Human iPS cell technologies are attracting a lot of interest in the nonclinical test for drug development for several years. Notably, it was demonstrated that proarrhythmia risk assessment using human iPS cell-derived cardiomyocytes and multi electrode array (MEA) system showed high clinical predictability. In the proarrhythmia risk assessment, it was well known that field potential duration (FPD) was a surrogate marker for QT duration and wave changes which showed early afterdepolarization (EAD) were observed by application of hERG channel blockers in MEA system. Arrhythmia risk were classified by FPD prolongation and occurrence of EAD. On the other hand, heart failure induced by anti-cancer drugs is an important issue in safety pharmacology. Many in vitro contractility platforms are introduced from some companies. In vivo echo and catheters assess whole heart contractility function. Parameters acquired by in vitro contractility platforms are absolutely different from parameters acquired by in vivo data. It is difficult to compare between in vivo data and in vitro data and to evaluate usefulness of in vitro platforms. In this symposium, I would like to discuss about evaluation of correlation between in vivo contractility data and in vitro contractility data.

Unexpected hepatotoxicity is a major obstacle to drug development, frequently underlying discontinuation of drug development and withdrawal from the market. Drug metabolism and pharmacokinetics are often involved in hepatotoxicity. Therefore, it is necessary to establish in vitro and in vivo models for predicting drug metabolism and related cases of hepatotoxicity. We focused on chimeric mice with humanized liver, consisting predominantly of human hepatocytes. Various human genes, encoding drug-metabolizing enzymes and transporters, are expressed in the liver. These chimeric mice were found to be useful for qualitative and quantitative prediction of human metabolite formation, pharmacokinetics, and drug-drug interactions. They are also useful for predicting hepatotoxicities, such as cholestasis and steatosis. Moreover, fresh hepatocytes isolated from the chimeric mice are useful for in vitro screening. It is possible to compare in vitro and in vivo profiles of the same chimeric mice. One possible drawback could be the contribution of residual mouse hepatocytes and extrahepatic tissues, which may detract from predictability. Recently, novel improved models which overcome these problems have been developed, and increased prediction accuracy is expected.

In recent years, oligonucleotide therapeutics (ONT), such as antisense oligonucleotides and siRNA therapeutics, have been attracting attention as a new modality to treat intractable and genetic diseases. The mechanisms of ONT-mediated toxicity can be conceptually divided into hybridization-dependent effects and hybridization-independent effects. The hybridization-dependent effects, also called off-target effects, are potentially caused by inadvertent binding of ONT to unintended RNAs. Toxicity induced by off-target effects could not be evaluated by conventional animal studies theoretically because of differences in the genome sequence between humans and other animals. Consequently, to predict toxicity induced by off-target effects, assessment of off-target effects with in silico analysis using a human RNA database and in vitro expression analysis using human cells has been proposed. The toxicity via hybridization-independent effects is likely to be due to interactions between the ONT and cellular proteins and it is assumed that the toxicity can be evaluated by animal experiments, though species differences are also observed in the hybridization-independent mechanism (e.g. innate immune activation via Toll-like receptors). Against this background, we develop and evaluate methods for safety assessment of ONT using human-derived cells and humanized animals. In this talk, I would like to outline the basis of ONT and discuss the IVIVE and species differences.

Modern society exposes us to social and environmental stress. Acute stress activates the sympathetic nervous and neuroendocrine systems as a survival mechanism. However, chronic stress is a risk factor for mental illnesses and induces behavioral changes such as social avoidance and elevated anxiety. 

Since several groups reported that inflammatory cytokines are increased in the peripheral blood from depressive patients, we are studying the role of inflammation in chronic stress-induced behavioral changes. We previously reported that acute stress activates mesocortical dopaminergic pathway to inhibit the induction of social avoidance. On the other hand, chronic stress suppresses this pathway by the inflammatory molecule to induce social avoidance. This finding suggests that repeated exposure to stress causes inflammation. Consistently, chronic stress activates microglia, an immune cell in the central nervous system, faster than acute stress.

I will show that chronic stress induces neuroinflammation originating from microglia and this inflammation plays a crucial role in behavioral changes in this symposium. Furthermore, I'd like to introduce our latest finding.

Postmortem brain analysis and PET imaging analysis are two major methods to estimate microglial activation in human subjects, and these studies have suggested activation of human microglia in the brain of patients with various neuropsychiatric disorders. However, by using the above methods, only limited aspects of microglial activation can be measured. We have originally developed a technique to create directly induced microglia-like (iMG) cells from fresh human peripheral blood monocytes adding GM-CSF and IL-34 for 2 weeks, instead of brain biopsy and iPS technique (Ohgidani, Kato et al. Sci Rep 2014). Using the iMG cells, dynamic morphological and molecular-level analyses such as phagocytosis and cytokine releases after cellular-level stress exposures are applicable. Just recently, we have confirmed the similarity between human iMG cells and brain primary microglia by RNAseq (Tanaka, et al. Front Immunology 2021). We believe that patients-derived iMG cells will take a role as one of the important surrogate markers to predict microglial activation in human.

Here I will report the latest insights on astrocyte signaling in the adult neural circuits, by using multiple integrated approaches, including calcium imaging, electrophysiology, opto/pharmaco-genetics, mouse behavioral tests, RNA-seq and new astrocyte manipulation tools that we recently developed. First, I will describe mechanisms of bi-directional neuron-astrocyte communications in the striatum that lead to hyperactivity and disrupted attention via a synaptogenic cue (ref 1). Second, I will present how astrocytes respond to distinct perturbations and how we can use the molecular signaling information for phenotypic benefits in neurodegenerative disease mouse models, e.g. Huntington's disease (ref 2). Third, I will report a validation work for a new effective, specific and consequential attenuation tool of astrocyte Gq-GPCR signaling in vivo (ref 3). Taken together, our findings show that signaling from astrocytes to neurons is sufficient per se to alter synapses, circuits and behavior. We also provide new tools to study such astrocyte-neuron dynamics.

