JP4395633B2 - Method for differentiation and maturation of non-neuronal cells into neurons, the composition and the method for searching for the composition - Google Patents

Method for differentiation and maturation of non-neuronal cells into neurons, the composition and the method for searching for the composition Download PDF

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JP4395633B2
JP4395633B2 JP2002252180A JP2002252180A JP4395633B2 JP 4395633 B2 JP4395633 B2 JP 4395633B2 JP 2002252180 A JP2002252180 A JP 2002252180A JP 2002252180 A JP2002252180 A JP 2002252180A JP 4395633 B2 JP4395633 B2 JP 4395633B2
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寅夫 石田
艶軍 張
浩代 近藤
志峰 於
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学校法人鈴鹿医療科学大学
寅夫 石田
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    • C12N2506/1353—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from bone marrow mesenchymal stem cells (BM-MSC)

Description

【０００１】
【発明が属する技術分野】
本発明は、（１）非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを有効成分とすることを特徴とする、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する組成物、（２）非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する能力を有する、薬学的許容範囲の毒性しかない低分子物質が３−ヒドロキシ−３−メチルグルタリル−補酵素Ａ（ＨＭＧ−ＣｏＡ）還元酵素阻害剤から選ばれる（１）に記載の組成物、（３）ＨＭＧ−ＣｏＡ還元酵素阻害組成物がアトルバスタチン、イタバスタチン、コンパクチン、シンバスタチン、ジヒドロコンパクチン、ダルバスタチン、ピタバスタチン、フルインドスタチン、フルバスタチン、プラバスタチン、ベルバスタチン、ベロスタチン、メバスタチン、ロスバスタチン、ロバスタチン、およびそれらの光学または幾何異性体、およびそれらのプロドラッグ、ならびに上記化合物又はそれらのプロドラッグの薬学的に許容することのできるそれらの塩ならびにそれらの水和物から選ばれる（２）に記載の組成物、（４）ＨＭＧ−ＣｏＡ還元酵素阻害組成物がアトルバスタチンカルシウム水和物、シンバスタチン、フルバスタチンナトリウム、プラバスタチンナトイウムから選ばれる（２）に記載の組成物、（５）非神経細胞が骨髄系非神経細胞と神経系非神経細胞から選ばれる（１）から（４）に記載の組成物、（６）骨髄系非神経細胞が骨髄幹細胞、間葉系細胞、骨髄間質細胞から選ばれる（５）に記載の組成物、（７）神経系非神経細胞が神経幹細胞と神経前駆細胞から選ばれる（５）に記載の組成物、（８）（１）から（７）に記載の組成物を有効成分とすることを特徴とする、要すれば活性後に、神経細胞に接触させ、該神経細胞を細胞分裂誘起物質で処理することなく成熟する組成物、（９）（１）から（７）に記載の組成物を有効成分とすることを特徴とする、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟する組成物、（１０）（１）から（７）に記載の組成物を有効成分とし、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することにより神経障害疾患を治療または悪性化抑制することを特徴とする、神経障害疾患を治療または悪性化抑制する組成物、（１１）（１）から（７）に記載の組成物を、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化することを特徴とする、非神経細胞を神経細胞へ分化する方法、（１２）（８）に記載の組成物を、要すれば活性後に、神経細胞に接触させ、該神経細胞を細胞分裂誘起物質で処理することなく成熟することを特徴