JPWO2012074043A1 - Traumatic neuropathy treatment - Google Patents
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Abstract
外傷性神経障害、即ち脳外傷、脳外科手術に伴う脳損傷及び／又は脊髄損傷を治療するための新規薬剤を提供する。脳に対する物理的外力とHMGB1との関係を調べたところ、正常な状態ではHMGB1は脳神経細胞の核内に存在するものの、傷害を受けた部位又はその近傍の組織ではHMGB1は神経細胞の核外に放出されることを初めて確認した。これにより、神経細胞の核外に放出されたHMGB1を捕捉することで、脳外傷、脳外科手術に伴う脳損傷及び／又は脊髄損傷により生じる運動障害を改善化しうることを見出し、本発明を完成した。Provided are novel agents for treating traumatic neuropathy, i.e. brain trauma, brain injury associated with brain surgery and / or spinal cord injury. When the relationship between the physical external force on the brain and HMGB1 was examined, HMGB1 is present in the nucleus of brain neurons in a normal state, but HMGB1 is outside the nucleus of nerve cells in the damaged site or in the vicinity thereof. It was confirmed for the first time that it was released. As a result, it was found that by capturing HMGB1 released outside the nucleus of nerve cells, it was possible to improve movement disorders caused by brain trauma, brain injury and / or spinal cord injury associated with brain surgery, and completed the present invention. .
Description
本発明は、外傷性神経障害、即ち脳外傷、脳外科手術に伴う脳損傷及び／又は脊髄損傷を治療するための薬剤に関するものである。 The present invention relates to an agent for treating traumatic neuropathy, that is, brain injury, brain injury associated with brain surgery, and / or spinal cord injury.
本出願は、参照によりここに援用されるところの日本出願、特願２０１０−２７０１３３号及び特願２０１１−１３１６７４号優先権を請求する。 This application claims the Japanese application which is used here by reference, Japanese Patent Application No. 2010-270133 and Japanese Patent Application No. 2011-131474.
脳外傷とは、外傷性脳損傷(Traumatic Brain Injury：TBI)ともいい、例えば何らかの事故（例えば交通事故、落下事故、衝突事故や転落事故など）により首から上の頭部に強い外力が加えられることにより外傷を負い、脳そのものが損傷した病態をいう。脳損傷には「閉鎖性頭部損傷：closed head injury」(CHI)と「開放性頭部損傷：open head injury」(OHI)の２つの基本的なタイプがあり、開放性頭部損傷は弾丸や何か突き刺さった物体によって引き起こされる。閉鎖性頭部外傷は、例えば上述のような事故により、急激な動きの間に脳が頭蓋骨の内側で急に動かされることによって引き起こされ、発生する。かかる急激な動きによる圧迫は神経の線維や軸索に損傷を与え、神経伝達経路を破壊する。 Brain trauma is also called traumatic brain injury (TBI). For example, a strong external force is applied to the head above the neck due to some kind of accident (such as a traffic accident, a fall accident, a collision accident, or a fall accident). This refers to a condition in which the brain itself has been injured and the brain itself has been damaged. There are two basic types of brain injuries: “closed head injury” (CHI) and “open head injury” (OHI). Or caused by something stuck. An obstructive head injury is caused and caused by a sudden movement of the brain inside the skull during a sudden movement, for example by an accident as described above. The compression caused by such rapid movement damages nerve fibers and axons and destroys the nerve transmission pathway.
脳外傷急性期には、生命維持に直接関連する呼吸と循環の管理による全身状態の機能維持が図られる。一方、脳外傷急性期には脳浮腫が発生するため、これを防ぐ目的で高浸透圧グリセロールやマンニトール点滴や代謝抑制を目指したバルビツール酸投与などが臨床的に用いられる場合がある。しかしながら、その有効性についての証拠・根拠（エビデンス）はない。低体温療法が急性期に用いられる場合もあるが、一般的でない。その他、脳損傷に起因する脳浮腫・脳障害に有効な薬物は見出されていない。つまり、脳外傷急性期の脳障害に対するエビデンスのある治療法は現在まで存在していない。 In the acute phase of brain injury, functional maintenance of the general state is achieved by managing respiration and circulation that are directly related to life support. On the other hand, cerebral edema occurs in the acute stage of brain trauma, and hyperosmolar glycerol, mannitol infusion, and barbituric acid administration aiming at metabolic inhibition may be clinically used to prevent this. However, there is no evidence or evidence for its effectiveness. Although hypothermia may be used in the acute phase, it is not common. In addition, no effective drug has been found for cerebral edema and brain damage caused by brain damage. In other words, there is no evidence of a cure for cerebral damage in the acute phase of brain injury.
脊髄は、脳と体の他の部分の情報を伝達する主要経路である。チューブ状の構造で、脳の基底部から下方へ伸びている。脊髄損傷(Spinal Cord Injury：SCI)とは、主として脊柱に強い外力が加えられることにより脊椎を損壊し、脊髄に損傷をうける病態である。かかる脊柱に加えられた強い外力により、神経の線維や軸索に損傷を与え、神経伝達経路を破壊する。 The spinal cord is the primary pathway for transmitting information about the brain and other parts of the body. A tube-like structure that extends downward from the base of the brain. Spinal Cord Injury (SCI) is a pathological condition that damages the spinal cord by damaging the spine mainly by applying a strong external force to the spinal column. The strong external force applied to the spinal column damages nerve fibers and axons and destroys the nerve transmission pathway.