References:

1. Nagai J et al., Hyperactivity with disrupted attention by activation of an astrocyte synaptogenic cue. Cell 2019

2. Yu X and Nagai J et al., Context-specific striatal astrocyte molecular responses are phenotypically exploitable. Neuron 2020

3. Nagai J et al., Specific and behaviorally consequential astrocyte Gq GPCR signaling attenuation in vivo with ibARK. Neuron 2021

Emerging evidence suggests that astrocytic GqPCR signaling play an important role in animal behavior such as cognition, learning and memory. It has been shown that impairments of GqPCR-mediated Ca²⁺ signaling in astrocytes and/or gliotransmitter release derived from astrocytes in the hippocampus and/or cortex cause depression-like behaviors in mice. However, it is not clear whether astrocytic GqPCR signaling is a target for driving behavior related to anxiety and motivation. We focused on the ventral hippocampus (vHIP), which contributes to anxiety and depression, and investigated the effect of chemogenetic manipulation of astrocytic GpPCR signaling in innate behaviors related to anxiety or depression. We expressed hM3Dq, a Gq-DREADD, selectively in astrocytes in the vHIP by injecting AAV bilaterally. In acute slices, Gq-DREADD activation caused robust Ca²⁺ signals in vHIP astrocytes. Gq-DREADD activation of vHIP astrocytes increased travel distance in the center of the open filed without affecting total travel distance and travel velocity, suggesting that GqPCR activation in vHIP astrocytes may be anxiolytic. Gq-DREADD activation of vHIP astrocytes did not affect immobility time in tail suspension test. The data suggest that astrocytes in vHIP play an important role in innate behavior which is related to anxiety.

While cell therapy has great potential to overcome unmet medical needs in a wide range of disease areas, it remains in its infancy in medical systems globally and is currently only being used to treat a small number of diseases. These two factors underlie Astellas’ involvement in cell therapy. Many avenues require exploration, including how the science of cell therapy will progress and how we can help patients for whom no treatment options exist.

Over the past few years, Astellas has acquired and developed technologies to build a broad cell therapy-related portfolio. In the research and development of cell therapy, an area that is attracting global attention and being undertaken by many companies and research institutions, Astellas is making steady progress toward practical application. Our approach centers on the use of differentiated cells derived from allogeneic pluripotent stem cells because of their potential to be mass-produced as off-the-shelf products and their suitability for practical use. We believe this approach will allow us to provide treatment options more rapidly than using autologous pluripotent stem cells and meet a wider range of unmet medical needs. To do this, however, we will need to overcome important challenges, one of which is the fact that differentiated cells derived from allogeneic pluripotent stem cells are susceptible to immune rejection. Other challenges are also expected as the cell therapy manufacturing-associated value chain moves toward mass production.

We envision that cell therapy will become a reality by around 2030, with many patients benefitting through expansion to a wider range of target diseases. In this session, I will introduce Astellas' initiatives for cell therapy.

Antibodies play an important role in the defense mechanism of the body by inducing antigen neutralization, complement-dependent cytotoxicity, or antibody-dependent cellular cytotoxicity / phagocytosis, to eliminate pathogens. Antibody therapeutics utilize the high specificity in antigen recognition and the originally equipped functions of the antibody. Since the first therapeutic antibody OKT3 was approved in 1986, more than 100 drugs have been approved and used in clinical practice. Antibody therapeutics have continued to evolve since its emergence, and at present, could be considered as a mature platform technology in the pharmaceutical industry. Antigenicity, one of the initial challenges in antibody therapeutics, were resolved as chimerization, humanization, and fully human antibody production technology were established. Attempts to enhance the effector function of the antibody, and in recent years, antibody-drug conjugates or bispecific antibodies have been developed. In this presentation, while looking back on the history and touching on recent topics, the future development of antibody therapeutics will be discussed.

Shionogi is enhancing our existing strengths in small-molecule drug discovery and also working on a wide range of therapeutic approaches (modalities) to meet the broad range of patient and societal needs. We aim to increase our drug discovery success rate by using the best drug discovery approach for each target therapeutic area.  That means trying medium-sized molecules such as peptides and nucleic acids. We are building new strengths from the base provided by this SAR engine for small-molecule drug discovery. Peptides do not readily pass through cell membranes because of their high molecular weight and chemical characteristics, so they can only be used to target extracellular molecules. To tackle this issue, we combined our small-molecule drug discovery capabilities with PDPS (Peptide Discovery Platform System) technology. After extensive research, we have made progress with a technology to optimize candidate peptide structures that results in a dramatic improvement in membrane permeability.  This has significantly broadened the scope for the discovery of pharmaceuticals to treat diseases where there are substantial unmet clinical needs, such as conditions with limited treatment options.

Alzheimer's disease (AD) is the most common dementia associated with brain inflammation. Aducanumab is the first disease-modifying therapeutics approved by the US-FDA for mild AD patients. Improvements of neuronal plasticity and neural networks in dementia are the next stage of mild AD drug development. However, none of the improvers in neurotransmission failed to prevent the progression to AD. We here introduced the novel improver of brain function through T-type calcium channel activation. Reduced T-type calcium channel levels are reported in aged brain and its inhibition by Abeta oligomers impair spine formation in the brain. We developed T-type calcium channel enhancer SAK3 which activates calcium currents in Neuro2A cells overexpressed Cav3.1 and Cav3.3 but not Cav3.2 channels. SAK3 promoted the hippocampal long-term potentiation (LTP) through CaMKII activation. CaMKII activation in the hippocampus and cortex were also associated with improvement of spine morphology in APP knock-in mice. The proteasome activation possibly mediated CaMKII dependent phosphorylation of Rpt-6 subunit of 19S proteasome in APP knock-in mouse brain. Enhancement of T-type calcium channel in neuroprogenitor cells in the hippocampus was accounted for improvement of neurogenesis, thereby ameliorating depressive behaviors. the diagnostic technology defined early mild dementia patients is critical for promoting clinical development of disease-modifying therapeutics. In the symposium, we also discuss the novel diagnostic technology for mild AD to define brain inflammation.

The science and technology infrastructure in drug discovery for central nervous system (CNS) diseases and disorders has been greatly developed over the past decade. Various hypotheses have been proposed about the pathogenesis and progression of CNS diseases and disorders, and efforts to develop new drugs for rare diseases are also becoming active. The drug modalities have expanded from small molecules to medium molecules, antibodies, proteins, nucleic acids, cells, and even digital, and the drug discovery process is changing dramatically. Drug discovery researchers require a wide variety of specialized knowledge and techniques. As research tools such as human induced pluripotent stem cells and genetically modified animals become increasingly complex, it is often difficult to prepare and procure them within a pharmaceutical corporation. Furthermore, patient stratification in clinical studies and translational research to predict the efficacy of drug candidates have become important pillars in drug discovery research. Under such circumstances, it is becoming difficult for pharmaceutical corporations to complete all elements necessary for drug discovery research on their own, and collaboration with partners with expertise is becoming essential to improve the quality and speed of research. In this presentation, I would like to introduce what corporations are seeking, hoping that this symposium will generate some ideas for new approaches and collaborations.

Psychiatric diseases are clearly different from other physical diseases in that the pathophysiology of the diseases is still unknown. Therefore, except for dementia and epilepsy, there are no objective biomarkers for diagnosis. A set of examinations is performed to rule out physical diseases, but there is no diagnostic evaluation tool for depression that corresponds to, for example, blood pressure, blood glucose, x-rays, or echocardiography.　The reasons for the poor understanding of the pathophysiology of psychiatric disorders include the difficulty in sampling the human brain and CNS, and there are no pure animal models for depression or bipolar disorder because the human and animal brains are very different. Another barrier regarding drug treatment is that high-molecular-weight drugs cannot reach the central nervous system due to the blood-brain barrier.