とする、神経細胞を成熟する方法、（１３）（９）に記載の組成物を、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することを特徴とする、非神経細胞を神経細胞へ分化し、かつ該神経細胞を成熟する方法、（１４）（１０）に記載の組成物を、要すれば活性化後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することにより神経障害疾患を治療または悪性化抑制することを特徴とする、神経障害疾患を治療または悪性化抑制する方法、（１５）移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞、（１６）（１２）に記載の方法において、癌化神経細胞混入の恐れがない神経細胞を成熟することにより得られる、移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞、（１７）（１１）または（１３）に記載の方法により得られる、移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞、（１８）要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない非神経細胞または神経細胞に接触させ、該非神経細胞の神経細胞への分化、および／または該神経細胞の成熟の有無を調べることを特徴とする、非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、そして／または神経細胞を細胞分裂誘起物質で処理することなく成熟する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法、（１９）（１８）に記載の方法において、要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない非神経細胞に接触させ、該非神経細胞の神経細胞への分化の有無を調べることを特徴とする、非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法、（２０）（１８）に記載の方法において、要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない神経細胞に接触させ、該神経細胞の成熟の有無を調べることを特徴とする、神経細胞を細胞分裂誘起物質で処理することなく成熟する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法、（２１）（１８）に記載の方法において、要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない非神経細胞に接触させ、該非神経細胞の神経細胞への分化、および該神経細胞の成熟の有無を調べることを特徴とする、非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法に関する。
【０００２】
【従来の技術】
アルツハイマー病、筋萎縮性側索硬化症、脳腫瘍、小脳変性症、交通性水頭症、ハンチントン病、前頭葉への照射、多発性硬化症、正常圧水頭症、パーキンソン病、ピック病、進行性多巣性白質脳症、進行性核上麻痺、拳闘家痴呆、脳外傷、外科手術、脳腫瘍、慢性硬膜下血腫、脳卒中、脳血管性痴呆、ウィルソン病細菌性心内膜炎、クロイツフェルト・ヤコブ病、ゲルストマン・シュトロイスラー・シャインカー病、HIV関連疾患、神経梅毒、結核性および真菌性髄膜炎、ウイルス性脳炎、無酸素症、B１２欠乏症、慢性的な薬物-アルコール-栄養性乱用、葉酸欠乏症、副甲状腺機能亢進症に伴う高カルシウム血症、低血糖、甲状腺機能低下症、肝性脳症、肺性脳症、尿毒素性脳症等の臓器系不全、ペラグラ等の神経疾患は繊維化、免疫反応、血管傷害、栄養と酸素欠乏、感染等により、神経細胞が退化、減少、細胞死し、または傷害、除外されることにより、組織や臓器がその機能を失い発症すると考えられている。したがって、これらの疾患を治療または悪化予防の１つの方法として失われた神経細胞を何らかの方法で補充するか再生することが考えられる。
【０００３】
そのため、ＥＳ細胞または神経幹細胞が注目された。ＥＳ細胞または神経幹細胞は未分化の細胞であり、神経細胞が死滅すると、その失われた細胞を補うように分化を始め、生体機能の維持に大きく貢献する可能性があるからであった。近年、機能を失った組織に対してＥＳ細胞や神経幹細胞を移植し、個々の生体機能を発現する細胞へ特異的に分化させることにより、当該病態を改善・治療する試みが行われた。しかし、ＥＳ細胞は胚性幹細胞であり、ヒトの胚を原料に用いるという倫理性の問題と移植抗原性の問題を有する。一方、神経幹細胞の場合も胎児由来の神経幹細胞しか入手が困難であり、胎児由来の神経幹細胞は倫理性の問題と移植抗原性の問題を有する。
【０００４】
