JP7502882B2 - Polyglutamine protein aggregation inhibitor and drug for preventing or treating polyglutamine disease - Google Patents
Polyglutamine protein aggregation inhibitor and drug for preventing or treating polyglutamine disease Download PDFInfo
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- JP7502882B2 JP7502882B2 JP2020062535A JP2020062535A JP7502882B2 JP 7502882 B2 JP7502882 B2 JP 7502882B2 JP 2020062535 A JP2020062535 A JP 2020062535A JP 2020062535 A JP2020062535 A JP 2020062535A JP 7502882 B2 JP7502882 B2 JP 7502882B2
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- hyaluronic acid
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- polyglutamine
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Description
本発明は、神経細胞の軸索の伸展剤に関する。また本発明は、軸索が伸展した神経細胞を製造する方法、神経細胞内におけるタンパク質凝集抑制剤、神経細胞内における凝集したタンパク質の分解促進剤、神経細胞内におけるタンパク質の凝集を抑制された神経細胞を製造する方法、および、神経変性疾患の予防または治療用医薬に関する。 The present invention relates to an agent for extending the axons of nerve cells. The present invention also relates to a method for producing nerve cells with extended axons, an agent for inhibiting protein aggregation in nerve cells, an agent for promoting the degradation of aggregated proteins in nerve cells, a method for producing nerve cells in which protein aggregation in nerve cells is inhibited, and a pharmaceutical agent for preventing or treating neurodegenerative diseases.
アルツハイマー病や前頭側頭型認知症などの神経変性疾患では、神経細胞の軸索の変性や神経細胞死が起き、その結果として認知症状をはじめとしたいくつかの神経症状が出現するが、いずれの神経変性疾患にも根治を可能にする薬剤の開発は成功していない。日本をはじめとした先進国では高齢化社会を迎え、神経変性疾患を根治する薬剤の開発が切望されている。 In neurodegenerative diseases such as Alzheimer's disease and frontotemporal dementia, degeneration of the axons of nerve cells and death of nerve cells occur, resulting in the appearance of several neurological symptoms, including cognitive symptoms, but the development of drugs that can cure any of these neurodegenerative diseases has not been successful. In developed countries such as Japan, as societies become aging, there is a strong demand for the development of drugs that can cure neurodegenerative diseases.
神経変性疾患には多くの疾患が分類されるが、それらの疾患には認知症状を伴うものが多い。現在の日本において、認知症患者は250万人にも上ると言われると同時に、そのうち7割はアルツハイマー病と考えられたことから、アルツハイマー病治療薬の開発競争が激しく行われた。しかしながら、これまで開発された約400にも達する薬剤は全て治療効果を示すことなく失敗に終わっている。
アルツハイマー病治療薬開発の失敗の原因としては諸説あるが、ヒトのアルツハイマー病の再現が非常に不十分なモデルマウスを用いた実験での成功を根拠にしたこと、アルツハイマー病は他の神経変性疾患と異なり、ベータアミロイドによる老人斑と高度にリン酸化されたタウタンパク質による神経原線維変化の2つの特徴的な構造物が形成されるが、開発された治療薬のターゲットがもっぱらベータアミロイドに偏っていたことも大きな原因と考えられている。治験においては、老人斑は減少したが、認知症状は改善されない、もしくは悪化したり副作用として悪性黒色腫の発症などが認められてしまった。老人斑はそもそもアルツハイマー病を発症していない高齢者や、認知症状を呈していない高齢者にも認められる構造物であるのに対し、変性したタウタンパク質を原因とする神経変性疾患は、アルツハイマー病以外にも複数認められ、現在はタウタンパク質に注目した治療薬開発が増えてきている。
There are many types of neurodegenerative diseases, many of which are accompanied by cognitive symptoms. Currently in Japan, it is said that there are as many as 2.5 million dementia patients, and 70% of these are thought to be suffering from Alzheimer's disease, leading to a fierce race to develop drugs to treat Alzheimer's disease. However, all of the approximately 400 drugs developed to date have failed without showing any therapeutic effect.
There are various theories as to why the development of Alzheimer's treatment drugs has failed, but one major reason is that they were based on success in experiments using model mice that do not adequately reproduce human Alzheimer's disease, and that unlike other neurodegenerative diseases, Alzheimer's disease forms two characteristic structures: senile plaques caused by beta-amyloid and neurofibrillary tangles caused by highly phosphorylated tau protein, but the target of the developed treatment drugs was biased toward beta-amyloid. In clinical trials, senile plaques were reduced, but cognitive symptoms did not improve or worsened, and side effects such as the development of malignant melanoma were observed. Senile plaques are structures that are found in elderly people who do not have Alzheimer's disease or do not exhibit cognitive symptoms, while multiple neurodegenerative diseases caused by denatured tau protein have been found other than Alzheimer's disease, and currently, the development of treatment drugs focusing on tau protein is increasing.
これまでに神経変性疾患において観察される神経細胞内の毒性凝集体形成の抑制を目的としたアプローチが報告されている。特許文献１は、ホルボールエステルを有効成分とするタウタンパク質の凝集抑制剤を開示しており、タウタンパク質の凝集を抑制することにより、タウタンパク質の凝集に起因する神経変性疾患の予防又は治療を行うことが可能である旨を記載している。なおホルボールエステル類は、神経細胞における軸索変性と軸索伸展阻害も改善でき、すでに変性を起こしかけている神経細胞の生存と機能回復、さらには神経細胞の機能の亢進まで起こし得ることが報告されている（特許文献２）。 Approaches aimed at suppressing the formation of toxic aggregates in nerve cells observed in neurodegenerative diseases have been reported so far. Patent Document 1 discloses a tau protein aggregation inhibitor containing phorbol ester as an active ingredient, and describes that by suppressing the aggregation of tau protein, it is possible to prevent or treat neurodegenerative diseases caused by the aggregation of tau protein. It has also been reported that phorbol esters can improve axon degeneration and inhibition of axon extension in nerve cells, and can restore the survival and function of nerve cells that are already beginning to degenerate, and even enhance the function of nerve cells (Patent Document 2).
本発明は神経細胞内の凝集タンパク質の形成を抑制可能な新たな物質の提供を課題とする。 The objective of the present invention is to provide a new substance that can suppress the formation of aggregated proteins in nerve cells.
発明者らは、治療薬開発研究のターゲットを最初からタウタンパク質やその他の神経細胞内に凝集体を形成するタンパク質に定め、このような凝集を抑制する薬剤の探索を行ってきた。また同時に、認知機能の回復に重要な軸索の伸展や保護、新たに軸索を生じさせる薬剤の探索も行った。その中で、多糖類であるヒアルロン酸が、「凝集体形成を抑制し」「軸索の伸展を促進する」両方の機能を持つことを見出した。
神経変性疾患には、共通して神経細胞の変性（特に軸索と呼ばれる長い突起物の変性や切断）と神経細胞死が認められる。従って、この両方を大幅に防御・あるいは回復させることが出来る薬剤が開発できれば、日本に限らず、また、アルツハイマー病や前頭側頭型認知症といった特定の疾患限定ではなく、世界中の神経変性疾患患者の治療が可能になるほか、巨大な市場を手に入れることが出来る。本発明は上記知見に基づき完成した発明であり、下記の態様を含む：
From the beginning, the inventors targeted tau protein and other proteins that form aggregates in nerve cells as the target of their therapeutic drug development research, and have been searching for drugs that suppress such aggregation. At the same time, they also searched for drugs that promote the extension and protection of axons, which are important for the recovery of cognitive function, and that generate new axons. In the process, they discovered that hyaluronic acid, a polysaccharide, has both the functions of "suppressing aggregate formation" and "promoting axon extension."
Neurodegenerative diseases commonly suffer from degeneration of nerve cells (especially degeneration or severing of long processes called axons) and death of nerve cells. Therefore, if a drug could be developed that could largely prevent or reverse both of these, it would be possible to treat patients with neurodegenerative diseases not only in Japan, but also around the world, not just in specific diseases such as Alzheimer's disease and frontotemporal dementia, and would also create a huge market. The present invention has been completed based on the above findings, and includes the following aspects:
本発明は一態様において、
〔１〕ヒアルロン酸またはその薬学的に許容される塩を含む、神経細胞内におけるタンパク質凝集抑制剤に関する。
ここで本発明のタンパク質凝集抑制剤は一実施の形態において、
〔２〕上記〔１〕に記載の神経細胞内におけるタンパク質凝集抑制剤であって、
前記タンパク質がタウタンパク質またはポリグルタミンタンパク質であることを特徴とする。
また本発明のタンパク質凝集抑制剤は一実施の形態において、
〔３〕上記〔２〕に記載の神経細胞内におけるタンパク質凝集抑制剤であって、
前記ポリグルタミンタンパク質がハンチンチンタンパク質であることを特徴とする。
また本発明は別の態様において、
〔４〕ヒアルロン酸またはその薬学的に許容される塩を含む、神経細胞内における凝集したタンパク質の分解促進剤に関する。
また本発明は別の態様において、
〔５〕神経細胞とヒアルロン酸またはその薬学的に許容される塩とを接触させる工程を含む、神経細胞内におけるタンパク質の凝集が抑制された神経細胞を製造する方法に関する。
また本発明は別の態様において、
〔６〕ヒアルロン酸またはその薬学的に許容される塩を含む、神経細胞の軸索の伸展剤に関する。
また本発明は別の態様において、
〔７〕神経細胞とヒアルロン酸またはその薬学的に許容される塩とを接触させる工程を含む、軸索が伸展した神経細胞を製造する方法に関する。
また本発明は別の態様において、
〔８〕ヒアルロン酸またはその薬学的に許容される塩を含む、タウタンパク質またはポリグルタミンタンパク質の凝集に起因する神経変性疾患の予防または治療用医薬に関する。
ここで本発明の神経変性疾患の予防または治療用医薬は一実施の形態において、
〔９〕上記〔８〕に記載の神経変性疾患の予防または治療用医薬であって、
前記神経変性疾患が、前頭側頭型認知症、アルツハイマー病、ポリグルタミン病、レビー小体型認知症、またはダウン症候群であることを特徴とする。
また本発明の神経変性疾患の予防または治療用医薬は一実施の形態において、
〔１０〕上記〔９〕に記載の神経変性疾患の予防または治療用医薬であって、
前記ポリグルタミン病がハンチントン病であることを特徴とする。
The present invention in one aspect comprises:
[1] The present invention relates to an agent for inhibiting protein aggregation in nerve cells, which comprises hyaluronic acid or a pharma- ceutically acceptable salt thereof.
In one embodiment, the protein aggregation inhibitor of the present invention comprises:
[2] The protein aggregation inhibitor in nerve cells according to [1] above,
The protein is characterized in that it is a tau protein or a polyglutamine protein.
In one embodiment, the protein aggregation inhibitor of the present invention comprises:
[3] The protein aggregation inhibitor in nerve cells according to [2] above,
The polyglutamine protein is characterized in that it is huntingtin protein.
In another aspect, the present invention comprises:
[4] The present invention relates to an agent for promoting the decomposition of aggregated proteins in nerve cells, which comprises hyaluronic acid or a pharma- ceutically acceptable salt thereof.
In another aspect, the present invention comprises:
[5] The present invention relates to a method for producing nerve cells in which protein aggregation in the nerve cells is suppressed, the method comprising the step of contacting the nerve cells with hyaluronic acid or a pharma- ceutically acceptable salt thereof.
In another aspect, the present invention comprises:
[6] A nerve cell axon extension agent comprising hyaluronic acid or a pharma- ceutically acceptable salt thereof.
