JPWO2003024485A1 - A therapeutic agent for β-amyloid-induced disease comprising an S-100β inhibitor as an active ingredient - Google Patents

A therapeutic agent for β-amyloid-induced disease comprising an S-100β inhibitor as an active ingredient Download PDF

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JPWO2003024485A1
JPWO2003024485A1 JP2003528579A JP2003528579A JPWO2003024485A1 JP WO2003024485 A1 JPWO2003024485 A1 JP WO2003024485A1 JP 2003528579 A JP2003528579 A JP 2003528579A JP 2003528579 A JP2003528579 A JP 2003528579A JP WO2003024485 A1 JPWO2003024485 A1 JP WO2003024485A1
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amyloid
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泰治 下田
泰治 下田
宜道 矢田
宜道 矢田
成人 立石
成人 立石
勝部　伸夫
伸夫 勝部
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Abstract

Ｓ−１００β阻害剤を有効成分とするβアミロイド起因疾患の治療および／または予防剤。Ｓ−１００β阻害剤はβアミロイド産生を抑制する作用を有するため、βアミロイドを起因物質とする疾患、すなわち神経変性疾患、ダウン症、ボクサー症、進行性核上麻痺、星状膠細胞腫、脳卒中や脳外傷後の神経機能障害、多発性硬化症、痴呆、精神***症、てんかん、不安、嘔吐、偏頭痛、神経細胞死、うつ病、睡眠障害、食欲不振などの摂食障害、尿失禁、低酸素症、脳梗塞、脳腫瘍、高酸素痙攣および高酸素毒症、炎症性もしくは神経障害性疼痛、髄膜炎等の治療および／または予防剤として有用である。An agent for treating and / or preventing β-amyloid-induced disease, comprising an S-100β inhibitor as an active ingredient. Since S-100β inhibitor has an action of suppressing β-amyloid production, diseases caused by β-amyloid, ie, neurodegenerative disease, Down syndrome, Boxer disease, progressive supranuclear palsy, astroglioma, stroke and Neurological dysfunction after brain trauma, multiple sclerosis, dementia, schizophrenia, epilepsy, anxiety, vomiting, migraine, nerve cell death, depression, sleep disorders, anorexia and other eating disorders, urinary incontinence, low It is useful as an agent for treating and / or preventing oxygen, cerebral infarction, brain tumor, hyperoxic convulsions and hyperoxin, inflammatory or neuropathic pain, meningitis and the like.

Description

技術分野
本発明は、Ｓ−１００β阻害剤を有効成分とするβアミロイド起因疾患の治療剤に関する。
背景技術
中枢神経系の実質を構成する細胞には神経細胞とグリア細胞があり、細胞数はグリア細胞が神経細胞よりはるかに多い。グリア細胞は神経細胞の働きを支持する役割を担っている。グリア細胞の一種であるアストロサイトは、神経細胞の支持細胞として細胞外のイオンおよび神経伝達物質の恒常性維持（Ｐｈａｒｍａｃｏｌｏｇｙ ａｎｄ ｆｕｎｃｔｉｏｎ，ｐｐ．１９３−２２８，Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ，Ｉｎｃ．，（１９９３））や神経栄養因子の供給（Ｐｈａｒｍａｃｏｌｏｇｙ ａｎｄ ｆｕｎｃｔｉｏｎ，ｐｐ．２６７−３０８，Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ，Ｉｎｃ．，（１９９３））などの機能を有していることから、脳機能を制御する重要な役割を演じていると考えられる。
従来、神経変性疾患（アルツハイマー病、筋萎縮性側策硬化症など）の成因は主に神経細胞の異常にあると考えられてきた。しかし、近年神経細胞を取り囲むグリア細胞、特にアストロサイトの機能的異常にあるとの考えが有力になってきた。
βアミロイドは神経変性疾患の一つであるアルツハイマー病の病理学的特徴の一つである老人斑の主構成物である。βアミロイドは正常人の脳内にも存在するが速やかに分解されるものと考えられている。このタンパクはアミロイド前駆体タンパク質の代謝過程においてβセクレターゼおよびγセクレターゼにより副次的に生成される。アミロイド前駆体タンパク質は通常αセクレターゼによって分解されるが、この場合βアミロイドは産生されないことが知られている。βアミロイドにはβアミロイド１−４０とβアミロイド１−４２（４３）の二つのアイソフォームが存在するが、転写レベルではなくβセクレターゼおよびγセクレターゼによってアミロイド前駆体タンパク質代謝段階で生成されると考えられている。脳で産生されるβアミロイドのほとんどはβアミロイド１−４０だが、難溶性のβアミロイド１−４２（４３）の方が凝集・沈着しやすく、現在のところどちらが病態に深く関与するのかは解明されていない。また産生されたβアミロイドは中性エンドペプチダーゼによって分解されることが知られている。
アルツハイマー病において、βアミロイドが凝集して不溶性の繊維形成がなされて脳に沈着し老人斑を形成する。現在、アルツハイマー病の根治的治療法としてアミロイド前駆体タンパク質およびβアミロイドの代謝系を制御しβアミロイド産生を抑制する方法が注目されている。