In this lecture, from the standpoint of being involved in several clinical trials as a medical expert, I would like to introduce some of the mood disorder-related drugs currently under clinical trial in Japan, despite the above limitations. I would like to prospect to solve the issue of psychiatric disorders described above from genomics and PGx and new technologies that are expected to overcome the barriers of psychiatry.

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 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, NMDA receptor antagonists, adenosine A2A receptor antagonists, COMT inhibitors, and MAO-B inhibitors have been marketed. With regard to the causal therapies, 57 out of 145 clinical trials for Parkinson's disease registered on ClinicalTrials.gov in January 2020 were related to disease-modifying drugs. Anti-a-synuclein antibodies, GLP-1 agonists, and kinase inhibitors have been examined in clinical trials as disease-modifying drugs, but no drug has been obviously demonstrated to inhibit the progression of Parkinson's disease to date. It is not easy to prove the beneficial results obtained from basic research in clinical trials. Especially for neurodegenerative disorders such as Parkinson's disease, it is more difficult to demonstrate clinical efficacy of disease-modifying 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.

IL-31 receptor blockade is a promising treatment strategy for chronic itch diseases. However, in vivo contributions of IL-31 receptor expressed by different cell types and the downstream JAK-STAT signaling pathway to the itch induction remain undetermined. Here we show that IL-31 receptor in sensory neurons but not in keratinocytes is required for the itch induction by IL-31. Surprisingly, unlike the previous report, our study indicated that the acute itch induction by an IL-31 injection as well as the chronic itch induction in the atopic dermatitis model was completely dependent on STAT3 in sensory neurons. In addition, we found that STAT3 in sensory neurons plays a suppressive role in inflammatory skin swelling. Thus, our study suggests the importance of STAT3-dependent gene expression in sensory neurons for the itch induction and regulation of inflammation.

Atopic dermatitis (AD) is a chronic inflammatory skin disease affecting 7–15% of the general population in Japan. AD is characterized by recurrent eczematous legions and intense itch. As itch sensation induces scratching behavior, which exacerbates the skin inflammation and disturbs the quality of life of affected individuals, chronic itch is a challenging clinical problem in the treatment of AD. IL-31 is a major pruritogen associated with AD, and mainly produced by helper T cells. However, the mechanism for IL-31 production remained unknown.

Mutations of DOCK8 in humans cause a combined immunodeficiency characterized by AD. We have previously reported that DOCK8-deficient (Dock8^–/–), but not Dock8^+/–, mice spontaneously develop AD-like skin disease, when crossed with transgenic mice expressing AND T-cell receptor (TCR). Importantly, upon stimulation with cognate antigen, helper T cells from Dock8^–/– AND Tg mice produce large amounts of IL-31 in a manner depending on the transcriptional factor EPAS1. Although EPAS1 is known to control hypoxic response through the interaction with arly hydrocarbon receptor nuclear translocator (ARNT), EPAS1-mediated IL31 promoter activation is independent of ARNT, but in collaboration with SP1. Therefore, EPAS1–SP1 axis could be a drug target for treatment of AD-associated itch. In my talk, I will discuss the molecular basis for IL-31 production in helper T cells, referring to its application to drug development.

Chronic itch is a cardinal symptom observed in patients with atopic and contact dermatitis for which existing treatments are largely ineffective.  A subset of excitatory neurons in the spinal dorsal horn (SDH) that expresses gastrin-releasing peptide receptors (GRPR) is critical for itch transmission.  Despite recent progress in our understanding of itch transmission circuitry, the mechanism underlying chronic itch remains largely unknown.  We have recently shown that GRP-induced excitability of GRPR⁺ SDH neurons is potentiated in a mouse model of chronic itch and that this sensitization requires a non-cell-autonomous signal from SDH astrocytes—glial cells that are activated under chronic itch conditions. In fact, suppressing the reactive astrocytes or decreasing LCN2 expression attenuates the potentiation of GRP-induced neuronal excitation.  Furthermore, glutamatergic excitatory inputs onto GRPR⁺ SDH neurons are also strengthened under chronic itch conditions.  This involves a DRG neuron-derived factor whose expression is upregulated by dermatitis. In my talk, I will show a model of the mechanism underlying the sensitization of GRPR⁺ itch transmission neurons in the SDH by signals from glia and primary afferents.

Atopic dermatitis (AD) is a common, chronic inflammatory skin disease that is characterized by skin barrier dysfunction, inflammation and intense itch. Although the exact mechanisms behind its pathogenesis remain unclear, it is evident that the complex interplay among barrier dysfunction, inflammation and itch are critical in its development, progression and chronicity. Therefore, the treatment described in the current guideline are based on the pathogenesis of the disease: 1) topical anti-inflammatory therapy for inflammation, 2) moisturizer skin care for physiological abnormalities of the skin, and 3) search for and countermeasures against aggravating factors. However, in AD, itch is the most common problem for patients and its control is often very difficult. This may be due to the fact that the mechanism of itch in AD is very complex and probably differs from patient to patient.

With the discovery of key pathways responsible for the atopic skin inflammation and atopic itch, a new era in the treatment of AD began. Various substances involved in the pathogenesis of AD have been identified and many novel therapeutic agents targeting them are under development. New drugs currently employ two strategies of inhibiting specific key mechanisms in the pathogenesis of AD. The first strategy is based on inhibition of the type2 cytokines such as IL-13/IL-4 and IL-31. This group is represented by dupilumab and nemolizumab. The second strategy is based on reducing nonspecific inflammation using JAK-STAT inhibition. The already available baricitinib and upadacitinib, and upcoming abrocitinib are representative.

In this symposium I would like to discuss the clinical trial results of many new drugs, the action points of new drugs based on the pathogenesis of AD, their expected effects, and the experience of their use in clinical practice. In addition, I would like to discuss the future issues in the new era of treatment with many new drugs.

A growing body of evidence suggests that gut microbiota-derived substances play a key role in the control of host body functions including cardiovascular function. Accordingly, various substances converted by gut microbiota can have beneficial or adverse effects on human health. Among them, indoxyl sulfate, which is metabolized in the liver from indole converted from dietary tryptophan by bacterial tryptophanases in the colon, is known as a protein-bound uremic toxin. Trimethylamine N-oxide (TMAO), which is generated via the oxidization of gut microbiota-derived trimethylamine by hepatic flavin monooxygenases, is known as an accelerator of atherosclerosis. Although some reports are investigated the relationship between gut microbiota-derived substances and vascular function, there are few studies focused on the direct effects of indoxyl sulfate or TMAO on vascular responses including endothelium-dependent relaxation. We recently found that these two substances could modulate endothelium-dependent relaxation in rat isolated arteries. In the present symposium, we will focus on the relationship between indoxyl sulfate and TMAO on vascular function. This information will provide a conceptual framework that would allow the development of novel preventive and/or therapeutic approaches against vasculopathies.

