induced zinc influx and oxidation of the copper chaperone ATOX1, leading to the impairment of the functional maturation of a copper-dependent enzyme dopamine-β-hydroxylase and the inhibition of noradrenaline biosynthesis. Thus, clioquinol-induced neurotoxicity in SMON seems to be mediated by multiple pathways.

Cardiotoxicity is one of the most significant adverse effects of anti-cancer agents, and is responsible for considerable morbidity and mortality. In particular, the most frequent and serious effect is heart failure with ventricular dysfunction. Thus, contractility is a key cardiac safety issue in drug development.

Human stem cell technology holds great promise as in vitro models to assess novel drug candidates during drug development. If human iPS cell-derived cardiomyocytes (iPSC-CMs) can be used to further improve the predictability of safety in humans, it is expected to revolutionize the drug discovery process by improving drug development efficiency, reducing cost and ensuring the patients' safety.

We have developed imaging-based method for cardiac contractility, which can detect and quantitate the strain of iPSC-CMs during their contraction and relaxation. As expected, treatment with anti-cancer agents, such as doxorubicin, inhibited contraction as well as relaxation speed using iPSC-CMs. Based on our experience about proarrhythmia risk assessment using iPSC-CMs, we try to get confidence with in vitro contractility assessment.

In this symposium, we would like to show our cardiac toxicity approach to predict clinical cardiotoxicity risk, and discuss future perspectives of cardio-oncology field.

Adverse drug reactions (ADRs) such as skin rash, drug-induced liver injury, and agranulocytosis have long been analyzed in relation to human leukocyte antigen (HLA) that is an important molecule involved in human immunity. HLA is composed of many genes, each of which has dozens of different types (alleles), and many HLA alleles associated with ADRs have been reported, starting with the report of HLA-DRB1*08:03:02 associated with agranulocytosis by methimazole. The odds ratios in the association with HLA alleles range from approximately 5 to several thousand, indicating a very large impact on the risk of ADRs. Thus, HLA genetic testing prior to initiation of drug therapy is expected to make a significant contribution to avoiding ADRs, but to demonstrate the clinical utility of genetic testing, it is necessary to prospectively show the effects of medical interventions based on the test results. We conducted the GENCAT study, a prospective, multicenter, single-arm clinical trial to investigate the impact of a therapeutic intervention based on the HLA-A*31:01 test on the incidence of carbamazepine-induced skin rash. HLA-A*31:01-positive patients were treated with an alternative drug such as valproic acid, and the study showed an approximately 60% reduction in the incidence of carbamazepine-induced skin rash. It is expected that the genetic test, which has demonstrated clinical utility, will lead to the establishment of safer and more appropriate stratified medicine by reflecting the information in clinical practice guidelines.

During the past decade, many high-alert medications have been developed for use in clinical practice. High-alert medications are defined by the Institute for Safe Medication Practices as drugs that bear a heightened risk of causing significant patient harm when used in error. In these situations, prescribers tend to be underdose to avoid the adverse events. Prompt pharmacotherapy, ideally without overdose and underdose, are required. Particularly, in the pharmacotherapy of high-alert medications with large individual differences, more attention is needed. It is a major mission of clinical pharmacology to determine what factors are responsible for individual differences, and how to translate the findings into clinical practice. In this symposium, we would like to introduce the research we have been engaged in and discuss how safe and secure pharmacotherapy should be practiced.

(1) Noda S., Morita S. and Terada T. Dose individualization of oral multi-kinase inhibitors for the implementation of therapeutic drug monitoring. Biol Pharm Bull 45, 814-823 (2022)

(2) Hira D. and Terada T. BCRP/ABCG2 and high-alert medications: biochemical, pharmacokinetic, pharmacogenetic and clinical implications. Biochem Pharmacol, 147, 201-210 (2018)

Although valuable findings are obtained in pharmacological research, the bridge between basic and clinical research is a barrier, since clinical applications are limited. Therefore, the development of a new research method is required to bridge the knowledge acquired from basic and clinical pharmacological researches. Furthermore, our laboratory realized the association between basic and clinical pharmacological findings utilizing medical big data. Therefore, by using the strategy to develop basic pharmacological experiments from useful findings obtained from clinical pharmacological research via various medical big data, the efficacy and human safety can be accurately predicted and applied to clinical applications.

