Anticancer drugs are most commonly administered via transvenous administration. However, the most serious local complication is extravasation because even a non-vesicant leak can cause inflammation of the vessel and surrounding tissue, narrowing of the vessel, and pathologic hardening of the surrounding tissue. Inflammatory findings such as thrombus and edema at the injection site are detected by subcutaneous observation using an ultrasound device immediately after treatment in the outpatient chemotherapy room, indicating that these findings were associated with the induration of delayed adverse events. To prevent extravasation and induration, the vessel wall must be preserved. Therefore, we aimed to minimize the stimulation of the vessel wall by administering drugs using a peripheral intravenous catheter. We observed that vessels can be preserved by using catheters made of soft materials, placing them in veins with large diameters, and inserting them in a single attempt. The effect of vessel preservation via catheter placement that meets these requirements will be presented.

創薬は難しい。大手製薬企業に在籍しても一生に一つ新しい薬を世に送り出せるとは限らない。そんな厳しい現実の中で、国も企業も大学もイノベーションを生み出すために様々な新しい取り組みをしている。私は2011年から5年間、京都大学と住友ファーマの産学連携プロジェクトであるDSKプロジェクトに参加した。このプロジェクトは若手の独立PIと企業の創薬部門を一つの器に入れてその化学反応から何かが生まれるのを期待したものだった。病気のメカニズムを知りたい基礎研究者と薬を作りたい企業研究者のマッチングから「白血病発症に必須のタンパク質間相互作用を阻害する分子標的薬」DSP-5336が生み出され、臨床試験が始まった。まだ道半ばであるが、産学連携プロジェクトから薬が生み出された一例としてその過程を振り返り、良かった点や難しかった点などを皆さんと共有したい。

私は、DSKプロジェクト以前は国立がん研究センターやスタンフォード大学にてMLL遺伝子異常を持つ白血病の発症メカニズムについての基礎研究に従事していた。独立PIポジションを探していた時にDSKプロジェクトの事を知り、縁あって参画の機会を得た。このプロジェクトは研究費が支給されるという若手PIにとっては大変魅力的なポジションだった。一方でこれは産学連携プロジェクトであり、何かしら産業貢献を求められるという条件があった。斯くして薬作りに携わったことのない基礎研究者が企業研究者と同じ建物の中で議論し、新しい薬作りに取り組む挑戦が始まった。私にとって幸運だったのは一緒に取り組む企業研究者が同年代であり、新しい薬を作ろうという意欲が高い人たちだったことだ。アカデミアとインダストリーの文化の違いもあったけれど、最終的にはお互いを認め合って一つの目標に向かっていくことができた。DSKプロジェクト期間中に特許申請に辿り着き、プロジェクト終了後もAMEDのACT-M研究費の支援を受けて適応拡大研究に進展した。この間、幾多の危機（社内評価など）を乗り越えて臨床試験に辿り着いた。産学連携プロジェクトにおいて重要なのは産と学が対等である事だと思う。お互いがお互いの要求を聞く必要がある。

最後に我々が開発した薬について紹介したい。MLLタンパク質は遺伝子のプロモーターに結合して転写を活性化する。染色体転座によってMLLと別の遺伝子が融合したMLL融合遺伝子が形成され、この遺伝子産物が「異常な活性化型MLLタンパク質」として働くことで白血病が引き起こされる。MLL融合タンパク質はMENINという共作用因子と結合することでプロモーターに作用するようになる。MENINとMLLタンパク質の分子間結合を阻害する低分子化合物DSP-5336はMLL融合タンパク質の機能を失わせ、白血病細胞の分化を誘導する。DSP-5336は優れた薬物動態と副作用の少なさを兼ね備えた化合物であり、現在日米加で急性骨髄性白血病について臨床試験が進行中である。

Chordia Therapeutics株式会社は2017年11月に武田薬品工業株式会社からスピンアウトして創業したバイオベンチャーです。当社は5つの低分子抗がん薬パイプラインの研究と開発を進めていますが、そのうちのMALT-1阻害剤は既に内資製薬メーカーに全世界での権利を導出し臨床試験が開始されています。MALT-1阻害剤のルーツは武田薬品時代に共同創業者が開始した京都大学との共同研究であり、製薬会社からのスピンアウトベンチャーであると同時に大学発ベンチャーの特徴も併せ持つことが当社の強みであります。当社のリードパイプラインであるCLK阻害剤CTX-712はスプライシングを変化させて過剰なRNA制御ストレスを発生させることによってがん細胞を死滅させる全く新しい作用機序を有する経口投与可能な抗がん剤候補です。現在、First in Humanの第1相臨床試験を本邦にて実施中で、複数のがん種における有効性と管理可能な安全性プロファイルが確認されており、Proof of Conceptが確立されています。また、将来的なグローバル展開を目指すべく、米国での第1/２相臨床試験も開始しています。私たちは、大学との共同研究を活用して、日本発で世界初の新しい抗がん薬を一日でも早く世界中の患者様にお届けすることを目指して、更なる研究開発を進めていきます。

人類にとって痛みは警告信号の一つであり、生命を維持するための生体防御機構として重要な役割を果たしている。一方、ケガやウイルス感染、糖尿病などの疾患が原因となって生じる慢性的な痛みは、人々のADLやQOLを著しく低下させ、現在でも多くの患者が慢性的な痛みに悩まされている。慢性疼痛に用いられる治療薬としてはNSAIDs、抗てんかん薬、抗うつ薬、オピオイド鎮痛薬などがあり、一定の効果が認められているものの、これら既存薬に対する治療満足度は十分ではない。特に神経障害性疼痛に対する治療薬は少なく、臨床現場では効果的な治療法の確立や新薬の登場が期待されている。創薬標的の一つとして、電位依存性ナトリウムチャネル（VGSC）が挙げられる。VGSCは痛みの神経伝達や神経細胞の興奮性に深く関与しており、これまでに9種類（Nav1.1～Nav1.9）が報告されている。その中で、末梢神経細胞や後根神経節（DRG）に発現するNav1.7、Nav1.8及びNav1.9は、各種疼痛疾患との関連性が報告されており、神経障害性疼痛治療薬の標的分子として注目を集めている。

AlphaNavi Pharma株式会社（アルファナビ）は、住友ファーマ株式会社で疼痛領域の創薬を行ってきた研究開発者によって設立されたカーブアウトベンチャーである。アルファナビは、住友ファーマが開発してきた神経障害性疼痛治療薬（ANP-230）に関するライセンス契約を締結し、2019年より研究開発を推進している。ANP-230は、Nav1.7、Nav1.8及びNav1.9を選択的に阻害する新薬候補であり、すでに米国、EU、日本でそれぞれ第I相試験を完了している。現在は国内において小児四肢疼痛発作症患者を対象としたANP-230の第I/Ⅱ相試験を実施している。小児四肢疼痛発作症は、2016年に京都大学と秋田大学のグループによって報告された家族性の希少疾患である。小児四肢疼痛発作症は四肢や手足の関節に疼痛発作を起こすが、それ以外に異常所見は観察されず、主に寒冷や悪天候、ストレス、疲労等で誘発されるのが特徴である。