In another aspect, the present invention comprises:
[7] The present invention relates to a method for producing nerve cells having extended axons, which comprises a step of contacting nerve cells with hyaluronic acid or a pharma- ceutically acceptable salt thereof.
In another aspect, the present invention comprises:
[8] A pharmaceutical agent for preventing or treating a neurodegenerative disease caused by aggregation of tau protein or polyglutamine protein, comprising hyaluronic acid or a pharma- ceutical acceptable salt thereof.
In one embodiment, the pharmaceutical composition for preventing or treating a neurodegenerative disease of the present invention comprises:
[9] The pharmaceutical composition for preventing or treating a neurodegenerative disease according to [8] above,
The neurodegenerative disease is characterized in that it is frontotemporal dementia, Alzheimer's disease, polyglutamine disease, dementia with Lewy bodies, or Down's syndrome.
In one embodiment, the pharmaceutical composition for preventing or treating a neurodegenerative disease of the present invention comprises:
[10] The pharmaceutical composition for preventing or treating a neurodegenerative disease according to [9] above,
The polyglutamine disease is characterized in that it is Huntington's disease.
本発明に係る神経細胞内におけるタンパク質凝集抑制剤によれば、神経細胞内における凝集タンパク質の形成を抑制することができる。 The protein aggregation inhibitor in nerve cells according to the present invention can inhibit the formation of aggregated proteins in nerve cells.
本発明は一態様として、ヒアルロン酸またはその薬学的に許容される塩を含む、神経細胞内におけるタンパク質凝集抑制剤を提供する。本発明に係る神経細胞内におけるタンパク質凝集抑制剤を用いれば、細胞内において凝集タンパク質の形成が抑制された神経細胞を得ることができる。 In one aspect, the present invention provides an inhibitor of protein aggregation in nerve cells, comprising hyaluronic acid or a pharma- ceutically acceptable salt thereof. By using the inhibitor of protein aggregation in nerve cells according to the present invention, nerve cells in which intracellular formation of aggregated proteins is suppressed can be obtained.
本明細書において「ヒアルロン酸」とは、Ｄ－グルクロン酸とＮ－アセチル－Ｄ－グルコサミンとがグリコシド結合してなる二糖単位を基本の構成単位とする直鎖状の多糖類である。ヒアルロン酸は上記基本の構成単位を有すればよく、神経細胞の軸索の伸展の促進または神経細胞内のタンパク質の凝集を抑制できる限りにおいて、その糖部分が修飾されていてもよい。例えば、糖の水酸基の一部またはすべては、エステル化、エーテル化等を受けていてもよい。グルクロン酸のカルボキシル基の一部またはすべては、エステル化、アミド化等を受けていてもよい。非還元末端に位置する糖は、飽和糖であっても、不飽和糖であってもよい。 In this specification, "hyaluronic acid" refers to a linear polysaccharide whose basic structural unit is a disaccharide unit formed by glycosidic bonds between D-glucuronic acid and N-acetyl-D-glucosamine. Hyaluronic acid may have the above basic structural unit, and the sugar portion may be modified as long as it promotes the extension of axons in nerve cells or inhibits protein aggregation in nerve cells. For example, some or all of the hydroxyl groups of the sugar may be esterified, etherified, etc. Some or all of the carboxyl groups of glucuronic acid may be esterified, amidated, etc. The sugar located at the non-reducing end may be a saturated sugar or an unsaturated sugar.
本発明に用いることのできるヒアルロン酸のサイズは、神経細胞の軸索の伸展の促進または神経細胞内のタンパク質の凝集を抑制できる限りにおいて制限されない。ヒアルロン酸のサイズは、好ましくは生体内における毒性を低減するために分子量の下限は１０万以上が好ましく、５０万以上、１００万以上がより好ましい。一方、ヒアルロン酸のサイズの上限は、以下に限定されないが、５００万以下、４００万以下、２００万以下とすることができる。
また一実施の形態において、本発明に用いるヒアルロン酸として好ましいのは、平均分子量５０万～３９０万のヒアルロン酸であり、さらに好ましくは平均分子量５０万～１２０万のヒアルロン酸である。
The size of hyaluronic acid that can be used in the present invention is not limited as long as it can promote the extension of the axon of nerve cell or suppress the aggregation of protein in nerve cell.The size of hyaluronic acid is preferably the lower limit of molecular weight is 100,000 or more, more preferably 500,000 or more, 1,000,000 or more in order to reduce toxicity in vivo.On the other hand, the upper limit of the size of hyaluronic acid is not limited to the following, but can be 5,000,000 or less, 4,000,000 or less, 2,000,000 or less.
In one embodiment, the hyaluronic acid used in the present invention is preferably hyaluronic acid having an average molecular weight of 500,000 to 3,900,000, and more preferably hyaluronic acid having an average molecular weight of 500,000 to 1,200,000.
ヒアルロン酸の薬学的に許容される塩としては、通常医薬として許容される塩であれば特に制限はなく、以下に限定されないが、塩酸、臭化水素酸、ヨウ化水素酸、硝酸、硫酸、リン酸などの無機酸との塩、酢酸、フマル酸、マレイン酸、コハク酸、クエン酸、酒石酸、アジピン酸、グルコン酸、グルコヘプト酸、グルクロン酸、テレフタル酸、メタンスルホン酸、乳酸、馬尿酸、１，２－エタンジスルホン酸、イセチオン酸、ラクトビオン酸、オレイン酸、パモ酸、ポリガラクツロン酸、ステアリン酸、タンニン酸、トリフルオロメタンスルホン酸、ベンゼンスルホン酸、ｐ－トルエンスルホン酸、硫酸ラウリルエステル、硫酸メチル、ナフタレンスルホン酸、スルホサリチル酸などの有機酸との塩；臭化メチル、ヨウ化メチルなどとの四級アンモニウム塩；臭素イオン、塩素イオン、ヨウ素イオンなどのハロゲンイオンとの塩；リチウム、ナトリウム、カリウムなどのアルカリ金属との塩；カルシウム、マグネシウムなどのアルカリ土類金属との塩；鉄、亜鉛などとの金属塩；アンモニアとの塩；トリエチレンジアミン、２－アミノエタノール、２，２－イミノビス（エタノール）、１－デオキシ－１－（メチルアミノ）－２－Ｄ－ソルビトール、２－アミノ－２－（ヒドロキシメチル）－１，３－プロパンジオール、プロカイン、Ｎ，Ｎ－ビス（フェニルメチル）－１，２－エタンジアミンなどの有機アミンとの塩などが挙げられる。ヒアルロン酸の塩として好ましくは、ヒアルロン酸ナトリウムである。また本発明における「ヒアルロン酸またはその薬学的に許容される塩」は、水和物または溶媒和物の形態をとっていてもよい。 Pharmaceutically acceptable salts of hyaluronic acid are not particularly limited as long as they are generally medicamentally acceptable salts, and include, but are not limited to, salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, and phosphoric acid, acetic acid, fumaric acid, maleic acid, succinic acid, citric acid, tartaric acid, adipic acid, gluconic acid, glucoheptic acid, glucuronic acid, terephthalic acid, methanesulfonic acid, lactic acid, hippuric acid, 1,2-ethanedisulfonic acid, isethionic acid, lactobionic acid, oleic acid, pamoic acid, polygalacturonic acid, stearic acid, tannic acid, trifluoromethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, lauryl sulfate, methyl sulfate, naphthalenesulfonic acid, sulfuric acid, methyl esters ... Examples of the salt include salts with organic acids such as salicylic acid; quaternary ammonium salts with methyl bromide, methyl iodide, etc.; salts with halogen ions such as bromide ion, chloride ion, and iodide ion; salts with alkali metals such as lithium, sodium, and potassium; salts with alkaline earth metals such as calcium and magnesium; metal salts with iron and zinc; salts with ammonia; and salts with organic amines such as triethylenediamine, 2-aminoethanol, 2,2-iminobis(ethanol), 1-deoxy-1-(methylamino)-2-D-sorbitol, 2-amino-2-(hydroxymethyl)-1,3-propanediol, procaine, and N,N-bis(phenylmethyl)-1,2-ethanediamine. The salt of hyaluronic acid is preferably sodium hyaluronate. In addition, the "hyaluronic acid or a pharma- ceutically acceptable salt thereof" in the present invention may be in the form of a hydrate or solvate.
ヒアルロン酸は、動物等の天然物から抽出されたもの、微生物を培養して得られたものであってよく、または、化学的もしくは酵素的に合成されたものを使用してもよい。好ましくは、例えば鶏冠、臍体、皮膚、関節液などの生体組織から公知の抽出法によって得ることができる（例えば、特開平１－１１５９０２号公報）。ヒアルロン酸としては、市販のＨｙａｌｕｒｏｎｉｃ Ａｃｉｄ Ｓｏｄｉｕｍ Ｓａｌｔ等を用いることができる。 Hyaluronic acid may be extracted from natural products such as animals, obtained by culturing microorganisms, or may be chemically or enzymatically synthesized. Preferably, it can be obtained by known extraction methods from biological tissues such as cockscomb, umbilicus, skin, and synovial fluid (for example, JP-A-1-115902). As hyaluronic acid, commercially available hyaluronic acid sodium salt, etc. can be used.
神経細胞とは、神経幹細胞から分化して生じる細胞群のうち、樹状突起と軸索という特有の構造を持ち、それぞれの神経細胞から伸びる軸索が、別の神経細胞の樹状突起とつながることによって、情報伝達を行える唯一の細胞であり、一般的にニューロンと呼ばれるものがそれに当たる。なお、樹状突起と軸索との間の隙間の構造をシナプスと呼ぶ。本明細書における神経細胞は、上記樹状突起と軸索という特有の構造を有する細胞であれば限定されず、脳などの生体内に存在する神経細胞や生体より分離または単離した神経細胞、神経前駆細胞や多能性幹細胞などの培養細胞より分化誘導して得られた神経細胞を含む。好ましくは脳に存在する神経細胞を挙げることができ、海馬神経細胞、大脳皮質神経細胞、中脳神経細胞、または線条体神経細胞を好適に挙げることができる。また、神経細胞の由来は特に制限されないが、哺乳動物、鳥類、魚類、爬虫類、両生類を挙げることができ、哺乳動物を好適に挙げることができ、ヒトをより好適に挙げることができる。 A nerve cell is a cell group that is differentiated from a neural stem cell, and is the only cell that has a unique structure of dendrites and axons, and can transmit information by connecting the axons extending from each nerve cell to the dendrites of another nerve cell, and is generally called a neuron. The structure of the gap between the dendrites and the axons is called a synapse. In this specification, the nerve cell is not limited to a cell that has the unique structure of the dendrites and axons, and includes nerve cells present in a living body such as the brain, nerve cells separated or isolated from a living body, and nerve cells obtained by inducing differentiation from cultured cells such as neural precursor cells and pluripotent stem cells. Preferably, nerve cells present in the brain can be exemplified, and hippocampal nerve cells, cerebral cortical nerve cells, midbrain nerve cells, or striatal nerve cells can be preferably exemplified. In addition, the origin of the nerve cells is not particularly limited, and can be exemplified by mammals, birds, fish, reptiles, and amphibians, preferably mammals, and more preferably humans.