Several medical information (receipt data) and life science (gene expression data) databases have been developed and maintained, but there are few databases for basic and clinical pharmacological researches. In this symposium, we have demonstrated research examples that connects basic and clinical pharmacological researches by utilizing medical big data and discussed future prospects.

Historically, basic pharmacology has long used adverse drug reactions to create models of human pathological conditions in experimental animals. This is because the phenotypes and/or pathogenic mechanisms of the adverse events share similarities with spontaneous human diseases. On the other hand, a large amount of real-world data (RWD), such as spontaneous reports of adverse drug reactions and insurance claims data, become available recently. Therefore statistical analysis of RWD has made it possible to accurately identify novel and unexpected confounding factors (e.g., concomitant medication) that influence the occurrence of adverse events or spontaneous disease. Such pharmacological drug-drug interactions not only lead to immediate drug repositioning, but also to the elucidation of adverse event mechanisms and the discovery of new drug targets. In addition, hypotheses derived from RWD analysis are expected to have extremely high clinical predictive value. In this presentation, we will show how RWD analysis can lead to the discovery of drug targets, by introducing examples of research from our previous reports, and discuss the potential of new research strategies in the future.

Comprehensive genomic profiling (CGP) for cancer has been introduced into health insurance practice in Japan since June 2019. The panel tests are indicated for those with advanced solid tumors who are in good condition to receive cancer treatment, typically as in a clinical trial, and who have completed or are likely to complete standard treatment soon. At government-designated core base hospitals or base hospitals, an intra-institutional molecular tumor board, consisting of a multidisciplinary medical team called an expert panel, annotates the CGP test results to propose appropriately targeted treatments for each patient. However, the treatment options are suggested in only approximately 10% of patients, implying that CGP testing is of no use to most patients. Furthermore, the options proposed are usually limited to treatments in clinical trials or that require the use of off-label drugs that are not covered by health insurance. Therefore, even the few privileged patients who receive treatment are faced with difficulties. Given this situation, it is clear that the most critical issue in current cancer genomic medicine is an exit strategy: how to link more patients to appropriate treatments. Realistically, CGP testing has made targeted therapy under clinical trials, with an unconfirmed efficacy and safety, a tempting treatment option for patients. Therefore, to benefit patients, medical oncologists must reasonably deal with such conflicts, distinguishing between evidence-based and experimental treatments.

Long COVID-19 is a public health concern globally. Recent clinical data suggest that COVID-19 can cause various complications that last weeks to months after initial recovery. SARS-CoV-2 are well known to infect many organs, not only lung tissue but also cardiomyocytes, intestine, and central nervous system. Thus, establishment of appropriate animal and in vitro models for COVID-19 is critical to elucidate pathological mechanisms and drug development.

We have made various organ models from human iPS cells and evaluated their usefulness as SARS-CoV-2 infection models. We found that SARS-CoV-2 infected human iPSC-derived cardiomyocytes efficiently. Imaging analysis revealed that SARS-CoV-2 induced contractile dysfunction in iPSC-cardiomyocytes. Next-generation sequencing analysis revealed that SARS-CoV-2 reduced the expression of proteins related to contractility, suggesting that SARS-CoV-2 directly infects the cardiomyocytes and induces dysfunction. These observations are consistent with the clinical observations. In addition to cardiomyocytes, we found that SARS-CoV-2 also infected into iPSC-based brain blood barrier, and small intestinal epithelial cells.

In the symposium, we would like to talk about the iPSC-based models of SARS-CoV-2 infection and discuss the advantages and drawbacks.