ANP-230は既存薬で懸念されている中枢性や循環器系への副作用がなく、ラット神経障害性疼痛モデルである脊髄神経結紮モデルや糖尿病性神経障害性疼痛モデル等に対して優れた鎮痛効果を示す。さらにANP-230は、患者に認められたNav1.9をコードする遺伝子変異（p.R222S）を導入したモデルマウスで観察される冷刺激や熱刺激、機械刺激による疼痛行動のいずれに対しても優れた抑制効果（鎮痛効果）を示し、神経細胞の過剰興奮を強力に抑制することが明らかになった。以上のことから、ANP-230は小児四肢疼痛発作症をはじめとする神経障害性疼痛を広くカバーする新規治療薬になると期待されている。本会では、カーブアウトベンチャー設立の経緯やANP-230に関する薬理プロファイルにフォーカスし、産業化を目指したこれまでの取り組みについて紹介する。

先天性無歯症は、通常6本以上の歯の欠如を認める症例が遺伝性とされ、その発症頻度は全人口の0.1％と報告されている。先天性無歯症の原因遺伝子としては、EDA1、MSX1、WNT10A、 PAX9、RUNX2等が同定され、その多くの原因遺伝子がマウスとヒトで共通である。先天性無歯症患者は、顎骨の発達期である幼少期から無歯症となるため、義歯や歯科インプラントの適応が困難である。また、成長期にオーラルフレイルの状態となり、栄養確保や成長に悪影響を及ぼす。既存治療としては、成人以降に義歯や歯科インプラントによる人工歯を用いた代替治療を施行するしかなく、根治的な治療として歯の再生治療の開発が強く望まれていた。これまで組織工学的な手法による歯の再生研究が数多く試みられてきたが、細胞リソース、コストや安全性などの問題で臨床応用までには至っていない。我々は、USAG-1タンパク（BMP / Wntのアンタゴニスト）の遺伝子欠損マウスにおいて、過剰歯を形成することを見出し、１種類のタンパク分子により歯の数を増やすことができることを明らかにした。また、各種先天性無歯症モデルマウスと過剰歯モデルマウスのUSAG-1遺伝子欠損マウスの交配により、歯の形成が回復することを見出した。AMED創薬支援推進事業、難治性実用化研究事業、京都大学GAPファンド、インキュベーションプログラムのご支援のもと中和抗体、siRNAを作製した。USAG-1はBMP/Wntシグナルのアンタゴニストであり、その機能ドメインが異なるため、その中和抗体は、BMP、WNTシグナルをそれぞれ活性化するまた両者を同時に活性化する3種類に分類された。それらの抗体のBMP、WNT signaling に対する活性化様式の違い、抗原認識部位の違いを考慮して、5個の中和抗体に絞り込み知財を取得した。その知財をもとに、研究成果活用事業として2020年５月にトレジェムバイオファーマ株式会社を設立した。USAG-1蛋白は、ヒト、マウス、ビーグル犬等の異種動物間で97%の高いアミノ酸相同性を有している。そのため、マウス抗USAG-1抗体は、先天性無歯症モデルマウス・ビーグル犬において単回腹腔内投与/静脈内投与にて欠損歯を回復できることを明らかにした。in vitro/in vivo活性、予備毒性試験より、ヒト抗USAG-1抗体の最終開発候補物TRG035を決定した。PMDA RS戦略相談対面助言にて非臨床試験の項目を確定した。トレジェムバイオファーマ株式会社、AMEDとの産官学連携を積極的推進することで、医師主導治験をすすめて行く予定としている。TRG035を先天性無歯症患者の治療薬として、有効性安全性を確立し、臨床応用への道筋をつけることを目指す。

潰瘍性大腸炎（ulcerative colitis: UC）および原発性硬化性胆管炎（primary sclerosing cholangitis: PSC）は、いずれも原因不明の慢性炎症性疾患である。PSCの約30%(欧米では76％)にUCを合併することから、両者には共通の発症機序が推測されてきた。UCは大腸粘膜にびらんや潰瘍を形成し、腹痛、発熱、血便などを呈し、若年者を中心に高齢者まで幅広い年齢層で発症する。罹患数は増加傾向にあり、国内で22万人以上とされている。寛解と再燃を繰り返し長期療養が必要となることは、患者のQuality of lifeの低下を招く。PSCは胆管に生じる線維性狭窄が進行性に増悪し、胆汁うっ滞性肝障害を呈し、発症から10年を経過すると約半数が肝硬変を経て死に至る。有効な治療薬がなく肝移植が唯一の救命法であるが、移植後も再発率が高く極めて予後不良の疾患である。UC/PSCの発症メカニズムとして遺伝的素因と環境因子を背景として、自己免疫学的機序を介して大腸/胆管上皮細胞が障害されると考えられているが、未だ原因解明には至っていない。このようにUC/PSCは原因不明のため、特異的診断法や根治療法がなく、我が国の難病に指定されている。UC/PSCともに特異的診断法や治療法の開発のため、病態解明が切望されてきた。

そのような中、申請者らはこれまでに、UC患者血清中に感度92.0%、特異度94.8%、PSC患者血清中に、感度89.1%、特異度96.7%の抗インテグリンαVβ6抗体を発見した。［Kuwada, Shiokawa(corresponding author). Gastroenterology. 2021. Yoshida Shiokawa(corresponding author). J Gastroenterology. 2023.］

また、ヒト大腸/胆管上皮細胞にインテグリンαVβ6が発現していることを確認し、同抗体が細胞外マトリックスタンパク質であるフィブロネクチンとインテグリンαVβ6との結合を阻害することも見出した。申請者らの報告以降、アメリカ、スウェーデン、イタリアの他のグループからも、同国のUC患者で抗インテグリンαVβ6抗体が高率に陽性であることが報告され、申請者らの発見の再現性が確認されたとともに、世界中で注目されている。

これまで課題であったUC/PSC診断、病勢把握に貢献できると考え、市販可能な同抗体測定キットを開発し、現在厚労省研究班と共同で本キットの有用性を検証するために大規模な前向き試験を行っている。2024年には薬事承認、2025年には保険適応を目指している。

また、本抗体は阻害作用を有することから病態への関与が考えられ、最適な治療標的になりうることが考えられる。2022年に京都大学内にベンチャー企業であるLink Therapeutics 株式会社を設立し、開発を進めている。

G protein-coupled receptors (GPCRs) are membrane proteins involved in a variety of physiological functions and disease progression. Research on GPCRs is fiercely competitive around the world using structural and molecular biological methods, and many drugs targeting GPCRs have been produced. However, most of them are based on the control mechanism by agonists and antagonists, and there have been few studies that delve into the diversity of receptor responses. In this study, we revealed that Cys residues in the intracellular third loop of GPCRs serve as target sites for oxidation and sulfuration, triggering internalization and proteolysis of GPCRs. We also found that transient receptor potential canonical (TRPC) 6 channels, which are permeable to metal ions (mainly Zn²⁺ and Fe²⁺), enhanced signal responses through zinc modification of GPCRs. In this presentation, I would like to introduce our recent findings and future directions for drug discovery research for inflammatory bowel disease targeting redox modification of GPCRs and drug discovery research for heart failure targeting receptor-operated cation channels.

Angiotensin II (AngII) regulates the circulation mainly through its type 1 receptors (AT₁R) coupled to G proteins and β-arrestins. Here, we demonstrate the recent findings on the roles of AT₁R/β-arrestins pathway in heart and kidney during developmental periods. We previously reported that a β-arrestin-biased AT₁ agonist (BBA), which activates β-arrestins but inhibits G proteins, induced a positive inotropic effect on neonatal mice almost without an increase in heart rate or oxygen consumption. This effect was also observed in neonatal mice harboring a cardiac troponin T mutation that causes dilated cardiomyopathy in humans. Although the majority of these homozygous littermates died before weaning, we found that BBA treatment alleviated their decline in cardiac function and improved the survival rate. In contrast, an AT₁R blocker (ARB), which inhibits both β-arrestins and G proteins, not only failed to rescue the survival rate but also severely hampered the postnatal metanephrogenesis in wildtype littermates. ARB-treated mice exhibited marked abnormalities of the cortex/medulla and renal failure symptoms. This renal toxicity was not observed with BBA. Therefore, it is likely that AngII exerts its prosurvival cardiovascular and renal effects on preweaning mammals mainly through β-arrestins, but not G proteins, coupled to AT₁R.