脳損傷防御剤及び頭部外傷や脳外科手術に伴う脳組織損傷防御剤について開示がある（特許文献１）。特許文献１では、（±）-N,N'-ロピレンジニコチンアミド（一般名：ニカラベン）又はその薬学上許容しうる塩を有効成分として含有することを特徴とし、脳損傷の進展に関連のあるマクロファージ及び反応性マイクログリアの動態を指標として評価している。他の報告では、外傷性脳損傷(TBI)又は低酸素性若しくは虚血性発作を治療する方法が開示されている（特許文献２）。特許文献２では、低酸素性若しくは虚血性発作を治療する方法であって、そのような治療を必要とする患者に、血栓崩壊剤と組合せてNMDAレセプター拮抗物質を投与することを含む方法について開示がある。具体的には、NR2Bサブタイプ選択的N-メチル-D-アスパラギン酸(NMDA)レセプター拮抗物質を、(a)ナトリウムチャネル拮抗物質；(b)一酸化窒素シンターゼ(NOS)阻害剤；(c)グリシン部位拮抗物質；(d)カリウムチャネルオープナー(opener)；(e)AMPA/カイニン酸レセプター拮抗物質；(f)カルシウムチャネル拮抗物質；(g)GABA-Aレセプターモジュレーター（例えばGABA-Aレセプター作用物質）；又は(h)抗炎症剤と組合せて投与することを含む方法が開示されている。また、別の報告では、外傷性脳損傷(TBI)、脊髄損傷(SCI)又はそれに関連する状態に起因する生理学的損傷を抑制又は治療するための方法について開示がある（特許文献３）。特許文献３では、補体副経路を選択的に阻害する作用剤及び組成物の使用が開示される。TBI又はSCIに起因する損傷の抑制に使用される好ましい試薬には、補体系のタンパク質であるB因子を阻害するものが含まれ、抗B因子抗体は特に好ましい作用剤として開示されている。副経路の活性化に関与する主要タンパク質は、B因子(fB)及びD因子(fD)である。 There is a disclosure of a brain injury protective agent and a brain tissue injury protective agent accompanying head trauma or brain surgery (Patent Document 1). Patent Document 1 is characterized by containing (±) -N, N′-ropyrenedinicotinamide (generic name: Nicaraben) or a pharmaceutically acceptable salt thereof as an active ingredient, and is related to the progress of brain damage. We evaluate the dynamics of certain macrophages and reactive microglia as an index. Another report discloses a method of treating traumatic brain injury (TBI) or hypoxic or ischemic stroke (Patent Document 2). Patent Document 2 discloses a method for treating hypoxic or ischemic stroke, comprising administering an NMDA receptor antagonist in combination with a thrombolytic agent to a patient in need of such treatment. There is. Specifically, an NR2B subtype-selective N-methyl-D-aspartate (NMDA) receptor antagonist, comprising: (a) a sodium channel antagonist; (b) a nitric oxide synthase (NOS) inhibitor; Glycine site antagonist; (d) potassium channel opener; (e) AMPA / kainic acid receptor antagonist; (f) calcium channel antagonist; (g) GABA-A receptor modulator (eg, GABA-A receptor agonist) Or (h) a method comprising administering in combination with an anti-inflammatory agent. Another report discloses a method for suppressing or treating physiological damage caused by traumatic brain injury (TBI), spinal cord injury (SCI) or a related condition (Patent Document 3). In US Pat. No. 6,057,059, the use of agents and compositions that selectively inhibit the alternative complement pathway is disclosed. Preferred reagents used to suppress damage caused by TBI or SCI include those that inhibit factor B, which is a protein of the complement system, and anti-factor B antibodies are disclosed as particularly preferred agents. The major proteins involved in the activation of the alternative pathway are factor B (fB) and factor D (fD).
HMGB1（High Mobility Group Box 1:以下、「HMGB1」という）タンパク質は、DNA の構造維持や転写調節に関わる非ヒストンクロマチン関連性タンパク質ファミリーの一員である。近年、細胞の壊死によりHMGB1が細胞外に遊離したり、血管炎症性シグナル応答で細胞外に能動分泌が見られるなど、新たな炎症マーカーとしても注目されている。HMGB1はげっ歯類からヒトまで99％以上のアミノ酸配列について相同性を有するタンパク質である。このHMGB1は正常細胞にも存在するが、敗血症（全身性炎症反応症候群）において放出される菌体内毒素であるLPS（リポ多糖）による刺激によって血中濃度が上昇し、最終的な組織障害をもたらす。このHMGB1に対する抗HMGB1モノクローナル抗体を有効成分とする薬剤について、脳血管攣縮抑制剤（特許文献４）及び脳梗塞抑制剤（特許文献５）がすでに特許されているが、脳外傷、脳外科手術に伴う脳損傷及び／又は脊髄損傷を治療するための薬剤については開示されていない。 HMGB1 (High Mobility Group Box 1: hereinafter referred to as “HMGB1”) protein is a member of a family of non-histone chromatin-related proteins involved in DNA structure maintenance and transcriptional regulation. In recent years, it has been attracting attention as a new inflammatory marker, such as HMGB1 being released extracellularly due to cell necrosis, or active secretion being observed extracellularly by vascular inflammatory signal response. HMGB1 is a protein having homology with 99% or more amino acid sequences from rodents to humans. Although this HMGB1 is also present in normal cells, the blood level is increased by stimulation with LPS (lipopolysaccharide), an endotoxin released in sepsis (systemic inflammatory response syndrome), resulting in final tissue damage . Regarding drugs containing anti-HMGB1 monoclonal antibody against HMGB1 as active ingredients, cerebral vasospasm inhibitor (Patent Document 4) and cerebral infarction inhibitor (Patent Document 5) have already been patented, but accompany brain injury and brain surgery. There is no disclosure of agents for treating brain injury and / or spinal cord injury.
本発明は、外傷性神経障害、即ち脳外傷、脳外科手術に伴う脳損傷及び／又は脊髄損傷を治療するための新規薬剤を提供することを課題とする。 An object of the present invention is to provide a novel drug for treating traumatic neuropathy, that is, brain injury, brain injury associated with brain surgery, and / or spinal cord injury.
本願発明者らは、上記課題を解決するために鋭意検討を重ね、脳に対する物理的外力とHMGB1との関係を調べたところ、正常な状態ではHMGB1は脳神経細胞の核内に存在するものの、傷害を受けた部位又はその近傍の組織ではHMGB1は神経細胞の核外に放出されることを初めて確認した。これにより、神経細胞の核外に放出されたHMGB1を捕捉することで、脳外傷、脳外科手術に伴う脳損傷及び／又は脊髄損傷により生じる運動障害を改善化しうることを見出し、本発明を完成した。 The inventors of the present invention have made extensive studies in order to solve the above problems and examined the relationship between the physical external force on the brain and HMGB1.In normal conditions, although HMGB1 is present in the nucleus of brain neurons, injury For the first time, it was confirmed that HMGB1 was released from the nucleus of nerve cells in the tissue at or near the site where it was exposed. As a result, it was found that by capturing HMGB1 released outside the nucleus of nerve cells, it was possible to improve movement disorders caused by brain trauma, brain injury and / or spinal cord injury associated with brain surgery, and completed the present invention. .