The nano-structured systems that transport bioactive substances to a target location in the body have recently attracted much attention and have been rapidly advancing. However, none of these systems have been able to efficiently enter the brain via a systemic route. Taking advantage of the phenomenon that GLUT1, which is expressed at a high level in the BCECs, migrates from the luminal to the abluminal side in response to an increase of blood glucose concentration from the hypoglycemic state, we succeeded in accumulating a self-assembled nanomachine with glucose integrated on the surface with optimal configuration in the brain, with significant efficiency. 

In this presentation, I firstly will introduce the development of BBB-crossing Nanomachine. I will also introduce the results of nanomachines that efficiently pass through nucleic acid and antibody drugs into the brain using this system as a platform. And finally, the concept of a "Hayabusa-type Nanomachine," inspired by the planetary probe Hayabusa, which efficiently passes through the BBB, samples molecules in the brain, and returns them to the bloodstream to provide information on brain pathology, will be introduced.

Major depressive disorder (MDD) is a highly prevalent psychiatric disorder that is associated with physical impairment, medical comorbidity, and mortality worldwide. Although the etiology underlying MDD remains unclear, stressful life events may cause depressive symptoms and the incidence of MDD. Neurotransmitters and their metabolites in the brain play crucial roles in the regulation of mood and emotion. In addition, exposure to stress alters the extracellular levels of these metabolites in the brain. Recently, metabolite levels in the blood have received attention as a possible marker of depression. Therefore, metabolomic alterations in the brain and the blood, especially under the stressful condition, and their relationship is important to resolve the etiology of depression. In this talk, I introduce the recent studies conducting the comprehensive analysis of extracellular metabolites in the brain and blood in mice.

Capillary electrophoresis-mass spectrometry (CE-MS) is a powerful tool for the comprehensive profiling of ionic biomolecules, including neurotransmitters such as glutamic acid (Glu), γ-aminobutyric acid (GABA), and other monoamines. However, CE-MS often lacks sensitivity due to the limitation in the sample injection volume (~ few nL), and it is not easy to analyze neurotransmitters of which amount is usually quite limited in the brain. In order to address this issue, we have developed highly efficient online sample preconcentration methods, which provide up to 2,000-fold higher sensitivity without losing separation performance [1,2]. By applying these techniques, we have achieved trace bioanalysis like single cell metabolome analysis [3], that shows the great potential of our CE-MS for exploring the trace brain molecules. In this symposium, I will introduce our recent studies on the ultra-sensitive analysis of brain molecules, especially focusing on neurotransmitters like Glu and GABA.

[1] T. Kawai et al., Chromatography, 2017, 38, 1.

[2] T. Kawai et al., J. Chromatogr. A 2018, 1565, 138.

[3] T. Kawai et al., Anal. Chem. 2019, 91, 10564.

Multiple stress-related psychiatric disorders exhibit emotional and behavioral disorders, and it is crucial to understand the specific machinery governing these behaviors at the subnuclear level in critical brain regions, such as the amygdala. Within the amygdala, the basolateral amygdala (BLA)-the central nucleus of the amygdala (CeA) circuit is known to mediate fear and anxiety responses. The bed nucleus of the stria terminalis (BNST) is also included in the central extended amygdala together with the CeA, comprising a functional-anatomical macrosystem. Both regions have complex subnuclear organizations. We recently discovered the differential engagement of distinct subnuclei of the central extended amygdala, critical for regulating fear- and anxiety-related behaviors in mice. In this symposium, I will introduce our recent studies and describe how we can dissect the function and molecular constituents of distinct subnuclei in the amygdala. Additionally, I will discuss how the technologies introduced in this symposium have the potential to be used in research on emotional regulation by the brain.