従って、ＥＳ細胞、胎児由来の神経幹細胞以外の非神経細胞で神経細胞に分化成熟し得る細胞が注目された。自己の体から容易に入手可能な骨髄間質細胞（骨髄ストロマ細胞ともいう）に細胞分裂誘起物質（マイトジェンともいう）で処理し、次いでレチノイン酸、成長因子、胎児ニューロン細胞またはその組合せから選択される分化剤を処理して得られる神経細胞（特表２００２−５１３５４５）が報告された。しかし、この骨髄細胞由来の神経細胞はその工程に細胞分裂誘起物質処理操作が必須のため、細胞分裂誘起物質を体内投与した場合、他の細胞の異常増殖をもたらし、細胞を癌化させる恐れがあり、また体外投与の場合でも、そういう処理操作で作られた神経細胞に癌化した神経細胞が混入している可能性があり、これをヒトに移植することは問題である。そこで細胞分裂誘起物質処理無しに骨髄間質細胞にメルカプトエタノール、ジブチリルサイクリックＡＭＰ等低分子の分化促進因子や骨形成因子（ＢＭＰ）、脳由来神経栄養因子（ＢＤＮＦ）、線維芽細胞成長因子（ｂＦＧＦ）等の高分子の分化促進因子や骨形成タンパク質阻害剤ノジン（Ｎｏｇｇｉｎ）と脱メチル化剤５−アザシチジンを作用させることにより神経細胞に分化することが報告されたことから（Ｍ．Ｄｅｚａｗａら、Ｅｕｒ．Ｊ．Ｎｅｕｒｏｓｃｉ．２００１年１４巻１７７１頁、Ｗ．Ｄｅｎｇら、Ｂｉｏｃｈｅｍ．Ｂｉｏｐｈｙｓ．Ｒｅｓ．Ｃｏｍｍｕｎ．２００１年２８２巻１４８頁、Ｄ．Ｗｏｏｄｂｕｒｙら、Ｊ．Ｎｅｕｒｏｓｃｉ．Ｒｅｓ．２０００年６１巻３６３頁、Ｊ．Ｓａｎｃｈｅｚ−Ｒａｍｏｓら、Ｅｘｐ．Ｎｅｕｒｏｌ．２０００年１６４巻２４７頁、Ｊ．Ｋｏｈｙｙａｍａら、Ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ、２００１年６８巻２３５頁）、これらを上記疾患の予防・治療に用いることも考えられるが、かかる高分子の分化促進因子も、分子量の大きいペプチドであるため、生体内で容易に分解を受け、また血液脳関門を通過できないことから、投与方法が著しく限定されるという問題があり、またメルカプトエタノール、ジブチリルサイクリックＡＭＰ等の分化促進因子、骨形成タンパク質阻害剤や脱メチル化剤等の低分子は毒性が高く、実用化は困難である。
【０００５】
一方、自家または自家と主要組織適合抗原を実質的に同じくする、神経幹細胞、神経前駆細胞に関しては体外で分化させて神経細胞にし、それを移植するよりも該細胞を分化し得る薬剤をヒトに投与して生体内に内在する神経幹細胞または神経前駆細胞を分化させて神経細胞にすることにより失われた神経の補充を行うことがより容易と考えられる。極最近、ラットの脳動脈を一時的に止めて脳虚血にして海馬の神経細胞に傷害を与えた後に、神経成長因子を注入すると、失われた神経細胞の4割が回復したという報告がある（Ｈ．Ｎａｋａｔｏｍｉら、Ｃｅｌｌ、２００２年１１０巻４２９頁）。研究としては素晴らしいが、神経成長因子というタンパク質を脳内に注入することは問題である。
【０００６】
従って、（１）非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを有効成分とすることを特徴とする、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する組成物、（２）非神経細胞が骨髄系非神経細胞と神経系非神経細胞から選ばれる（１）に記載の組成物、（３）骨髄系非神経細胞が骨髄幹細胞、間葉系細胞、骨髄間質細胞から選ばれる（２）に記載の組成物、（４）神経系非神経細胞が神経幹細胞と神経前駆細胞から選ばれる（２）に記載の組成物、（５）（１）から（４）に記載の組成物を有効成分とすることを特徴とする、要すれば活性後に、神経細胞に接触させ、該神経細胞を細胞分裂誘起物質で処理することなく成熟する組成物、（６）（１）から（４）に記載の組成物を有効成分とすることを特徴とする、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟する組成物、（７）（１）から（４）に記載の組成物を有効成分とし、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することにより神経障害疾患を治療または悪性化抑制することを特徴とする、神経障害疾患を治療または悪性化抑制する組成物、（８）（１）から（４）に記載の組成物を、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化することを特徴とする、非神経細胞を神経細胞へ分化する方法、（９）（５）に記載の組成物を、要すれば活性後に、神経細胞に接触させ、該神経細胞を細胞分裂誘起物質で処理することなく成熟することを特徴とする、神経細胞を成熟する方法、（１０）（６）に記載の組成物を、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することを特徴とする、非神経細胞を神経細胞へ分化し、かつ該神経細胞を成熟する方法、（１１）（７）に記載の組成物を、要すれば活性化後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することにより神経障害疾患を治療または