すなわち本発明は、以下よりなる。
１．抗HMGB1モノクローナル抗体を有効成分とすることを特徴とする、外傷性神経障害治療剤。
２．外傷性神経障害が、脳外傷、脳外科手術に伴う脳損傷及び／又は脊髄損傷である、前項１に記載の外傷性神経障害治療剤。
３．外傷性障害を受けた後に投与するものである前項１又は２に記載の外傷性神経障害治療剤。
４．抗HMGB1モノクローナル抗体を、１回当たり0.2〜5 mg/kg投与するものである前項１〜３のいずれか１に記載の外傷性神経障害治療剤。
５．前項１〜５のいずれか１に記載の外傷性神経障害治療剤を用いることを特徴とする、外傷性神経障害の治療方法。That is, this invention consists of the following.
1. A therapeutic agent for traumatic neuropathy, comprising an anti-HMGB1 monoclonal antibody as an active ingredient.
2. 2. The therapeutic agent for traumatic neuropathy according to item 1 above, wherein the traumatic neuropathy is brain injury, brain injury associated with brain surgery, and / or spinal cord injury.
3. 3. The therapeutic agent for traumatic neuropathy according to 1 or 2 above, which is administered after receiving traumatic disorder.
4). 4. The therapeutic agent for traumatic neuropathy according to any one of 1 to 3 above, wherein the anti-HMGB1 monoclonal antibody is administered at 0.2 to 5 mg / kg at a time.
5. 6. A method for treating traumatic neuropathy, comprising using the therapeutic agent for traumatic neuropathy according to any one of 1 to 5 above.
本発明の抗HMGB1モノクローナル抗体を有効成分とすることを特徴とする、外傷性神経障害治療剤は、パーカッション傷害モデルラットにおいて、行動学的にも、組織学的にも優れた効果を示した。また、現在使用されている抗体薬剤を考慮すれば、重篤な副作用を生じる可能性は極めて少ないと考えられる。従って、本発明の外傷性神経障害治療剤は、これまで特に有効な処置手段のなかった外傷性神経障害を治療できるものとして、極めて有用である。 The therapeutic agent for traumatic neuropathy, which comprises the anti-HMGB1 monoclonal antibody of the present invention as an active ingredient, showed excellent behavioral and histological effects in a percussion injury model rat. In addition, considering the currently used antibody drugs, it is very unlikely that serious side effects will occur. Therefore, the therapeutic agent for traumatic neuropathy of the present invention is extremely useful as a therapeutic agent for traumatic neuropathy that has not been particularly effective until now.
本発明の外傷性神経障害治療剤は、抗HMGB1モノクローナル抗体を有効成分とする。抗HMGB1モノクローナル抗体は、原則としてHMGB1のみに結合して無毒化し、他の化合物等には作用しない。よって、副作用が生じる可能性はないか、極めて少ないと考えられる。本発明において、外傷性神経障害治療剤として、有効成分を抗HMGB1モノクローナル抗体とするのは、健常な状態ではHMGB1は神経細胞核内に存在するのに対し、外傷性神経障害に伴い、傷害部位又は傷害部位周辺において、HMGB1は神経細胞核内から、細胞質や細胞外に放出されることを初めて確認したことによる。抗HMGB1モノクローナル抗体を有効成分とする治療剤を投与することで、脳組織や血中に放出されたHMGB1を捕捉し、HMGB1に起因する外傷性神経障害を治療しうる。 The therapeutic agent for traumatic neuropathy of the present invention comprises an anti-HMGB1 monoclonal antibody as an active ingredient. In principle, the anti-HMGB1 monoclonal antibody binds only to HMGB1 to be detoxified and does not act on other compounds. Therefore, it is thought that there is no possibility that a side effect will occur or very little. In the present invention, as an agent for the treatment of traumatic neuropathy, the active ingredient is an anti-HMGB1 monoclonal antibody, whereas HMGB1 is present in the nerve cell nucleus in a healthy state, whereas with traumatic neuropathy, This is because it was confirmed for the first time that HMGB1 was released from the nucleus of the nerve cell to the cytoplasm and extracellular area around the injury site. By administering a therapeutic agent comprising an anti-HMGB1 monoclonal antibody as an active ingredient, it is possible to capture HMGB1 released into brain tissue or blood and treat traumatic neuropathy caused by HMGB1.
本発明において、外傷性神経障害とは、脳外傷、脳外科手術に伴う脳損傷及び／又は脊髄損傷をいう。背景技術の欄でも説明したように、脳外傷とは、外傷性脳損傷(TBI)ともいい、首から上の頭部に強い外力が加えられることにより外傷を負い、脳そのものが損傷した病態をいう。また、脳外科手術に伴う脳損傷とは、例えば腫瘍摘出、血腫除去、その他の脳領域における外科的手術の際に、不可避的に脳に損傷を与えてしまう場合があり、そのような場合をいう。また、脊髄損傷(SCI)とは、主として脊柱に強い外力が加えられることにより脊椎を損壊し、脊髄に損傷をうける病態である。かかる首から上の頭部や脊柱に加えられた強い外力により、神経の線維や軸索に損傷を与え、神経伝達経路を破壊するため、本発明において脳外傷、脳外科手術に伴う脳損傷及び／又は脊髄損傷を、外傷性神経障害ということとする。 In the present invention, traumatic neuropathy refers to brain injury, brain damage associated with brain surgery, and / or spinal cord injury. As explained in the background section, brain trauma is also called traumatic brain injury (TBI), which is a pathological condition in which a strong external force is applied from the neck to the upper head, resulting in injury and damage to the brain itself. Say. In addition, brain damage associated with brain surgery may inevitably damage the brain during, for example, tumor removal, hematoma removal, or other surgical operations in the brain region. . Spinal cord injury (SCI) is a pathological condition that damages the spinal cord by damaging the spine mainly by applying strong external force to the spinal column. In order to damage nerve fibers and axons and destroy nerve transmission pathways by the strong external force applied to the head and spine above the neck, the brain injury and / or brain surgery associated with brain surgery in the present invention and / or Or spinal cord injury is referred to as traumatic neuropathy.
抗HMGB1モノクローナル抗体の調製は、常法に従えばよい。例えば、市販のHMGB1を用いてマウスやラット等を免疫し、その抗体産生細胞や脾細胞と骨髄腫細胞とを融合させてハイブリドーマを得る。このハイブリドーマをクローニングし、HMGB1へ特異的に反応する抗体を産生しているクローンをスクリーニングする。このクローンを培養し、分泌されるモノクローナル抗体を精製すればよい。 The anti-HMGB1 monoclonal antibody may be prepared according to a conventional method. For example, mice, rats, etc. are immunized using commercially available HMGB1, and hybridomas are obtained by fusing the antibody-producing cells, spleen cells and myeloma cells. The hybridoma is cloned, and a clone producing an antibody specifically reacting with HMGB1 is screened. This clone may be cultured and the secreted monoclonal antibody may be purified.