Why do we age? This physiological phenomenon, which is experienced by everyone, is still largely unknown and remains a great mystery even today with the advancement of science and technology. Aging is a major risk factor for the development of many diseases such as cancer, but there is no clear answer as to why the elderly are more susceptible to cancer and various other diseases. Although little is currently known about the mechanisms that determine maximum lifespan, the mechanisms that control aging are gradually becoming clearer. In other words, it is now known that inflammation-inducing cells, such as senescent cells, accumulate in various organs and tissues with aging and adversely affect the microenvironment, which is the pathological basis for the decline of organ functions and the development of diseases such as cancer. Cellular senescence is induced by various senescence-inducing stimuli, such as DNA damage, telomere length reduction, and oncogene activation. However, senescent cell research to date has mainly been conducted using cultured cells, so it is difficult to determine whether senescent cells in individuals have the same characteristics as cultured cells. When and where do they exist? Also, can accumulation of senescent cells really induce age-related changes in individuals? Can senescent cells be efficiently removed from individuals? Many questions remain unanswered. Thus, in order to understand integrated changes in individuals such as senescent traits, it is essential to analyze them at the individual level using laboratory animals. In this symposium, I would like to introduce our recent findings and discuss whether it is possible to control human aging in the future.

Aging is the gradual but progressive decline of physiological function in cellular and tissue levels, eventually leading to the disruption of homeostasis in an individual organism. Advancing age is known to be one of the risk factors for various diseases and mortality, but the quantification and intervention are challenging. Aging hallmarks are well characterized while they are not exclusively specific to aging, and dependent on context, cell type and disease state. In addition, aging process is used to be considered as unidirectional. However, the generation of iPS cells demonstrates that terminally differentiated somatic cells can be reverted to embryonic like state while keeping genetic information. Owing to going through the iPS cell state, we can obtain rejuvenated phenotype of cells and tissues. Intriguingly, vigorous research for understanding the iPS cell reprogramming process has also demonstrated that the partial reprogramming reverses many age-associated changes without going through iPS cell state. In this presentation, I will introduce and discuss the potential of partial reprogramming against age-associated changes and the process which gives insight for the mechanisms.

During the past half century, more than 25 viral diseases including Ebola virus disease, SFTS and AIDS, appeared in human population as novel infectious diseases. Current pandemic of COVID-19 reminded us of the risk of emerging viral diseases again. In addition to emerging viral diseases, many other viral diseases including dengue, chikungunya, monkeypox and influenza, are still serious public health concerns in the world. One of major strategies to overcome these viral diseases is to develop the antiviral drugs. Several effective antiviral drugs have been developed for some viral diseases. However, there is no approved effective antivirals for most viral diseases. To improve this situation and also prepare for the emerging viral diseases which may appear in future, the novel antiviral drugs with a wide-antiviral spectrum are required.We recently observed that Mitochonic acid-5 (MA-5) significantly suppress the growth of SARS-CoV-2 in Calu-3 cells. MA-5 is known to facilitates ATP production in mitochondria and reduces ROS independent of electron transfer complex (ETC) by facilitating ATP synthase oligomerization and supercomplex formation with mitofilin. In some viral diseases including COVID-19, it has been reported that the dysfunction of mitochondria is involved in severe disease progression. The improvement of the mitochondrial function damaged by virus infection may be important for the therapeutics of viral diseases.We are now analysing the antiviral mechanism of MA-5 and also investigating the possibilities of MA-5 and its derivatives as a wide-spectrum antiviral.

Space travel has advanced significantly over the last six decades with astronauts spending up to usually 6 months (up to 12 months) at the International Space Station (ISS). The living environment on the ISS is challenging to astronauts because microgravity (μG) induces various health problems such as bone demineralization, muscle atrophy, cardiovascular deconditioning, and vestibular and sensory imbalance including visual acuity, altered metabolic and nutritional status, and immune system dysregulation. In addition, isolated and limited habitability causes psychological stress, and exposure to space radiation potentially endangers the health of the astronauts. The next challenging steps for humankind include new missions to the Moon followed by human exploration of Mars. Deep space human exploration has become more realistic.In a recent study, a multi-omics, systemic biological approach revealed a major mitochondrial dysfunction using spaceflight samples. It was also found the evidence of altered mitochondrial function using the urine and blood metabolic data compiled from the astronaut cohort and NASA Twin Study data, indicating mitochondrial stress as a consistent phenotype of spaceflight. In the future, drugs to maintain mitochondrial function are expected to enable safe space odyssey and space planet habitation. In this lecture, I would like to review the trend of space experiments in the world, and introduce our platform for simulated space environments.