神経細胞傷害時、活性化したアストロサイトにおいてアミロイド前駆体タンパク質発現が増大していることが知られている（Ｎｅｕｒｏｎ，３，２７５−２８５，（１９８９））。またβアミロイドは神経毒性を示すことから、アミロイド前駆体タンパク質の異常代謝によるβアミロイドの過剰産生、蓄積がアルツハイマー病の発症および進展に関与していると考えられる（Ｓｃｉｅｎｃｅ，２４５，４１７−４２０，（１９８９））。さらに、βアミロイドの神経毒性においてＮ−メチル−Ｄ−アスパラギン酸受容体と一酸化窒素合成酵素を介した活性酸素産生亢進の関与が報告されている（Ｊ．Ｎｅｕｒｏｃｈｅｍ．，７６（４），１０５０−１０５６，（２００１））。したがって、βアミロイドが活性酸素起因疾患に関与している可能性が考えられる。
Ｓ−１００βはアストロサイト特異的タンパク質であり、カルシウム結合部位を有し細胞内カルシウム濃度の調節、種々の細胞骨格蛋白と結合し細胞の形態変化や神経伝達物質の遊離等に関与している（ＴＩＢＳ，１３，４３７−４４３，（１９８８））。Ｓ−１００βはアストロサイトの活性化により産生され細胞外に放出される。放出されたＳ−１００βは、神経細胞に対し栄養因子的な作用および傷害因子的な作用（Ｐｒｏｇｒｅｓｓ ｉｎ Ｎｅｕｒｏｂｉｏｌｏｇｙ，４６，７１−８２，（１９９５））を示し、またアストロサイトに対しては増殖促進作用や誘導型一酸化窒素合成酵素の発現作用を有することが報告されている（Ｊ．Ｂｉｏｌｏｇｉｃａｌ Ｃｈｅｍｉｓｔｒｙ，２７１，２５４３−２５４７，（１９９６））。
また、Ｓ−１００βは脳傷害時、活性化されたアストロサイトにおいて過剰発現することが知られている。アルツハイマー患者の脳内でＳ−１００β量が増加しており（Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，８６，７６１１−７６１５，（１９８９））、その増加量はアルツハイマー患者の脳における老人斑密度と相関があることが報告されている（Ｊ．Ｎｅｕｒｏｓｃｉ．Ｒｅｓ．，３９，３９８−４０４，（１９９４））。さらにアルツハイマー患者の脳における老人斑近傍にはＳ−１００βの過剰発現を伴った活性化したアストロサイトが存在し、その発現強度は神経の異常突起伸展との間に相関が見られることが報告されている（Ｊ．Ｎｅｕｒｏｐａｔｈｏｌ．Ｅｘｐ．Ｎｅｕｒｏｌ．，５５，２７３−２７９，（１９９６））。その他にも、脳卒中（Ｓｔｒｏｋｅ，３０，１１９０−１１９５，（１９９９））、頭部外傷（Ｎｅｕｒｏｓｕｒｇｅｒｙ，４５，４７７−４８３，（１９９９））、多発性硬化症（Ａｃｔａ Ｎｅｕｒｏｌ Ｓｃａｎｄ，９６，１４２−１４４，（１９９７））、ダウン症（Ｊ．Ｔｈｏｒａｃ．Ｃａｒｄｉｏｖａｓｃ．Ｓｕｒｇ．，１１６，２８１−２８５，（１９９８））などの患者の血清中もしくは脳髄液中でＳ−１００β量の増加がみられる。
これまでに、Ｓ−１００β、βアミロイドおよびアミロイド前駆体タンパク質の関係については以下のことが報告されている。
１）ラット神経細胞において、Ｓ−１００βはアミロイド前駆体タンパク質とそれをコードするｍＲＮＡ量を増加させた（Ｊ．Ｎｅｕｒｏｃｈｅｍ．，７１（４），１４２１−１４２８，（１９９８））。
２）トランスジェニックマウス（アミロイド前駆体タンパク質が過剰発現した家族性アルツハイマー病マウス）において、βアミロイド斑が出現する前にＳ−１００βが過剰発現した（Ｊ．Ｎｅｕｒｏｃｈｅｍ．，７４（１），２９５−３０１，（２０００））。
３）ラット神経細胞において、βアミロイドはＳ−１００βの遺伝子発現を増加させた（Ｍｏｌ．Ｂｒａｉｎ Ｒｅｓ．，３４（１），１１８−１２６，（１９９５））。
アルツハイマー病などの神経変性疾患において、βアミロイドおよびＳ−１００βの過剰発現が確認されている。しかしながら、これらのタンパク質の相互関係には諸説がありいまだ明らかになっていない。
発明の開示
アルツハイマーをはじめとする神経変性疾患の治療における最終目標は、脳内病理過程の進行を遮断し、発症および進行を完全に抑制することにある。しかし、現在使用されている治療薬はコリンエステラーゼ阻害剤などの対症療法薬である。また現在臨床応用検討中の抗炎症薬や女性ホルモンなども内在する病変悪化を修正する根治治療ではない。したがって、神経変性疾患の原因を解明し、根治治療薬を提供することが急務である。
本発明者らは、神経変性疾患におけるＳ−１００βの発現とβアミロイドの生成との関係について種々検討を行った結果、Ｓ−１００βがβアミロイド産生量を増加させることを明らかにし、本発明を完成した。
すなわち、本発明は下記のβアミロイド起因疾患の治療および／または予防剤、被験化合物のβアミロイド産生抑制作用を測定することによる化合物のスクリーニング方法、およびその方法により得られる化合物を提供する。
１．Ｓ−１００β阻害剤を有効成分とするβアミロイド起因疾患の治療および／または予防剤。
２．Ｓ−１００β阻害剤が以下の（１）〜（６）の少なくともひとつから選択される機構に作用してβアミロイド産生を抑制することを特徴とする前項１記載のβアミロイド起因疾患治療および／または予防剤：
（１）βセクレターゼおよびγセクレターゼによるβアミロイド産生、
（２）αセクレターゼによるアミロイド前駆体タンパク質の代謝、
（３）中性エンドペプチダーゼによるβアミロイドの代謝、
（４）Ｓ−１００βの細胞内への取り込み機構、
（５）糖付加、
（６）タンパク質成熟化。
３．βアミロイドに起因する疾患が、神経変性疾患、ダウン症、ボクサー症、進行性核上麻痺、星状膠細胞腫、脳の神経機能障害、多発性硬化症、痴呆、精神***症、てんかん、不安、嘔吐、偏頭痛、神経細胞死、うつ病、睡眠障害、摂食障害、尿失禁、低酸素症、脳梗塞、脳腫瘍、高酸素痙攣および高酸素毒症、炎症性もしくは神経障害性疼痛、髄膜炎である前項１に記載のβアミロイド起因疾患治療および／または予防剤。
４．ヒト由来グリア細胞に（１）Ｓ−１００βを添加しβアミロイド産生を亢進させた条件下において、被験化合物を添加して、細胞から放出されたβアミロイドを検出するか、または（２）被験化合物を添加し、Ｓ−１００βを添加して、細胞から放出されたβアミロイドを検出することによって、被験化合物のβアミロイド産生抑制作用を決定することを特徴とするβアミロイド産生抑制作用を有する化合物のスクリーニング方法。
５．βアミロイド産生抑制作用が以下の作用から選択される前項４記載のスクリーニング方法；
（１）βセクレターゼまたはγセクレターゼ阻害作用、
（２）αセクレターゼ活性化作用、
（３）中性エンドペプチダーゼ活性化作用、
（４）Ｓ−１００β取り込み阻害作用、
（５）糖付加阻害作用、
（６）タンパク質成熟化阻害作用。
６．前項４記載のスクリーニング方法によって得られた化合物。
７．前項６記載の化合物を有効成分とする前項１記載のβアミロイド起因疾患の治療および／または予防剤。
以下、本発明を詳細に説明する。
従来技術によれば、ラット神経細胞においてはＳ−１００βはアミロイド前駆体タンパク質とそれをコードするｍＲＮＡ量を増加させることが報告されている。しかし、本発明者らの実験によってヒト由来グリア細胞においては、意外にもＳ−１００βはアミロイド前駆体タンパク質をコードするｍＲＮＡ量を変化させず、Ｓ−１００βはアミロイド前駆体タンパク質量には影響を与えないことが確認された。すなわち、アミロイド前駆体タンパク質量は変化せずβアミロイド量のみが増加していることから、アミロイド前駆体タンパク質が代謝されβアミロイドを産生する過程もしくはβアミロイドが代謝される過程でＳ−１００βが作用することが新たに示唆された。