Chronic kidney disease (CKD) is an emerging national health issue in Japan. Recently, sodium-glucose co-transporter 2 inhibitors and the mineralocorticoid antagonist Finerenone have been approved for treatment against CKD, including diabetic nephropathy. However, the current pharmacotherapy options for patients with kidney diseases remain limited. Moreover, the molecular mechanisms underlying the pathogenesis of CKD remain unclear.

Podocytes are highly differentiated cells that play a critical role in the ultrafiltration system, together with endothelial cells and the glomerular basement membrane in the glomerulus. We have provided the first evidence that transcription factor old astrocyte specifically induced substance (OASIS) in myofibroblasts promotes kidney fibrosis. Additionally, we discovered that OASIS is also expressed in podocytes. We demonstrated that the upregulation of OASIS in podocytes contributes to kidney injury. This presentation will focus on our findings regarding the role of OASIS in podocytes.

We hope that our findings will contribute to the development of novel therapeutic strategies against CKD.

Chromatin regulation such as dynamic changes in gene expression and chromatin structure occurs in host cells upon virus infection. Host cells activate anti-viral processes to protect themselves and viruses also invade the host cell system to create an environment useful for virus survival. Coronavirus diseas-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), has been spreading all over the world since 2019, causing many kinds of severe symptoms. However, it remains unclear how SARS-CoV2 affect host cells and what chromatin regulation occurs in COVID-19. We have investigated changes in chromatin structure in the cells of COVID-19 patient with various degrees of severity, using chromatin structure analysis such as Hi-C and ChIP analysis. Severe patients tended to show severely affected chromatin patterns especially around genes in signaling pathways associated with virus infection. These results suggest that SARS-CoV2 may affect critical host cell systems and cause severe symptoms. In this symposium, we would like to discuss the relationship between COVID-19 pathogenesis and chromatin regulation.

Transposons, constituting approximately 50% of the human genome, can be considered genome modifiers due to their ability to transpose themselves. However, the reasons for their existence and their significant impact on phenotypes still remain unclear. Our research has brought new insights into the regulation of transposon expression and transposition by piRNAs (PIWI-interacting RNAs). piRNAs, small non-coding RNAs of 24-30 nucleotides, are exclusively expressed in animal gonads and loaded into the PIWI clade members of the Argonaute protein family. These piRNAs act as guides for the PIWI protein and its associated complexes to induce co-transcriptional regulation of their target transposons. Through our investigations, we have revealed that PIWI-piRNA-mediated regulation not only governs the expression of transposons but also exerts a profound influence on genome structure and compartmentalization, often facilitated by the formation of heterochromatin. This dynamic regulatory mechanism profoundly shapes the landscape of the genome. Moreover, our findings have uncovered a novel enhancer-mediated regulation mechanism governing Drosophila telomeric transposons. This mechanism complements piRNA-mediated regulation, especially in somatic cells. Taking these into account, we propose a hypothesis that genomic plasticity emerges from the intricate interplay and antagonism between transposons and the regulatory mechanism.

Chromatin fibers, as the substance of the genome, are biopolymers packed inside eukaryotic cells. The recent development of Hi-C has provided insights into the hierarchical organization of chromosomes as 3D genome structures. These structures include A/B compartments at the megabase scale, TADs spanning hundreds of kilobases, and smaller contact/loop domains. However, Hi-C technology relies on the chemical fixation of cells and chromatin, limiting its ability to capture the dynamic nature of chromatin. In contrast, live-cell imaging experiments have demonstrated the dynamic nature of chromatin. To bridge the gap between the 3D genome structures and chromatin dynamics, we have developed a computational tool called PHi-C for interpreting Hi-C data as the dynamic 3D genome state in terms of polymer physics. PHi-C constructs a polymer model from an input Hi-C matrix. Analyzing a segment's dynamics reveals that it follows the generalized Langevin equation. Then, the segment's linear-response function relates to mean-square displacement. Thus, we established a theoretical approach to unveil the linear viscoelasticity of the 3D genome itself from Hi-C data or stochastic motion of a labeled locus.

During the post-meiotic stage, male germ cells called spermatids undergo dynamic chromatin alterations. This is induced by histone-protamine exchange, in which more than 90% of histones are replaced by protamine in mature sperm, resulting in highly condensed sperm chromatin. In mice, spermatids are morphologically divided into 16 steps according to their differentiation state, while no method has been established to separate the spermatids at each step with high purity. Thus, it’s difficult to analyze the stepwise chromatin condensation quantitatively.

We previously generated a transgenic mouse line named “VC mouse” that carries two transgenes, H4-Venus (H4V) and H3.3-mCherry (H33C) in testicular germ cells. This mouse line allows us to separate the spermatids in different steps according to the expression level of H4V and H33C in combination with the cell size. By taking advantage of this property, we successfully isolated the spermatids at each step and subjected them to NGS analyses to track changes in chromatin dynamics and gene expression. We also apply the VC allele to various KO mouse analyses with defective sperm nuclei to compare their phenotypes regarding chromatin condensation level. My presentation will include examples of such use of the VC mouse.

Although there are sex-based differences in pain regulation, the mechanisms underlying such differences are poorly understood. Here, we investigated whether androgens are responsible for sex-dependent differences in neurons and microglia that play an important role in abnormal pain in the spinal dorsal horn (SDH). In male mice, androgen receptor (AR) was expressed in the superficial area of the SDH, and most were localized in either excitatory or inhibitory neurons. Moreover, AR was also expressed in C-fibers and A-fibers in the dorsal root ganglia (DRG). AR expression level in SDH and DRG, and mechanical pain threshold were higher in normal male mice than that in female mice or gonadectomized (GDX) male mice. On the other hand, peripheral nerve injury-induced mechanical hypersensitivity was suppressed by treatment with microglial inhibitors in male mice but not female or GDX male mice. These results suggest that androgens may largely affect pain regulatory mechanisms by determining the sex-dependent characteristics of the pain-responsive neurons and spinal microglia.

Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect of cytotoxic anticancer drugs. Clinically, there are no effective medicines to prevent CIPN, and duloxetine is the only recommended drug to treat the developed CIPN. We have demonstrated the involvement of high mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) protein, in the pathogenesis of CIPN. Our clinical and reverse translational research evidence clearly shows that estrogen decline after menopause or ovariectomy promotes the severity of CIPN following paclitaxel treatment, which involves macrophage-derived HMGB1. Paclitaxel directly stimulates HMGB1 release from macrophages, an effect blocked by estrogen via activation of nuclear and membrane estrogen receptors. Ovariectomy also aggravates CIPN following oxaliplatin treatment, which involves HMGB1 derived from non-macrophage cells. Ovariectomized mice exhibit nociceptive hypersensitivity in response to intraplantar injection of HMGB1, which is prevented by repeated treatment with elcatonin, known to restore the ovariectomy-induced downregulation of 5-HT_1A receptors in the spinal cord. We thus suggest that postmenopausal estrogen decline aggravates CIPN possibly through the impairment of estrogen modulation of HMGB1 release and the downstream signals.