Currently more than 50% of clinical trial notifications submitted to the PMDA are for multi-national trials. Industry, academia and regulatory authorities have been seeking efficient, streamlined and low-cost ways of conducting clinical development in order to cope with complexity of clinical trials in recent years especially in multi-national settings.

In addition, since 2020, COVID-19 pandemic brought several challenges in clinical development of drugs, such as difficulties for patients and monitors/CRCs in visiting trial sites during the pandemic, need for large-scale trials involving tens of thousands subjects planned and conducted in a very short time for vaccine development.

While, digital transformation (DX) is now called for in various areas of society, the incorporation of online technology in each process of clinical trials (e.g. informed consent, data collection, SDV/monitoring etc.) can be a way to respond to the current challenges. 

The Ministry of Health, Labour and Welfare (MHLW) has been investigating and collecting information on actual cases of clinical trials using online technology including decentralized clinical trials (DCTs) as well as relevant guidelines in Japan and abroad, in order to provide certain guidance/points to consider for ensuring reliability in clinical trials using online technology

In this presentation, such situations will be outlined from the regulator's point of view for further discussion at the symposium.

In the drug development, manufacturers or investigators need to demonstrate the effectiveness of their products through the conduct of adequate and well-controlled studies. On the other hand, the success probability of clinical trials is not high in some disease areas. This is because even for treatments that are expected to be effective, the method of conduct and study design may affect the outcome of clinical trials. For example, in clinical trials of neuropsychiatric disorders, there is substantial improvement and variability in placebo groups, and/or in which the effects of therapy are small or variable. In addition, efficacy evaluation is generally done by subjective evaluation using a symptom rating scale, which results in a certain degree of variability in the evaluation. This can lead to higher-than-expected response to placebo and failure to achieve the expected results in clinical trials. In other words, clinical trials present issues specific to disease areas. Therefore, in planning and conducting clinical trials, it is important to identify and control factors that influence efficacy and safety assessment. In this symposium, important points of clinical trials and how to examine factors affecting efficacy assessment will be introduced.

One of the mission of regulatory science is to promote human longevity by providing safer and more effective drugs and ensuring human health. At present, various in vitro and in vivo evaluation methods are used for drug development, and no major problems have been observed. Although the conventional non-clinical testing methods can predict clinical outcomes, there is still some room for improvement in terms of risk prediction in humans. Thus, new approach methodologies (NAMs) have been expected to predict adverse events in humans more accurately. Based on the international trend on animal alternative methods and the current COVID-19 pandemic, in vitro methods and computational approach are accelerated to improve the efficiency of drug development, ensure the patients' safety and speed up the review process.

In this session, we would like to summarize the current status and future perspectives of pharmacological assay system using NAM in drug development.

Pathology-dependent protein-protein interactions (PPIs) have been attracted attention as a new drug target, since this strategy is expected to reduce the risk of adverse effect caused by chronic drug treatment. Our group investigated the molecular mechanism underlying the development of cardiac remodeling, especially focusing on myocardial stiffness associated with fibrosis and myocardial early senescence associated with mitochondrial abnormality and oxidative stress. The pathology-dependent formation of PPIs is revealed to involve post-translational (redox-dependent) modifications of target proteins or the escape of target proteins from endoplasmic reticulum-associated degradation (ERAD) system. We performed drug screenings to inhibit these PPIs, using approved drugs whose efficacy and safety are guaranteed by Japanese government. In fact, pharmacological disruption of pathology-dependent PPIs was found to improve chronic heart failure in mice. These results strongly suggest that targeting pathology-dependent PPIs using approved drugs will become a breakthrough strategy for the treatment of intractable cardiovascular diseases.