アミロイド前駆体タンパク質代謝過程においては、α、βおよびγセクレターゼの関与が知られており、βアミロイドはβおよびγセクレターゼの作用により産生され、αセクレターゼで代謝された場合産生されないことが知られている。したがって、Ｓ−１００βは（１）βおよびγセクレターゼを活性化もしくは（２）αセクレターゼを抑制していると考えられる。また産生されたβアミロイドは中性エンドペプチダーゼで代謝されることが報告されていることから、Ｓ−１００βが（３）中性エンドペプチダーゼを阻害していることも考えられる。さらに、アミロイド前駆体タンパク質の糖付加や成熟化を抑制すると、βアミロイド産生が抑制されることが知られている。したがって、Ｓ−１００βが（４）糖付加またはタンパク質成熟化を促進している可能性も考えられる。アルツハイマーをはじめとする神経変性疾患の根治的治療において、アミロイド前駆体タンパク質およびβアミロイド代謝過程を制御しβアミロイド産生を抑制することが注目されてきた。しかしながら、Ｓ−１００βがアミロイド前駆体タンパク質およびβアミロイド代謝過程に作用する事実は今回本発明者らが実験により初めて確認したことであり、神経変性疾患の根治治療薬を開発するうえで有用な事実である。
βアミロイドは、先に述べたように神経変性疾患等に関与している。今回、Ｓ−１００βが（１）βおよびγセクレターゼ活性化、（２）αセクレターゼ抑制、（３）中性エンドペプチダーゼ阻害、（４）糖付加促進、（５）タンパク質成熟化促進の中から選ばれる少なくともひとつの作用によってβアミロイドの発現を促進することが本発明者らによって明らかにされた。したがって、Ｓ−１００βを阻害することによりβアミロイドの過剰発現が関与していると考えられる疾患、特に神経細胞の欠落を特徴とする神経変性疾患等の治療に用いることができると考えられる。
すなわち、Ｓ−１００β阻害剤はβアミロイドに起因する疾患の治療に用いることができると考えられる。βアミロイドに起因する疾患とはβアミロイドが増加することによって起こる疾患であり、具体的にはアルツハイマー病をはじめとする神経変性疾患（アルツハイマー病、クロイツフェルトヤコブ病、パーキンソン病、ハンチントン病、オリーブ橋小脳萎縮症、筋萎縮性側策硬化症等）、ダウン症、ボクサー症、進行性核上麻痺、星状膠細胞腫、脳卒中や脳外傷後の神経機能障害、多発性硬化症、痴呆、精神***症、てんかん、不安、嘔吐、偏頭痛、神経細胞死、うつ病、睡眠障害、食欲不振などの摂食障害、尿失禁、低酸素症、脳梗塞、脳腫瘍、高酸素痙攣および高酸素毒症、炎症性もしくは神経障害性疼痛、髄膜炎などが考えられる。
また、本発明にはＳ−１００βで処置したヒト由来細胞を用いてβアミロイドを検出することを特徴とするβアミロイド起因疾患治療剤のスクリーニング方法も含まれる。
現在までに知られている一般的なβアミロイド抑制作用測定方法は、サイトカイン、ケモカインおよびＣ−キナーゼ等によってβアミロイド産生を亢進させる方法であるが、今回Ｓ−１００βとβアミロイドの関係が明らかとなったことにより本発明のβアミロイド起因疾患治療剤のスクリーニングが可能となった。
より具体的には、ヒト由来グリア細胞にＳ−１００βを添加し、βアミロイド産生を亢進させ、被験化合物を添加して、一定時間後細胞培養上清中のβアミロイド量を測定することにより、βアミロイドの産生を抑制することのできる化合物を選択することができる。
ラットにはアミロイド前駆体タンパク質代謝酵素であるセクレターゼがほとんど存在しないことからβアミロイドの検出が不可能である。このような動物種によるタンパク質発現の差を考慮すると、本発明のスクリーニング方法では、ヒト由来細胞を用いることが好ましい。またスクリーニングのための細胞は、ヒト由来のグリア細胞であれば何でもよい。具体的には、ヒトグリオブラストーマ、アストロサイトーマ由来細胞株であるＵ−３７３ＭＧ、ヒトグリオブラストーマ由来細胞株のＴ９８Ｇ、Ａ−１７２、ヒトアストロサイトーマ由来細胞株のＣＣＦ−ＳＴＴＧ１等があるが、種々検討した結果、ヒトグリオブラストーマ、アストロサイトーマ由来細胞株であるＵ−３７３ＭＧ細胞が好ましい。
被験化合物は、Ｓ−１００β添加直前、直後または同時に加えてもよく、場合に応じて化合物の添加タイミングを変更してもよい。
βアミロイド検出方法は、βアミロイドを検出することができればどのような方法を用いてもよいが、例えば酵素免疫測定法、放射免疫測定、ウエスタンブロット等が挙げられる。
さらに本発明には前記のスクリーニング方法によって得られる化合物が含まれる。本スクリーニング系によりβアミロイド産生を抑制する化合物、具体的にはβおよびγセクレターゼを抑制する化合物、αセクレターゼを活性化する化合物、中性エンドペプチダーゼを活性化する化合物、糖付加を阻害する化合物、またはタンパク質成熟化を阻害する化合物などが選定できる。またアストロサイトの活性化によって産生され細胞外に放出されたＳ−１００βは、アストロサイトの特定部位によって再取り込みされアストロサイト自身に作用する。したがって本スクリーニング系ではＳ−１００βの取り込みを抑制する化合物をインビトロの系で簡便に選定することができる。これまでに、Ｓ−１００βがアストロサイトに作用することを阻害する化合物は報告されておらず、本発明によって初めてβアミロイド産生を抑制する化合物として認識されるものである。
発明を実施するための最良の形態
以下にＳ−１００βがアミロイド前駆体タンパク質の遺伝子量を変化させずβアミロイド量を増加させることを確認した実験例を挙げて、本発明をより具体的に説明するが、これらは本発明の範囲を制限するものではない。
実験例１：Ｓ−１００βによるβアミロイド産生量測定
１）Ｓ−カルボキシメチル化Ｓ−１００β調製
ヨード酢酸を用い、ラットリコンビナントＳ−１００β（東洋紡績（株））をＳ−カルボキシメチル化した。
すなわち５０ｍＬチューブ内でリコンビナントＳ−１００β溶液３４．５ｍＬ（リコンビナントＳ−１００β含量：１００ｍｇ）に炭酸水素ナトリウム１．４５ｇを溶解した。次に，１−プロパノール１１．５ｍＬを加えよく撹拌した（１−プロパノール最終濃度２５％）。さらにトリブチルホスフィン５０μＬを加え撹拌した後，室温で１時間静置した。その後、ヨード酢酸試薬（ヨード酢酸を１ｍｏｌ／Ｌとなるように１ｍｏｌ／Ｌ水酸化ナトリウムに溶解、用時調製）を１ｍＬ加え、遮光下、室温で１時間３０分静置した。
反応液を透析膜（三光純薬）に移し、あらかじめ冷やしておいたトリスバッファー（４０ｍｍｏｌ／Ｌ，Ｔｒｉｓ−ＨＣｌ、ｐＨ７．５）で４℃下透析を行なった。６時間以上経過した後、バッファー交換を行ない透析した。
透析回収サンプルをセントリプラスＹＭ−１０（アミコン）を用い濃縮した。濃縮後、０．２２μｍフィルター（ミリポア）を通してろ過滅菌した。これをＳ−カルボキシメチル化Ｓ−１００β（以下、ＣＭ−Ｓ−１００βと略記することがある。）とし、−８０℃で保存した。
２）ラットリコンビナントＳ−１００βの調製
ラットリコンビナントＳ−１００β（以下、ｒＳ−１００βと記すことがある。）は、東洋紡績（株）より入手したものを、透析を行ない濃縮およびろ過滅菌したものを使用した。操作は前記の「１）Ｓ−カルボキシメチル化Ｓ−１００β調製」に準じて行なった。
３）Ｓ−１００βによるβアミロイド産生量測定