本発明の抗HMGB1モノクローナル抗体を有効成分とする外傷性神経障害治療剤は、その剤形や投与形態は特に問わない。有効成分としての抗HMGB1モノクローナル抗体とともに、溶液、マイクロエマルジョン、分散液、リポソーム、又は高薬物濃度に好適なその他の適確な形状として、製剤化することができる。必要量の抗HMGB1モノクローナル抗体を、必要に応じて上に列挙した成分の１つ又は組み合わせとともに、適切な溶媒中に組み入れた後、ろ過滅菌することによって、調製することができる。 The dosage form and administration form of the therapeutic agent for traumatic neuropathy comprising the anti-HMGB1 monoclonal antibody of the present invention as an active ingredient is not particularly limited. Along with the anti-HMGB1 monoclonal antibody as an active ingredient, it can be formulated as a solution, microemulsion, dispersion, liposome, or other suitable shape suitable for high drug concentrations. The requisite amount of anti-HMGB1 monoclonal antibody can be prepared by incorporating in a suitable solvent, optionally with one or a combination of the components listed above, and then filter sterilizing.
本発明の外傷性神経障害治療剤は、当分野で知られている多様な方法によって投与することができる。投与形態は特に問わないが、外傷性神経障害に対する緊急性を考慮すれば、注射剤として静脈内投与することが好ましい。その場合、溶媒としては、pHを調整した生理食塩水やグルコース水溶液など、血漿の等張液を用いることができる。また、抗体を塩類等と共に凍結乾燥した場合には、純水、蒸留水、滅菌水等も使用できる。その濃度も通常の抗体製剤のものとすればよく、0.2〜5 mg/mLとすることができる。但し、注射剤の浸透圧は、血漿と同等にする必要がある。 The therapeutic agent for traumatic neuropathy of the present invention can be administered by various methods known in the art. The administration form is not particularly limited, but it is preferably administered intravenously as an injection in consideration of the urgency for traumatic neuropathy. In that case, as the solvent, an isotonic solution of plasma such as a physiological saline adjusted with pH or an aqueous glucose solution can be used. In addition, when the antibody is lyophilized with salts or the like, pure water, distilled water, sterilized water, or the like can also be used. The concentration may be that of a normal antibody preparation, and can be 0.2 to 5 mg / mL. However, the osmotic pressure of the injection must be equivalent to that of plasma.
本発明の外傷性神経障害治療剤の投与時期は特に制限されないが、外傷性神経傷害後、早ければ早いほど好ましく、具体的は、傷害後１２時間以内、好ましくは３時間以内、より好ましくは１時間以内、最も好ましくは１０分以内である。 The timing of administration of the therapeutic agent for traumatic neuropathy of the present invention is not particularly limited, but it is preferably as early as possible after traumatic nerve injury, specifically within 12 hours after injury, preferably within 3 hours, more preferably 1 Within hours, most preferably within 10 minutes.
後述する実施例で示す通り、体重約300gのラットに対して１回当たり200μgの抗HMGB1モノクローナル抗体を投与した場合に、脳外傷による脳障害の顕著な抑制効果が得られた。かかる結果から考えると、ヒトに対する投与量は、１回当たり抗HMGB1モノクローナル抗体を0.2〜5 mg/kgとし得、より好適には0.2〜2 mg/kgとすることができる。但し、これら薬剤の投与量は、患者の年齢や性別、疾患の重篤度等によって適宜変更すべきである。また、本発明の外傷性神経障害の治療剤は、複数回にわたって或いは持続的に投与することができる。 As shown in Examples described later, when 200 μg of anti-HMGB1 monoclonal antibody was administered to a rat having a body weight of about 300 g at a time, a remarkable effect of suppressing brain damage due to brain injury was obtained. Considering these results, the dose for humans can be 0.2 to 5 mg / kg, more preferably 0.2 to 2 mg / kg of anti-HMGB1 monoclonal antibody per dose. However, the dosage of these drugs should be changed as appropriate according to the age and sex of the patient, the severity of the disease, and the like. Further, the therapeutic agent for traumatic neuropathy of the present invention can be administered multiple times or continuously.
さらには、本発明の外傷性神経障害治療剤と組み合わせて、他の治療薬を用いることもできる。特定の実施形態において、本発明の抗HMGB1モノクローナル抗を、１以上の別の治療薬とともに製剤化し、及び／又は同時投与してもよい。本発明は、上記本発明の外傷性神経障害の治療剤を用いることを特徴とする、外傷性神経障害の治療方法にも及ぶ。 Furthermore, other therapeutic agents can be used in combination with the therapeutic agent for traumatic neuropathy of the present invention. In certain embodiments, the anti-HMGB1 monoclonal antibodies of the invention may be formulated and / or co-administered with one or more other therapeutic agents. The present invention also extends to a method for treating traumatic neuropathy, which comprises using the therapeutic agent for traumatic neuropathy of the present invention.
以下、実施例を挙げて本発明をより具体的に説明するが、本発明はもとより下記実施例により制限を受けるものではなく、前・後記の趣旨に適合し得る範囲で適当に変更を加えて実施することも可能であり、それらはいずれも本発明の技術的範囲に含まれる。 EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. It is also possible to implement, and they are all included in the technical scope of the present invention.
（実施例１） 抗HMGB1モノクローナル抗体の調製
特許文献４に記載の方法と同手法により、抗HMGB1モノクローナル抗体を調製した。具体的には、以下のとおりである。(Example 1) Preparation of anti-HMGB1 monoclonal antibody An anti-HMGB1 monoclonal antibody was prepared by the same method as described in Patent Document 4. Specifically, it is as follows.