Angiotensin-converting enzyme (ACE) 2 is a multi-functional protein, including carboxypeptidase, virus receptor and regulator of amino acid transporter. ACE2 is an essential receptor for cell entry of SARS-CoV-2, resulting in explosive increase of infected people and COVID-19 patients. On the other hand, ACE2 is a carboxypeptidase which degrades angiotensin II, and thereby is a critical regulator of cardiovascular physiology and pathology. The enzymatic activity of ACE2 is protective against acute respiratory distress syndrome (ARDS). Upon infection, both SARS-CoV-2 and SARS coronaviruses downregulates ACE2 expression, likely associated with the pathogenesis of ARDS. Soluble forms of recombinant ACE2 can be utilized as a decoy to neutralize SARS-CoV-2 and a supplementation of ACE2 carboxypeptidase activity, whereas the cost for mass-production of recombinant ACE2 in mammalian cells is high and thus being obstacle for clinical development. We have identified B38-CAP as a bacteria-derived ACE2-like enzyme, which degrades angiotensin II in mice and is easily prepared in E. coli protein expression system (Nat Commun. 2020). Importantly, B38-CAP protects from SARS-CoV-2 infection-induced lung injury in mice and hamsters (Nat Commun. 2021). Our results indicate that increasing ACE2-like enzymatic activity is a potential therapeutic strategy to alleviate lung pathologies in COVID-19 patients and also implicate the utility of bacteria-derived generic recombinant protein in drug development.

In cancer chemotherapy, new drugs such as immune checkpoint inhibitors have been introduced into clinical practice, improving treatment outcomes compared to previous years. As more and more patients continue anticancer treatment for a long period, the management of side effects caused by anticancer drugs has become a more important issue. Therefore, the development of treatments for side effects associated with cancer chemotherapy may contribute significantly to the completion of anticancer treatment, as well as improving patients' quality of life. 

In recent years, a drug discovery strategy called drug repositioning (DR) has been implemented, in which a new pharmacological action of an already approved drug used in clinical practice is discovered and developed as a treatment for another disease.

The major advantage is that the time and cost required for drug development can be significantly reduced because approved drugs have already undergone clinical trials and information on safety and pharmacokinetics in humans has been accumulated. To conduct DR research efficiently and rationally, we are conducting "database-driven drug repositioning research," a strategy to narrow down drugs, diseases, and molecules to be targeted for DR by utilizing medical big data, life science databases, and drug discovery support AI system, and to validate them in basic and clinical research.

At this symposium, I would like to present our recent studies and discuss the current status and issues of database-driven pharmacological research.

Various animal models have been created for elucidating the pathophysiological mechanisms of pulmonary hypertension (PH) and developing new therapeutics. Each model has its unique characteristics. Comparative transcriptome analysis of two models, in which a phenotype of interest is observed in one but not the other model, can be used to get clues related to the phenotype. In this study, we first compared the transcriptome of lung tissues from two different rat PH models. One model used Su5416, an agonist for aryl hydrocarbon receptor (AhR) and hypoxia to induce PH with the development of neointimal hyperplasia and plexiform layer. Both neointimal hyperplasia and plexiform layer are characteristics in PH patients. The other model used hypoxia only to induce PH in which neointimal hyperplasia and plexiform layer seldom occur. The comparison revealed a cluster of genes with increased expression only in Su5416/hypoxia model. We then compared the transcriptome of lung tissues from Su5416/hypoxia model with and without the treatment of an antagonist for AhR. Integration of these analyses identified several genes that were increased in the Su5416/hypoxia model and regulated by AhR signaling. These genes may be involved in the mechanisms underlying the formation of neointimal hyperplasia and plexiform layer in PH. In this symposium, we would like to demonstrate our study with the validation of these genes using PH model rats and discuss the possibility of these genes as novel therapeutic targets.