悪性化抑制することを特徴とする、神経障害疾患を治療または悪性化抑制する方法、（１２）移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞、（１３）（９）に記載の方法において、癌化神経細胞混入の恐れがない神経細胞を成熟することにより得られる、移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞、（１４）（８）または（１０）に記載の方法により得られる、移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞、（１５）要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない非神経細胞または神経細胞に接触させ、該非神経細胞の神経細胞への分化、および／または該神経細胞の成熟の有無を調べることを特徴とする、非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、そして／または神経細胞を細胞分裂誘起物質で処理することなく成熟する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法、（１６）（１５）に記載の方法において、要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない非神経細胞に接触させ、該非神経細胞の神経細胞への分化の有無を調べることを特徴とする、非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法、（１７）（１５）に記載の方法において、要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない神経細胞に接触させ、該神経細胞の成熟の有無を調べることを特徴とする、神経細胞を細胞分裂誘起物質で処理することなく成熟する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法、（１８）（１５）に記載の方法において、要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない非神経細胞に接触させ、該非神経細胞の神経細胞への分化、および該神経細胞の成熟の有無を調べることを特徴とする、非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法の開発が強く望まれている。
【０００７】
一方、ＨＭＧ−ＣｏＡ還元酵素阻害剤はコレステロール合成阻害作用、脂質低下作用、動脈硬化進展抑制作用、外因性コレステロール吸収抑制作用が認められ、かつ長期投与でも毒性が低いため、高コレステロール血症、家族性高コレステロール血症、高脂血症の治療または悪性化抑制に使われている。一方、実用化には至っていないが、特定のＨＭＧ−ＣｏＡ還元酵素阻害剤とアンジオテンシン２受容体拮抗剤の組み合せ物による心不全の予防若しくは治療（特開２００２−１４５７７０）、ＨＭＧ−ＣｏＡ還元酵素阻害剤とアンジオテンシン変換酵素（ＡＣＥ）抑制剤の組み合せ物による二次心臓発作の危険予防（特開平８−２９１０８２）、ＨＭＧ−ＣｏＡ還元酵素阻害剤とインスリン抵抗性改善薬の組み合せ物によるＴＮＡ−ａ抑制（特開２００１−２９４５３７）、同組み合せ物による動脈硬化症予防または治療（特開平９−７１５４０）、ＨＭＧ−ＣｏＡ還元酵素阻害剤のスタチンによる造血幹細胞由来細胞から内皮細胞への分化（Ｊ．Ｌｌｅｖａｄｏｔら、Ｊ．Ｃｌｉｎｉｃａｌ Ｉｎｖｅｓｔｉｇａｔｉｏｎ、２００１年１０８巻３９９頁）スタチンとポリホスホネートの組み合せ物による骨形成促進とアテローム動脈硬化症治療（特開２００１−２５３８２７）、ＨＭＧ−ＣｏＡ還元酵素阻害剤とプロスタグランジン・アゴニストの組み合せ物による骨形成増強作用（特開２００２−１７９５９５）、ＨＭＧ−ＣｏＡ還元酵素阻害剤による骨吸収阻害（特表２００２−５０６０３０）、スタチンによる骨形成刺激作用（Ｇ．Ｍｕｎｄｙら、ＳＣＩＥＮＣＥ、１９９９年２８６巻１９４６頁）、セリバスタチンナトリウムの慢性ＨＩＶ−１に対する効果（特開２００２−２９９７３）、ＨＭＧ−ＣｏＡ還元酵素阻害剤のアンドロゲン活性アンタゴニスト作用による拒食症の処置および予防（特表２００１−５１３５７２）等の報告がある。
【０００８】