（ａ）ラットの免疫
市販のウシ胸腺由来HMGB1とHMGB2との混合物（和光純薬工業社製、コード番号：080-070741）1 mg/mLを2 mLガラス製注射筒にとり、別の2 mLガラス製注射筒にとった等容量のフロイント完全アジュバンドと連結管を通じて徐々に混和することによって、エマルションとした。セボフルレンにより麻酔したラットの後肢足蹠に、得られたエマルションを0.05 mLずつ、計0.1 mL注射投与した。2週間後、頚静脈から試験採血し、抗体価の上昇を確認した。次いで、腫大した腸骨リンパ節を前記注射投与から3週間後に無菌的に取り出した。得られた2個のリンパ節から、約6×107 個の細胞を回収することができた。(A) Immunization of rats A mixture of commercially available bovine thymus-derived HMGB1 and HMGB2 (manufactured by Wako Pure Chemical Industries, Ltd., code number: 080-070741) 1 mg / mL in a 2 mL glass syringe and another 2 mL glass An emulsion was prepared by gradually mixing with an equal volume of Freund's complete adjuvant taken in a syringe barrel through a connecting tube. 0.05 mL of the obtained emulsion was injected by 0.1 mL in total into the hind footpads of rats anesthetized with sevoflurane. Two weeks later, test blood was collected from the jugular vein, and an increase in antibody titer was confirmed. The swollen iliac lymph nodes were then aseptically removed 3 weeks after the injection administration. Approximately 6 × 10 7 cells could be recovered from the two lymph nodes obtained.
（ｂ）細胞融合とクローニング
上記腸骨リンパ節細胞とマウスミエローマSP2/O-Ag14(SP2)細胞を、ポリエチレングリコールを用いて融合させ、得られた融合細胞を96穴マイクロプレートに蒔いた。1週間後、最初のELISAスクリーニングを行ない、陽性ウェルについて、ウェスタンブロットにより二次スクリーニングを行なった。陽性を示すウェル細胞を24穴マイクロプレートに移し、細胞をほぼコンフルエントな状態（約2×105）に殖やしてから、0.5 mLの凍結培地（GIT培地にウシ胎児血清を10%とジメチルスルホキシドを10%添加したもの）を用いて、液体窒素中で凍結保存した。この凍結保存細胞を解凍した後、96穴マイクロプレートでクローニングした。(B) Cell fusion and cloning The iliac lymph node cells and mouse myeloma SP2 / O-Ag14 (SP2) cells were fused using polyethylene glycol, and the resulting fused cells were seeded in a 96-well microplate. One week later, the first ELISA screening was performed and the positive wells were secondary screened by Western blot. Transfer positive well cells to a 24-well microplate, grow the cells to an almost confluent state (approximately 2 x 10 5 ), and then add 0.5 mL of freezing medium (GIT medium with 10% fetal bovine serum and dimethyl sulfoxide). 10% added) was stored frozen in liquid nitrogen. The cryopreserved cells were thawed and then cloned in a 96-well microplate.
（ｃ）抗体の精製
回転培養装置（SARSTEDT社製）により上記陽性細胞を2週間大量培養し、濃度2〜3 mg/mLの抗体液を得た。この抗体液をアフィニティゲル（MEP-HyperCelTM、インビトロジェン社製）と中性pH下で混和し、抗HMGB1抗体をゲルへ特異的に結合させた。特異的にゲルに結合した抗体を、酢酸ナトリウムバッファー（pH 4）により溶出した。溶出液を限外濾過装置により濃縮した後、セファロースCL6Bゲル濾過カラム（直径2 cm×長さ97 cm）によって、さらに精製した。(C) Purification of antibody The positive cells were cultured in a large amount for 2 weeks using a rotating culture apparatus (SARSTEDT) to obtain an antibody solution having a concentration of 2 to 3 mg / mL. This antibody solution was mixed with an affinity gel (MEP-HyperCel ™ , manufactured by Invitrogen) under a neutral pH to specifically bind the anti-HMGB1 antibody to the gel. The antibody specifically bound to the gel was eluted with sodium acetate buffer (pH 4). The eluate was concentrated by an ultrafiltration device and further purified by a Sepharose CL6B gel filtration column (diameter 2 cm × length 97 cm).
（実施例２）外傷性神経障害モデルラットに対する抗HMGB1抗体の作用
１）外傷性神経障害モデルラットの作製
外傷性神経障害モデルラットとして、パーカッション傷害作製装置(Dragonfly HPD-1700：ドラゴンフライ社）を用いたパーカッション傷害ラットを作製した。ウィスター系雄性ラット（体重：約300 g）をバルビツレート（30 mg/kg）で麻酔し、ラットの頭蓋骨のブレグマ（Bregma：冠状縫合と矢状縫合の交点）から後方約3 mm、右側方約3 mmに専用の穿頭器を用いて穿頭孔 （burr-hole）を穿ち、筒型のプラスチックを瞬間接着剤・骨セメントを用いて装着した。皮膚を軽く縫合した。翌日、イソフルラン（導入2%、随時漸減）を用いて、麻酔導入し、動物用定位脳手術台に固定したうえで、脳圧測定した。その後、パーカッション傷害作製装置により、出力が2.2 atmとなるようにセットし、脳硬膜上から衝撃を加えて頭部外傷のパーカッション傷害ラットを作製した。以降、頭部外傷のパーカッション傷害を、単に「TBI」という場合がある。パーカッション傷害（TBI)の概要は、図１を参照されたい。(Example 2) Action of anti-HMGB1 antibody on traumatic neuropathy model rat 1) Preparation of traumatic neuropathy model rat As a traumatic neuropathy model rat, a percussion injury preparation device (Dragonfly HPD-1700: Dragonfly) was used. The percussion injured rat used was prepared. Wistar male rats (weight: approx. 300 g) are anesthetized with barbiturate (30 mg / kg), about 3 mm posterior from the bregma of the rat's skull (Bregma: the intersection of coronary and sagittal sutures), about 3 to the right A burr-hole was pierced using a special crusher for mm, and a cylindrical plastic was attached using instant adhesive and bone cement. The skin was lightly sutured. On the next day, anesthesia was introduced using isoflurane (2% introduced, gradually reduced as needed), and the brain pressure was measured after fixing to a stereotaxic table for animals. Then, the percussion injury preparation device was set so that the output would be 2.2 atm, and an impact was applied from above the dura mater to produce a percussion injury rat with head injury. Hereinafter, the head percussion injury may be simply referred to as “TBI”. See Figure 1 for an overview of percussion injury (TBI).
抗HMGB1モノクローナル抗体（実施例１参照）投与群１５匹と対照抗体（抗Keyhole Limpet hemocyanin(KLH) 抗体：自家製）投与群１５匹に分けた。TBIラットを作製してから５分後に再び脳圧測定した。さらに、５分後に別の術者が尾静脈より薬物（抗HMGB1抗体又は抗KLH抗体）を200μg投与した。 The group was divided into 15 groups administered with anti-HMGB1 monoclonal antibody (see Example 1) and 15 groups administered with control antibody (anti-Keyhole Limpet hemocyanin (KLH) antibody: homemade). The brain pressure was measured again 5 minutes after the generation of the TBI rat. Furthermore, another operator administered 200 μg of drug (anti-HMGB1 antibody or anti-KLH antibody) from the tail vein 5 minutes later.