Traumatic brain injury (TBI) can cause the development of posttraumatic epilepsy (PTE) characterized by delayed onset. Increased convulsion risk persists for a long period, from a few months to several years after TBI. The late-onset PTE is often pharmacoresistant and occurs after an unpredictable latency. Thus, the latent period from TBI to the occurrence of the first unprovoked seizure may offer a window of opportunity for preventing the late-onset PTE. To clarify TBI pathology leading to the late-onset PTE, we observed changes of each cell type constituting neurovascular unit (NVU) in mice subjected to controlled cortical impact (CCI), which is an experimental traumatic brain injury at postoperative day 0-28. CCI mice showed that　increased PDGFRβ expression in pericytes precedes increased Iba1 and GFAP expression in glial cells and neuronal hyperexcitability indicated by pilocarpine-induced convulsive behavior. Treatment with an inhibitor of PDGFRβ in the early phase after CCI suppressed microglial activation and neuronal hyperexcitability at postoperative day 28. Our results indicate that TBI-induced activation of pericytes characterized by increased PDGFRβ expression may drive the development of dysregulated NVU coordination including glial activation and neuronal hyperexcitability after TBI leading to the onset of PTE.

Febrile seizures are caused by an abrupt increase in body temperature. They are sometimes recurrent, and the more seizures are triggered, the higher the risk of epilepsy and psychiatric disorders increase after growing up. Prevention of febrile seizure is considered to be one of the effective countermeasures in protecting the future health of children. Docosahexaenoic acid (DHA) is an important nutrient especially during pregnancy and childhood and is reported to suppress several types of epilepsy. Therefore, we challenged maternal DHA intake to prevent febrile seizures and secondary epilepsy after growth in mice. We used a heat chamber for febrile seizure induction. Intake of DHA during pregnancy and infancy increased the amount of DHA in the brain of offspring. DHA prolonged the seizure latency and increased body temperature at which the first seizure occurred, indicating that maternal DHA intake decreases febrile seizure sensitivity. The mice with febrile status epilepticus showed increased susceptibility to seizures induced by pentylenetetrazol. DHA intake during infancy suppressed the appearance of excitatory electroencephalography and prolonged the seizure latency. Collectively, DHA intake during pregnancy and infancy is of significance in protecting infant from seizures as well as conserving health after growth.

This presentation focuses on the strategies to prevent West syndrome (WS), an epileptic encephalopathy, wherein the brain is damaged not by "trauma" but by "inflammation." The most effective treatment for WS is ACTH, which has potent anti-inflammatory effects, suggesting that inflammatory responses, including cytokines, are involved. Interleukin-1β (IL-1β) is a proconvulsant, and its antagonist, Interleukin-1 receptor antagonist (IL-1RA), has potent anticonvulsant effects. Notably, serum IL-1RA increased after the improvement of WS, suggesting that IL-1RA may be involved in the antiepileptic effect of ACTH, which is yet to be clarified. Using flow cytometry analysis of patients with refractory epilepsy, we observed higher levels of intracellular IL-1β in monocytes but not in the plasma, peripheral T cells, or NK cells and also found a correlation with the frequency of convulsions. Peripheral blood cells have been found to invade the central nervous system and aid the pathogenesis of epilepsy. Thus, we focused on pericytes to prevent peripheral cells from invading the central nervous system. Pericytes might function not only as tight junctions of the blood-brain barrier but also as key regulators of brain inflammation. This presentation highlights our study results on affected children, even though our efforts are limited.

Stroke is a prevalent medical condition, a leading cause of disability and the third most common cause of death. Post-stroke seizure/epilepsy (PSS/E) is a common complication, accounting for 30–40% of epilepsy cases in older adults. In this study, we have discussed GABA_A receptor-mediated seizure susceptibility after photothrombotic (PT) cerebral stroke in aged mice. Generally, young adult mice aged around 10 weeks are widely used in stroke experiments. Our study is based on the facts that most strokes are diagnosed in the older adults and PSS/E is a common complication of stroke. Hence, we utilized the PT model of cerebral ischemia in aged mice. For this purpose, we examined susceptibility to seizures to use low-doses of pentylenetetrazol (PTZ) injections, and seizures were monitored by video and EEG. Susceptibility to PTZ in aging mice increased relative to that in younger adults. A month after PT stroke, older mice still exhibited severe late-onset convulsive seizures. Anti-seizure medications like levetiracetam showed temporary seizure control in such cases. Thus, GABA_A receptor-mediated seizures susceptibility post-PT stroke in aging mice may be an effective method for examining the effects of anti-seizure medication.