本明細書において「凝集タンパク質」または「タンパク質の凝集」とは、神経細胞内においてタンパク質または変異したタンパク質同士が複数凝集したものをいい、特に神経変性疾患に関連して神経細胞内に生じる凝集タンパク質を含む。このような凝集タンパク質を構成するタンパク質としては、タウタンパク質、ハンチンチンタンパク質などのポリグルタミンタンパク質、アミロイドβ、α－シヌクレイン、プリオン、ＴＤＰ－４３などを挙げることができる。前記に列挙するタンパク質の構造に変異もしくは変性が生じた変異タンパク質が細胞内で凝集し凝集タンパク質を形成することが知られている。 As used herein, "aggregated proteins" or "protein aggregation" refers to the aggregation of multiple proteins or mutated proteins within a nerve cell, and particularly includes aggregated proteins that occur within nerve cells in association with neurodegenerative diseases. Examples of proteins that constitute such aggregated proteins include polyglutamine proteins such as tau protein and huntingtin protein, amyloid beta, α-synuclein, prion, TDP-43, etc. It is known that mutant proteins in which mutations or denaturation have occurred in the structure of the proteins listed above aggregate within cells to form aggregated proteins.
例えば、タウタンパク質はその構造内のセリン、スレオニン、チロシがリン酸化されるが、そのうちの特定の領域の異常リン酸化が凝集タンパク質形成に関与すると報告されている（Noble, W. et al, “The importance of tau phosphorylation for neurodegenerative diseases”. Front. Neurol. 4, 1-11(2013)）。また以下の実施例に示すように、タウタンパク質の４０６番目のアミノ酸であるアルギニンがトリプトファンに置換した変異型タウタンパク質も神経細胞内で凝集することが知られている。本明細書において「変異型タウタンパク質」というとき、神経細胞内で凝集タンパク質を形成する変異を有するタウタンパク質を意味し、変異の種類は制限されない。このような変異型タウタンパク質としては、例えば、その構造内に異常リン酸化を有するタウタンパク質や４０６番目のアルギニンがトリプトファンに置換したタウタンパク質が含まれる。
また例えばハンチンチンタンパク質はその構造内のポリグルタミン（ｐｏｌｙＱ）鎖が異常伸長することで変異タンパク質（ポリグルタミンタンパク質）となり不溶性の線維構造（凝集体）を形成することが知られている。本明細書において「異常ハンチンチンタンパク質」というとき、神経細胞内で凝集タンパク質を形成する変異を有するハンチンチンタンパク質を意味し、変異の種類は制限されない。このような異常ハンチンチンタンパク質としては、その構造内のポリグルタミン（ｐｏｌｙＱ）鎖が異常伸長したハンチンチンタンパク質が含まれる。ポリグルタミン（ｐｏｌｙＱ）鎖が異常伸長したハンチンチンタンパク質としては、例えば、連続する４１以上のグルタミンを有するハンチンチンタンパク質を挙げることができる。
また本明細書において「ポリグルタミンタンパク質」とは、その構造内にポリグルタミン（ｐｏｌｙＱ）鎖を繰り返して複数有するタンパク質であって、神経細胞内で凝集を形成するタンパク質を意味する。ポリグルタミンタンパク質としては上記異常ハンチンチンタンパク質を含むがこれに限定されない。現在確認されている９つのポリグルタミン病の原因タンパク質は全て異なっているほか、正常なタンパク質でもいくつかのグルタミンが連続したポリグルタミン鎖を有しているものの、その正常な長さはそれぞれの原因タンパク質ごとに異なっている。
For example, tau protein is phosphorylated at serine, threonine, and tyrosine in its structure, and it has been reported that abnormal phosphorylation of a specific region among these is involved in the formation of aggregated proteins (Noble, W. et al., "The importance of tau phosphorylation for neurodegenerative diseases". Front. Neurol. 4, 1-11(2013)). In addition, as shown in the following examples, mutant tau proteins in which the 406th amino acid, arginine, of tau protein is replaced with tryptophan are also known to aggregate in neurons. In this specification, the term "mutant tau protein" refers to a tau protein having a mutation that forms an aggregated protein in neurons, and the type of mutation is not limited. Examples of such mutant tau proteins include tau proteins having abnormal phosphorylation in their structure and tau proteins in which the 406th arginine is replaced with tryptophan.
It is also known that, for example, the polyglutamine (polyQ) chain in the huntingtin protein structure is abnormally extended to become a mutant protein (polyglutamine protein) and form an insoluble fibrous structure (aggregate). In this specification, the term "abnormal huntingtin protein" refers to a huntingtin protein having a mutation that forms an aggregate protein in a nerve cell, and the type of mutation is not limited. Such abnormal huntingtin proteins include huntingtin proteins having an abnormally extended polyglutamine (polyQ) chain in the structure. An example of a huntingtin protein having an abnormally extended polyglutamine (polyQ) chain is a huntingtin protein having 41 or more consecutive glutamines.
In addition, in this specification, "polyglutamine protein" refers to a protein that has multiple repeating polyglutamine (polyQ) chains in its structure and forms aggregates in nerve cells. Polyglutamine proteins include, but are not limited to, the abnormal huntingtin protein. The causative proteins of the nine polyglutamine diseases currently identified are all different, and even normal proteins have polyglutamine chains with several consecutive glutamines, but the normal length of the chain varies depending on each causative protein.
本発明のタンパク凝集抑制剤は、神経細胞内でタンパク質または変異タンパク質が凝集体を形成するのを抑制する。細胞内の凝集タンパク質はその凝集の程度に応じて様々な状態で存在する場合がある。すなわちタンパク質または変異タンパク質は細胞内において、凝集以前の単量体、凝集初期のオリゴマー、多量体、凝集が進んだ状態である線維、高度に凝集した封入体として存在し得る。本発明のタンパク質または変異タンパク質凝集抑制剤は、これらの変異タンパク質のオリゴマー、多量体、線維および封入体の形成の少なくとも一つを抑制することができる。好ましい実施の形態において、これらのタンパク質または変異タンパク質のオリゴマー、多量体、線維および封入体の形成を全て抑制することができる。 The protein aggregation inhibitor of the present invention inhibits the formation of aggregates by proteins or mutant proteins in nerve cells. Aggregated proteins in cells may exist in various states depending on the degree of aggregation. That is, proteins or mutant proteins may exist in cells as monomers before aggregation, oligomers or multimers in the early stages of aggregation, fibrils in advanced stages of aggregation, or inclusion bodies in highly aggregated states. The protein or mutant protein aggregation inhibitor of the present invention can inhibit at least one of the formation of oligomers, multimers, fibrils, and inclusion bodies of these mutant proteins. In a preferred embodiment, it can inhibit the formation of all of the oligomers, multimers, fibrils, and inclusion bodies of these proteins or mutant proteins.
本発明は別の態様として、ヒアルロン酸またはその薬学的に許容される塩を含む、神経細胞内における凝集したタンパク質の分解促進剤を提供する。本発明に係る神経細胞内における凝集したタンパク質の分解促進剤を用いれば、すでに凝集したタンパク質を、その細胞内に有する神経細胞であっても当該凝集タンパク質の分解を促進させることができる。以下の理論に縛られないが、ヒアルロン酸は細胞内の異常な構造を有するタンパク質の分解に関与する遺伝子や細胞内のタンパク質の構造を正常な構造に保つように機能する遺伝子の発現の増加に影響し、神経細胞内における凝集したタンパク質の分解を促すと考えられる。 In another aspect, the present invention provides an agent for promoting the degradation of aggregated proteins in nerve cells, comprising hyaluronic acid or a pharma- ceutically acceptable salt thereof. By using the agent for promoting the degradation of aggregated proteins in nerve cells according to the present invention, it is possible to promote the degradation of aggregated proteins even in nerve cells that already have aggregated proteins in the cells. Without being bound by the following theory, it is believed that hyaluronic acid promotes the degradation of aggregated proteins in nerve cells by influencing the increase in expression of genes involved in the degradation of proteins with abnormal intracellular structures and genes that function to maintain the intracellular protein structure in a normal structure.
本発明は別の態様として、神経細胞とヒアルロン酸またはその薬学的に許容される塩とを接触させる工程を含む、神経細胞内におけるタンパク質の凝集が抑制された神経細胞を製造する方法を提供する。本態様に係る製造方法によれば、タンパク質の凝集を抑制された神経細胞を得ることができる。 In another aspect, the present invention provides a method for producing nerve cells in which protein aggregation within the nerve cells is suppressed, the method comprising the step of contacting nerve cells with hyaluronic acid or a pharma- ceutically acceptable salt thereof. According to the production method of this aspect, nerve cells in which protein aggregation is suppressed can be obtained.
神経細胞とヒアルロン酸またはその薬学的に許容される塩とを接触させる方法は、ヒアルロン酸の作用により神経細胞内におけるタンパク質の凝集を抑制できる限りにおいて限定されず公知の手法により行うことができる。以下に限定されないが、例えば、１０％ウシ胎児血清（ＦＢＳ）を含むＤｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ（ＤＭＥＭ）中、３７℃の条件で培養することができる。ヒアルロン酸の濃度もタンパク質の凝集を抑制された神経細胞を得られる限りにおいて限定されず、培地中のヒアルロン酸の濃度が１～２５０ｎＭ、より好ましくは１～１５０ｎＭ、さらに好ましくは１～１００ｎＭの濃度になるようにヒアルロン酸またはその塩を添加すればよい。培養期間は例えば０．５日～６０日とすることができる。 The method of contacting nerve cells with hyaluronic acid or a pharma- ceutically acceptable salt thereof is not limited, and can be performed by a known method, so long as the action of hyaluronic acid can suppress protein aggregation in nerve cells. For example, but not limited to, the nerve cells can be cultured at 37°C in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS). The concentration of hyaluronic acid is also not limited, so long as nerve cells in which protein aggregation is suppressed can be obtained, and hyaluronic acid or a salt thereof may be added so that the concentration of hyaluronic acid in the medium is 1 to 250 nM, more preferably 1 to 150 nM, and even more preferably 1 to 100 nM. The culture period can be, for example, 0.5 to 60 days.
本発明は別の態様において、ヒアルロン酸またはその薬学的に許容される塩を含む神経細胞の軸索の伸展剤を提供する。本発明に係る神経細胞の軸索の伸展剤を用いれば、神経細胞における軸索の伸展を可能とし、ひいてはアルツハイマー病や前頭側頭型認知症等に大きく関与する軸索の変性を改善し得る。また例えば、神経細胞を対象患者に移植した後、当該神経細胞へヒアルロン酸またはその薬学的に許容される塩を含む神経細胞の軸索の伸展剤を作用させることによって、患者の脳内で、軸索が十分に伸展した神経細胞を得ることが出来、アルツハイマー病や前頭側頭型認知症等の神経変性疾患を改善し得る。 In another aspect, the present invention provides a neuronal axon extension agent comprising hyaluronic acid or a pharma- ceutically acceptable salt thereof. Use of the neuronal axon extension agent according to the present invention enables axon extension in neuronal cells, and can improve axon degeneration that is closely related to Alzheimer's disease, frontotemporal dementia, and the like. For example, after transplanting neuronal cells into a subject patient, a neuronal axon extension agent comprising hyaluronic acid or a pharma-ceutically acceptable salt thereof can be applied to the neuronal cells, thereby obtaining neuronal cells with sufficiently extended axons in the brain of the patient, and improving neurodegenerative diseases such as Alzheimer's disease and frontotemporal dementia.
本発明における軸索とは、神経細胞から延びている細長い突起状の構造体であって、神経細胞において他の神経細胞への情報を伝達する機能を有するものである。 In the present invention, an axon is a long, thin, projection-like structure extending from a nerve cell, which has the function of transmitting information from the nerve cell to other nerve cells.