Emerging evidences indicate that a microbial imbalance (dysbiosis) is linked to several diseases including metabolic cardiovascular diseases. A fecal microbiota transplantation from hypertensive human donor to germ-free mice causes blood pressure elevation, suggesting that gut microbiome may mediate development of hypertension. One recent report demonstrated that an increasing number of gram-positive Streptococcus was found in the feces of adult spontaneously hypertensive rats (SHR) with an increased intestinal permeability (Santisteban et al., 2017). However, detailed mechanism by which the dysbiosis induces an increased blood pressure remains unknown. We recently found an elevated plasma level of streptolysin O (SLO), a streptococcal exotoxin, in younger SHR aged 10-14 week-old. Treatment of vascular tissues with SLO in vitro impaired endothelial-dependent vasorelaxation, which was mediated by PKCβ-induced phosphorylation of an inhibitory site of endothelial nitric oxide synthase, possibly through TLR4. Intravenous administration of SLO in normal rats impaired an acetylcholine-induced decrease in blood pressure. We confirmed that intestinal permeability was increased with a decreased tight junction protein in younger SHR (10-14-week-old). We conclude that streptococcal exotoxin causes vascular endothelial dysfunction and may contribute to a dysregulation of blood pressure control. This finding might contribute to further understanding of the potential roles of circulating enteric toxin in the pathogenesis of hypertension.

Sudden cardio death (SCD) is a sudden, unexpected death caused by loss of heart function. The cause of SCD is not well understood, and there is no effective medicine. GPx4 is the antioxidant enzyme which reduces the oxidized phospholipid using glutathione. We recently established new sudden cardio death mouse model with lipid peroxidation in heart GPx4 KO mice (KO mice) induced by change of vitamin E diet to normal diet. Next we screened the inhibitor of sudden cardio death using this mouse model and found that drinking of antibiotic cefoperazone (CPZ) in water completely could suppress the sudden cardio death in mice without direct antioxidant activity, but not the abdominal cavity dosage. To clarify whether CPZ resistant gut bacteria could suppress the lipid peroxidation dependent sudden cardio death in mice or not, we generated the germ free KO mice and a para-sterility KO mouse pretreated by four antibiotics. These KO mice drinking CPZ could not rescue sudden cardio death induced by decrease of the content of vitamin E in diet, indicating that CPZ resistant gut bacteria could suppress sudden cardio death in mice. We next examined the changes of enterobacterial flora by treatment of CPZ using the next generation sequencer. We found that CPZ treatment dramatically changed enterobacterial flora to almost only one bacteria in mice. This bacteria transplanted KO mice could rescue the cardio sudden death in mice. From these results, we first identified one enterobacteria that could suppress the sudden cardio death with lipid peroxidation in mice.

Severe asthma, a part of difficult-to-treat asthma, represents a poorly controlled asthma despite maximally optimized treatment and its adherence. Uncontrolled asthma is judged by poor symptom control, frequent exacerbations requiring oral corticosteroids (OCS), or severe exacerbations requiring hospitalization. It is considered to be 5 percent or less of all asthmatics (GINA 2019).

In the treatment of severe asthma, in parallel with progress in the elucidation of the it pathogenesis at the end of the 20th century, biologics targeting particular molecular pathways (such as IgE, IL-5,) have become key players over the past two decades, and our humanized anti IL-5 monoclonal antibody, mepolizumab, has also become a part of it.

It was already evident by the end of the 20th century that eosinophils have a key role in asthma pathologies, partly based on mouse models.

Attempts to show efficacy of humanized anti IL-5 monoclonal antibodies in asthma were initially unsuccessful in mild asthma, but a proof-of-Concept study targeting moderate-to- severe asthma in Phase IIa, and other POC studies for severe eosinophilic asthma to show efficacy in exacerbation reduction and reducing OCS have led to the identification of populations that respond to this asset.

In addition, now efficacy (exacerbation, OCS-reduction, QOL) and safety data including relatively longer period one are shown with phase IIb-III studies including DREAM, MENSA, SIRIUS, MUSCA, COSMOS, COSMEX, COLUMBA, and now available in a clinical setting in Japan.

Overseas, REALITI-A studies have been reported on effectiveness and safety as a real-world data. In the meantime, evidence generation mainly based on database-based studies have been currently conducted and published to show effectiveness also in Japan.

Recent advance of medications brings more effective treatment intervention to the patients with asthma. As far as referring guidelines, approximately 90% of patients with asthma can acquire good control. However, some of them resist conventional treatment. Most of them have corticosteroid-resistant airway inflammation. There are two ways to deal with this problem. The first one is biologics. Biologics are introduced for some patients with difficult asthma and outstanding results are also seen. The second one is a treatment focusing on cancellation of corticosteroid-resistance. In early 2010s, a new-intracellular signaling pathway (phosphatydilinositol-3-kinese pathway: PI3K pathway) is proven to be closely associated with corticosteroid-resistance (CR). PI3K-inhibitor should be promising to attenuate CR. But PI3K-inhibitor also has a toxicity when it is given systemically. To deal with this problem, drug-repositioning (DR) is a good option. To find out PI3K-inhibitor from numerous existing medicines enables us to inhibit PI3K pathway safely and effectively. The presenter has found both low-dose theophylline1) and long-acting beta-2 agonist2) have a potential to antagonize PI3K. These medicines have been prescribed for long time and their safety has already been proved. DR obtains cheap and safe options to treat specific intracellular signaling pathways. The presenter will reveal that the fundamentals of this strategy and show how to adjust it to real patients with difficult asthma in this talk.