（実験例２−１）行動学的評価
実施例２で作製したTBIラットについて、行動学的評価を行った。行動学的評価として、協調運動を観察するためにロータロッド(rotarod)テスト（０−１０分間程）、及び上肢の自発運動を観察するためのシリンダーテスト（３分間）を、各々頭部外傷直前と外傷後２４時間後に行った。 (Experimental example 2-1) Behavioral evaluation About the TBI rat produced in Example 2, behavioral evaluation was performed. As behavioral evaluations, a rotarod test (for about 0-10 minutes) to observe coordinated movements and a cylinder test (for 3 minutes) to observe spontaneous movements of the upper limbs were performed immediately before head injury. And 24 hours after the injury.
（Ａ）ロータロッドテスト結果
ロータロッドテストは、TBIによる脳外傷前及び脳外傷後について、ラットを回転するロッドに乗せ、当該ラットがロッドから転落するまでの時間を計測することにより実施した。その結果を表１及び図２に示した。(A) Rotarod test results The rotarod test was performed by placing a rat on a rotating rod before and after brain injury caused by TBI, and measuring the time until the rat fell from the rod. The results are shown in Table 1 and FIG.
表１において、TBI(atm)とはTBI作製時の脳硬膜上に与えた圧力を示し、pre(sec)はTBI前のラットがロッドから転落するまでの時間（秒）を、post(sec)は同様にTBI後のラットがロッドから転落するまでの時間（秒）を示した。ラットにより個体差はあるものの、抗HMGB1モノクローナル抗体投与群（表１では、HM群）では、TBI前(pre)に比べて転落するまでの時間がやや伸びる傾向であった。一方、抗KLH抗体（自家製）投与群（対照群：表１ではKL群）では、転落までの時間が明らかに短くなっていることが観察された。この結果より、抗HMGB1モノクローナル抗体投与群では、TBI後であってもラットがバランスをとってうまく行動することができ、協調運動能力が維持できていることが認められた。 In Table 1, TBI (atm) indicates the pressure applied to the dura mater at the time of TBI preparation, pre (sec) indicates the time (seconds) until the rat before TBI falls from the rod, post (sec ) Similarly shows the time (seconds) until the rat after TBI falls from the rod. Although there were individual differences depending on rats, in the anti-HMGB1 monoclonal antibody administration group (in Table 1, HM group), the time to fall slightly tended to be slightly longer than that before TBI (pre). On the other hand, in the anti-KLH antibody (homemade) administration group (control group: KL group in Table 1), it was observed that the time to fall was clearly shortened. From these results, it was confirmed that in the anti-HMGB1 monoclonal antibody administration group, even after TBI, rats were able to balance and behave well and maintain their ability to coordinate.
図２では、TBI前の運動能（ラットのロッドから転落するまでの時間）に対するTBI後の運動能（post TBI/pre TBI）を比較した結果を示した。疑似手術群(sham群）は、実施例２に示すTBIラットの作製方法において、脳硬膜上から衝撃を与える前の状態のもの、即ちラットの頭蓋骨のブレグマから後方約3 mm、右側方約3 mmに専用の穿頭器を用いて穿頭孔を穿ち、筒型のプラスチックを瞬間接着剤・骨セメントを用いて装着し、皮膚を軽く縫合したものとした。対照の抗KLH抗体（自家製）投与群では、TBIにより運動能力の著明な低下がみられたが、抗HMGB1モノクローナル抗体投与群では低下が認められなかった。なお、sham群では、２回目のロータロッドテストの結果、学習によりロッドから転落するまでの時間が伸びていることが確認されたが、抗HMGB1モノクローナル抗体投与群では、sham群と同程度に伸びており、現行治療薬の50%グリセロール投与群と比べても有意に優れた運動能を示した（図２）。 FIG. 2 shows the results of comparing the post-TBI motility (post TBI / pre TBI) to the pre-TBI motility (time to fall from the rat rod). The sham operation group (sham group) is the TBI rat production method shown in Example 2 in the state before impact from the brain dura mater, that is, about 3 mm posterior from the bregma of the rat's skull, about the right side A burr hole was drilled at 3 mm using a special burr, and a cylindrical plastic was attached using instant adhesive and bone cement, and the skin was lightly sutured. In the control anti-KLH antibody (homemade) group, TBI showed a significant decrease in exercise capacity, but no decrease was observed in the anti-HMGB1 monoclonal antibody group. In the sham group, as a result of the second rotarod test, it was confirmed that the time to fall from the rod was increased by learning, but the anti-HMGB1 monoclonal antibody administration group increased to the same extent as the sham group. Compared with the 50% glycerol administration group of the current therapeutic drug, it showed significantly superior motility (FIG. 2).
（Ｂ）シリンダーテスト（３分間）
シリンダーテストは、パーカッション傷害により脳の右部位にTBIを与えた脳外傷モデルラットをシリンダー内（直径17.5cm)に入れ、健常側と患側について探索的に前肢を壁にかける動作の左右差を確認することにより行った。具体的には、左右の前肢をついた全回数を分母とし、患側肢（左）をつく回数を分子として計算し、前肢を壁にかける動作の左右差を確認した。TBI前の左右前肢を同等の頻度で壁にかける健常動物では、ほぼ0.5の値を示したが、TBI後２４時間では、対照の抗KLH抗体（自家製）投与群では、患側肢をつく頻度に著明な低下がみられたが、抗HMGB1モノクローナル抗体投与群では低下が認められなかった（図３）。(B) Cylinder test (3 minutes)
In the cylinder test, a brain trauma model rat that gave TBI to the right part of the brain due to percussion injury was placed in the cylinder (diameter: 17.5 cm), and the difference between the left and right movements of exploring the forelimb on the wall on the healthy side and the affected side was confirmed. It was done by doing. Specifically, the total number of left and right forelimbs was used as the denominator, and the number of times the affected side limb (left) was attached was calculated as a numerator, and the difference between the left and right movements of the forelimb on the wall was confirmed. Healthy animals with the same frequency on the left and right forelimbs before TBI showed a value of approximately 0.5. However, at 24 hours after TBI, the control anti-KLH antibody (homemade) group showed the frequency of applying the affected limb. Although a significant decrease was observed, no decrease was observed in the anti-HMGB1 monoclonal antibody administration group (FIG. 3).