ヒトグリオブラストーマ、アストロサイトーマ由来細胞株であるＵ−３７３ＭＧ細胞（住商ファーマインターナショナル（株））を１０％ウシ胎児血清含有高グルコースダルベッコ改変イーグル培地（高グルコースダルベッコ改変イーグル培地（ＤＭＥＭ）に１０％ウシ胎児血清、２０ｍｍｏｌ／ＬのＨＥＰＥＳ、２ｍｍｏｌ／ＬのＬ−グルタミン、１００Ｕ／ｍＬペニシリンおよび１００μｍｏｌ／Ｌのストレプトマイシンを添加した培地。以下、１０％ＦＣＳ−ＤＭＥＭと記す。）にて、３７℃５％ＣＯ２下７５ｃｍ３のフラスコで継代培養した。
培養細胞をトリプシン／エチレンジアミン四酢酸（ＥＤＴＡ）により剥離した。剥離した細胞は遠心後、２×１０５ｃｅｌｌｓ／ｍＬとなるよう１０％ＦＣＳ−ＤＭＥＭ培地で希釈し、２４穴プレートに１ｍＬ／ｗｅｌｌで播種した。播種した細胞は３７℃５％ＣＯ２下で培養した。
播種後二日目の細胞を無血清のＤＭＥＭ１ｍＬで洗浄後、無血清のＤＭＥＭ３６０μＬに培地を置換した。次にトリス緩衝溶液で既知の濃度に希釈したＣＭ−Ｓ−１００βあるいはｒＳ−１００β溶液を４０μＬ添加し、３７℃５％ＣＯ２下で培養した。
Ｓ−１００β処置４８時間後に２４穴プレートより培養上清を３００μＬ回収した。回収した培養上清は、１５，０００ｒｐｍ、４℃で５分間遠心して上清を回収し、測定用サンプルとした。サンプルは測定時まで氷上に保存した。
サンプル中のβアミロイド１−４０およびβアミロイド１−４２の蓄積量をそれぞれヒューマンβアミロイド１−４０イライザキット（バイオスコア）、ヒューマンβアミロイド１−４２イライザキット（バイオスコア）を用いて測定した。測定法はキットの説明書に従った。その際サンプルはキット付属の希釈液を用い希釈した。それぞれのスタンダードの検量線よりβアミロイド１−４０およびβアミロイド１−４２量を算出した。その結果を表１および図１〜２に示す。
ＣＭ−Ｓ−１００βおよびｒＳ−１００βは１０および３０μｍｏｌ／ＬでＵ−３７３ＭＧ細胞においてβアミロイド１−４０の蓄積量をビヒクル処置群と比較して有意に増加させた（図１、表１）。同様に、ＣＭ−Ｓ−１００βは１０および３０μｍｏｌ／Ｌで、またｒＳ−１００βは３、１０および３０μｍｏｌ／Ｌでβアミロイド１−４２蓄積量を有意に増加させた（図２、表１）。
この結果から、Ｓ−１００βはβアミロイド産生促進作用を有していることが確認された。

Figure 2003024485
実験例２：アミロイド前駆体タンパク質ｍＲＮＡ発現量測定
１）ｍＲＮＡサンプルの調製
前記と同様にＳ−１００β処置したプレートを作成した。細胞上清を回収した後の細胞からクイックプレップｍＲＮＡ精製キット（アマシャム・ファルマシア・バイオテック）を用いてｍＲＮＡを精製し、アミロイド前駆体タンパク質（以下、ＡＰＰと略記することがある。）ｍＲＮＡ発現量測定用サンプルとした。ｍＲＮＡサンプルは−８０℃で保存した。
２）ｃＤＮＡの調製
８０ｎｇのｍＲＮＡからファースト−ストランド ｃＤＮＡ合成キット（アマシャム・ファルマシア・バイオテック）を用いてｃＤＮＡを合成し、さらに蒸留水で３倍希釈した後ｃＤＮＡサンプルとした。
３）ＲＴ−ＰＣＲ法
表２に示すＰＣＲ反応溶液を調製し、変性を９８℃１秒、アニーリングを６８もしくは７０℃１５秒の条件でＰＣＲを行なった。反応液の一部を１％アガロースゲルで電気泳導を行なった。
Figure 2003024485
使用したプライマーの塩基配列を表３に示す。
Figure 2003024485
４）結果
アミロイド前駆体タンパク質（ＡＰＰ６９５、ＡＰＰ７５１およびＡＰＰ７７０）のｍＲＮＡ発現量を検討した。その結果を対照（βアクチンの発現量変化）と共に図３に示す。図３中、（１）はＣＭ−Ｓ−１００βによるβアクチン（陰性対照）発現量変化、（２）はＣＭ−Ｓ−１００βによるＡＰＰ７７０発現量変化、（３）はＣＭ−Ｓ−１００βによるＡＰＰ７５１発現量変化、（４）はＣＭ−Ｓ−１００βによるＡＰＰ６９５発現量変化、（５）はｒＳ−１００βによるβアクチン（陰性対照）発現量変化、（６）はｒＳ−１００βによるＡＰＰ７７０発現量変化、（７）はｒＳ−１００βによるＡＰＰ７５１発現量変化、（８）はｒＳ−１００βによるＡＰＰ６９５発現量変化を示す。
図３から明らかなように、ＣＭ−Ｓ−１００βおよびｒＳ−１００βいずれの処置においてもｍＲＮＡ発現量に変化はみられなかった。したがって、前記のＳ−１００β処置群におけるβアミロイド産生量増加にその前駆体であるアミロイド前駆体タンパク質の発現量増加は関与していないことが示唆された。このことから、Ｓ−１００βは転写レベルではなく、βセクレターゼおよびγセクレターゼによるアミロイド前駆体タンパク質代謝の過程に作用している可能性が推定された。
実験例３：βアミロイド産生量測定による化合物評価
前記の実験例１と同様にＵ−３７３ＭＧ細胞を調製し、２４穴プレートに２×１０５ｃｅｌｌｓ／ｍＬとなるよう１ｍＬ／ｗｅｌｌで播種した。播種した細胞は３７℃５％ＣＯ２下で培養した。
播種後二日目の細胞を無血清のＤＭＥＭ１ｍＬで洗浄後、無血清のＤＭＥＭ３２０μＬに培地を置換し、化合物の濃縮液（処置濃度の１０倍濃度）を４０μＬ添加した。次にトリス緩衝溶液で既知の濃度に希釈したｒＳ−１００β溶液を４０μＬ添加し、３７℃５％ＣＯ２下で培養した。
前記の実験例１と同様に、測定用サンプルを作成し、サンプル中のβアミロイド１−４０の蓄積量を測定した。スタンダードの検量線よりβアミロイド１−４０量を算出した。その結果を表４に示す。
ｒＳ−１００β処置における化合物存在下のβ−アミロイド１−４０産生作用を検討したところ，γ−セクレターゼ阻害剤１により抑制作用が認められた。また、抑制作用はＡＥＢＳＦ（β−セクレターゼ阻害作用）、ＰＭＡ（α−セクレターゼ活性化作用）、ツニカマイシン（糖付加阻害剤）、ブレフェルジンＡ（ｂｒｅｆｅｌｄｉｎ Ａ）、モネシン（ゴルジ、小胞体におけるタンパク質成熟化阻害作用）でも認められた。一方、中性エンドペプチダーゼ阻害剤のホスホラミドンでは増強作用が認められた。
Figure 2003024485
【配列表】
Figure 2003024485
Figure 2003024485
Figure 2003024485

【図面の簡単な説明】
図１（Ａ）はＵ−３７３ＭＧ細胞におけるＣＭ−Ｓ−１００βのβアミロイド１−４０産生促進作用を示し、（Ｂ）はＵ−３７３ＭＧ細胞におけるｒＳ−１００βのβアミロイド１−４０産生促進作用を示す。
図２（Ａ）はＵ−３７３ＭＧ細胞におけるＣＭ−Ｓ−１００βのβアミロイド１−４２産生促進作用を示し、（Ｂ）はＵ−３７３ＭＧ細胞におけるｒＳ−１００βのβアミロイド１−４２産生促進作用を示す。
図３（１）〜（４）は、各々ＣＭ−Ｓ−１００βによる、βアクチン（陰性対照）発現量変化、ＡＰＰ７７０発現量変化、ＡＰＰ７５１発現量変化、およびＡＰＰ６９５発現量変化を示し、（５）〜（８）は、各々ｒＳ−１００βによる、βアクチン（陰性対照）発現量変化、ＡＰＰ７７０発現量変化、ＡＰＰ７５１発現量変化、およびＡＰＰ６９５発現量変化を示す。TECHNICAL FIELD The present invention relates to a therapeutic agent for a β-amyloid-induced disease comprising an S-100β inhibitor as an active ingredient.