1) To Y et al. Am J Respir Crit Care Med 2010

2)Rossios C et al. Br J Pharmacol 2012

Asthma therapy in general has improved a lot in recent years, but it is still a major problem that severe asthma, which accounts for 10 to 20%, still suffers from strong symptoms on a daily basis despite all therapeutic agents used in combination. American SARP and European ENFUMOSA started in 2000 to advance pathophysiological insights of severe asthma. Clinical usage of antibodies and inhibitors against IgE, TNF, IL-5, IL-4, IL-13, and TSLP are also accumulating. Some of these molecular-targeted drugs improve respiratory function and reduce acute exacerbations in patients with severe asthma. Until now, cytokines have been assumed to be involved in chronic inflammation, but it is also interesting to elucidate the pathways of how cytokines are involved in respiratory function and acute exacerbations. We registered approximately 100 steroid-dependent asthma patients in Japan. Although long-lasting poor control of the disease was considered the cause of severe asthma in the past, steroid dependence in one third of the cases occurred within 2-3 years after the onset. Steroid resistance seems a key process from the early stage of the disease. Steroid resistance of T cell level was induced by extracellular co-stimulation and cytokine signals. The inhibition may improve steroid sensitivity and treat steroid-resistant asthma. Therefore, we established a steroid-resistant asthma model for the first time by transferring steroid resistant T cell clones, and analyzed the steroid sensitivity recovery effect of CTLA4-Ig. In addition, a multicenter, double-blind, placebo-controlled exploratory trial was performed as a POC study investigating the efficacy of abatacept in treatment-resistant severe asthma. Elucidation of the pathophysiology and mechanism by which steroids do not work is expected to be a breakthrough for the prevention and treatment of severe asthma.

Glucocorticosteroids are principal drugs for the treatment of bronchial asthma. However, around 10 % of asthmatic patients do not respond adequately to the steroid therapy. The intractable asthma is considered to associate with dysregulated Th17 cells and resulting neutrophil invasion. In this presentation, we show that the inhibition of LAT1, a transporter of large neutral amino acids, provides a possible option to improve steroid-resistant asthma. We investigated the effect of JPH203, a LAT1 specific inhibitor, on allergic airway inflammation developed in BALB/c mice transferred with in vitro-differentiated ovalbumin-specific Th17 cells. JPH203 administration suppressed the allergen-induced bronchial hyperresponsiveness and neutrophil accumulation in the lungs, whereas no significant effects were observed by dexamethasone treatment. JPH203 inhibited cytokine production of Th17 cells along with reducing mTOR activity, in contrast to the less effect of dexamethasone, suggesting the possible mechanisms underlying the suppression of Th17-dependent and steroid-resistant airway inflammation by LAT1 inhibition. Our results propose LAT1 as a novel target for the treatment of steroid-resistant asthma.

The rapid development of big data and groundbreaking AI technology in recent years has brought about major changes in the medical field. Pharmaceutical companies are also promoting digital transformation by using big data to improve productivity, including more accurate drug discovery in a shorter period of time. Astellas has launched Advanced Informatics and Analytics function as a part of its digital transformation. We aim to further accelerate this effort as valuable innovation is increasingly being driven by data and analytics. In drug discovery research, we are promoting data-driven drug discovery that uses AI and analytics.

In this presentation, we will introduce an example of analysis of literature information. We believe that text mining will enable us to find new insights and hypotheses for drug discovery from a large amount of articles and intellectual property information. However, there are still many issues to be addressed in order to fully utilize the information in drug discovery research, and there are also many technologies to be incorporated. We will introduce our efforts with examples such as the analysis of disease mechanisms.

For improving low R&D efficiency in drug discovery and development, digital transformation (DX) based on big-data and data science technologies, such as machine learning, artificial intelligence and modeling & simulation, has been implemented in pharmaceutical and biotech industries. The process innovation by DX, called as data-driven drug discovery and development (D5), is supporting the rational decision making from exploratory research to late-stage clinical development, and enables identification of novel target molecules, optimization of lead compounds, clarification of pharmacological or toxicological mechanism-of-action, prioritization of development indications, facilitating of translational research, optimization of clinical trials, and others. In this presentation, I will explain the effectiveness and the technical issues of D5 approach by showing some case-studies related to computational drug repurposing as a good practice in D5 approach. And also, I will show the current application of deep learning and transfer learning for automation and speed-up of image data analytics in non-clinical pharmacological studies and drug screening. The D5 approach is an innovative research framework for leading to successful drug discovery and development by improving the R&D efficiency and changing of research process.

How quickly reach the goal / establish the platform of artificial intelligence (AI) for drug development is one of the biggest issue for most of pharmaceutical company. Model informed drug development (MIDD) is applied across the drug development phase, and biology / physiology based sciences. One of the key expected outcomes by MIDD is to estimate 3 view points of RIGHT which are "RIGHT dose", "RIGHT patients", and "RIGHT timing". To obtain three RIGHT, it is required to demonstrate drug exposure, drug penetration, pharmacodynamic biomarker response, and clinical outcomes. Quantitative system pharmacology (QSP) model is one the tool find these "RIGHT" and is give us the hypothetical resolution against the research/clinical questions. Integrated into wet experimental data, genetic analysis, drug binding, metabolism, polymorphisms, biological pathways. Accurate computational power is required to establish the appropriate quality of QSP model, therefore abilities of AI is required. To implementation of AI to resolve the dedicated model, it is expected to accelerated the speed of drug development and QSP model primed to change the landscape of drug development.

Alzheimer's disease is the most common agerelated neurodegenerative disease. The current treatment objectives for dementia are mainly to suppress the progression of dementia and to treat BPSD such as hallucinations symptomatically. Clinicians aim to reduce the dose as much as possible because drug therapy always has problems of limited effects and side effects, and also problems of polypharmacy.

On the other hand, spatial memory and ability of discriminating smells are correlated, and the sense of smell is important for establishing spatial memory. Smell promotes appetite and improves memory and learning through ghrelin. This may be one of the rationale for the effectiveness of aromatherapy for dementia. Nose is not only the start point of smell sensation, but also a transporting route to the brain. There are several aroma oils that are said to be good for dementia. Thus, the sense of smell and nose using aroma oil will become more important in the future as a route for early diagnosis of dementia and development of the preventive and therapeutic measure. 

Aromatherapy using essential oils is one of the complementary therapies that fills the gap between such drug treatments in the treatment of dementia. 

In this symposium, I would review essential oils that are used complementarily for the treatment of dementia and consider their purpose and usage.

Until now, aromatherapy using essential oils with many physiological andpharmacological effects has been introduced in various medical fields anddeveloped as medical aromatherapy because of its action to ameliorate mental andphysical disorders.In the meantime, in recent years, there have been manyreports on the effects of aromatherapy of essential oils on mental and physicalfatigue recovery, performance improvement, and appetite control. Against thisbackground, "sports aromatherapy" using the scent ofessential oils as well as sports massage using essential oils has attractedattention in the field of exercise. However, the current state of sportsaromatherapy has rarely been reported physiologically and pharmacologically,such as medical aromatherapy, and there are many unclear points about itseffects. To date, we have made evidence for developing sports aromatherapy inanimals and humans. In this symposium, we present our experimental data on thefunctional and morphological analysis of the regulation of feeding in thearomatherapy of essential oils by animal experiments and the effect of restoringfatigue during exercise by scent exposure in humans.