また、ＨＭＧ−ＣｏＡ還元酵素阻害剤に関し、神経変性病患者がアポリボ蛋白質（ＡｐｏＥ）が低いことを根拠に神経変性病の治療法法として神経変性病患者にＡｐｏＥのレベルを増加させる組成物を投与する方法と神経細胞と検定化合物を接触させ、神経細胞のＡｐｏＥ上昇の有無を測定するＡｐｏＥ上昇化合物の探索法（特表２００１−５１７６１７）、アルツハイマー病を罹病しているヒトの脳の総コレステロール（ＴＣ）とＬＤＬの濃度が健常人より高いことを根拠にアルツハイマー病を罹病しているヒトに血漿トリグリセライドレベル低下剤を投与し、あるいはＬＤＬ−Ｃレベルを低下させ、ＨＤＬレベルを上昇させるために、所望によりコレステロールレベル低下剤を共投与して血漿トリグリセライドレベルを低下させることによるアルツハイマー病の発症防止を憶測し（特表２００２−５０１８８７）、アルツハイマー病の危険のあるコレステロールのレベル上昇を伴う個人に対して、血中コレステロールレベルを減少させる組成物を投与し、アミロイドタンパク質の産生を減少させる方法（特表２００２−５０７５６４）の報告がある。これらの報告はＨＭＧ−ＣｏＡ還元酵素阻害剤の高脂血症治療に用いられたＡｐｏＥ上昇、コレステロールレベル低下、ＬＤＬレベル低下等の作用を応用すればアルツハイマー病等神経変性病が予防または治療可能と考えているにすぎない。アルツハイマー病等神経変性病により神経細胞が退化、減少、細胞死し、または傷害、除外されることにより、組織や臓器がその機能を失い発症することを直視していない。いくらＡｐｏＥ上昇、コレステロールレベル低下、ＬＤＬレベル低下させても退化、減少、細胞死し、または傷害、除外された神経細胞は再生されない。神経を再生する能力のある、薬学的に許容することのできる毒性しかない低分子物質を有効成分とする組成物の開発が強く望まれる。
【０００９】
【発明が解決しようとする課題】
本発明の目的は、（１）非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを有効成分とすることを特徴とする、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する組成物、（２）非神経細胞が骨髄系非神経細胞と神経系非神経細胞から選ばれる（１）に記載の組成物、（３）骨髄系非神経細胞が骨髄幹細胞、間葉系細胞、骨髄間質細胞から選ばれる（２）に記載の組成物、（４）神経系非神経細胞が神経幹細胞と神経前駆細胞から選ばれる（２）に記載の組成物、（５）（１）から（４）に記載の組成物を有効成分とすることを特徴とする、要すれば活性後に、神経細胞に接触させ、該神経細胞を細胞分裂誘起物質で処理することなく成熟する組成物、（６）（１）から（４）に記載の組成物を有効成分とすることを特徴とする、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟する組成物、（７）（１）から（４）に記載の組成物を有効成分とし、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することにより神経障害疾患を治療または悪性化抑制することを特徴とする、神経障害疾患を治療または悪性化抑制する組成物、（８）（１）から（４）に記載の組成物を、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化することを特徴とする、非神経細胞を神経細胞へ分化する方法、（９）（５）に記載の組成物を、要すれば活性後に、神経細胞に接触させ、該神経細胞を細胞分裂誘起物質で処理することなく成熟することを特徴とする、神経細胞を成熟する方法、（１０）（６）に記載の組成物を、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することを特徴とする、非神経細胞を神経細胞へ分化し、かつ該神経細胞を成熟する方法、（１１）（７）に記載の組成物を、要すれば活性化後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することにより神経障害疾患を治療または悪性化抑制することを特徴とする、神経障害疾患を治療または悪性化抑制する方法、（１２）移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞、（１３）（９）に記載の方法において、癌化神経細胞混入の恐れがない神経細胞を成熟することにより得られる、移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞、（１４）（８）または（１０）に記載の方法により得られる、移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞、（１５）要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない非神経細胞または神経細胞に接触させ、該非神経細胞の神経細胞への分化、および／または該神経細胞の成熟の有無を調べることを特徴とする、非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、そして／または神経細胞を細胞分裂誘起物質で処理することなく成熟する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法、（１６）（１５）に記載の方法において、要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない非神経細胞に接触させ、該非神経細胞の神経細胞への分化の有無を調べることを特徴とする、非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