BACKGROUND ART Cells constituting the parenchyma of the central nervous system include nerve cells and glial cells, and the number of glial cells is much larger than that of nerve cells. Glial cells play a role in supporting the function of nerve cells. Astrocytes, which are a kind of glial cells, maintain extracellular ions and neurotransmitter homeostasis (Pharmacology and function, pp. 193-228, Academic Press, Inc., (1993)) as supporting cells for neurons. It has functions such as supply of neurotrophic factors (Pharmacology and function, pp. 267-308, Academic Press, Inc., (1993)), and thus plays an important role in controlling brain functions. Conceivable.
Conventionally, it has been considered that neurodegenerative diseases (Alzheimer's disease, amyotrophic lateral sclerosis, etc.) are mainly caused by abnormalities of nerve cells. However, in recent years, the idea that glial cells surrounding nerve cells, particularly astrocytes, are functionally abnormal has become influential.
β-amyloid is a major constituent of senile plaques, one of the pathological features of Alzheimer's disease, a neurodegenerative disease. β-amyloid is also present in the brain of normal humans, but is thought to be rapidly degraded. This protein is produced secondarily by β-secretase and γ-secretase in the process of metabolizing amyloid precursor protein. Amyloid precursor protein is normally degraded by α-secretase, but it is known that β-amyloid is not produced in this case. There are two isoforms of β-amyloid, β-amyloid 1-40 and β-amyloid 1-42 (43). Have been. Most β-amyloid produced in the brain is β-amyloid 1-40, but poorly soluble β-amyloid 1-42 (43) is more likely to aggregate and deposit, and it has been elucidated which one is deeply involved in the pathology at present. Not. It is known that the produced β-amyloid is degraded by neutral endopeptidase.
In Alzheimer's disease, β-amyloid aggregates to form insoluble fibrils and deposits in the brain to form senile plaques. At present, attention has been focused on a method for controlling the metabolic system of amyloid precursor protein and β-amyloid to suppress β-amyloid production as a radical treatment for Alzheimer's disease.
It is known that amyloid precursor protein expression is increased in activated astrocytes upon nerve cell injury (Neuron, 3 , 275-285, (1989)). Since β-amyloid exhibits neurotoxicity, it is considered that the overproduction and accumulation of β-amyloid due to abnormal metabolism of amyloid precursor protein is involved in the development and progression of Alzheimer's disease (Science, 245 , 417-420, (1989)). Furthermore, it has been reported that β-amyloid neurotoxicity involves an increase in active oxygen production via N-methyl-D-aspartate receptor and nitric oxide synthase (J. Neurochem., 76 (4) , 1050 ) . -1056, (2001)). Therefore, it is possible that β-amyloid is involved in active oxygen-induced diseases.
S-100β is an astrocyte-specific protein, which has a calcium binding site and is involved in regulating intracellular calcium concentration, binding to various cytoskeletal proteins, changing cell morphology and releasing neurotransmitters ( TIBS, 13 , 437-443, (1988)). S-100β is produced by the activation of astrocytes and is released extracellularly. The released S-100β exhibits trophic and traumatic effects on nerve cells (Progress in Neurobiology, 46 , 71-82, (1995)), and promotes proliferation on astrocytes. It has been reported that it has an action and an expression action of inducible nitric oxide synthase (J. Biological Chemistry, 271 , 2545-247, (1996)).
S-100β is known to be overexpressed in activated astrocytes during brain injury. The amount of S-100β is increased in the brain of Alzheimer's patients (Proc. Natl. Acad. Sci. USA, 86 , 7611-7615, (1989)). It has been reported that there is a correlation (J. Neurosci. Res., 39 , 398-404, (1994)). Furthermore, it has been reported that activated astrocytes with overexpression of S-100β are present near the senile plaques in the brain of Alzheimer's patients, and that the expression intensity correlates with abnormal neuronal process extension. (J. Neuropathol. Exp. Neurol., 55 , 273-279, (1996)). In addition, stroke (Stroke, 30 , 1190-1195, (1999)), head trauma (Neurosurgery, 45 , 477-483, (1999)), multiple sclerosis (Acta Neurol Scan, 96 , 142-144). , (1997)), and Down syndrome (J. Thorac. Cardiovasc. Surg., 116 , 281-285, (1998)) show an increase in S-100β in serum or cerebrospinal fluid.
So far, the following has been reported on the relationship between S-100β, β-amyloid and amyloid precursor protein.
1) In rat nerve cells, S-100β increased the amount of amyloid precursor protein and mRNA encoding it (J. Neurochem., 71 (4) , 1421-1428, (1998)).
2) In transgenic mice (familial Alzheimer's disease mice overexpressing amyloid precursor protein), S-100β was overexpressed before β-amyloid plaques appeared (J. Neurochem., 74 (1) , 295- ) . 301, (2000)).
3) In rat nerve cells, β-amyloid increased the gene expression of S-100β (Mol. Brain Res., 34 (1) , 118-126, (1995)).
In neurodegenerative diseases such as Alzheimer's disease, overexpression of β-amyloid and S-100β has been confirmed. However, there are various theories regarding the interrelationship between these proteins, and it has not been clarified yet.
DISCLOSURE OF THE INVENTION The ultimate goal in the treatment of neurodegenerative diseases, including Alzheimer's, is to block the progression of pathological processes in the brain and completely suppress the onset and progression. However, currently used therapeutic agents are symptomatic therapeutic agents such as cholinesterase inhibitors. In addition, anti-inflammatory drugs and female hormones, which are currently under clinical application, are not curative treatments to correct the underlying lesion deterioration. Therefore, it is urgently necessary to elucidate the cause of a neurodegenerative disease and to provide a curative drug.