In the life cycle of women, itching (pruritus) is commonly and clinically known to occur during periods such as pregnancy and menopause. However, the underlying causes of variations in this itching were previously unknown. We hypothesized that female hormones are involved in modulating itch sensitivity and investigated the effects of main female hormones, 17β-estradiol and progesterone, on the itch threshold using female rats. We demonstrated that 17β-estradiol increased scratching behavior induced by one of the peripheral itch mediators, histamine, in skin, indicating an increase in itch sensitivity. On the other hand, progesterone did not show an increase in itch sensitivity. To further elucidate the mechanism by which 17β-estradiol regulates itch sensitivity, we focused on the gastrin-releasing peptide receptor (GRPR), an itch-specific mediator in the spinal cord. 17β-estradiol revealed an increase in the firing frequency and sustained activity of spinal GRPR-expressing neurons upon itch stimulation of the skin. Moreover, the increased scratching behavior induced by 17β-estradiol was suppressed by a GRPR inhibitor. Taken together, our findings suggest that 17β-estradiol modulates the expression and activity of spinal GRPR-expressing neurons, leading to changes in itch sensitivity.

In Japan, the number of patients with kidney diseases is increasing due to the westernization of lifestyles and aging societies. Chronic kidney disease (CKD) is a risk factor for the development of end-stage kidney disease and the incidence of cardiovascular diseases. More than 1 in 8, or 13.3 million people are estimated to have CKD in Japan. Therefore, CKD is recognized as a novel national disease.

Kidney fibrosis is a final common pathway for almost all kidney diseases, and its progression is correlated with impaired renal function. Although inhibiting kidney fibrosis could be a potential therapeutic approach to halt the progression of CKD, no specific therapeutic strategies are available for kidney fibrosis. In addition, the molecular mechanisms by which kidney fibrosis progresses are poorly understood.

We have found some novel regulators of kidney fibrosis (Yamamoto et al., FASEB J. 2021, Miyake et al., Biochem Biophys Res Commun. 2021). In this presentation, we will discuss our findings on the involvement of transcription factor old astrocyte specifically induced substance (OASIS) in kidney fibrosis. Specifically, OASIS in myofibroblasts facilitates kidney fibrosis, at least partially through increased bone marrow stromal antigen 2. I hope the findings will contribute to the development of novel therapies against CKD.

Heart failure is a leading cause of death in developed countries. The role of mRNA regulation in the pathology of heart failure remains elusive. CCR4-NOT complex is a multi-subunit protein complex constituting exonuclease-mediated degradation of mRNA’s poly(A) tails, a process called deadenylation. Here we analyzed the roles of deadenylase subunit in heart failure. After 2 weeks of transverse aortic constriction (TAC)-induced pressure overload, expression of CNOT6L deadenylase subunit was markedly upregulated in the hearts. Loss of CNOT6L significantly decreased cardiac contractility and enhanced fibrosis at 2 weeks after TAC. Transcriptome analyses elucidated that CNOT6L targets mRNA of the GeneX, which stimulates tissue fibrosis. Furthermore, CNOT6L markedly suppressed geneX expression in cardiac fibroblasts. Poly(A) tail length and luciferase reporter analyses revealed that CNOT6L regulates deadenylation via the cis-element in the geneX 3'UTR. Double knockout of GeneX and CNOT6L improved cardiac fibrosis and dysfunction in single CNOT6 knockout mice. Thus, CNOT6L deadenylase prevents the progression of heart failure by suppressing the expression of fibrotic geneX in cardiac fibroblasts, implicating a potential therapeutic strategy of targeting mRNA deadenylation.

Cardiac tissue is composed of cardiomyocytes, vascular cells, fibroblasts, and extracellular matrix (ECM). Myocardial cell death due to cellular injury, such as acute myocardial infarction, triggers a multistage repair response in which the damaged tissue is replaced by a fibrotic scar, mainly composed of ECM produced by myofibroblasts. In the heart, it is believed that most myofibroblasts are transdifferentiated from resident cardiac fibroblasts. Although the initial reparative fibrosis is critical to prevent ventricular wall rupture, an excessive fibrotic response is detrimental. This is because sustained activation of myofibroblasts leads to excessive ECM deposition, which reduces cardiac contractile properties and disturbs cardiac electrical signaling, resulting in heart failure. Therefore, myofibroblast regulation may be a potential target for heart failure therapy. In this section, we discuss cardiac fibrosis with a focus on myofibroblasts and present our work on 2,5-dimethylecoxib which is a promising new anti-fibrotic agent. We hope that our discussion of the relationship between cardiac fibrosis and myofibroblasts will lead to the development of new anti-fibrotic agents and the treatment of heart failure.

Among many pathological and radiological subtypes of pulmonary fibrosis, one of typical pathological types is usual interstitial pneumonia (UIP), followed by nonspecific interstitial pneumonia and organizing pneumonia. These subtypes are related to treatment responses to corticosteroids and/or immunosuppressants. Pathologically, proliferation of fibroblasts and abnormal deposition of extracellular matrix (ECM) are mainly distributed in the margins of pulmonary lobules, destroying the normal alveolar structure in pulmonary fibrosis. In UIP, scattered fibroblastic foci with immature fibroblasts with abnormal cuboidal epithelial cells covering the surface of fibroblast foci are assumed to be the forefront of the process of pulmonary fibrosis, and alterations in which the alveolar epithelium of alveolar walls and small airways is replaced by metaplastic bronchiolar epithelium are also observed. Mild inflammatory cell infiltration is often seen at fibrotic sites.

Among antifibrotic agents in treating pulmonary fibrosis, nintedanib is a PDGF/VEGF/FGF receptor tyrosine kinase inhibitor that suppresses ECM production from fibroblasts induced by TGF-β stimulation, and pirfenidone inhibits ECM production and growth of fibroblasts by TGF-β stimulation, and suppresses reactive oxygen species. The crosstalk between bronchial and alveolar epithelium and fibroblasts may be involved in the pathogenesis of pulmonary fibrosis, and blocking these molecular pathways in the crosstalk can be the potential therapeutic target of pulmonary fibrosis.

Fibrosis occurs in all organs and tissues except the brain, and its progression leads to dysfunction of affected organs. Fibrosis-induced organ dysfunction results from the loss of elasticity, strength, and functionality of tissues due to the extracellular matrix secreted by myofibroblasts that express smooth muscle-type actin as a marker. Myofibroblasts, which play a major role in fibrosis, were once thought to originate exclusively from activated fibroblasts; however, it is now clear that myofibroblasts are diverse in origin, from epithelial cells, endothelial cells, adipocytes, macrophages, and other cells. Fibrosis in the cardiovascular systems, such as the kidneys, heart, and lungs, is a serious chronic disease that ultimately leads to death. Indeed, fibrotic diseases are an important cause of morbidity and mortality worldwide and are responsible for up to 45% of all deaths in developed countries. This symposium will introduce the latest topics on the therapeutic strategies against fibrosis in the heart, kidneys, and lungs from basic research to clinical practice. We hope that this symposium will lead to the development of novel therapeutic strategies against tissue fibrosis.