法、（１７）（１５）に記載の方法において、要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない神経細胞に接触させ、該神経細胞の成熟の有無を調べることを特徴とする、神経細胞を細胞分裂誘起物質で処理することなく成熟する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法、（１８）（１５）に記載の方法において、要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない非神経細胞に接触させ、該非神経細胞の神経細胞への分化、および該神経細胞の成熟の有無を調べることを特徴とする、非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法を提供することにある。
【００１０】
【課題を解決するための手段】
本発明者らは、斯かる実状に鑑み、非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する能力を有する、薬学的許容範囲の毒性しかない低分子物質を種々探索した結果、ＨＭＧ−ＣｏＡ還元酵素阻害剤を有効成分とする組成物に、非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する作用と該神経細胞を成熟させる作用があることを見出し、さらに種々研究した結果、（１）非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを有効成分とすることを特徴とする、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する組成物、（２）非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する能力を有する、薬学的許容範囲の毒性しかない低分子物質が３−ヒドロキシ−３−メチルグルタリル−補酵素Ａ（ＨＭＧ−ＣｏＡ）還元酵素阻害剤から選ばれる（１）に記載の組成物、（３）ＨＭＧ−ＣｏＡ還元酵素阻害組成物がアトルバスタチン、イタバスタチン、コンパクチン、シンバスタチン、ジヒドロコンパクチン、ダルバスタチン、ピタバスタチン、フルインドスタチン、フルバスタチン、プラバスタチン、ベルバスタチン、ベロスタチン、メバスタチン、ロスバスタチン、ロバスタチン、およびそれらの光学または幾何異性体、およびそれらのプロドラッグ、ならびに上記化合物又はそれらのプロドラッグの薬学的に許容することのできるそれらの塩ならびにそれらの水和物から選ばれる（２）に記載の組成物、（４）ＨＭＧ−ＣｏＡ還元酵素阻害組成物がアトルバスタチンカルシウム水和物、シンバスタチン、フルバスタチンナトリウム、プラバスタチンナトイウムから選ばれる（２）に記載の組成物、（５）非神経細胞が骨髄系非神経細胞と神経系非神経細胞から選ばれる（１）から（４）に記載の組成物、（６）骨髄系非神経細胞が骨髄幹細胞、間葉系細胞、骨髄間質細胞から選ばれる（５）に記載の組成物、（７）神経系非神経細胞が神経幹細胞と神経前駆細胞から選ばれる（５）に記載の組成物、（８）（１）から（７）に記載の組成物を有効成分とすることを特徴とする、要すれば活性後に、神経細胞に接触させ、該神経細胞を細胞分裂誘起物質で処理することなく成熟する組成物、（９）（１）から（７）に記載の組成物を有効成分とすることを特徴とする、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟する組成物、（１０）（１）から（７）に記載の組成物を有効成分とし、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することにより神経障害疾患を治療または悪性化抑制することを特徴とする、神経障害疾患を治療または悪性化抑制する組成物、（１１）（１）から（７）に記載の組成物を、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化することを特徴とする、非神経細胞を神経細胞へ分化する方法、（１２）（８）に記載の組成物を、要すれば活性後に、神経細胞に接触させ、該神経細胞を細胞分裂誘起物質で処理することなく成熟することを特徴とする、神経細胞を成熟する方法、（１３）（９）に記載の組成物を、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することを特徴とする、非神経細胞を神経細胞へ分化し、かつ該神経細胞を成熟する方法、（１４）（１０）に記載の組成物を、要すれば活性化後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することにより神経障害疾患を治療または悪性化抑制することを特徴とする、神経障害疾患を治療または悪性化抑制する方法、（１５）移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞、（１