Sensorineural olfactory dysfunction is caused by damage in the olfactory neuroepithelium and central olfactory pathway. Although many types of drugs, such as zinc preparations, Chinese medicine, topical and systemic steroids, vitamins, and metabolic agents have been tested for the treatment of sensorineural olfactory dysfunction, none of them have been shown to be effective in placebo-controlled randomized controlled trials. In 2009, Hummel et al. reported that olfactory training using odorants was effective in improving sensorineural olfactory dysfunction. In their study, the patient group which did olfactory training with four odorants (phenylethyl alcohol (rose), eucalyptol (eucalyptus), citronellal (lemon), eugenol (clove) twice a day for 12 weeks showed better improvement of the olfactory function after the intervention period than the patient group which did not do the training. Since then, various protocols to change the intervention period, number of odorants, and intensity of odors have been tested for improving the efficacy. The cellular mechanism of why olfactory training is effective for the treatment of sensorineural olfactory dysfunction in humans is unknown, but based on the results of animal studies, it may reflect the olfactory input-dependent survival of olfactory receptor neurons in the neuroepithelium and granule cells in the olfactory bulb. A multi-center study of medical institutions in Japan is currently underway to establish a Japanese-style olfactory training program.

Odors elicit many kinds of emotion and emotion-based behaviors. While some odors induce genetically-inherited behaviors, many odors are initially neutral and odor responses are learned through experience. Odor information received in the olfactory epithelium is transferred to the olfactory cortex. Olfactory cortex can be divided into several areas, and we are interested in one area, olfactory tubercle (OT). When mice associate a particular odor to food reward and become liking the odor, anteromedial subarea of the OT is activated. On the other hand, when mice associate the same odor to electrical shock punishment and become disliking the odor, lateral subarea of the OT is activated. Given that activation of brain areas depends on synaptic inputs, plastic property of synaptic inputs to the OT was examined. By associating optogenetically-stimulated synaptic inputs to the OT with either reward or punishment, the synaptic inputs were strengthened in an OT subarea-specific manner. Further, the OT expresses a variety of neuromodulatory signal molecules, and manipulation of the signals altered olfactory behaviors. Through these topics I wish to introduce the highly plastic and adaptive nature of the olfactory system to determine olfactory behaviors by experience. A possible relation between odor, emotion and pain is also discussed.

Environmental odors are often composed of multiple types of odorants. However, it remains unclear how the mixtures of odorants are detected and processed in the olfactory system. Using in vivo calcium imaging of mouse olfactory bulb and olfactory epithelium, we show that odors produce not only excitatory but also inhibitory responses in olfactory sensory neurons (OSNs). Heterologous assays indicate that odorants can act as agonists to some but inverse agonists to other odorant receptors. We also demonstrate that responses to odor mixtures are extensively suppressed or enhanced in OSNs. When high concentrations of odors are mixed, widespread antagonism suppresses the overall response amplitudes and density. In contrast, a mixture of low concentrations of odors often produces synergistic effects and boosts the faint odor inputs. Thus, odor responses are extensively tuned by inhibition, antagonism, and synergy at the most peripheral level. These mechanisms may explain why the mixed odors are perceived as more than just a simple sum of their components.

Due to recent technical advances in instrumentation and sample preparation protocols, current imaging mass spectrometry (IMS) is now able to visualize the localization of neurotransmitters, including monoamines. Although monoamine-producing neurons, as well as their projections and synapses, have been thoroughly characterized, localization and fluctuation of monoamines within these circuits remains unclear. We addressed this problem with generation of the in situ monoamine concentration maps obtained with IMS, which allow us to study fluctuations in local monoamine concentration in response to physiological stimuli, drug administration, and neurodegenerative disease progression. Our recent studies have shown that monoamines accumulate not only in cell bodies expressing enzymes that produce these transmitters (such as TH and TPH), but also in distant nerve terminals. This indicates that they are either actively transported along the axon or synthesized locally at the terminals. Furthermore, since the pharmacokinetics of exogenous drugs can also be visualized with IMS, the development of a method for simultaneous imaging of selective serotonin reuptake inhibitors (SSRIs) and monoamines could reveal where long-term SSRIs accumulate and how they affect local monoamine metabolism. I'll also show that how the stimulated immune metabolism alters brain monoamine signaling, by visualization of deficiency of the serotonin and dopamine in the brain, resulting in behavioral changes.

Levodopa (L-DOPA) therapy is the gold standard for the treatment with Parkinson's disease. The pharmacological actions of L-DOPA have been believed to be mediated through its conversion to dopamine. On the contrary, we propose that L-DOPA is a neurotransmitter. We identified G-protein coupled receptor (GPCR) GPR143, a gene product of ocular albinism-1, as a receptor for L-DOPA. In the course of our phenotypic analysis of Gpr143 gene-deficient (Gpr143^-/y) mice, we found that behavioral response to quinpirole, a dopamine D2 receptor (D2R) agonist, was attenuated in Gpr143^-/y mice when compared to wild type (WT) mice. This phenotype was also observed in the striatal indirect pathway specific Gpr143 gene-deficient (A2A-cre;Gpr143^flox/y) mice. To investigate the physiological role of GPR143, we performed anxiety behavior using zero maze test. The anxiety behavior was attenuated in Gpr143^-/y and A2A-cre;Gpr143^flox/y mice when compared to corresponding control animals, as was reported in the striatal indirect pathway specific D2R deficient mice. These results suggest that L-DOPA-GPR143 fine-tunes the dopamine D2R in the striatal indirect pathway.

Evidence is growing that the exposure to anesthetics and sedatives in the neonatal period, which is the "window of vulnerability" during which they are particularly susceptible to disturbance factors, induce neurodevelopmental disorders long after the causative event. We found that rats neonatally treated with midazolam, a common sedative using in pediatric clinical settings, showed changes in sociality and deficits in social novelty discrimination behavior in adulthood. The decrement of the number of positive cells for tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, in the ventral tegmental area and substantia nigra pars compacta observed in these rats suggested that long-lasting decrease of dopamine release is involved in the mechanisms of deficits in social behavior. Furthermore, we revealed that correction of dopamine homeostasis by acute systemic administration of methylphenidate, a dopamine and noradrenaline reuptake inhibitor, restored the impaired social novelty discrimination behavior in neonatal midazolam treated rats. In this symposium, we would like to introduce a new probable mechanism concerning hypofunction of dopaminergic system underlies the impairment induced by exposure to anesthetics in neonatal period.