Allergy immunotherapy (AIT) is a well-documented, safe, effective treatment option for respiratory allergic disease, including AR. It has been demonstrated that AIT can provide relief from clinical symptoms and that AIT has the potential to provide long-term post-treatment effect. Although the mechanism of AIT is not fully understood, it can actively modulate protective allergen-reactive pathways of the immune system and alter the natural course of disease. Unlike pharmacotherapy, AIT addresses the basic immunological mechanisms that are responsible for the development and persistence of allergic conditions. Currently two main routes of AIT administration are commonly available, subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT). Both SCIT and SLIT are clinically effective, and SLIT is particularly well tolerated, with a lower risk of systemic allergic reactions compared with SCIT. In Japan, SLIT-tablets for treatment of house dust mite AR or Japanese cedar pollinosis, without any age limitation, have been developed, and SLIT-tablets are now the best-documented form of AIT. Here we introduce the development of a fast dissolving SLIT tablet (Zydis formulation) for treatment of Japanese cedar pollinosis.

The prevalence of Allergic rhinitis (AR) is reached 49.2% in 2019. In particular, the prevalence of Japanese cedar (JC) pollinosis is 38.8%, and the onset age of pollinosis is becoming younger. AR is known to be a risk factor for the development of allergic asthma, a potentially life-threatening condition. Allergy immunotherapy (AIT) is a well-documented, safe, effective treatment option for respiratory allergic disease. It has been demonstrated that AIT can provide relief from clinical symptoms and that AIT has the potential to provide long-term post-treatment effect. Unlike pharmacotherapy, AIT addresses the basic immunological mechanisms that are responsible for the development and persistence of allergic conditions. Currently two main routes of AIT administration are commonly available, subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT).

In Japan, house dust mite (HDM) SLIT tablets have been available since 2015, and JC SLIT tablet is approved by 2018 without any age limitation. The randomized DBPC trials that included pediatric patients have been conducted in Japan. In phase II/III trail with JC SLIT tablets, treatment effect-size (improvement of clinical symptoms compared to placebo) was 46.3% after three years treatment. In addition, AR was improved in 45.3% (1 year) and 34.0% (2 years) after discontinuation of SLIT. Adverse reactions in the first season were 50.3%, mostly modulate to mild reactions such as oral edema and pharyngeal irritation.

In April 2023, several future government initiatives against cedar pollen allergies were announced as follows; (1) Measures to control the source of cedar pollen, (2) Improvement in the accuracy of pollen dispersal forecasts, and (3) Measures to prevent the onset and exposure to cedar pollen including the AIT. This presentation will be covering the findings to date, including immunotherapy not only for JC pollinosis but also for HDM induced perennial AR.

In recent years, the number of patients with allergic diseases has been increased, mainly in developed countries. In Japan, about half of the population reportedly have some kinds of allergic diseases, such as hay fever and food allergies. Therefore, allergic diseases have become a social and health economic problem. Since many of the therapeutic agents for allergy target mediators involved in the induction of inflammation, treatment using these agents does not eliminate the underlying cause of inflammation, but only be a symptomatic treatment. Allergen-specific immunotherapy, including sublingual immunotherapy, is a treatment that can be expected to modulate human immune responses to specific allergens, and is the only treatment that has the potential to change the natural history of allergic diseases, thereby attracting enormous attention. On the other hand, there are still many issues to be solved regarding the persistence of the effect and the underlying mechanisms. In this presentation, I will talk about the mechanisms of allergic inflammation and allergen-specific immunotherapy, and discuss the future prospects.

The tendency of animals to interact with others and to live in groups is called sociality. Animals show sociality toward novel individuals, mates (partners), and offspring. Sociality toward novel individuals has been extensively studied. However, sociality with specific members of the opposite sex, such as spouses, has not been well studied. In particular, it is still unclear how the abstract concept of sociality with one's spouse is represented in the brain.

In this study, we used prairie voles, a naturally monogamous rodent, to investigate the neural activity corresponding to socialization with a partner. Previous research has investigated the hippocampal CA1 area as the brain region representing abstract concepts. Therefore, we hypothesized that partner representations also take place in the hippocampus.

We chronically implanted silicon probes into the CA1 area of the hippocampus of male prairie voles to record the CA1 neural activity while they interacted with partner or non-partner females. The males huddled with their partner females significantly earlier than their non-partners. Moreover, some CA1 neurons of the subject voles exhibited specific firing patterns when the subject interacted with the partner.

Oxytocin (oxt) is required for facilitating mate preferences for familiar individuals in a monogamous rodent (prairie vole), irrespective of sex. While the role of oxt in mate choice is understood in a few monogamous species, its function in non-monogamous species, comprising the vast majority of vertebrate species, remains unclear. To address this issue, we evaluated the involvement of oxt in mate choice in medaka fish (Oryzias latipes), which is a model organism commonly used for molecular genetics. Female medaka prefer to choose familiar males as their mating partner. We generated mutants of the oxt ligand (oxt) and receptor genes (oxtr1, and oxtr2), and revealed that the oxt-oxtr1 signaling pathway was essential for eliciting female mate preference for familiar males. In addition, since our group previously reported that the high pacemaker frequency of terminal nerve GnRH3-neruon (TN-GnRH3 neurons) is required for the female mating preference in medaka, we measured the pacemaker frequency of TN-GnRH3 neurons in mutant females. 

Furthermore, RNA-seq analysis revealed that the transcription of three genes that comprise all components of C1q are downregulated in both oxt and oxtr1 mutants, potentially explaining the mutant phenotype.

Aggressive behavior is motivated by conflicts such as territory, food, and mate. However, males need be adaptive for the reproduction of their own pups. It is previously reported that the central part of the medial preoptic area (cMPOA) and the rhomboid nucleus of the bed nucleus of the stria terminalis (BSTrh) are involved in paternal and infanticidal behaviors, respectively. In this study, we demonstrate that this behavioral shift is mediated by synaptoplastic changes induced by social experience with a female partner and pups. The cMPOA was involved in the synaptic inhibition in the BSTrh, which was enhanced by parental experiences. In contrast, the excitatory–inhibitory balance of synaptic inputs from the medial amygdala (Me) to the cMPOA was shifted toward the excitation ahead of pup delivery. The optogenetic manipulation of the Me−cMPOA synapses promoted affiliative behavior toward pups. These findings provide possible neurocellular mechanisms for the inhibition of infanticide and the emergence of paternal behavior in mice.

The adult brain can flexibly adapt behaviors to specific life-stage demands. For example, while sexually naïve male mice are aggressive to the conspecific infants, father mice provide caregiving to them. How this behavioral shift from infanticidal to parental state is implemented at the level of neural circuits remains poorly understood. Vasopressin is a neural hormone produced in the paraventricular nucleus of the hypothalamus. We herein show that vasopressin suppresses pup-directed aggression in male mice. Vasopressin is known to activate oxytocin receptors (OTRs) as well as its canonical receptors, a phenomenon called ligand-receptor crosstalk. We found that vasopressin-mediated suppression of aggression is impaired by pharmacological blockage of OTRs or conditional knockout of the OTR gene from the preoptic regions, suggesting that vasopressin-to-OTR signaling contributes to the inhibition of pup-directed aggression. The chemogenetic activation of OTR+ neurons in the preoptic regions is sufficient to induce paternal caregiving behaviors. Collectively, our study demonstrates that vasopressin-derived suppression of aggression is, in part, mediated via OTR signaling in preoptic regions. This is, to our knowledge, the first in vivo functional characterization of non-canonical vasopressin-to-OTR crosstalk.