軸索が伸展することによって、神経細胞同士の情報交換および伝達物質の交換が非常に活性化されることにより、前頭側頭型認知症、アルツハイマー病、レビー小体型認知症、またはダウン症候群の予防または治療が可能となる。 By extending axons, the exchange of information and neurotransmitters between nerve cells becomes highly activated, making it possible to prevent or treat frontotemporal dementia, Alzheimer's disease, dementia with Lewy bodies, or Down's syndrome.
本発明は別の態様として、神経細胞とヒアルロン酸またはその薬学的に許容される塩とを接触させる工程を含む、軸索が伸展した神経細胞を製造する方法を提供する。本態様に係る製造方法によれば、軸索が伸展した神経細胞を得ることができる。当該神経細胞は、以下に限定されないが、例えば生体から分離または単離した神経細胞、神経前駆細胞や多能性幹細胞などの培養細胞より分化誘導して得られた神経細胞を用いることができる。 In another aspect, the present invention provides a method for producing nerve cells having extended axons, comprising the step of contacting nerve cells with hyaluronic acid or a pharma- ceutically acceptable salt thereof. According to the production method of this aspect, nerve cells having extended axons can be obtained. The nerve cells may be, but are not limited to, nerve cells separated or isolated from a living body, or nerve cells obtained by inducing differentiation from cultured cells such as neural progenitor cells or pluripotent stem cells.
神経細胞とヒアルロン酸またはその薬学的に許容される塩とを接触させる方法は、ヒアルロン酸の作用により神経細胞の軸索が伸展する限りにおいて限定されず公知の手法により行うことができる。例えば神経細胞が培養細胞である場合、神経細胞を培養している培養液中にヒアルロン酸またはその塩を添加すればよい。神経細胞の培養方法は公知の条件を採用することができ、軸索が伸展した神経細胞を得られる限りにおいて制限されない。以下に限定されないが、例えば、１０％ＦＢＳを含むＤＭＥＭ中、３７℃の条件で培養することができる。ヒアルロン酸の濃度も軸索が伸展した神経細胞を得られる限りにおいて限定されず、１～２５０ｎＭ、より好ましくは１～１５０ｎＭ、さらに好ましくは１～１００ｎＭの濃度となるように培地にヒアルロン酸またはその塩を添加すればよい。培養期間は例えば０．５日～６０日とすることができる。 The method of contacting nerve cells with hyaluronic acid or a pharma- ceutically acceptable salt thereof is not limited as long as the axons of the nerve cells are extended by the action of hyaluronic acid, and can be performed by a known method. For example, when the nerve cells are cultured cells, hyaluronic acid or a salt thereof may be added to the culture medium in which the nerve cells are cultured. The method of culturing the nerve cells may adopt known conditions, and is not limited as long as nerve cells with extended axons can be obtained. For example, the nerve cells may be cultured in DMEM containing 10% FBS at 37°C, but is not limited thereto. The concentration of hyaluronic acid is also not limited as long as nerve cells with extended axons can be obtained, and hyaluronic acid or a salt thereof may be added to the medium to a concentration of 1 to 250 nM, more preferably 1 to 150 nM, and even more preferably 1 to 100 nM. The culture period may be, for example, 0.5 to 60 days.
また本発明は別の態様として、ヒアルロン酸またはその薬学的に許容される塩を含む、タウタンパク質またはポリグルタミンタンパク質の凝集に起因する神経変性疾患の予防または治療用医薬を提供する。本態様に係る神経変性疾患の予防または治療用医薬によれば、タウタンパク質またはポリグルタミンタンパク質の凝集に起因する神経変性疾患を予防または治療することができる。 In another aspect, the present invention provides a pharmaceutical for preventing or treating a neurodegenerative disease caused by the aggregation of tau protein or polyglutamine protein, comprising hyaluronic acid or a pharma- ceutically acceptable salt thereof. According to the pharmaceutical for preventing or treating a neurodegenerative disease according to this aspect, a neurodegenerative disease caused by the aggregation of tau protein or polyglutamine protein can be prevented or treated.
本明細書において「タウタンパク質の凝集に起因する神経変性疾患」とは、神経細胞内においてタウタンパク質の凝集によって認知機能が低下する疾患を意味する。また本明細書において「ポリグルタミンタンパク質の凝集に起因する神経変性疾患」とは、神経細胞内においてポリグルタミンタンパク質の凝集によって認知機能が低下する疾患を意味する。かかる神経変性疾患としては、前頭側頭型認知症（ＦＴＬＤ：ｆｒｏｎｔｏｔｅｍｐｏｒａｌ ｌｏｂａ ｄｅｎｇｅｒａｔｉｏｎ）、アルツハイマー病、ポリグルタミン病（ハンチントン病や脊髄小脳変性症）、レビー小体型認知症、ダウン症候群などを挙げることができる。また、「タウタンパク質の凝集に起因する神経変性疾患」としてはアルツハイマー病または前頭側頭型認知症（ＦＴＬＤ）を好適に挙げることができ、「ポリグルタミンタンパク質の凝集に起因する神経変性疾患」としてはポリグルタミン病（ハンチントン病や脊髄小脳変性症）を好適に挙げることができる。また「ポリグルタミンタンパク質の凝集に起因する神経変性疾患」は「ハンチンチンタンパク質の凝集に起因する神経変性疾患」を含み、「ハンチンチンタンパク質の凝集に起因する神経変性疾患」としてハンチントン病を好適に挙げることができる。前頭側頭型認知症（ＦＴＬＤ）は、臨床病型として主にＦＴＬＤ－ｔａｕ、ＦＴＬＤ－ＴＤＰ、およびＦＴＬＤ－ＦＵＳに分類され、ＦＴＬＤ－Ｔｔａｕにはピック病、ＦＴＬＤ－１７、皮質基底核変性症（ＣＢＤ）、進行性核上性麻痺（ＰＳＰ）、嗜銀顆粒性認知症（ＡＧＤ）、神経原線維変化型老年認知症（ＳＤ－ＮＦＴ）、ＭＳＴＤ、ＷＭＴ－ＧＧＩが含まれ、ＦＴＬＤ－ＴＤＰにはＦＴＤ－ＭＮＤ、ＦＴＤ－ＴＤＰが含まれ、ＦＴＬＤ－ＦＵＳにはａｔｙｐｉｃａl ＦＴＬＤ－Ｕ、ＢＩＢＤ、ＮＩＦＩＤが含まれる。 In this specification, "neurodegenerative disease caused by tau protein aggregation" means a disease in which cognitive function is reduced due to the aggregation of tau protein in nerve cells. In addition, in this specification, "neurodegenerative disease caused by polyglutamine protein aggregation" means a disease in which cognitive function is reduced due to the aggregation of polyglutamine protein in nerve cells. Examples of such neurodegenerative diseases include frontotemporal dementia (FTLD), Alzheimer's disease, polyglutamine diseases (Huntington's disease and spinocerebellar degeneration), dementia with Lewy bodies, Down's syndrome, etc. In addition, Alzheimer's disease or frontotemporal dementia (FTLD) can be preferably cited as "neurodegenerative disease caused by tau protein aggregation", and polyglutamine diseases (Huntington's disease and spinocerebellar degeneration) can be preferably cited as "neurodegenerative disease caused by polyglutamine protein aggregation". Furthermore, "neurodegenerative diseases caused by polyglutamine protein aggregation" includes "neurodegenerative diseases caused by huntingtin protein aggregation," and Huntington's disease is a suitable example of "neurodegenerative diseases caused by huntingtin protein aggregation." Frontotemporal dementia (FTLD) is primarily classified into FTLD-tau, FTLD-TDP, and FTLD-FUS as clinical disease types, with FTLD-TTau including Pick's disease, FTLD-17, corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), argyrophilic grain dementia (AGD), senile dementia with neurofibrillary tangles (SD-NFT), MSTD, and WMT-GGI, FTLD-TDP including FTD-MND and FTD-TDP, and FTLD-FUS including atypical FTLD-U, BIBD, and NIFID.
本発明の神経細胞内におけるタンパク質凝集抑制剤や、神経細胞内における凝集したタンパク質の分解促進剤、軸索の伸展剤、および本発明のタウタンパク質の凝集またはポリグルタミンタンパク質の凝集に起因する神経変性疾患の予防または治療用医薬には、製剤化のために通常使用され薬学的に許容される賦形剤、結合剤、滑沢剤、崩壊剤、防腐剤、等張化剤、安定化剤、分散剤、酸化防止剤、着色剤、香味剤、緩衝剤等の添加物を含んでいてもよい。製剤の剤型としては、注射剤、経口剤、外用剤など投与形態に合わせて適宜好ましい財型を採用することができ、例えば、散剤、顆粒剤、錠剤、液剤、乾燥粉末剤、注射剤、点滴剤、外用剤、貼付剤、気化投与用の液剤、挿入剤等であってもよい。この場合、１種または２種以上の薬学的に許容される添加物と組み合わせて調製してもよい。 The protein aggregation inhibitor in nerve cells, the agent for promoting the degradation of aggregated proteins in nerve cells, the agent for extending axons, and the pharmaceutical for preventing or treating neurodegenerative diseases caused by the aggregation of tau proteins or the aggregation of polyglutamine proteins of the present invention may contain additives such as excipients, binders, lubricants, disintegrants, preservatives, isotonicity agents, stabilizers, dispersants, antioxidants, colorants, flavoring agents, and buffers that are commonly used for formulation and are pharma- ceutically acceptable. The dosage form of the formulation may be an appropriate, preferred dosage form according to the mode of administration, such as injections, oral preparations, and topical preparations, and may be, for example, powders, granules, tablets, liquids, dry powders, injections, drips, topical preparations, patches, liquids for vapor administration, and inserts. In this case, the formulation may be prepared in combination with one or more pharma-ceutical acceptable additives.
本発明のタウタンパク質の凝集またはポリグルタミンタンパク質の凝集に起因する神経変性疾患の予防または治療用医薬の投与方法としては、所望の神経変性疾患の予防または治療効果が得られる限り特に制限されず、静脈内投与、経口投与、筋肉内投与、皮下投与、経皮投与、経鼻投与、経肺投与等を挙げることができる。また、本発明のタウタンパク質の凝集またはポリグルタミンタンパク質の凝集に起因する神経変性疾患の予防または治療用医薬の投与量は特に制限されず、被検者や被検動物の体調、病状、体重、年齢、性別等によって適宜調整することができる。１日あたりの投与量は、上記の投与方法のうちいずれを用いるかによって異なるが、感受性の違いを踏まえ、１０～５００μｇ、好ましくは２０～１００μｇ、より好ましくは４０～６０μｇを挙げることができる。さらに、本発明のタウタンパク質の凝集またはポリグルタミンタンパク質の凝集に起因する神経変性疾患の予防または治療用医薬を他の神経変性疾患の予防または治療医薬と併用してもよい。本発明のタウタンパク質の凝集またはポリグルタミンタンパク質の凝集に起因する神経変性疾患の予防または治療用医薬の投与回数や投与期間等も特に制限されず、１日あたりの投与量を１日１回または数回に分けて投与することもできる。また投与対象の細胞、組織の由来や生体は特に制限されず、好ましくは哺乳類であり、例えばヒト、サル、ウシ、ウマ、ヒツジ、ブタ、イヌ、ネコ、ラット、マウス、ハムスター等を例示することができ、好ましくはヒトを例示することができる。 The method of administration of the present invention for preventing or treating neurodegenerative diseases caused by tau protein aggregation or polyglutamine protein aggregation is not particularly limited as long as the desired preventive or therapeutic effect for neurodegenerative diseases is obtained, and examples of the method include intravenous administration, oral administration, intramuscular administration, subcutaneous administration, transdermal administration, nasal administration, and pulmonary administration. The dosage of the present invention for preventing or treating neurodegenerative diseases caused by tau protein aggregation or polyglutamine protein aggregation is not particularly limited, and can be appropriately adjusted depending on the physical condition, disease state, weight, age, sex, etc. of the subject or test animal. The daily dosage varies depending on which of the above administration methods is used, but taking into account differences in sensitivity, examples of the dosage are 10 to 500 μg, preferably 20 to 100 μg, and more preferably 40 to 60 μg. Furthermore, the present invention for preventing or treating neurodegenerative diseases caused by tau protein aggregation or polyglutamine protein aggregation may be used in combination with other neurodegenerative disease preventive or therapeutic drugs. There are no particular limitations on the number of doses or duration of administration of the pharmaceutical agent for preventing or treating neurodegenerative diseases caused by tau protein aggregation or polyglutamine protein aggregation of the present invention, and the daily dose can be administered once or in divided doses. There are also no particular limitations on the origin of cells or tissues or the living organisms to which the agent is administered, and the preferred examples are mammals, such as humans, monkeys, cows, horses, sheep, pigs, dogs, cats, rats, mice, and hamsters, with humans being preferred.