（実験例２−２）組織学的評価
実施例２で作製したTBIラットについて、TBI作製２４時間後に脳圧測定の上で、バルビツレート(50 mg/kg)で麻酔し、パラホルムアルデヒドにて心還流固定を行い、脳組織を取り出した。パラフィン包埋脳組織から薄切切片を作製し、組織学的に脳障害を評価し、TBI時のHMGB1動態について評価した。(Experimental example 2-2) Histological evaluation About the TBI rat produced in Example 2, anesthesia with barbiturate (50 mg / kg) was carried out on brain pressure measurement 24 hours after TBI production, and cardiac reflux was performed with paraformaldehyde. Fixation was performed and brain tissue was removed. Slices were prepared from paraffin-embedded brain tissue, histologically assessed for brain damage, and evaluated for HMGB1 dynamics during TBI.
（Ａ）組織学的評価
ラット脳組織のうち、受傷中心部分を含む冠状断切片について組織切片を作製し、ヘマトキシリン-エオシン染色により組織学的評価を行った。コントロール群（抗KLH抗体：自家製）に比べて、抗HMGB1モノクローナル抗体投与群では、傷害の広がりが明らかに抑制されていた（図４）。(A) Histological evaluation Among rat brain tissues, tissue sections were prepared for coronal sections including the central part of the wound, and histological evaluations were performed by hematoxylin-eosin staining. Compared with the control group (anti-KLH antibody: homemade), the spread of injury was clearly suppressed in the anti-HMGB1 monoclonal antibody administration group (FIG. 4).
（Ｂ）分子生物学的評価（参考）
ラット脳組織のうち、大脳皮質部分の組織切片を作製し、HMGB1を抗HMGB1モノクローナル抗体（赤）、神経細胞マーカーであるMAP-2を抗MAP-2ウサギポリクローナル抗体（緑）で染色し、細胞核をDAPI（青）で染色した。その結果、傷害を与えた部位（傷害部位）とは反対の部位（正常領域）では、HMGB1は神経細胞の核内に存在することが示された（図５）。一方、傷害部位では細胞核内にHMGB1は認められず、神経細胞外に分散していることが確認された（図６）。また、傷害部位よりやや内部の組織についても確認した結果、HMGB1は神経細胞質部分には認められるものの、核内にはとどまっていないことが観察された。HMGB1は、傷害により神経細胞核外に放出されることが確認された（図７）。(B) Molecular biological evaluation (reference)
Slices of cerebral cortex from rat brain tissue were prepared, and HMGB1 was stained with anti-HMGB1 monoclonal antibody (red) and neuronal marker MAP-2 was stained with anti-MAP-2 rabbit polyclonal antibody (green). Was stained with DAPI (blue). As a result, it was shown that HMGB1 is present in the nucleus of the nerve cell at the site (normal region) opposite to the site of injury (damage site) (FIG. 5). On the other hand, HMGB1 was not observed in the cell nucleus at the injury site, and it was confirmed that it was dispersed outside the nerve cell (FIG. 6). In addition, as a result of confirming the tissue slightly inside from the injury site, it was observed that HMGB1 was found in the neurocytoplasmic part but not in the nucleus. It was confirmed that HMGB1 is released outside the nucleus of nerve cells due to injury (FIG. 7).
（実験例２−３）脳血管透過性の評価
実施例２で作製したTBIラットについて、傷害作製３時間後に尾静脈よりエバンスブルー（EB）を投与し、その３時間後に脱血・灌流して、脳内に移行したエバンスブルーを定量した。その結果、対照群では、エバンスブルーの漏出が認められたが、抗HMGB1モノクローナル抗体投与群では、顕著に漏出量が低かった（図８）。(Experimental Example 2-3) Evaluation of cerebral vascular permeability About TBI rats prepared in Example 2, Evans Blue (EB) was administered from the tail vein 3 hours after injury preparation, and blood was removed and perfused 3 hours later. The amount of Evans blue that migrated into the brain was quantified. As a result, Evans blue leakage was observed in the control group, but the leakage was significantly lower in the anti-HMGB1 monoclonal antibody administration group (FIG. 8).
（実施例３）外傷性神経障害モデルラットに対する抗HMGB1モノクローナル抗体の効果
本実施例では、外傷性神経障害モデルラットとして実施例２と同手法により作製したTBIラットに対して、抗HMGB1モノクローナル抗体を投与したときの効果を、実験例２−１と同様に行動学的評価（ロータロッドテスト）により確認した。TBI後１０分目に抗HMGB1モノクローナル抗体を尾静脈より200μg経静脈投与した。抗体の対照として抗KLH抗体（抗Keyhole Limpet hemocyanin(KLH) 抗体：自家製）を用いた。また、標準治療方法として10%グリセロールを含む0.9％生理食塩液を0.5ml経静脈投与した。対照として、sham群についても確認した。Example 3 Effect of Anti-HMGB1 Monoclonal Antibody on Traumatic Neuropathy Model Rat In this example, anti-HMGB1 monoclonal antibody was administered to a TBI rat prepared by the same method as in Example 2 as a traumatic neuropathy model rat. The effect at the time of administration was confirmed by behavioral evaluation (Rotarod test) in the same manner as in Experimental Example 2-1. Ten minutes after TBI, anti-HMGB1 monoclonal antibody was intravenously administered at 200 μg from the tail vein. As an antibody control, an anti-KLH antibody (anti-Keyhole Limpet hemocyanin (KLH) antibody: homemade) was used. In addition, 0.5 ml of 0.9% physiological saline containing 10% glycerol was intravenously administered as a standard treatment method. As a control, the sham group was also confirmed.
行動学的評価は、TBI直前とTBI後３、６及び２４時間後についてロータロッドテストにより行った。ロータロッドテストは、実験例２−１（Ａ）と同手法により行い、各時間における脳外傷作製前の運動能（ラットのロッドから転落するまでの時間）に対する脳外傷作製後の運動能（post TBI/pre TBI）を比較した（図９−１１）。
その結果、抗HMGB1モノクローナル抗体投与群では、経時的に行動学的改善効果が認められ、２４時間目ではsham群と同程度に伸びており、現行治療薬のグリセロール投与群と比べても有意に優れた運動能を示した（図１２）。Behavioral evaluation was performed by rotarod test just before TBI and 3, 6 and 24 hours after TBI. The rotarod test was performed in the same manner as in Experimental Example 2-1 (A), and the motility after the preparation of the brain injury (post time to fall from the rod of the rat) for each time before the preparation of the brain injury (post) TBI / pre TBI) were compared (FIGS. 9-11).