Antibody drugs have been widely applied to various diseases such as cancer and rheumatoid arthritis. Recently, antibody drugs against amyloid-β have attracted attention as a therapeutic agent for Alzheimer's disease, and are also expected to be applied to central nervous system diseases. On the other hand, delivery of antibody drugs to the central nervous system is not easy due to the existence of the blood-brain barrier and other barriers. Intranasal administration is a method of direct drug delivery from the nasal cavity to the brain without blood flow, and drugs are transported into the brain through the olfactory and trigeminal nerves, or the intercellular spaces and parenchyma of the olfactory epithelium. This method is considered to be particularly suitable for the administration of macromolecular compounds, and several biopharmaceuticals have already been marketed as nasal drop formulations.

We have focused on the role of high mobility group box-1 (HMGB1), one of the damage-associated molecular patterns, and the proinflammatory cytokine interleukin-6 (IL-6) in chronic pain and investigated their potential as novel drug targets. In this symposium, we will present the effects of intranasal administration of HMGB1 and IL-6 antibody on nociceptive hypersensitivity and emotional and cognitive dysfunction associated with long lasting pain in chronic pain models.

The development of chimeric antigen receptor (CAR) T-cell therapies has been opening a new era in cell-based immunotherapy. However, like classical medicine-based therapies, efficiency of functional cell delivery toward target organs and cells is crucial for producing beneficial and adverse-effects of the cell-based therapy. To explore the pathogenesis of allergy diseases, we have been developing new mouse models of allergic inflammation, especially focusing the role of antigen-specific T cells. Antigen administration to mice transferred with in vitro-differentiated antigen-specific T cells evokes inflammatory responses at the site of antigen exposure. In response to chemokines produced and with the usage of several cell adhesion molecules upregulated, allergen-specific T cells efficiently recruit and proliferate in the inflammation sites. T cell has capacity to obtain various immune-regulatory properties following in vitro culture under specific conditions. Large number of T cells expressing T cell receptors reactive to specific antigens can be prepared by employing somatic cell nuclear transfer techniques. I would like to provide these data probably helpful for considering new cell delivery system in cell-based immunotherapy.

The concept of "Precision Medicine", in that genomic information in the individual patient are used in treatment and dosing protocols, is rapidly expanding. In particular, a series of RNA vaccine against COVID-19 developed by BioNTech / Pfizer and Moderna are now widely used worldwide with extremely high preventive results.

Since the mRNA molecule per se is overwhelmingly large. In addition, the mRNA is easily degradable. Thus, the development of proper DDS to stabilize mRNA in extracellular environment, and to delivery it to the cytoplasm of target cells is extremely needed in mRNA-based medicine.

In this presentation, we will summarize the current development of lipid nanoparticles (LNP) and lipid materials for mRNA/nucleic acid drug discovery. Among them, we will focus on our materials (SS-cleavable and pH-activated lipid-like material), those are designed so that nanoparticles are actively disintegrated by being cleaved in a cytoplasmic reducing environment. Recently, we are also using our ssPalm as a delivery vehicle of small molecule drugs. In the presentation, I will report on the research to use ssPalm for the delivery of drugs to regulate the cancer immunity cycle.

　Tanaka H et al. Self-Degradable Lipid-Like Materials Based on "Hydrolysis accelerated by the intra-Particle Enrichment of Reactant (HyPER)" for Messenger RNA Delivery. Advanced Functional Materials, 30, 1910575 (2020).