本発明は別の態様として、ヒアルロン酸またはその薬学的に許容される塩を含む薬剤の治療有効量を、タウタンパク質またはポリグルタミンタンパク質の凝集に起因する神経変性疾患の予防または治療を必要とする対象に投与することを含む、神経変性疾患の予防または治療方法を提供する。 In another aspect, the present invention provides a method for preventing or treating a neurodegenerative disease, comprising administering a therapeutically effective amount of a drug containing hyaluronic acid or a pharma- ceutically acceptable salt thereof to a subject in need of prevention or treatment of a neurodegenerative disease caused by aggregation of tau protein or polyglutamine protein.
本発明はさらに別の態様として、タウタンパク質またはポリグルタミンタンパク質の凝集に起因する神経変性疾患の予防または治療のためのヒアルロン酸またはその薬学的に許容される塩を提供する。 In yet another aspect, the present invention provides hyaluronic acid or a pharma- ceutically acceptable salt thereof for the prevention or treatment of a neurodegenerative disease caused by the aggregation of tau protein or polyglutamine protein.
本発明はまた別の態様として、ヒアルロン酸またはその薬学的に許容される塩の、タウタンパク質またはポリグルタミンタンパク質の凝集に起因する神経変性疾患の予防または治療用医薬の製造における使用を提供する。 In another aspect, the present invention provides the use of hyaluronic acid or a pharma- ceutically acceptable salt thereof in the manufacture of a medicament for the prophylaxis or treatment of a neurodegenerative disease caused by the aggregation of tau protein or polyglutamine protein.
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。 The present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.
（実施例１．ヒアルロン酸による神経細胞の軸索の伸展）
１０％ウシ胎児血清（ＦＢＳ）を含むＤｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ （ＤＭＥＭ；メルク社製）培地を加えた６ｃｍ ｄｉｓｈにヒト神経芽細胞腫ＳＨ－ＳＹ５Ｙ株を１.０×１０４となるようにまいて３７℃で２４時間培養した。その後１０ｎＭの濃度となるようにリン酸緩衝生理食塩水（ＰＢＳ）で溶解したヒアルロン酸（Ｈｙａｌｕｒｏｎｉｃ Ａｃｉｄ Ｓｏｄｉｕｍ Ｓａｌｔ ｆｒｏｍ Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｆｏｒ Ｂｉｏｃｈｅｍ：コードＮｏ．ＨＡ００２００５ 長良サイエンス社製）を培地に加え、さらに３７℃で７日間培養した。またコントロールとして、ヒアルロン酸含有ＰＢＳと等量のＰＢＳのみを培地に加え、同様に３７℃で７日間培養した。７日間培養後の細胞の写真を図１に示す。
(Example 1. Extension of nerve cell axons by hyaluronic acid)
Human neuroblastoma SH-SY5Y strain was seeded at 1.0 x 10 4 on a 6 cm dish containing Dulbecco's Modified Eagle Medium (DMEM; Merck) containing 10% fetal bovine serum (FBS) and cultured at 37 ° C for 24 hours. Then, hyaluronic acid (Hyaluronic Acid Sodium Salt from Streptococcus for Biochem: Code No. HA002005 Nagara Science Co., Ltd.) dissolved in phosphate buffered saline (PBS) to a concentration of 10 nM was added to the medium and further cultured at 37 ° C for 7 days. As a control, only PBS equivalent to the hyaluronic acid-containing PBS was added to the medium and similarly cultured at 37 ° C for 7 days. A photograph of the cells after 7 days of culture is shown in FIG.
図１に示すように、ヒアルロン酸を加えることでヒト神経芽細胞腫の軸索が３０μｍと細胞体のおおよそ６倍に大きく伸展していることが明らかとなった。なお、ＰＢＳのみで処理したコントロール群では、ヒト神経芽細胞腫の軸索は長いものでも２～１０μｍであった。かかる結果より、ヒアルロン酸を投与することで、神経細胞の軸索を伸展することが可能であることが明らかとなった。 As shown in Figure 1, it was revealed that the addition of hyaluronic acid caused the axons of human neuroblastoma to extend to 30 μm, approximately six times the length of the cell body. In the control group treated with PBS only, the longest axons of human neuroblastoma were only 2-10 μm long. These results demonstrate that the administration of hyaluronic acid makes it possible to extend the axons of nerve cells.
（実施例２．ヒアルロン酸の濃度変化に対する神経細胞の軸索の伸展への影響）
上記実施例１において、ヒアルロン酸は神経細胞の軸索を伸展させる作用を有することが明らかとなった。本実施例では、神経細胞を培養する培養液中のヒアルロン酸濃度を変化させたときの軸索の進展への影響を検証した。
１０％ウシ胎児血清（ＦＢＳ）を含むＤｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ（ＤＭＥＭ；メルク社製）培地を加えた６ｃｍ ｄｉｓｈにヒト神経芽細胞腫ＳＨ－ＳＹ５Ｙ株を１.０×１０４となるようにまいて３７℃で２４時間培養した。その後、１．０ｎＭ、２．５ｎＭ、５．０ｎＭ、または、１２．５ｎＭの濃度となるようにＰＢＳで溶解したヒアルロン酸（コードＮｏ．ＨＡ００２００５ 長良サイエンス社製）を培地に加え、さらに３７℃で７日間培養した。またコントロールとして、ヒアルロン酸含有ＰＢＳと等量のＰＢＳのみを培地に加え、同様に３７℃で７日間培養した。７日間の培養後、神経細胞の軸索が細胞体の大きさの５倍以上になった細胞の割合を図２のグラフに示す。
(Example 2. Effect of changes in hyaluronic acid concentration on the extension of nerve cell axons)
In the above-mentioned Example 1, it was revealed that hyaluronic acid has the effect of extending the axon of nerve cells. In this Example, the effect on the extension of axons when the concentration of hyaluronic acid in the culture medium for culturing nerve cells is changed was verified.
Human neuroblastoma SH-SY5Y strain was seeded at 1.0 x 10 4 on a 6 cm dish containing Dulbecco's Modified Eagle Medium (DMEM; Merck) containing 10% fetal bovine serum (FBS) and cultured at 37 ° C for 24 hours. Then, hyaluronic acid (Code No. HA002005, Nagara Science Co., Ltd.) dissolved in PBS to a concentration of 1.0 nM, 2.5 nM, 5.0 nM, or 12.5 nM was added to the medium and further cultured at 37 ° C for 7 days. As a control, only PBS equivalent to the hyaluronic acid-containing PBS was added to the medium and similarly cultured at 37 ° C for 7 days. After 7 days of culture, the percentage of cells whose axons of nerve cells were 5 times or more the size of the cell body is shown in the graph of FIG. 2.
図２に示すように、ヒアルロン酸を添加した区では１．０ｎＭ～１２．５ｎＭのいずれの濃度となるように添加した区においても、コントロールと比較して軸索が伸展した細胞の割合が増加していた。特にヒアルロン酸の濃度が１．０ｎＭ～５．０ｎＭの区では、コントロールと比較して軸索が伸展した細胞の割合がより増加していた（ｐ＜０．０１）。 As shown in Figure 2, the percentage of cells with extended axons was increased in the sections where hyaluronic acid was added at concentrations between 1.0 nM and 12.5 nM compared to the control. In particular, the percentage of cells with extended axons was increased more in the sections where hyaluronic acid was added at concentrations between 1.0 nM and 5.0 nM compared to the control (p<0.01).
（実施例３．ヒアルロン酸によるタウタンパク質の凝集抑制）
前頭側頭型認知症（ＦＴＬＤ）の原因となる変異型タウタンパク質（タウタンパク質の４０６番目のアミノ酸であるアルギニンがトリプトファンに置換）を発現した神経細胞を作製し、変異型タウタンパク質の発現および凝集に対するヒアルロン酸の作用を検証した。
(Example 3. Inhibition of tau protein aggregation by hyaluronic acid)
We created neural cells expressing mutant tau protein (the 406th amino acid in tau protein is replaced with tryptophan) that causes frontotemporal dementia (FTLD), and examined the effect of hyaluronic acid on the expression and aggregation of mutant tau protein.
３－１．変異型タウタンパク質（ＴａｕＲ４０６Ｗ）をコードするプラスミドベクターの作製
配列番号１に示す塩基配列からなるヒトのタウタンパク質をコードするｃＤＮＡをヒト子宮頸がん細胞のＨｅＬａ細胞からクローニングした後、これをテンプレートとして、二段階ＰＣＲ法を用いて、配列番号２に示すアミノ酸配列からなるタウタンパク質の４０６番目のアミノ酸であるアルギニンがトリプトファンに置き換わるように塩基の置換を行い、ヒトの変異型タウタンパク質をコードするｃＤＮＡを得た。置換のためのＰＣＲに用いたフォワードプライマーの塩基配列を配列番号３に、リバースプライマーの塩基配列を配列番号４に示す。ＰＣＲの条件は、９４℃１分、６５℃２分、７２℃３分を１サイクルとして３０サイクル繰り返し、その後７２℃５分処理し、その後４℃とした。ポリメラーゼはＥｘ－Ｔａｑ（タカラバイオ社製）を用いた。ＰＣＲ産物は、ｃＤＮＡの塩基配列を調べて正しく変異が入っていることを確認した。かかるヒトの変異型タウタンパク質をコードするｃＤＮＡをｐＥＧＦＰ－Ｎ１ベクターに組み込み、ｐＥＧＦＰ－Ｎ１－ｔａｕＲ４０６Ｗベクターを作製し、前記ｃＤＮＡのＣ末端側に緑色蛍光タンパク質（ＧＦＰ）のｃＤＮＡをつなげ、ＧＦＰの蛍光によって可視化出来る変異型タウタンパク質が発現するようにした。さらに上記ｐＥＧＦＰ－Ｎ１－ｔａｕＲ４０６Ｗ－ＧＦＰベクターをｐＳｈｕｔｔｌｅ－ＣＭＶベクターに組み込み、トランスフェクション用のプラスミドベクターを作製した。なお、配列番号１における１２４０番目の塩基は正常型では「ｃ」であるが、これが「ｔ」に変わることによって配列番号２に示すアミノ酸配列からなるタウタンパク質の４０６番目のアミノ酸のアルギニン（Ｒ）がトリプトファン（Ｗ）に変化し（Ｒ４０６Ｗ：タウタンパク質はアイソフォームが６種類知られており、慣例によりアミノ酸番号は４４１アミノ酸型最長アイソフォームに準じる）、前頭側頭型認知症（ＦＴＬＤ）の原因となることが知られている。
3-1. Preparation of a plasmid vector encoding mutant tau protein (TauR406W) After cloning a cDNA encoding human tau protein consisting of the base sequence shown in SEQ ID NO: 1 from HeLa cells, a human cervical cancer cell, this was used as a template to perform base substitution using a two-step PCR method so that the 406th amino acid, arginine, of the tau protein consisting of the amino acid sequence shown in SEQ ID NO: 2 was replaced with tryptophan, thereby obtaining a cDNA encoding a human mutant tau protein. The base sequence of the forward primer used in the PCR for substitution is shown in SEQ ID NO: 3, and the base sequence of the reverse primer is shown in SEQ ID NO: 4. The PCR conditions were 94°C for 1 minute, 65°C for 2 minutes, and 72°C for 3 minutes, which were repeated for 30 cycles, followed by treatment at 72°C for 5 minutes, and then at 4°C. Ex-Taq (manufactured by Takara Bio Inc.) was used as the polymerase. The PCR product was confirmed to have the correct mutation by examining the base sequence of the cDNA. The cDNA encoding the human mutant tau protein was incorporated into the pEGFP-N1 vector to prepare the pEGFP-N1-tauR406W vector, and the cDNA encoding green fluorescent protein (GFP) was connected to the C-terminus of the cDNA to express the mutant tau protein that can be visualized by the fluorescence of GFP. Furthermore, the pEGFP-N1-tauR406W-GFP vector was incorporated into the pShuttle-CMV vector to prepare a plasmid vector for transfection. Note that the 1240th base in SEQ ID NO: 1 is "c" in the normal form, but when this is changed to "t", the arginine (R) at the 406th amino acid of the tau protein consisting of the amino acid sequence shown in SEQ ID NO: 2 is changed to tryptophan (W) (R406W: tau protein has six known isoforms, and by convention the amino acid number conforms to the longest isoform of the 441 amino acid type), which is known to cause frontotemporal dementia (FTLD).