The present inventors have conducted various studies on the relationship between the expression of S-100β and the production of β-amyloid in neurodegenerative diseases, and as a result, revealed that S-100β increases the amount of β-amyloid production. completed.
That is, the present invention provides the following therapeutic and / or prophylactic agents for β-amyloid-induced diseases, a method for screening a compound by measuring the inhibitory action of a test compound on β-amyloid production, and a compound obtained by the method.
1. An agent for treating and / or preventing β-amyloid-induced disease, comprising an S-100β inhibitor as an active ingredient.
2. The S-100β inhibitor acts on a mechanism selected from at least one of the following (1) to (6) to suppress β-amyloid production: Prophylactic:
(1) β-amyloid production by β-secretase and γ-secretase,
(2) Metabolism of amyloid precursor protein by α-secretase,
(3) metabolism of β-amyloid by neutral endopeptidase,
(4) a mechanism of S-100β uptake into cells,
(5) sugar addition,
(6) Protein maturation.
3. Diseases caused by β-amyloid include neurodegenerative disease, Down's syndrome, boxer's disease, progressive supranuclear palsy, astroglioma, neurological dysfunction of the brain, multiple sclerosis, dementia, schizophrenia, epilepsy, anxiety, Vomiting, migraine, neuronal death, depression, sleep disorders, eating disorders, urinary incontinence, hypoxia, cerebral infarction, brain tumors, hyperoxic convulsions and hyperoxia, inflammatory or neuropathic pain, meninges 2. The therapeutic and / or preventive agent for β-amyloid-induced disease according to the above item 1, which is inflammation.
4. Under the condition that (1) S-100β is added to human-derived glial cells to increase β-amyloid production, a test compound is added to detect β-amyloid released from the cells, or (2) the test compound Is added, S-100β is added, and β-amyloid released from the cells is detected, thereby determining the β-amyloid production inhibitory effect of the test compound. Screening method.
5. the screening method according to the above 4, wherein the β-amyloid production inhibitory action is selected from the following actions;
(1) β-secretase or γ-secretase inhibitory action,
(2) α-secretase activating action,
(3) neutral endopeptidase activating action,
(4) S-100β uptake inhibitory action,
(5) sugar addition inhibitory action,
(6) Protein maturation inhibitory action.
6. A compound obtained by the screening method according to the above item 4.
7. 7. The therapeutic and / or prophylactic agent for the β-amyloid-induced disease according to the above item 1, which comprises the compound according to item 6 as an active ingredient.
Hereinafter, the present invention will be described in detail.
According to the prior art, it has been reported that S-100β increases the amount of amyloid precursor protein and mRNA encoding it in rat nerve cells. However, according to experiments by the present inventors, in human-derived glial cells, S-100β did not unexpectedly change the amount of mRNA encoding the amyloid precursor protein, and S-100β did not affect the amount of amyloid precursor protein. It was confirmed not to give. That is, since the amount of amyloid precursor protein does not change and only the amount of β-amyloid increases, S-100β acts during the process of metabolizing the amyloid precursor protein to produce β-amyloid or the process of metabolizing β-amyloid. It is newly suggested to do so.
In the process of amyloid precursor protein metabolism, the involvement of α, β and γ secretase is known, and β amyloid is known to be produced by the action of β and γ secretase and not produced when metabolized by α secretase. I have. Therefore, it is considered that S-100β activates (1) β and γ secretase or (2) suppresses α secretase. In addition, it has been reported that the produced β-amyloid is metabolized by neutral endopeptidase, and thus it is conceivable that S-100β inhibits (3) neutral endopeptidase. Furthermore, it is known that β-amyloid production is suppressed when sugar addition or maturation of the amyloid precursor protein is suppressed. Therefore, it is also possible that S-100β promotes (4) glycosylation or protein maturation. In the curative treatment of neurodegenerative diseases such as Alzheimer's disease, attention has been focused on controlling amyloid precursor protein and β-amyloid metabolic processes and suppressing β-amyloid production. However, the fact that S-100β acts on the amyloid precursor protein and the β-amyloid metabolism process was confirmed by the present inventors for the first time through experiments, and was useful in developing a curative therapeutic drug for neurodegenerative diseases. It is.
β-amyloid is involved in neurodegenerative diseases and the like as described above. This time, S-100β is selected from (1) β and γ secretase activation, (2) α secretase inhibition, (3) neutral endopeptidase inhibition, (4) promotion of sugar addition, and (5) promotion of protein maturation. It has been found by the present inventors that the expression of β-amyloid is promoted by at least one of the actions described above. Therefore, it can be used for the treatment of a disease in which the overexpression of β-amyloid is involved by inhibiting S-100β, particularly a neurodegenerative disease characterized by a loss of nerve cells.
That is, it is considered that the S-100β inhibitor can be used for treating a disease caused by β-amyloid. A disease caused by β-amyloid is a disease caused by an increase in β-amyloid, and specifically, neurodegenerative diseases such as Alzheimer's disease (Alzheimer's disease, Creutzfeldt-Jakob disease, Parkinson's disease, Huntington's disease, Olive Bridge Cerebellar atrophy, amyotrophic lateral sclerosis, etc.), Down's syndrome, boxer's disease, progressive supranuclear palsy, astroglioma, neurological dysfunction after stroke or brain injury, multiple sclerosis, dementia, schizophrenia Disorders, epilepsy, anxiety, vomiting, migraine, neuronal death, depression, sleep disorders, eating disorders such as anorexia, urinary incontinence, hypoxia, cerebral infarction, brain tumors, hyperoxic convulsions and hyperoxia, Inflammatory or neuropathic pain, meningitis, etc. are considered.
The present invention also includes a screening method for a therapeutic agent for a β-amyloid-induced disease, wherein β-amyloid is detected using human-derived cells treated with S-100β.
The general method of measuring β-amyloid inhibitory action known to date is a method of increasing β-amyloid production by cytokines, chemokines, C-kinases and the like, but the relationship between S-100β and β-amyloid has now become clear. As a result, screening for the therapeutic agent for a β-amyloid-induced disease of the present invention has become possible.
More specifically, by adding S-100β to human-derived glial cells to enhance β-amyloid production, adding a test compound, and measuring the amount of β-amyloid in the cell culture supernatant after a certain period of time, A compound capable of suppressing the production of β-amyloid can be selected.
In rats, β-amyloid cannot be detected because secretase, which is an amyloid precursor protein metabolizing enzyme, is scarcely present. In consideration of such a difference in protein expression between animal species, it is preferable to use human-derived cells in the screening method of the present invention. The cells for screening may be any cells as long as they are human-derived glial cells. Specific examples include human glioblastoma, U-373MG, a cell line derived from astrocytoma, T98G and A-172, a cell line derived from human glioblastoma, and CCF-STTG1, a cell line derived from human astrocytoma. However, as a result of various studies, U-373MG cells, which is a cell line derived from human glioblastoma and astrocytoma, are preferable.