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 enter the brain via a systemic route beyond the blood-brain barrier (BBB) to reach the neurons efficiently. Taking advantage of the phenomenon that transporter-1, which is expressed at a very high level in the brain capillary endothelial cells (BCECs), migrates from the luminal side to the abluminal side in response to a rapid increase of blood glucose concentration from the hypoglycemic state, we succeeded in accumulating a self-assembled supramolecular nanocarrier with glucose integrated on the surface with optimal configuration in the brain, with significant efficiency. Precise control of glucose density on the surface of the nanocarrier allowed selection of the accumulation point in the brain, and a particularly high degree of accumulation in the neurocytes was achieved. In this presentation, we will introduce recent research that is developing a treatment for neurological diseases together with nanocarrier that passes through the BBB with nearly 100 times higher efficiency than existing technologies.

New drug candidate modalities for central nervous system (CNS) diseases with high unmet medical needs has expanded from small molecule drugs to medium and high molecular weight drugs, and expectations for therapeutic drug development are increasing. However, these drug modalities delivery into the brain via systemic blood circulation is limited by the blood-brain barrier (BBB), and the development of a drug delivery system (DDS) that overcomes the BBB is key to the development of new drugs for CNS diseases.

　In recent years, the administration routes have divaricated along with the diversification of modalities. Among above, intranasal administration has been noted as a method of non-invasive delivery of a drug modality to the brain by bypassing the BBB via the nose-to-brain direct route. This route has the potential to be novel administration route into the CNS for all drug modalities that are not expected to penetrate the BBB. However, intranasal administration of a modality alone is often insufficient in terms of its delivery efficiency and delivery deeper site into the brain, in addition, there are very few reports on drug delivery into the spinal cord. Therefore, to achieve efficient nose-to-brain drug delivery to the CNS, including the spinal cord, it is necessary to design DDS nanocarriers based on an understanding of nose-to-brain/spinal cord pharmacokinetics. In this situation, the nose-to-brain drug delivery research using nanoparticles as DDS nanocarriers (nanoDDS) has recently become more active and diversified.

In this symposium, we will review the representative mechanisms as well as the types and characteristics of typical nanoparticles in nose-to-brain delivery, and introduce our latest research on nanoDDS of nucleic acid drugs based on nose-to-brain route.

Schizophrenia affects more than 20 million people worldwide, and approximately one third of patients have a limited response to antipsychotic medication treatment and are assessed to have treatment-resistant schizophrenia. Clozapine is the only drug with proven efficacy in schizophrenia that does not respond to other antipsychotics. However, potentially serious side effects such as agranulocytosis have limited clozapine treatment. Using comprehensive combined gene expression analysis and proteomics analysis, we have identified connective tissue growth factor (CTGF), a modular secreted protein, involved in the ameliorative effects of clozapine on cognitive impairments. CTGF, also known as CCN2, is a 38 kDa, cysteine-rich, extracellular matrix protein. We have found that coadministering peptides and proteins with the cell-penetrating peptide L-penetratin intranasally significantly increased transport to the brain and enhanced pharmacological effects. We thus demonstrated that chronic administration of intranasal CTGF with L-penetratin improved cognitive impairments. Additionally, concomitant treatment with intranasal injection of the neutralizing antibody against CTGF and L-penetratin blocked the effects of clozapine. In this symposium, we would like to discuss about new treatment perspectives in treatment-resistant schizophrenia.

Drug targets for the treatment of neurodegenerative and neuropsychiatric disorders reside in the central nervous system (CNS). However, the routes of drug transfer from the periphery to the CNS include the blood-brain barrier and the blood-cerebrospinal fluid barrier, which stand as big walls in the construction of treatment strategies. A number of antibodies targeting various biological molecules have recently been developed. Antibodies targeting amyloid-β have attracted attention as a treatment for Alzheimer's disease, and are expected to be applied to other CNS diseases in the future. By contrast, antibodies do not easily pass through those barriers due to their characteristics. Therefore, the development of efficient delivery methods for antibodies to the CNS is an important topic. We have focused on intranasal administration as a means of delivery to the CNS and have confirmed its usefulness in various antibodies. This talk will present an antibody therapy targeting type I interferon receptor (IFNR), a molecule that contributes to emotional abnormalities in mice with chronic pain stress. In conclusion, we suggest that antibodies against IFNR may be useful for the treatment of depression and anxiety disorders and present a new therapeutic strategy for neuropsychiatric disorders based on intranasal administration.

Real-time recording of the kinetics of systemically administered drugs in vivo may improve medical therapies. Nonetheless, conventional methods for drug detection require considerable analyte quantities and have poor sampling rates. Additionally, they cannot address how drug kinetics correlates with target function over time. Here, we describe a microsensing system equipped with two different sensors. One is a needle-type microsensor composed of boron-doped diamond, and the other is a glass microelectrode. We first tested bumetanide, a diuretic that can induce deafness. In the guinea-pig cochlea, our microsensing system measured, simultaneously and in real-time, changes in bumetanide concentration and the electrical activity underlying hearing. In the rat brain, we tracked the kinetics of the drug and neuronal activity. The system also detected the actions of the antiepileptic drug lamotrigine and its effect on neurons. Clearly, the local pharmacokinetics of bumetanide differed from that of lamotrigine. Finally, the diamond microsensor successfully detected the behavior of doxorubicin, an anticancer reagent, in the skin. Our microsensing system may detect pharmacological and physiological responses of other chemical compounds and contribute to advances in next-generation pharmacological interventions and on-site drug monitoring.

With the establishment of Modernization 2.0 and the OECD's in vitro battery tests for developmental neurotoxicity, there has been a growing demand for compound evaluation methods using in vitro neuronal cells. Assessment methods utilizing microelectrode array (MEA) are increasingly becoming a primary approach for evaluating the safety and efficacy of compounds, taking the functionality of neural networks as a benchmark. In this presentation, we will introduce compound evaluation methods based on high spatiotemporal resolution measurements using the next-generation MEA, the high-density complementary metal-oxide semiconductor (CMOS)-MEA, and an MEA system capable of simultaneously measuring extracellular potentials and electrochemical measurements. Using the CMOS-MEA, we detected compound responses based on detailed propagation pattern analysis in brain slices and brain organoids, as well as single-cell firing pattern analysis in cultured neurons. We have also developed an enzyme-modified carbon nanotube microelectrode array (CNT-MEA) capable of measuring glutamate at the nM level. By establishing an MEA system that can simultaneously measure extracellular potentials and electrochemical measurements, we successfully achieved real-time measurements of extracellular potentials and glutamate release from acute brain slices. CMOS-MEA and CNT-MEA provides a new understanding of the basic mechanisms of brain circuits in vitro and ex vivo, on human neurological diseases for drug discovery, and compound toxicity assessment.

Micro- and nanoscale microelectrodes can directly oxidise or reduce cellular metabolites and quantify the concentration of metabolites based on the current value. It is also possible to quantify chemicals that are difficult to oxidise or reduce, such as glucose, by measuring the chemicals produced via enzymatic reactions. We have developed a system that enables the measurement of metabolites on the cell surface by miniaturising the size of these microelectrodes to the level of 1/100 of a single cell. Furthermore, scanning electrochemical microscopy (SECM), which utilises scanning probe microscopy technology to scan the microelectrode on the cell, has developed a simultaneous imaging technique for cell surface topography and metabolites.

　Furthermore, a similar cell surface imaging technique was established for nanopipettes, which are miniaturised glass pipettes used for patch clamping. Nanopipettes are easier to miniaturise than microelectrodes, and a nanopipette with a radius of 50 nm can be used for topography imaging of live cell surfaces with a resolution of 20 nm in the XY direction and 5 nm in the Z direction. Furthermore, a system for simultaneous imaging of pH and topography has been developed by modifying the tip of a glass nanopipette with a pH-sensitive polymer.