３－２．ヒアルロン酸投与による変異型タウタンパク質（ＴａｕＲ４０６Ｗ）の凝集抑制
１０％ウシ胎児血清（ＦＢＳ）を含むＤｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ（ＤＭＥＭ；メルク社製）培地を加えた６ｃｍ ｄｉｓｈにヒト神経芽細胞腫ＳＨ－ＳＹ５Ｙ株を１.０×１０４となるようにまいて３７℃で２４時間培養した。２４時間の培養後、ヒト神経芽細胞腫ＳＨ－ＳＹ５Ｙ株を、上記ｐＥＧＦＰ－Ｎ１－ｔａｕＲ４０６Ｗ－ＧＦＰベクターを組み込んだｐＳｈｕｔｔｌｅ－ＣＭＶベクターによってトランスフェクションし、変異型タウタンパク質（ＴａｕＲ４０６Ｗ）を発現するヒト神経芽細胞腫ＳＨ－ＳＹ５Ｙ株を作製し、さらに３７℃で２４時間培養した。
その後１０ｎＭの濃度となるようにリン酸緩衝生理食塩水（ＰＢＳ）で溶解したヒアルロン酸（コードＮｏ．ＨＡ００２００５ 長良サイエンス社製）を培地に加え、さらに３７℃で７日間培養した。またコントロールとして、ヒアルロン酸含有ＰＢＳと等量のＰＢＳのみを培地に加え、同様に３７℃で７日間培養した。７日間培養後に蛍光顕微鏡を用いて、細胞中のＧＦＰの蛍光を観察した。図３Ａは細胞を蛍光顕微鏡および光学顕微鏡下で撮像した写真を示す。
また７日間培養後の細胞を回収してウェスタンブロット解析を行った。ウェスタンブロット解析には各レーンにタンパク質をアプライし、電気泳動後、ニトロセルロース膜に転写されたものを解析した。変異型タウタンパク質の検出には、抗ＧＦＰ抗体（ＧＦ２００、ナカライテスク社製）を用い、内部コントロールのベータアクチンには、Ｍ１７７－３抗体（MBL社製）を用いた。結果を図３Ｂに示す。
3-2. Inhibition of Mutant Tau Protein (TauR406W) Aggregation by Hyaluronic Acid Administration Human neuroblastoma SH-SY5Y strain was seeded at 1.0 x 10 4 on a 6 cm dish containing Dulbecco's Modified Eagle Medium (DMEM; Merck) medium containing 10% fetal bovine serum (FBS) and cultured at 37°C for 24 hours. After 24 hours of culture, the human neuroblastoma SH-SY5Y strain was transfected with the pShuttle-CMV vector incorporating the pEGFP-N1-tauR406W-GFP vector to prepare a human neuroblastoma SH-SY5Y strain expressing mutant tau protein (TauR406W), which was then further cultured at 37°C for 24 hours.
Then, hyaluronic acid (Code No. HA002005, Nagara Science Co., Ltd.) dissolved in phosphate buffered saline (PBS) to a concentration of 10 nM was added to the medium, and the medium was further cultured at 37°C for 7 days. As a control, PBS alone was added to the medium in an amount equal to that of the hyaluronic acid-containing PBS, and the medium was similarly cultured at 37°C for 7 days. After 7 days of culture, the fluorescence of GFP in the cells was observed using a fluorescent microscope. Figure 3A shows a photograph of the cells taken under a fluorescent microscope and an optical microscope.
After 7 days of culture, cells were collected and subjected to Western blot analysis. For Western blot analysis, proteins were applied to each lane, electrophoresed, and then transferred to a nitrocellulose membrane for analysis. Anti-GFP antibody (GF200, Nacalai Tesque) was used to detect mutant tau protein, and M177-3 antibody (MBL) was used to detect beta-actin as an internal control. The results are shown in Figure 3B.
図３Ａに示すように、コントロール群のヒト神経芽細胞腫ＳＨ－ＳＹ５Ｙ株ではその細胞中に多くの変異型タウタンパク質の凝集体が観察された。一方、ヒアルロン酸を添加した群では、変異型タウタンパク質の凝集体が減少していた。図３Ｂのウェスタンブロット解析の結果をみると、ヒアルロン酸を添加した群では、可溶性の（凝集していないため、細胞質中に存在する；分解も可能）変異型タウタンパク質の発現の減少が示されたが、同時に、不溶性の（凝集し、細胞内に蓄積する；分解されにくい）変異型タウタンパク質は、可溶性に比べ、より顕著に発現量が減少していた。これは、ヒアルロン酸が変異型タウタンパク質自体の発現量を減少させたか、または、可溶性の変異型タウタンパク質の分解を促進した、あるいは不溶性の変異型タウタンパク質の分解も誘導したことが推察される。 As shown in Figure 3A, many mutant tau protein aggregates were observed in the cells of the human neuroblastoma SH-SY5Y strain in the control group. On the other hand, the number of mutant tau protein aggregates was reduced in the group to which hyaluronic acid was added. The results of the Western blot analysis in Figure 3B showed that the expression of soluble (not aggregated, present in the cytoplasm; can be degraded) mutant tau protein was reduced in the group to which hyaluronic acid was added, but at the same time, the expression level of insoluble (aggregated and accumulated in cells; difficult to degrade) mutant tau protein was reduced more significantly than that of soluble mutant tau protein. This suggests that hyaluronic acid reduced the expression level of mutant tau protein itself, promoted the degradation of soluble mutant tau protein, or induced the degradation of insoluble mutant tau protein.
（実施例４．ヒアルロン酸の濃度変化に対するポリグルタミンタンパク質封入体形成への影響）
ポリグルタミンタンパク質を発現した神経細胞を作製し、変異型タウタンパク質の凝集に対するヒアルロン酸の作用を検証した。
４－１．ポリグルタミンタンパク質をコードするプラスミドベクターの作製
ポリグルタミンタンパク質をコードするｃＤＮＡをｐＥＧＦＰ－Ｎ１に組み込んだｐｏｌｙＱ８１－ｐＥＧＦＰ－Ｎ１の供与を受け（Nagai et al., Experimental Neurology, 1999）、このベクターからｐｏｌｙＱ８１－ｐＥＧＦＰをコードする領域のｃＤＮＡをｐＳｈｕｔｔｌｅ－ＣＭＶベクターに組み込んだ。
(Example 4. Effect of changes in hyaluronic acid concentration on the formation of polyglutamine protein inclusion bodies)
We created neural cells expressing polyglutamine proteins and examined the effect of hyaluronic acid on the aggregation of mutant tau protein.
4-1. Preparation of a plasmid vector encoding a polyglutamine protein PolyQ81-pEGFP-N1, which was prepared by incorporating a cDNA encoding a polyglutamine protein into pEGFP-N1, was provided (Nagai et al., Experimental Neurology, 1999), and the cDNA encoding the region of polyQ81-pEGFP from this vector was incorporated into the pShuttle-CMV vector.
４－２．ヒアルロン酸投与によるポリグルタミンタンパク質の凝集抑制
１０％ウシ胎児血清（ＦＢＳ）を含むＤｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ（ＤＭＥＭ；メルク社製）培地を加えた６ｃｍ ｄｉｓｈにヒト神経芽細胞腫ＳＨ－ＳＹ５Ｙ株を１.０×１０４となるようにまいて３７℃で２４時間培養した。２４時間の培養後、ヒト神経芽細胞腫ＳＨ－ＳＹ５Ｙ株を、上記ｐｏｌｙＱ８１－ｐＥＧＦＰをコードする領域のｃＤＮＡを組み込んだｐＳｈｕｔｔｌｅ－ＣＭＶベクターによってトランスフェクションし、ポリグルタミンタンパク質を発現するヒト神経芽細胞腫ＳＨ－ＳＹ５Ｙ株を作製し、さらに３７℃で２４時間培養した。
その後、１.０ｎＭ、２.５ｎＭ、５.０ｎＭ、または、１２.５ｎＭの濃度となるようにリン酸緩衝生理食塩水（ＰＢＳ）で溶解したヒアルロン酸（コードＮｏ．ＨＡ００２００５ 長良サイエンス社製）を培地に加え、さらに３７℃で７日間培養した。またコントロールとして、ヒアルロン酸含有ＰＢＳと等量のＰＢＳのみを培地に加え、同様に３７℃で７日間培養した。７日間培養後に光学顕微鏡下でポリグルタミンタンパク質封入体を形成している細胞をカウントした。図４は、ポリグルタミンタンパク質封入体を形成している細胞の割合を示す。
4-2. Inhibition of polyglutamine protein aggregation by administration of hyaluronic acid Human neuroblastoma SH-SY5Y strain was seeded at 1.0 x 10 4 on a 6 cm dish containing Dulbecco's Modified Eagle Medium (DMEM; Merck) containing 10% fetal bovine serum (FBS) and cultured for 24 hours at 37°C. After 24 hours of culture, the human neuroblastoma SH-SY5Y strain was transfected with the pShuttle-CMV vector incorporating the cDNA of the region encoding the polyQ81-pEGFP to prepare a human neuroblastoma SH-SY5Y strain expressing polyglutamine protein, which was then cultured for another 24 hours at 37°C.
Thereafter, hyaluronic acid (Code No. HA002005, Nagara Science Co., Ltd.) dissolved in phosphate buffered saline (PBS) to a concentration of 1.0 nM, 2.5 nM, 5.0 nM, or 12.5 nM was added to the medium, and the medium was further cultured at 37°C for 7 days. As a control, PBS alone was added to the medium in an amount equal to that of the hyaluronic acid-containing PBS, and the medium was similarly cultured at 37°C for 7 days. After 7 days of culture, the cells that formed polyglutamine protein inclusion bodies were counted under an optical microscope. Figure 4 shows the percentage of cells that formed polyglutamine protein inclusion bodies.