As a result, the anti-HMGB1 monoclonal antibody administration group showed a behavioral improvement effect over time, and increased to the same extent as the sham group at 24 hours. Excellent motor ability was shown (FIG. 12).
（実施例４）外傷性神経障害モデルラットに対する抗HMGB1モノクローナル抗体の効果
本実施例では、外傷性神経障害モデルラットとして実施例２と同手法により作製したTBIラットに対して、抗HMGB1モノクローナル抗体を投与したときの効果を、実験例２−１と同様に行動学的評価（シリンダーテスト）により確認した。TBI後１０分目に抗HMGB1モノクローナル抗体を尾静脈より200μg経静脈投与した。抗体の対照として抗KLH抗体（抗Keyhole Limpet hemocyanin (KLH) 抗体：自家製）を用いた。行動学的評価は、TBI後３、６及び２４時間後について実験例２−１（Ｂ）と同手法でのシリンダーテストにより行った（図１３−１５）。その結果、抗HMGB1モノクローナル抗体投与群では、TBI後経時的に行動学的改善効果が認められた（図１６）。(Example 4) Effect of anti-HMGB1 monoclonal antibody on traumatic neuropathy model rat In this example, anti-HMGB1 monoclonal antibody was applied to TBI rat prepared by the same method as in Example 2 as traumatic neuropathy model rat. The effect at the time of administration was confirmed by behavioral evaluation (cylinder test) in the same manner as in Experimental Example 2-1. Ten minutes after TBI, anti-HMGB1 monoclonal antibody was intravenously administered at 200 μg from the tail vein. As an antibody control, an anti-KLH antibody (anti-Keyhole Limpet hemocyanin (KLH) antibody: homemade) was used. The behavioral evaluation was performed by a cylinder test in the same manner as in Experimental Example 2-1 (B) after 3, 6 and 24 hours after TBI (FIGS. 13-15). As a result, in the anti-HMGB1 monoclonal antibody administration group, a behavioral improvement effect was observed over time after TBI (FIG. 16).
（実施例５）パーカッション傷害ラットにおけるHMGB1
本実施例では、外傷性神経障害モデルラットとして実施例２と同手法により作製したTBIラットについて、抗HMGB1モノクローナル抗体を投与したときの組織中のHMGB1について、ウエスタンブロッティングにより評価した。実施例３と同手法により、TBI後１０分目に抗HMGB1モノクローナル抗体を尾静脈より200μg経静脈投与した。抗体の対照として抗KLH抗体（抗Keyhole Limpet hemocyanin(KLH) 抗体：自家製）を用いた。また、標準治療方法として10%グリセロールを含む0.9％生理食塩液を0.5mL経静脈投与した。
受傷中心部から前側方の大脳皮質を受傷後２４時間後に3mm×3mmの大きさでサンプリングした。非障害側についても対称の位置からサンプリングした。大脳皮質組織を１ml のRIPPA buffer (50 mM Tris-HCl, pH 8.0, 0.15M NaCl, 0.1% SDS, 0.1% Triton X-100, 0.5% Sodium deoxycholate)でホモジナイズした。10,000×g ２０分遠心後、上清を12% ポリアクリルアミドSDS-PAGEで泳動分離し、ニトロセルロース膜に転写した。ニトロセルロース膜上HMGB1をウエスタンブロッティング法によりHRP標識抗HMGB1ラット単クローン抗体を用いて化学発光法で検出した（図１７）。
上記の結果、抗HMGB1モノクローナル抗体群及びグリセロール投与群ではTBI側及び非障害側で、組織中のHMGB1の存在について差を認めなかったが、抗KLH抗体投与群では非障害側に比べて、TBI側のほうがHMGB1のバンドが薄かった。このことより、大脳皮質中のHMGB1の存在については、抗HMGB1モノクローナル抗体は既存のグリセロールと同等の効果を示したことが確認された（図１８、１９）。Example 5 HMGB1 in percussion-injured rats
In this example, TBI rats produced as traumatic neuropathy model rats by the same method as in Example 2 were evaluated by Western blotting for HMGB1 in tissues when anti-HMGB1 monoclonal antibody was administered. In the same manner as in Example 3, 200 μg of anti-HMGB1 monoclonal antibody was intravenously administered via the tail vein 10 minutes after TBI. As an antibody control, an anti-KLH antibody (anti-Keyhole Limpet hemocyanin (KLH) antibody: homemade) was used. Moreover, 0.9 mL of physiological saline containing 10% glycerol was intravenously administered as a standard treatment method.
The cerebral cortex from the center of the injury to the anterior side was sampled at a size of 3 mm × 3 mm 24 hours after the injury. The non-disturbed side was also sampled from a symmetrical position. Cerebral cortical tissue was homogenized with 1 ml of RIPPA buffer (50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 0.1% SDS, 0.1% Triton X-100, 0.5% Sodium deoxycholate). After centrifugation at 10,000 × g for 20 minutes, the supernatant was electrophoresed on 12% polyacrylamide SDS-PAGE and transferred to a nitrocellulose membrane. HMGB1 on the nitrocellulose membrane was detected by chemiluminescence using Western blotting with an HRP-labeled anti-HMGB1 rat monoclonal antibody (FIG. 17).
As a result, the anti-HMGB1 monoclonal antibody group and the glycerol administration group showed no difference in the presence of HMGB1 in the tissue between the TBI side and the non-injured side, but the anti-KLH antibody administration group showed no difference in the TBI side. The HMGB1 band was thinner on the side. From this, it was confirmed that the anti-HMGB1 monoclonal antibody showed the same effect as the existing glycerol for the presence of HMGB1 in the cerebral cortex (FIGS. 18 and 19).
以上詳述したように、本発明の抗HMGB1モノクローナル抗体を有効成分とすることを特徴とする、外傷性神経障害の治療剤は、パーカッション傷害ラットにおいて、行動学的にも、組織学的にも優れた効果を示した。これまで特に有効な処置手段のなかった外傷性神経障害、特に急性期の外傷性神経障害を治療できるものとして、極めて有用である。 As described above in detail, the therapeutic agent for traumatic neuropathy, which comprises the anti-HMGB1 monoclonal antibody of the present invention as an active ingredient, is both behaviorally and histologically in percussion-injured rats. Excellent effect. The present invention is extremely useful as a treatment for traumatic neuropathy that has not been particularly effective until now, especially for acute traumatic neuropathy.
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