The test compound may be added immediately before, immediately after, or simultaneously with the addition of S-100β, and the addition timing of the compound may be changed as occasion demands.
As a β-amyloid detection method, any method may be used as long as β-amyloid can be detected, and examples thereof include an enzyme immunoassay, a radioimmunoassay, and a Western blot.
The present invention further includes a compound obtained by the above-mentioned screening method. Compounds that inhibit β-amyloid production by this screening system, specifically, compounds that inhibit β and γ secretase, compounds that activate α secretase, compounds that activate neutral endopeptidase, compounds that inhibit sugar addition, Alternatively, a compound that inhibits protein maturation can be selected. S-100β produced by the activation of astrocytes and released outside the cells is re-uptaken by a specific site of the astrocytes and acts on the astrocytes themselves. Therefore, in the present screening system, a compound that suppresses the uptake of S-100β can be easily selected in an in vitro system. To date, no compound that inhibits S-100β from acting on astrocytes has been reported, and it is recognized by the present invention as a compound that suppresses β-amyloid production for the first time.
BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described more specifically with reference to experimental examples in which it was confirmed that S-100β increases the amount of β-amyloid without changing the gene amount of amyloid precursor protein. However, they do not limit the scope of the invention.
Experimental Example 1: Measurement of β-amyloid production amount by S-100β 1) Preparation of S-carboxymethylated S-100β Rat recombinant S-100β (Toyobo Co., Ltd.) was S-carboxymethylated using iodoacetic acid.
That is, 1.45 g of sodium hydrogencarbonate was dissolved in 34.5 mL of the recombinant S-100β solution (content of the recombinant S-100β: 100 mg) in a 50 mL tube. Next, 11.5 mL of 1-propanol was added and stirred well (final concentration of 1-propanol 25%). Further, 50 μL of tributylphosphine was added and stirred, and then left at room temperature for 1 hour. Thereafter, 1 mL of an iodoacetic acid reagent (iodoacetic acid was dissolved in 1 mol / L sodium hydroxide to 1 mol / L, prepared at the time of use) was added, and the mixture was allowed to stand at room temperature for 1 hour and 30 minutes under light shielding.
The reaction solution was transferred to a dialysis membrane (Sanko Junyaku), and dialyzed at 4 ° C. against a previously cooled Tris buffer (40 mmol / L, Tris-HCl, pH 7.5). After 6 hours or more, the buffer was exchanged and dialyzed.
The dialyzed sample was concentrated using Centriplus YM-10 (Amicon). After concentration, the solution was sterilized by filtration through a 0.22 μm filter (Millipore). This was designated as S-carboxymethylated S-100β (hereinafter sometimes abbreviated as CM-S-100β) and stored at -80 ° C.
2) Preparation of rat recombinant S-100β Rat recombinant S-100β (hereinafter sometimes referred to as rS-100β) was obtained by dialysis, concentrated, and sterilized by filtration from Toyobo Co., Ltd. used. The operation was performed according to the above-mentioned "1) Preparation of S-carboxymethylated S-100β".
3) Measurement of β-amyloid production by S-100β Human glioblastoma and astrocytoma-derived cell line U-373MG cells (Sumisho Pharma International Co., Ltd.) were modified with a high glucose Dulbecco's modified Eagle medium containing 10% fetal bovine serum ( Medium containing 10% fetal bovine serum, 20 mmol / L HEPES, 2 mmol / L L-glutamine, 100 U / mL penicillin and 100 μmol / L streptomycin added to high glucose Dulbecco's modified Eagle's medium (DMEM). -DMEM) at 37 ° C under 5% CO 2 in a 75 cm 3 flask.
The cultured cells were detached with trypsin / ethylenediaminetetraacetic acid (EDTA). After centrifugation, the detached cells were diluted with 10% FCS-DMEM medium to a concentration of 2 × 10 5 cells / mL, and seeded on a 24-well plate at 1 mL / well. The seeded cells were cultured at 37 ° C. under 5% CO 2 .
The cells on the second day after seeding were washed with 1 mL of serum-free DMEM, and then the medium was replaced with 360 μL of serum-free DMEM. Next, 40 μL of a CM-S-100β or rS-100β solution diluted to a known concentration with a Tris buffer solution was added, and the cells were cultured at 37 ° C. in 5% CO 2 .
48 hours after the S-100β treatment, 300 μL of the culture supernatant was collected from the 24-well plate. The collected culture supernatant was centrifuged at 15,000 rpm at 4 ° C. for 5 minutes to collect the supernatant, which was used as a sample for measurement. Samples were stored on ice until the time of measurement.
The accumulation amounts of β-amyloid 1-40 and β-amyloid 1-42 in the sample were measured using a human β-amyloid 1-40 ELISA kit (Bioscore) and a human β-amyloid 1-42 ELISA kit (BioScore), respectively. The measuring method followed the instructions of the kit. At that time, the sample was diluted using a diluent included in the kit. The amounts of β-amyloid 1-40 and β-amyloid 1-42 were calculated from the calibration curves of the respective standards. The results are shown in Table 1 and FIGS.
CM-S-100β and rS-100β significantly increased the accumulation of β-amyloid 1-40 in U-373MG cells at 10 and 30 μmol / L compared to the vehicle-treated group (FIG. 1, Table 1). Similarly, CM-S-100β significantly increased β-amyloid 1-42 accumulation at 10 and 30 μmol / L and rS-100β at 3, 10 and 30 μmol / L (FIG. 2, Table 1).
From these results, it was confirmed that S-100β has a β-amyloid production promoting effect.
Figure 2003024485
Experimental Example 2: Measurement of Amyloid Precursor Protein mRNA Expression Level 1) Preparation of mRNA Sample A plate treated with S-100β was prepared in the same manner as described above. MRNA is purified from the cells after collecting the cell supernatant using a QuickPrep mRNA purification kit (Amersham Pharmacia Biotech), and the amyloid precursor protein (hereinafter sometimes abbreviated as APP) mRNA expression level. A sample for measurement was used. mRNA samples were stored at -80 ° C.
2) Preparation of cDNA cDNA was synthesized from 80 ng of mRNA using a first-strand cDNA synthesis kit (Amersham Pharmacia Biotech), and further diluted three times with distilled water to obtain a cDNA sample.
3) RT-PCR method A PCR reaction solution shown in Table 2 was prepared, and PCR was performed under the conditions of denaturation at 98 ° C for 1 second and annealing at 68 or 70 ° C for 15 seconds. A part of the reaction solution was electrophoresed with 1% agarose gel.
Figure 2003024485
Table 3 shows the nucleotide sequences of the primers used.
Figure 2003024485
4) Results The mRNA expression levels of amyloid precursor proteins (APP695, APP751 and APP770) were examined. The results are shown in FIG. 3 together with the control (change in the expression level of β-actin). In FIG. 3, (1) changes in β-actin (negative control) expression levels by CM-S-100β, (2) changes in APP770 expression levels by CM-S-100β, and (3) APP751 changes by CM-S-100β. (4) change in APP695 expression level by CM-S-100β, (5) change in β-actin (negative control) expression level by rS-100β, (6) change in APP770 expression level by rS-100β, (7) shows the change in APP751 expression level by rS-100β, and (8) shows the change in APP695 expression level by rS-100β.