図４に示すように、ヒアルロン酸を投与することによりコントロールと比較してポリグルタミンタンパク質封入体の形成を抑制することができた。また、ヒアルロン酸の投与濃度が高いほどポリグルタミンタンパク質封入体の形成抑制効果は高かった。 As shown in Figure 4, administration of hyaluronic acid was able to suppress the formation of polyglutamine protein inclusion bodies compared to the control. Furthermore, the higher the concentration of hyaluronic acid administered, the greater the effect of suppressing the formation of polyglutamine protein inclusion bodies.
（実施例５．ヒアルロン酸によるポリグルタミンタンパク質凝集体の分解促進）
ポリグルタミンタンパク質の凝集体を形成している細胞に対してヒアルロン酸投与による効果を検証した。
１０％ウシ胎児血清（ＦＢＳ）を含むＤｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ（ＤＭＥＭ；メルク社製）培地を加えた６ｃｍ ｄｉｓｈにヒト神経芽細胞腫ＳＨ－ＳＹ５Ｙ株を１.０×１０４となるようにまいて３７℃で２４時間培養した。２４時間の培養後、ヒト神経芽細胞腫ＳＨ－ＳＹ５Ｙ株を、上記ｐｏｌｙＱ８１－ｐＥＧＦＰをコードする領域のｃＤＮＡを組み込んだｐＳｈｕｔｔｌｅ－ＣＭＶベクターによってトランスフェクションし、ポリグルタミンタンパク質を発現するヒト神経芽細胞腫ＳＨ－ＳＹ５Ｙ株を作製し、さらに３７℃で２４時間培養した。２４時間培養後の細胞中には、ポリグルタミンタンパク質の封入体が形成されていることを光学顕微鏡下で確認した。
その後、１.０ｎＭ、２.５ｎＭ、５.０ｎＭ、または、１２.５ｎＭの濃度となるようにリン酸緩衝生理食塩水（ＰＢＳ）で溶解したヒアルロン酸（コードＮｏ．ＨＡ００２００５ 長良サイエンス社製）を培地に加え、さらに３７℃で７日間培養した。またコントロールとして、ヒアルロン酸含有ＰＢＳと等量のＰＢＳのみを培地に加え、同様に３７℃で７日間培養した。
７日間培養後の細胞を回収してウェスタンブロット解析を行った。ウェスタンブロット解析には各レーンに３０μｇのタンパク質をアプライし、電気泳動後、ニトロセルロース膜に転写されたものを解析した。ポリグルタミンタンパク質の検出には、抗ＧＦＰ抗体（ナカライテスク社製）を用い、内部コントロールのベータアクチンには、Ｍ１７７－３抗体（MBL社製）を用いた。結果を図５に示す。
また図５のウェスタンブロット解析結果が示すように、ヒアルロン酸の投与濃度が高いほど不溶性（凝集）ポリグルタミンタンパク質が減少していた。特に、５．０ｎＭ以上を添加した区では、不溶性（凝集）ポリグルタミンタンパク質の減少が顕著であった。
Example 5. Hyaluronic acid promotes degradation of polyglutamine protein aggregates
The effect of administering hyaluronic acid to cells forming polyglutamine protein aggregates was examined.
Human neuroblastoma SH-SY5Y strain was seeded at 1.0 x 10 4 on a 6 cm dish containing Dulbecco's Modified Eagle Medium (DMEM; Merck) medium containing 10% fetal bovine serum (FBS) and cultured at 37 ° C. for 24 hours. After 24 hours of culture, the human neuroblastoma SH-SY5Y strain was transfected with a pShuttle-CMV vector incorporating the cDNA of the region encoding the polyQ81-pEGFP to prepare a human neuroblastoma SH-SY5Y strain expressing polyglutamine protein, which was further cultured at 37 ° C. for 24 hours. It was confirmed under an optical microscope that inclusion bodies of polyglutamine protein were formed in the cells after 24 hours of culture.
Thereafter, hyaluronic acid (Code No. HA002005, Nagara Science Co., Ltd.) dissolved in phosphate buffered saline (PBS) to a concentration of 1.0 nM, 2.5 nM, 5.0 nM, or 12.5 nM was added to the medium, and the medium was further cultured for 7 days at 37° C. As a control, PBS alone was added to the medium in an amount equal to that of the hyaluronic acid-containing PBS, and the medium was similarly cultured for 7 days at 37° C.
After 7 days of culture, the cells were collected and subjected to Western blot analysis. For Western blot analysis, 30 μg of protein was applied to each lane, electrophoresed, and then transferred to a nitrocellulose membrane for analysis. Anti-GFP antibody (Nacalai Tesque) was used to detect polyglutamine protein, and M177-3 antibody (MBL) was used to detect beta-actin as an internal control. The results are shown in Figure 5.
In addition, as shown in the results of Western blot analysis in Figure 5, the amount of insoluble (aggregated) polyglutamine protein decreased with increasing concentration of hyaluronic acid. In particular, the decrease in insoluble (aggregated) polyglutamine protein was remarkable in the group where 5.0 nM or more of hyaluronic acid was added.
（実施例６．ヒアルロン酸によるタンパク質分解に関与する遺伝子の発現への影響１）
神経細胞に対するヒアルロン酸投与が、変性タンパク質の構造正常化または分解に働くタンパク質の発現に影響するか検証した。具体的には、細胞の生存を維持する多くの遺伝子発現を誘導し、ポリグルタミンタンパク質の分解を担うＨＳＦ１およびＨＳＦ２、ならびに、ＨＳＦ２と結合してＨＳＦ２の発現誘導を行う必須因子であるＷＤＲ５（Hayashida, Biochem Biophys Res Commun, 2015）の発現に対するヒアルロン酸投与の影響をウェスタンブロットにより解析した。
(Example 6. Effect of hyaluronic acid on the expression of genes involved in protein degradation 1)
We investigated whether administration of hyaluronic acid to nerve cells affects the expression of proteins that normalize the structure or degrade denatured proteins. Specifically, we analyzed the effects of administration of hyaluronic acid on the expression of HSF1 and HSF2, which induce the expression of many genes that maintain cell survival and are responsible for the degradation of polyglutamine proteins, and WDR5, an essential factor that binds to HSF2 and induces its expression (Hayashida, Biochem Biophys Res Commun, 2015), by Western blot analysis.
１０％ウシ胎児血清（ＦＢＳ）を含むＤｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ（ＤＭＥＭ；メルク社製）培地を加えた６ｃｍ ｄｉｓｈにヒト神経芽細胞腫ＳＨ－ＳＹ５Ｙ株を１.０×１０４となるようにまいて３７℃で２４時間培養した。その後、１．０ｎＭ、２．５ｎＭ、５．０ｎＭ、または、１２．５ｎＭの濃度となるようにＰＢＳで溶解したヒアルロン酸（コードＮｏ．ＨＡ００２００５ 長良サイエンス社製）を培地に加え、さらに３７℃で７日間培養した。またコントロールとして、ヒアルロン酸含有ＰＢＳと等量のＰＢＳのみを培地に加え、同様に３７℃で７日間培養した。７日間培養後の細胞を回収してウェスタンブロット解析を行った。ウェスタンブロット解析には各レーンに３０μｇのタンパク質をアプライし、電気泳動後、ニトロセルロース膜に転写されたものを解析した。ＨＳＦ１とＨＳＦ２の検出には、過去に本発明者が作製した抗ＨＳＦ１抗体と抗ＨＳＦ２抗体を用い、ＷＤＲ５の検出には抗ＷＤＲ５抗体（本発明者が独自に作製）を用い、内部コントロールのベータアクチンには、Ｍ１７７－３抗体（MBL社製）を用いた。結果を図６に示す。 Human neuroblastoma SH-SY5Y strain was seeded at 1.0 x 10 4 on a 6 cm dish containing Dulbecco's Modified Eagle Medium (DMEM; Merck) containing 10% fetal bovine serum (FBS) and cultured at 37 ° C for 24 hours. Then, hyaluronic acid (Code No. HA002005, Nagara Science Co., Ltd.) dissolved in PBS to a concentration of 1.0 nM, 2.5 nM, 5.0 nM, or 12.5 nM was added to the medium and further cultured at 37 ° C for 7 days. As a control, only PBS equivalent to the hyaluronic acid-containing PBS was added to the medium and similarly cultured at 37 ° C for 7 days. After 7 days of culture, the cells were collected and subjected to Western blot analysis. For Western blot analysis, 30 μg of protein was applied to each lane, electrophoresed, and then transferred to a nitrocellulose membrane for analysis. HSF1 and HSF2 were detected using anti-HSF1 and anti-HSF2 antibodies previously prepared by the present inventors, WDR5 was detected using anti-WDR5 antibody (originally prepared by the present inventors), and the internal control beta-actin was detected using M177-3 antibody (MBL). The results are shown in FIG. 6.
図６に示すように、ヒアルロン酸の投与によりＨＳＦ１およびＷＤＲ５の発現が増加した。ヒアルロン酸の投与濃度が高いほど、ＨＳＦ１およびＷＤＲ５の発現もより増加していた。ＨＳＦ１及びＨＳＦ２は活性化しても発現量は変わらないというのがこの分野の研究者のコンセンサスであるが、このデータからヒアルロン酸による神経細胞保護機構はこれまでに知られたものとは異なる機構であると考えられる。 As shown in Figure 6, administration of hyaluronic acid increased the expression of HSF1 and WDR5. The higher the concentration of hyaluronic acid administered, the greater the increase in the expression of HSF1 and WDR5. The consensus among researchers in this field is that the expression levels of HSF1 and HSF2 do not change even when activated, but this data suggests that the mechanism by which hyaluronic acid protects nerve cells is different from previously known mechanisms.
（実施例７．ヒアルロン酸によるタンパク質分解または正常化に関与する遺伝子の発現への影響２）
１０％ウシ胎児血清（ＦＢＳ）を含むＤｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ（ＤＭＥＭ；メルク社製）培地を加えた６ｃｍ ｄｉｓｈにヒト神経芽細胞腫ＳＨ－ＳＹ５Ｙ株を１.０×１０４となるようにまいて３７℃で２４時間培養した。その後、１．０ｎＭ、２．５ｎＭ、５．０ｎＭ、または、１２．５ｎＭの濃度となるようにＰＢＳで溶解したヒアルロン酸（コードＮｏ．００２００５ 長良サイエンス社製）を培地に加え、さらに３７℃で７日間培養した。またコントロールとして、ヒアルロン酸含有ＰＢＳと等量のＰＢＳのみを培地に加え、同様に３７℃で７日間培養した。７日間培養後の細胞を回収してＲＴ－ＰＣＲを行った。
本実施例においては、ＮＦＡＴｃ２、ＣＲＹＡＢおよび、ＰＩＭＴの発現に対するヒアルロン酸投与の影響をさらにＲＴ－ＰＣＲ法より解析した。なおＮＦＡＴｃ２はＣＲＹＡＢの発現誘導に重要な転写因子であり、ＣＲＹＡＢはユビキチンリガーゼ複合体を形成し、ポリグルタミンタンパク質の分解を担う（Hayashida et al., EMBO J, 2010）。ＰＩＭＴは酵素を媒介せずに生じるイソアスパラギン酸をアスパラギン酸に変換し、タンパク質の構造を正常化する。
(Example 7. Effect of hyaluronic acid on the expression of genes involved in protein degradation or normalization 2)
Human neuroblastoma SH-SY5Y strain was seeded at 1.0 x 10 4 on a 6 cm dish containing Dulbecco's Modified Eagle Medium (DMEM; Merck) containing 10% fetal bovine serum (FBS) and cultured at 37 ° C for 24 hours. Then, hyaluronic acid (Code No. 002005, Nagara Science Co., Ltd.) dissolved in PBS to a concentration of 1.0 nM, 2.5 nM, 5.0 nM, or 12.5 nM was added to the medium and further cultured at 37 ° C for 7 days. As a control, only PBS equivalent to the hyaluronic acid-containing PBS was added to the medium and similarly cultured at 37 ° C for 7 days. After 7 days of culture, the cells were collected and RT-PCR was performed.