A better understanding of the biological functions of microorganisms is required to reduce their threats and increase their usefulness. Therefore, the importance of real-time evaluation of bacterial activity increase for various purposes such as hygiene management, development of antibacterial agents, and effective utilization of bacterial resources. This necessitates a quantitative assessment of metabolic processes, including growth and respiration. Here we would like to introduce the development of electrochemical methods for assessing bacterial activity. 

Electrochemical detection of viable bacterial cells was performed using a cell membrane permeable electron mediator and redox-active pigment. A tetrazolium salt was converted to an insoluble reduction form through the respiration of microbial cells. The insolubility of this formazan was effectively exploited as a surface-confined oxidation event. The electrochemical oxidation of formazan was effectively coupled with the thermal lysis of microbes. The sensitivity of the present technique is up to 10,000-fold higher than that of colorimetry and requires an incubation time of only 1 h. Furthermore, the measurement of the reduction current of tetrazolium salt provides an effective means for assessing the bacterial activity in suspension.

With the experience of the COVID-19 pandemic and the importance of health is the renewed awareness, the relationship between food and health is attracting attention. In particular, advances in analytical techniques have revealed the biological functions of metabolites produced from fatty acids in dietary oils, and the function of dietary oils in relation to health has been elucidated. In this presentation, I would like to introduce recent findings about the possibility of precision nutrition based on the individual differences in the regulation of function and health maintenance/promotion through the metabolism of dietary oils by the host and commensal bacteria.

GPx4 is an enzyme that reduces oxidized phospholipids in a glutathione-dependent manner. GPx4 is also a regulator of lipid oxidation-dependent cell death ferroptosis (iron-dependent) and lipoxytosis (iron-independent), which have recently attracted much attention. Mice deficient in cardiac-specific GPx4 are lethal during development, but grow normally when fed a vitamin E-supplemented diet. Conversely, when heart-specific GPx4-deficient mice that have grown normally are switched from a vitamin E-supplemented diet to a normal diet, sudden cardiac death is induced in about 15 days by increase of lipid peroxidation in heart. Using these mice as a model of lipid oxidation-dependent heart failure, we screened for drugs and food ingredients that can suppress heart failure. As a result, we found several heart failure inhibitors with lipid oxidation inhibitory activity. Surprisingly, however, we also found that food ingredients and antibiotics without lipid oxidation inhibitory activity could suppress lethality. Analysis of the mechanism revealed that these antibiotics inhibit cardiac lipid oxidation by altering the intestinal microbiota. It was also found that administration of this intestinal bacterium also suppressed lipid oxidation-dependent heart failure. This finding opens the way for the development of new therapeutic agents for heart failure.

Currently, the development of functional foods that take advantage of the bio-regulatory effects of foods has been active. The phytochemicals, which are derived from plants, play an important role as a factor in the functionality of such foods. Phytochemicals are substances produced by plants to protect themselves from ultraviolet rays, harmful substances, pests, and foreign enemies, and are a general term for chemical substances contained in plants. With the development of analytical techniques, the functionality of various phytochemicals and their mechanisms of action are becoming clearer. Since phytochemicals cannot be produced in the human body, they must be supplemented from foods and supplements. Therefore, the phytochemicals should be taken for the purpose of health maintenance by understanding which phytochemicals should be taken for what purpose. We have been engaged in research on the effects of carotenoids and polyphenols, which are classified as phytochemicals, on the central nervous system, such as the brain and eyes, as well as their molecular mechanisms. In this presentation, we will focus on the pharmacological effects of carotenoids such as astaxanthin and crocetin, and discuss their anti-aging and preventive effects on central nervous system diseases.

Aging has long been an issue, and approximately one of four Japanese will be 75 or older by 2065. Therefore, it is expected that medical costs will increase, and locomotive disorders (locomotive syndrome) and dementia will become more serious. Aging is a combination of such disorders of memory, movement, and emotion. These motor, memory, and emotional disorders need to be improved collectively in order to achieve a healthy and long-lived society. We found that knockout (KO) mice of DGKgamma, a lipid-metabolizing enzyme, exhibit memory, motor, and emotional deficits due to aging. Using these aged model mice, we found that the memory, motor, and emotional deficits were caused by an increase in the activity of PKCgamma in respective brain-specific regions. We also found that PKCgamma activity in the brain is increased in SAM mice, which are famous model mouse of aging. More surprisingly, the memory, emotional, and motor deficits were ameliorated by oral administration of certain flavonoid for 2 weeks, along with normalization of PKCgamma activity. These facts suggest that relatively short-term dietary application can improve memory, emotional, and motor impairments associated with aging.

Antimicrobial resistance (AMR) in bacteria is one of the most critical public health challenges humanity is facing. In our country, infections caused by AMR bacteria, such as multidrug-resistant Gram-negative rods and non-tuberculous mycobacteria, are spreading without effective treatment options, leading to the increasing complexity of bacterial infections. Given this context, there is a strong need to establish efficient treatment strategies for AMR bacterial infections. Understanding the impact of patient background factors and disease pathophysiology on treatment outcomes is crucial for advancing personalized medicine in infectious diseases and selecting more effective treatment options. However, this aspect remains insufficiently elucidated at present. In recent years, medical big data analysis has made it possible to conduct clinical evaluations of drug treatments that reflect real-world clinical settings, encompassing diverse patient populations, and various factors, and such analyses have commenced across various diseases. We are conducting research to comprehensively explore factors and drug interactions that influence the efficacy of infectious disease treatments using medical big data, which encompasses the diversity and complexity of diseases. To establish a system for conducting rapid clinical research during future outbreaks of emerging infectious diseases, it is desirable to establish an analysis platform utilizing big medical data.

Trimethoprim (TMP)-sulfamethoxazole (SMX) is a synergistic antimicrobial drug combination used to treat various bacterial and certain fungal infections. It was previously understood that this drug combination is synergistic because these two drugs inhibit sequential steps of the bacterial folate biosynthesis pathway. SMX inhibits the production of the tetrahydrofolate (THF) precursor dihydropteroate, and TMP inhibits the conversion of dihydrofolate (DHF) to THF. Consequently, SMX potentiates TMP by limiting de novo DHF production, and this mono-potentiation mechanism was the previous explanation for their synergistic action. We demonstrated that this model is insufficient to explain the potent synergy of TMP-SMX. Using genetic and biochemical approaches, we characterize a metabolic feedback loop in which THF is critical for producing the folate precursor dihydropterin pyrophosphate (DHPPP). We reveal that TMP potentiates SMX activity through the inhibition of DHPPP synthesis. Our study demonstrates that the mutual potentiation of the action of each drug on the other drives the TMP-SMX synergy. These findings highlight the importance of metabolic pathway structure in understanding antimicrobial drug interactions. In this talk, I will introduce our strategy to identify additional pathways that can be explored for potently synergistic drug targets.

Following the discovery of the world's first antibiotic, penicillin, by Alexander Fleming in 1928, there was a time when numerous antimicrobials were developed, and it appeared as if humanity had conquered bacterial infections. However, the use of antimicrobials and the emergence of antimicrobial-resistant bacteria have led to repetitive cycles, resembling a cat-and-mouse game, and bacterial infections continue to pose a global public health threat. In the 2000s, when the development of novel antimicrobials stagnated, the battle between humans and bacteria tilted in favor of rapidly evolving multidrug-resistant bacteria. According to the latest epidemiological study, it was estimated that approximately 1.27 million people worldwide died from antimicrobial-resistant bacterial infections in 2019. This number surpasses the deaths caused by malaria and HIV infections, thus being internationally recognized with significant impact. Here, I will provide the overview of antimicrobial resistance mechanisms in clinically relevant bacterial pathogens as well as discuss our research and development of antimicrobial adjuvants that enhance the activity of existing antimicrobials.