Placebo analgesia is a kind of pain relief that follows the administration of a pharmacologically inert treatment and is thought to result from the activation of endogenous pain modulatory system by psychological processes such as expectation and implication. Neuroimaging studies in humans have suggested that the hierarchical brain regions and neurochemical systems, such as endogenous opioid system, are involved in the placebo analgesia. However, because of the lack of proper approaches to perform molecular and cellular manipulations, the detailed molecular processes behind it have not been clarified. Recently, we have developed a small-animal neuroimaging method that combines FDG-PET imaging with SPM analysis for investigating regional brain activity in the entire rat brain. Using this method, we have identified that widely distributed brain regions, which are quite similar to those of human neuroimaging studies could be involved in Pavlovian conditioning-induced placebo analgesia, and demonstrated that the endogenous µ opioid system in the PrL, causally contributed to placebo analgesia in rats. In this symposium, I will introduce the neurobiological mechanism of placebo analgesia how the endogenous opioid system activates the descending pain modulatory pathway.

The morphology of microglia, which are immune cells in the brain, is strongly linked to their function. In particular, when phagocytosing dead cells or debris, the morphology of microglia is altered from their normal ramified structure. We have recently found that microglia with characteristic morphology cluster around hemorrhagic region in the brains of healthy newborn mice. The perinatal period is a critical period for brain development, and intracerebral hemorrhage during this period can cause irreversible brain damage. In particular, erythrocytes leaking into the brain parenchyma are cytotoxic and need to be removed rapidly. We found that erythrocytes are mainly phagocytosed by microglia. Pharmacological removal of microglia increased the number of leaking erythrocytes in the hemorrhagic region. Therefore, under physiological conditions, microglia may contribute to normal brain development by rapidly phagocytosing and removing leaking erythrocytes. Next, to examine the changes in microglial properties mediated by erythrophagocytosis, we performed a transcriptome analysis of erythrophagocytic microglia, finding that these microglia highly expressed several genes that are considered essential for erythrocyte processing. The expression of these genes may lead to the heterogeneity of microglia function in the mature brain.

When sense environmental changes, astrocytes become "reactiveastrocytes" and encode them into unique Ca²⁺ signals thataffect greatly contribute to physiological and pathophysiological brainfunctions. Here, we show that reactive astrocyte-mediated Ca²⁺signalsare decoded into synapse remodeling in the primary somatosensory cortex (S1) andthe hippocampus. After peripheral nerve injury, we found that S1 corticalastrocytes become reactive and show frequent Ca²⁺ signals. TheCa²⁺ responses were initiated by upregulation of mGluR5, followedby release of multiple synaptogenic molecules such as TSP-1 and Glypican4, andexcess uncontrolled synapse formation. Then, S1 astrocytes caused misconnectionof tactile- and pain-networks, thereby leading to sustained mechanicalallodynia. In the epileptogenesis model, hippocampal astrocytes also becomereactive, and encode them into interesting Ca²⁺ signals, which werealso triggered by mGluR5. The Ca²⁺ signals in the reactive astrocytesare decoded into synaptogenesis and network remodeling in the hippocampus,thereby leading to epileptogenesis. Taken together, the uniqueCa²⁺signals seen in reactive astrocytes could be decoded into excesssynaptogenesis, which are greatly involved in brain diseases.

Microglia are the sole immune responding cells in the central nervous system. Their role as neuro-immune cells in the pathogenesis of various neurodegenerative and infectious diseases of the brain have been extensively studied. In addition to the pathological function of microglia, recent developments in molecular probes and optical imaging in vivo have revealed that microglia are highly motile cell in the healthy brain, extending and retracting their process that extend from a largely stationary cell soma. We used in vivo two photon microscopy to reveal their physiological and pathological function on synapse and vessels. We particularly showed the functional consequence of microglial contact on synapse and vessels to indicate their role in neurological or psychiatric brain. 

In this session, we will show 1. Microglial regulation of blood brain barrier, 2. Microglial role for cross modal plasticity that indicate their pathological role in schizophrenia. 

Acute nociceptive pain is a key defense system for detecting danger signals. In contrast, chronic pain (like neuropathic pain occurring after damage of the nervous system) persists for a long period even in the absence of dangerous stimuli or after injuries have healed.  In animal models of neuropathic pain, peripheral nerve injury has been shown to cause a variety of plastic modifications in the somatosensory system at the levels of neuronal synapses, connections, and networks, which leads to the development of neuropathic pain. A number of studies including ours have demonstrated that these pathologically alterations require signals from glial cells, especially microglia (a type of tissue-resident macrophages in the CNS), and have significantly advanced our understanding of the mechanism underlying neuropathic pain development.  On the other hand, the role of microglia after the development remains unknown, but our recent study provide evidence indicating that a subset of microglia contributes to spontaneous recovery from neuropathic pain. These new findings suggest that microglia play a highly dynamic role during the course of neuropathic pain. In my talk, I will show our data including the new role of microglial subset and discuss the new mechanism of neuropathic pain.

Humans and animals adapt their behavior to acquire or avoid reward and aversive stimuli, respectively. However, the precise neural mechanisms that underly the signaling of such stimuli, as well as learnt associations between environmental cues and the rewarding/aversive stimuli they predict, are still unclear. Within the nucleus accumbens (NAc), a key neural substrate for limbic processing, dopamine D1 and D2 receptor-expressing medium spiny neuron (D1-/D2-MSNs) populations have been revealed to play important roles in controlling reward and aversion learning, respectively. Interestingly, recent reports have also suggested that within these populations there may be more complex neural circuit mechanisms contributing to the processing of limbic information. In this study, we expressed a calcium indicator in either NAc D1- or D2-MSNs and recorded neural activity at the single-cell level by miniature microscopy during reward and aversive Pavlovian learning. Diverse activity patterns were found in response to direct rewards (sucrose) and punishments (air puff), as well as environmental cues (auditory tones) paired with their delivery. These results suggest novel complexity in the control of reward and aversive learning by diverse activity patterns in subpopulations of NAc D1- and D2-MSNs.

Measurements of the rewarding or discriminative stimulus effects, and sensitization to locomotor activity induced by abused drugs give us an available information for understanding of abuse liability and mechanism of abused drugs. Furthermore, genetic factors are known to play a role in the likelihood of drug abuse by humans. Psychostimulants, such as methamphetamine and cocaine, induce robust discriminative stimulus effects mediated through an activation of dopaminergic system without induction of the rewarding effects in Fischer 344 rats, unlike in the case of Lewis rats indicating gene expressions in the medium spiny neurons (MSN) could be determinants to establish the rewarding effects induced by psychostimulants in spite dopaminergic systems and sensing to the discriminative stimulus are properly worked. There are two types of MSNs (e.g., D₁- and D₂-receptor containing MSN, so called D₁-MSN and D₂-MSN) in the basal ganglia. Repeated administration of methamphetamine produced a sensitization to locomotor activity in mice. We found that the A_2A-receptor antagonist and d-opioid receptor agonist, which directly influence the D₂-MSN, but not D₁-receptor agonist, cross-sensitized to locomotor activity in methamphetamine sensitized mice. Since activation D₁-receptpr plays core role in the addictive behaviors themselves, our findings indicate that regulation of D₂-MSN play the regulatory role in the expression of such behaviors. Thus, regulations of MSNs are taking a part to express several addictive behaviors.