In this example, the effects of hyaluronic acid administration on the expression of NFATc2, CRYAB, and PIMT were further analyzed by RT-PCR. NFATc2 is an important transcription factor for inducing the expression of CRYAB, and CRYAB forms a ubiquitin ligase complex and is responsible for the degradation of polyglutamine proteins (Hayashida et al., EMBO J, 2010). PIMT converts the resulting isoaspartic acid to aspartic acid without the aid of an enzyme, normalizing the structure of the protein.
ＨＳＦ１、ＨＳＦ２、ＮＦＡＴｃ２、ＣＲＹＡＢおよび、ＰＩＭＴの検出には、それぞれの遺伝子の発現を検出できるプライマーを用いた（Hayashida et al., EMBO J, 2010）。内部コントロールにはＳ１８を用いた。結果を図７に示す。 To detect HSF1, HSF2, NFATc2, CRYAB, and PIMT, primers capable of detecting the expression of each gene were used (Hayashida et al., EMBO J, 2010). S18 was used as an internal control. The results are shown in Figure 7.
図７に示すように、ヒアルロン酸の添加により、ＨＳＦ１、ＨＳＦ２、ＮＦＡＴｃ２、ＣＲＹＡＢ、および、ＰＩＭＴのいずれもヒアルロン酸無添加区と比較して発現が増加した。なお、ＨＳＦ２、ＣＲＹＡＢは、１２．５ｎＭのヒアルロン酸添加区においては発現の増加を示さなかった。ＲＴ－ＰＣＲの結果とウェスタンブロットの結果とは必ずしも一致するものではないが、ＲＴ－ＰＣＲによって遺伝子発現の増加が見られた場合、それが一時的なものであっても、少なくともその遺伝子がコードするタンパク質も発現が増加する場合が多い。ＨＳＦ２、ＣＲＹＡＢは、１２．５ｎＭのヒアルロン酸添加区においては発現の増加を示さなかったが、１２．５ｎＭのヒアルロン酸は軸索の伸展を強力に促進するため、軸索の形成に関係する遺伝子の発現は大きく増加したと考えられるが、ＨＳＦ２、ＣＲＹＡＢのように、神経細胞の軸索では発現せず、細胞体でのみ発現する遺伝子は発現量が相対的に低く検出された可能性が高い。リボゾームは軸索の先端まで輸送されそこでタンパク質の合成に関わるため、ここでコントロールに用いたＳ１８では大きな減少がなかったと考えられる。 As shown in Figure 7, the addition of hyaluronic acid increased the expression of HSF1, HSF2, NFATc2, CRYAB, and PIMT compared to the group without hyaluronic acid. HSF2 and CRYAB did not show an increase in expression in the group with 12.5 nM hyaluronic acid added. Although the results of RT-PCR and Western blot do not necessarily coincide, when an increase in gene expression is observed by RT-PCR, even if it is temporary, the expression of at least the protein encoded by that gene is often also increased. HSF2 and CRYAB did not show an increase in expression in the group with 12.5 nM hyaluronic acid added, but since 12.5 nM hyaluronic acid strongly promotes axon extension, it is thought that the expression of genes related to axon formation was greatly increased, but it is highly likely that genes that are not expressed in the axons of nerve cells and are expressed only in the cell bodies, such as HSF2 and CRYAB, were detected at relatively low expression levels. Ribosomes are transported to the tip of the axon where they are involved in protein synthesis, which is why there was probably no significant decrease in S18, which was used as a control here.
（実施例８．ヒアルロン酸の毒性試験）
本実施例では、ヒアルロン酸（分子量１００～１４０万）をマウスの腹腔内へ注射することによりその毒性について検証した。
１６、４０、８０、または２００μｍｏｌのヒアルロン酸（コードＮｏ．ＨＡ００２００５ 長良サイエンス社製）を含むＰＢＳを調製した。各濃度のヒアルロン酸含有ＰＢＳ１ｍＬをＣ５７ＢＬ／６マウスに腹腔内注射した。またコントロールとしてヒアルロン酸を含まないＰＢＳ １ｍＬをマウスの腹腔内へ注射した。腹腔内への注射は週１回行い、２０週まで投与を継続した。２０週まで各濃度のヒアルロン酸含有ＰＢＳを投与したマウスの生存率を図８に示す（ｎ＝３）。
(Example 8. Toxicity test of hyaluronic acid)
In this example, the toxicity of hyaluronic acid (molecular weight: 1,000,000 to 1,400,000) was examined by injecting it into the abdominal cavity of a mouse.
PBS containing 16, 40, 80, or 200 μmol of hyaluronic acid (Code No. HA002005, Nagara Science Co., Ltd.) was prepared. 1 mL of PBS containing hyaluronic acid at each concentration was intraperitoneally injected into C57BL/6 mice. As a control, 1 mL of PBS without hyaluronic acid was injected into the abdominal cavity of the mice. Intraperitoneal injection was performed once a week, and administration was continued for 20 weeks. The survival rate of mice administered PBS containing hyaluronic acid at each concentration for 20 weeks is shown in FIG. 8 (n=3).
図８に示すように、１６～２００μｍｏｌのヒアルロン酸を腹腔内投与したマウスは全頭行動異常もなく全て生存しており、ヒアルロン酸投与による毒性は認められなかった。 As shown in Figure 8, all mice that received intraperitoneal administration of 16 to 200 μmol of hyaluronic acid survived without any abnormal behavior, and no toxicity due to administration of hyaluronic acid was observed.
（実施例９．ヒアルロン酸によるハンチンチンタンパク質の凝集抑制）
本実施例では、ハンチントン病モデルマウスに対してヒアルロン酸を投与することにより神経細胞内のハンチンチンタンパク質の凝集体形成を抑制可能であるか検証した。
具体的には、８週齢のハンチントン病モデルマウスＲ６／２に、１週間毎日ＰＢＳに溶解した５ｍｇのヒアルロン酸を腹腔内投与し、その後は１週間ごとに同様の投与を行い、１２週齢になった時点で臓器を回収し、凍結切片を作製した。切片は１０ｍｍ厚とした。この切片に抗ハンチンチンタンパク質抗体Ｎ―１８（Santa Cruz社製）とＦＩＴＣ結合２次抗体を用いて、蛍光免疫染色を行い、その後ＤＡＰＩで核を染色した後、蛍光顕微鏡を用いて、細胞中のＦＩＴＣおよびＤＡＰＩの蛍光を観察した。図９は細胞を蛍光顕微鏡で撮影した写真を示す。
Example 9. Inhibition of huntingtin protein aggregation by hyaluronic acid
In this example, we examined whether the formation of huntingtin protein aggregates in nerve cells can be suppressed by administering hyaluronic acid to Huntington's disease model mice.
Specifically, 5 mg of hyaluronic acid dissolved in PBS was intraperitoneally administered to 8-week-old Huntington's disease model mice R6/2 every day for one week, and then the same administration was performed every week. When the mice reached 12 weeks of age, the organs were collected and frozen sections were prepared. The sections were 10 mm thick. The sections were subjected to fluorescent immunostaining using anti-huntingtin protein antibody N-18 (manufactured by Santa Cruz) and FITC-conjugated secondary antibody, and then the nuclei were stained with DAPI, and the fluorescence of FITC and DAPI in the cells was observed using a fluorescent microscope. Figure 9 shows a photograph of cells taken with a fluorescent microscope.
図９に示すように、コントロールのヒアルロン酸無添加（ＰＢＳのみ）群ではその細胞中に異常ハンチンチンタンパク質の発現および凝集が観察された。一方、ヒアルロン酸を添加した群では、ハンチンチンタンパク質の凝集体が減少していた。これは、ヒアルロン酸が異常ハンチンチンタンパク質自体の発現量を減少させたか、または、異常ハンチンチンタンパク質の分解を促進したと推察される。 As shown in Figure 9, in the control group where no hyaluronic acid was added (PBS only), expression and aggregation of abnormal huntingtin protein was observed in the cells. On the other hand, in the group where hyaluronic acid was added, the amount of huntingtin protein aggregates was reduced. This suggests that hyaluronic acid either reduced the expression level of the abnormal huntingtin protein itself or promoted the decomposition of the abnormal huntingtin protein.
軸索は、神経細胞が他の神経細胞や他の細胞（筋肉細胞など）と連結して情報を伝えるのに不可欠なものであり、軸索の数が少なくなり、あるいは変性・切断によって機能を失えば、生物個体の行動に重篤な影響をもたらす。今回の実験で、ヒアルロン酸は、軸索を伸展させている神経細胞の数を約5倍にまで増やすことが出来たほか、軸索の長さも伸ばすことに成功した。アルツハイマー病をはじめとした多くの神経変性疾患の患者は認知症状を呈し、これが大きな問題となっているが、認知機能の基盤には、神経細胞同士が軸索で密につながっていることが上げられる。このことから、ヒアルロン酸は、認知機能の回復に働く可能性を持っている。神経変性疾患のもう1つの大きな特徴である凝集体形成については、細胞レベル及び個体レベル（動物モデル）で、ヒアルロン酸は凝集体の形成を抑制出来ることが示された。さらに長期間の接種（連続投与）を行っても、接種された動物において異常は認められなかった。
ヒアルロン酸はすでに長い間サプリメントや化粧品などで人に使用されており、特に大きな問題は起きていないことから安全性は高いと想定はしていたが、動物実験によって、それがさらに裏付けられ、また、投与量を上げても毒性を生じない可能性も示唆された。今回の結果は、ヒアルロン酸が、複数の神経変性疾患の治療に非常に効果的な薬剤として、あるいは根治にまでつながる薬剤の開発に、他の新規の薬剤に比べ短期間でつながる可能性を示している。
Axons are essential for nerve cells to connect with other nerve cells and other cells (such as muscle cells) and transmit information. If the number of axons decreases or they lose their function due to degeneration or cutting, it will have a serious effect on the behavior of the individual organism. In this experiment, hyaluronic acid was able to increase the number of nerve cells extending axons by about five times, and also succeeded in extending the length of the axons. Many patients with neurodegenerative diseases, including Alzheimer's disease, exhibit cognitive symptoms, which is a major problem, but the basis of cognitive function is that nerve cells are densely connected to each other by axons. For this reason, hyaluronic acid has the potential to work in restoring cognitive function. Regarding aggregate formation, another major characteristic of neurodegenerative diseases, it has been shown that hyaluronic acid can suppress the formation of aggregates at the cellular and individual levels (animal models). Furthermore, no abnormalities were observed in the inoculated animals even after long-term inoculation (continuous administration).
Hyaluronic acid has been used in humans for a long time in supplements and cosmetics, and since no major problems have occurred, it was assumed to be highly safe, but this has been further supported by animal experiments, and it has also been suggested that even increased dosages may not cause toxicity. The results of this study indicate that hyaluronic acid may be used as a highly effective drug for the treatment of multiple neurodegenerative diseases, or may even lead to the development of drugs that can cure the diseases, in a shorter period of time than other new drugs.
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