As is clear from FIG. 3, there was no change in the mRNA expression level in any of the treatments with CM-S-100β and rS-100β. Therefore, it was suggested that the increase in the amount of β-amyloid produced in the S-100β-treated group was not related to the increase in the amount of expression of the amyloid precursor protein as a precursor thereof. From this, it was presumed that S-100β was acting not at the transcriptional level but in the process of amyloid precursor protein metabolism by β-secretase and γ-secretase.
Experimental Example 3: Evaluation of Compound by Measurement of Production of β-Amyloid U-373MG cells were prepared in the same manner as in Experimental Example 1 described above, and seeded on a 24-well plate at 1 mL / well at 2 × 10 5 cells / mL. The seeded cells were cultured at 37 ° C. under 5% CO 2 .
The cells on the second day after seeding were washed with 1 mL of serum-free DMEM, the medium was replaced with 320 μL of serum-free DMEM, and 40 μL of a compound concentrate (10 times the treatment concentration) was added. Next, 40 μL of an rS-100β solution diluted to a known concentration with a Tris buffer solution was added, and the cells were cultured at 37 ° C. under 5% CO 2 .
In the same manner as in Experimental Example 1, a measurement sample was prepared, and the accumulated amount of β-amyloid 1-40 in the sample was measured. The amount of β-amyloid 1-40 was calculated from the standard calibration curve. Table 4 shows the results.
When the action of producing β-amyloid 1-40 in the presence of the compound in the treatment with rS-100β was examined, an inhibitory action was observed by γ-secretase inhibitor 1. The inhibitory action is AEBSF (β-secretase inhibitory action), PMA (α-secretase activator action), tunicamycin (sugar addition inhibitor), brefeldin A (brefeldin A), monesin (Golgi, inhibition of protein maturation in the endoplasmic reticulum) Action). On the other hand, the phosphoramidone, a neutral endopeptidase inhibitor, had an enhancing effect.
Figure 2003024485
[Sequence list]
Figure 2003024485
Figure 2003024485
Figure 2003024485

[Brief description of the drawings]
1 (A) shows the action of promoting the production of β-amyloid 1-40 of CM-S-100β in U-373MG cells, and FIG. 1 (B) shows the action of promoting the production of β-amyloid 1-40 of rS-100β in U-373MG cells. Show.
FIG. 2 (A) shows the action of promoting the production of β-amyloid 1-42 by CM-S-100β in U-373MG cells, and FIG. 2 (B) shows the action of promoting the production of β-amyloid 1-42 by rS-100β in U-373MG cells. Show.
3 (1) to 3 (4) show changes in β-actin (negative control) expression level, APP770 expression level change, APP751 expression level change, and APP695 expression level change by CM-S-100β, respectively (5) (8) shows changes in the expression levels of β-actin (negative control), APP770, APP751, and APP695 due to rS-100β.

Claims (7)

Ｓ−１００β阻害剤を有効成分とするβアミロイド起因疾患の治療および／または予防剤。An agent for treating and / or preventing β-amyloid-induced disease, comprising an S-100β inhibitor as an active ingredient. Ｓ−１００β阻害剤が以下の（１）〜（６）の少なくともひとつから選択される機構に作用してβアミロイド産生を抑制する請求の範囲１記載のβアミロイド起因疾患治療および／または予防剤：
（１）βセクレターゼおよびγセクレターゼによるβアミロイド産生、
（２）αセクレターゼによるアミロイド前駆体タンパク質の代謝、
（３）中性エンドペプチダーゼによるβアミロイドの代謝、
（４）Ｓ−１００βの細胞内への取り込み機構、
（５）糖付加、
（６）タンパク質成熟化。The therapeutic and / or prophylactic agent for a β-amyloid-induced disease according to claim 1, wherein the S-100β inhibitor acts on a mechanism selected from at least one of the following (1) to (6) to suppress β-amyloid production:
(1) β-amyloid production by β-secretase and γ-secretase,
(2) Metabolism of amyloid precursor protein by α-secretase,
(3) metabolism of β-amyloid by neutral endopeptidase,
(4) a mechanism of S-100β uptake into cells,
(5) sugar addition,
(6) Protein maturation. βアミロイドに起因する疾患が、神経変性疾患、ダウン症、ボクサー症、進行性核上麻痺、星状膠細胞腫、脳の神経機能障害、多発性硬化症、痴呆、精神***症、てんかん、不安、嘔吐、偏頭痛、神経細胞死、うつ病、睡眠障害、摂食障害、尿失禁、低酸素症、脳梗塞、脳腫瘍、高酸素痙攣および高酸素毒症、炎症性もしくは神経障害性疼痛、髄膜炎である請求の範囲１に記載のβアミロイド起因疾患治療および／または予防剤。Diseases caused by β-amyloid include neurodegenerative disease, Down syndrome, Boxer disease, progressive supranuclear palsy, astroglioma, neurological dysfunction of the brain, multiple sclerosis, dementia, schizophrenia, epilepsy, anxiety, Vomiting, migraine, neuronal death, depression, sleep disorders, eating disorders, urinary incontinence, hypoxia, cerebral infarction, brain tumors, hyperoxic convulsions and hyperoxia, inflammatory or neuropathic pain, meninges The agent for treating and / or preventing β-amyloid-induced disease according to claim 1, which is inflammation. ヒト由来グリア細胞に（１）Ｓ−１００βを添加しβアミロイド産生を亢進させた条件下において、被験化合物を添加して、細胞から放出されたβアミロイドを検出するか、または（２）被験化合物を添加し、Ｓ−１００βを添加して、細胞から放出されたβアミロイドを検出することによって、被験化合物のβアミロイド産生抑制作用を決定することを特徴とするβアミロイド産生抑制作用を有する化合物のスクリーニング方法。Under conditions where (1) S-100β is added to human-derived glial cells to enhance β-amyloid production, a test compound is added to detect β-amyloid released from the cells, or (2) the test compound Is added, S-100β is added, and β-amyloid released from the cells is detected to determine the β-amyloid production inhibitory action of the test compound. Screening method. βアミロイド産生抑制作用が以下の作用から選択される請求の範囲４記載のスクリーニング方法；
（１）βセクレターゼまたはγセクレターゼ阻害作用、
（２）αセクレターゼ活性化作用、
（３）中性エンドペプチダーゼ活性化作用、
（４）Ｓ−１００β取り込み阻害作用、
（５）糖付加阻害作用、
（６）タンパク質成熟化阻害作用。The screening method according to claim 4, wherein the β-amyloid production inhibitory action is selected from the following actions:
(1) β-secretase or γ-secretase inhibitory action,
(2) α-secretase activating action,
(3) neutral endopeptidase activating action,
(4) S-100β uptake inhibitory action,
(5) sugar addition inhibitory action,
(6) Protein maturation inhibitory action. 請求の範囲４記載のスクリーニング方法によって得られた化合物。A compound obtained by the screening method according to claim 4. 請求の範囲６記載の化合物を有効成分とする請求の範囲１記載のβアミロイド起因疾患の治療および／または予防剤。The therapeutic and / or prophylactic agent for a β-amyloid-induced disease according to claim 1, comprising the compound according to claim 6 as an active ingredient.
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