Against the backdrop of inappropriate use of antimicrobials, the number of antimicrobial-resistant (AMR) bacteria is increasing worldwide. On the other hand, the development of new antimicrobials with efficacy to counter them is on the decline, making this a major issue internationally. Infections caused by AMR bacteria are considered to be “silent pandemic” as this is spreading steadily and global threat.

In the “Global Action Plan on Antimicrobial Resistance” by the World Health Organization (WHO), donor countries are required to support the achievement of the Global Action Plan. In particular, it is considered important to ensure the availability of antimicrobials with measures to maintain their effectiveness, and to work on new prevention, diagnosis, and treatment methods. In Japan, “National Action Plan on AMR (2023-2027)” was released in April 2023. It emphasizes the importance of collaborating with various fields from an international perspective to promote countermeasures against drug resistance.

In this presentation, I will show the characteristics and global development strategy of novel siderophore cephalosporin, cefiderocol, which has activity against Gram-negative bacteria, including multidrug-resistant bacteria. In addition, I will introduce GARDP-led efforts toward the clinical use of flomoxef, which has activity against ESBL-producing bacteria, for sepsis in neonates. I hope that this will be an opportunity to think about the importance of fusion of various fields and international cooperation to combat AMR.

Digital transformation (DX) is now being touted in various areas of drug discovery research. However, automation of non-clinical studies and basic research using laboratory animals is highly challenging. In our laboratory, we have been developing an animal behavior analysis system using images and machine learning for six years. We are now in the process of developing a technology that not only automates animal experiments, but also captures new animal phenotypes over long periods of time, day and night, that have not been visible to humans before, and reduces them to structural values. It is also very significant in that it allows us to evaluate animal phenotypes in an unbiased manner. Currently, it is possible to 1) identify individual mice and rats, 2) evaluate their spontaneous movements including eating and drinking, 3) identify and track tissue sites, 4) detect specific behaviors such as scratching, grooming, and standing up, and 5) detect facial expressions such as pain. At the same time, we are also working to generalize these technologies so that they can be used in your laboratories in the near future. In this symposium, we would like to introduce current technologies and future challenges. We hope that these technologies will contribute to animal welfare and provide a new impact for pharmacotoxicological research.

Pain is a fundamental sensation to perceive tissue injury. Since various diseases induce tissue injury, many patients are suffering from it. Therefore, intensive research has been carried out using experimental animal models to investigate the mechanism and to develop the effective therapy. Recent methods for pain assessment like grimace scale scoring and von Frey test depend on researcher’s manual observation. These human-powered tests are laborious, time-consuming, and low-throughput, and lack in the objectivity and reproducibility. The technology of artificial intelligence especially neural network achieved a remarkable progress. Among them, convolutional neural network (CNN) has been the de facto standard method for image recognition tasks. We here established an automated pain assessment method from the face image of mice using CNN. CNN trained with hundreds of thousands face images could accurately predict “pain” or “no pain” from a face image (sensitivity: 97%, specificity: 99%). We also revealed that trained CNN was applicable for the assessment of pain killer. In this section, I would like to introduce the detailed method, results, and application of our methods.

The pharmaceutical industry is currently facing the challenge of optimizing and streamlining the drug discovery process. Despite the increasing use of artificial intelligence (AI) in drug target identification, compound exploration, and optimization, most non-clinical animal experiments still rely on human manual assessments. In each company and laboratory, animal experiments involve varied systems and observation criteria relying on visual assessments, resulting in discrepancies in test outcomes and imprecise evaluations. To address these challenges, we propose standardizing experimental systems and implementing AI-driven video analysis technology. Leveraging collaborative research with The University of Tokyo, Revamp Corporation. is now developing these AI-based animal behavior analysis systems, including environmental designs. Our company aims to optimize and improve the efficiency of drug development by building animal behavior analysis  systems with high accuracy, reproducibility, and versatility. In this presentation, I would like to introduce some of those attempts with specific examples.

Traditional evaluations of human disease model mice for drug development have primarily focused on hematological and biochemical analyses, as well as pathological assessments. Recently, non-invasive live imaging techniques such as MRI and CT have been introduced. However, there is a pressing need to identify early disease pathogenesis promptly when utilizing model animals for drug research. Thus, the development of a new animal experimental method is essential, which involves continuous, comprehensive monitoring of animal behavior through digital analysis.  We propose the use of high-resolution IR cameras and RFID readers in custom-designed cages to acquire continuous and comprehensive behavioral data for disease model mice. By embedding chips in individual mice, we can analyze both individual and group behavior among multiple animals. Furthermore, this approach allows for the quantitative capture of symptom exacerbation and improvement over time. Additionally, the behavioral data obtained in this study will be subjected to "unsupervised learning" using artificial intelligence (AI). We expect that AI-based analysis will enable the detection of subtle behavioral changes in disease model mice that may go unnoticed to the human eye.

Oral tolerance is an immune regulatory system for foods. Foods are nutrition in healthy people. However, foods are immune exclusion material in patients of food allergy. We thought two possibilities for immune intolerance for foods, failing acquisition of oral tolerance and breaking acquired tolerance. Then, we made murine models for two possibilities.

First, we made the food allergy model and oral tolerance model for ovalbumin (OVA). Experimental oral tolerance was induced by previously oral treatment with OVA solution before the sensitization by intraperitoneal injection of OVA. In the oral tolerance model, elevation of OVA-specific IgE was suppressed completely and anaphylaxis was not induced. Next, as the model of failing acquisition of oral tolerance, we added the food additives into the OVA solution for tolerance. As results, intake of the OVA solution with food additives prevented acquiring oral tolerance and induced anaphylaxis for OVA. Recently, “dual-allergen exposure hypothesis” was suggested. The hypothesis proposed allergic sensitization to food could occur through cutaneous sensitization and that consumption of food protein induced oral tolerance. We attempted to override oral tolerance by sensitization via skin. We found that epicutaneous sensitization or sensitization by intradermal injection of OVA could override acquired oral tolerance. As two common denominators for immune intolerance for foods, we confirmed the change of migration of dendritic cells and prevention of inducing regulatory T cells.

Development of allergic diseases is mainly dominated by the type 2 immune response governed by Th2 cells and group 2 innate lymphoid cells (ILC2), both of which produce interleukin (IL)-4, IL-5 and IL-13. Allergen immunotherapy (AIT) is an only causative treatment for allergic diseases by modification of the type 2 immune response. Although regulatory mechanisms of Th2 cells by AIT have been reported to date, those of ILC2 have not been fully elucidated. Although 50-70% of patients with allergies respond favorably to AIT, these therapies are ineffective for the remaining ~30%. The non-responders exhibit persistent ILC2 in peripheral blood despite long-term treatment of AIT. On the other hand, it has been clinically demonstrated that AIT amplified production of "blocking antibody" IgG4 in peripheral blood. In a murine model of asthma, we demonstrated that AIT-increased IgG1 corresponding to human IgG4 significantly suppresses the proliferation of ILC2. It was found that the murine ILC2 did express mRNA of the inhibitory Fc gamma receptor, FcγRIIB, and the expression level correlated with the severity of allergic responses. These data suggested that AIT-increased IgG1 directly suppresses the proliferation of ILC2 via binding FcγRIIB. Therefore, the IgG1-FcγRIIB axis could be a therapeutic target for the non-responders to AIT.