Cortico-basal ganglia loop circuits play important roles in the acquisition, performance, and switching of learning behaviors. The loop circuits are composed of some subloop structures mediating cognitive, motor, and limbic functions, and these substructures are considered to interact with each other and shift in response to environmental changes. Our research group used positron emission tomography (PET) for small animals to monitor the time course of brain activity during the learning processes, and found that the activity in the anterior dorsolateral striatum (aDLS) was increased at the early phase of auditory discrimination learning and then the activity of the posterior ventrolateral striatum (pVLS) was elevated at the late phase of the learning. Pharmacological inhibition of each subregion at the corresponding timing impaired the acquisition of learning, and multiunit recording of neural activity indicated event-related changes in the firing between the aDLS and pVLS at different learning phases. These data suggest that the aDLS and pVLS have distinct roles in different phases of auditory discrimination and that learning-related activity shift from the aDLS to the pVLS during the progress of learning. Although we previously reported the importance of striatal direct and indirect pathways in the dorsal striatum in reaction time modulation and response accuracy in stimulus discrimination learning, we need to reconsider the function of striatal projections in the subregions of the striatum. In this symposium, we will present the roles of striatal projections originating from each subregion in the acquisition of discrimination learning.

Levodopa is known as the most effective drug for the treatment of Parkinson's disease. However, long-term use of levodopa usually is associated with levodopa-induced dyskinesia, which is mimicked chorea and dystonia. The abnormal involuntary movements sometimes develop troublesome for the patient's quality of life. Thus, revealing the pathomechanisms of levodopa-induced dyskinesia will be useful for the treatment of Parkinson's disease. It is considered that the key pathology of levodopa-induced dyskinesia is dopaminergic neuronal degeneration in the substantia nigra pers compacta. We revealed that the loss of dopaminergic neurons will be associated with levodopa-induced dyskinesia in vivo by using MRI neuromelanin imaging. Furthermore, we also revealed that the preservation of white matter structures, which are the connecting tracts between cortices and basal ganglia, might be important for developing levodopa-induced dyskinesia by using diffusion MRI. Finally, we revealed that the increasing GFAP positive astroglia in the striatum plays important role in the development of levodopa-induced dyskinesia in a rat model. In this symposium, we will show our data and discuss the pathomechanisms of levodopa-induced dyskinesia.

Nonlinear Raman scattering has been widely used as one of the methods for label-free molecular imaging in recent years. Among them, coherent anti-Stokes Raman scattering (CARS) has the capability of obtaining the broad spectral bandwidth of >3000 cm^-1, which is the broadest spectral bandwidth ever reported. In this talk, I will introduce our development of CARS microscopy and its applications to living cell imaging.

In the study of the working mechanisms of multicellular systems, the measurement of chemical quantities (gene expression and metabolism) has been the main approach. However, in tissue formation and force generation, mechanical quantities (force, elasticity) inside and outside the cells are involved. To understand the complex network intervening between chemical and mechanical quantities, it is necessary to measure both chemical and mechanical quantities in living cell systems. In recent years, bioimaging methods using Brillouin scattering have been attracting attention as a method to measure the mechanical quantities in living cells. Brillouin scattering is a type of inelastic scattering, and the frequency shift due to Brillouin scattering is proportional to the propagation velocity of acoustic phonons in the medium. Since the speed of acoustic phonon depends on the elastic constants, the elastic properties can be estimated from the frequency shift. We have been developing Brillouin scattering microscopy for applications in developmental biology and mechanobiology. The imaging system was designed and constructed with the target of observing the inside of living cells. We have achieved not only imaging of single cells, but also of ES cell colonies, tissues and organs in model organisms. We are also working on simultaneous measurements with other optical modalities (fluorescent protein, Raman scattering, etc.) to advance biological interpretation. Correlation between multiple features related to cellular state will be discussed.

Spatiotemporal dynamics of drugs in tissues is a key determinant of their pharmacological actions. While such pharmacokinetics research has been performed at macroscopic level, those of microenvironment at cellular and subcellular scales could not have been assessed due to technical limitations. In this regard, nonlinear microscopic techniques have great potentials. With high spatiotemporal resolution provided by tissue penetrating ability of near infrared light and spatial restriction of nonlinear optical effects, multiphoton microscopy can reveal hidden phenomena inside tissues. In addition, various optical phenomena other than fluorescence can be utilized to image targets that cannot be labeled with and/or visualized by conventional fluorescence techniques. In this talk, I will introduce the basics of such imaging techniques as well as our attempts to utilize them for characterizations of pharmacokinetics as well as cellular responses in microenvironment at subcellular resolution.

Regarding the circadian rhythm of glucose metabolism in humans and animals, glucose tolerance and insulin sensitivity show circadian rhythm with higher in the morning than in the evening. Blood glucose levels are higher in oral glucose tolerance tests or a mixed meal consumption test conducted in the afternoon and evening than in those conducted in the morning, whereas insulin secretions, especially early-phase of insulin secretion, are higher in the morning than in the evening. Epidemiological studies demonstrate that eating behaviors such as time of meal and number of chew food are associated with risk of obesity and type2 diabetes. Slow eating and chewing food well increase dietary induced thermogenesis (DIT) and appetite-reducing hormone secretions (PYY and GLP1), and decrease hunger hormone (ghrelin). In recently, we could reveal that the effect of mastication on postprandial glucose metabolism is dependent on the time of day and frequency of mastication. In this study, healthy male participants consumed a high carbohydrate food (200g rice) with 10 or 40 chews per mouthful at 0800 h and 2000 h. The results reveal that morning mastication but not evening decreases postprandial blood glucose concentrations and increases insulin secretion at 30 min and so-called the insulinogenic index as a marker of early-phase β-cell function. Clinically, impairment of early-phase insulin secretion is considered an early marker of β-cell dysfunction and the development of type 2 diabetes. Thus, the present findings suggest that morning mastication improves early-phase insulin secretion after rice consumption.

Numerous studies have previously revealed the various mechanisms of insulin resistance in vivo and in vitro, in which insulin receptor functions and receptor substrates played important roles. For example, inflammatory mediators induce serine/threonine phosphorylation and subsequent degradation of insulin receptor substrate-1 (IRS-1), which causes interference of insulin receptor signaling and insulin resistance. Here, we show the additional findings of insulin receptor function, suggesting the novel mechanism of insulin resistance in adipose tissues and stem cells.