６）（１２）に記載の方法において、癌化神経細胞混入の恐れがない神経細胞を成熟することにより得られる、移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞、（１７）（１１）または（１３）に記載の方法により得られる、移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞、（１８）要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない非神経細胞または神経細胞に接触させ、該非神経細胞の神経細胞への分化、および／または該神経細胞の成熟の有無を調べることを特徴とする、非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、そして／または神経細胞を細胞分裂誘起物質で処理することなく成熟する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法、（１９）（１８）に記載の方法において、要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない非神経細胞に接触させ、該非神経細胞の神経細胞への分化の有無を調べることを特徴とする、非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法、（２０）（１８）に記載の方法において、要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない神経細胞に接触させ、該神経細胞の成熟の有無を調べることを特徴とする、神経細胞を細胞分裂誘起物質で処理することなく成熟する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法、（２１）（１８）に記載の方法において、要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない非神経細胞に接触させ、該非神経細胞の神経細胞への分化、および該神経細胞の成熟の有無を調べることを特徴とする、非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法を開発し、本発明を完成した。
【００１１】
すなわち、本発明は、（１）非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを有効成分とすることを特徴とする、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する組成物、（２）非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する能力を有する、薬学的許容範囲の毒性しかない低分子物質が３−ヒドロキシ−３−メチルグルタリル−補酵素Ａ（ＨＭＧ−ＣｏＡ）還元酵素阻害剤から選ばれる（１）に記載の組成物、（３）ＨＭＧ−ＣｏＡ還元酵素阻害組成物がアトルバスタチン、イタバスタチン、コンパクチン、シンバスタチン、ジヒドロコンパクチン、ダルバスタチン、ピタバスタチン、フルインドスタチン、フルバスタチン、プラバスタチン、ベルバスタチン、ベロスタチン、メバスタチン、ロスバスタチン、ロバスタチン、およびそれらの光学または幾何異性体、およびそれらのプロドラッグ、ならびに上記化合物又はそれらのプロドラッグの薬学的に許容することのできるそれらの塩ならびにそれらの水和物から選ばれる（２）に記載の組成物、（４）ＨＭＧ−ＣｏＡ還元酵素阻害組成物がアトルバスタチンカルシウム水和物、シンバスタチン、フルバスタチンナトリウム、プラバスタチンナトイウムから選ばれる（２）に記載の組成物、（５）非神経細胞が骨髄系非神経細胞と神経系非神経細胞から選ばれる（１）から（４）に記載の組成物、（６）骨髄系非神経細胞が骨髄幹細胞、間葉系細胞、骨髄間質細胞から選ばれる（５）に記載の組成物、（７）神経系非神経細胞が神経幹細胞と神経前駆細胞から選ばれる（５）に記載の組成物、（８）（１）から（７）に記載の組成物を有効成分とすることを特徴とする、要すれば活性後に、神経細胞に接触させ、該神経細胞を細胞分裂誘起物質で処理することなく成熟する組成物、（９）（１）から（７）に記載の組成物を有効成分とすることを特徴とする、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟する組成物、（１０）（１）から（７）に記載の組成物を有効成分とし、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することにより神経障害疾患を治療または悪性化抑制することを特徴とする、神経障害疾患を治療または悪性化抑制する組成物、（１１）（１）から（７）に記載の組成物を、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化することを特徴とする、非神経細胞を神経細胞へ分化する方法、（１２）（８）に記載の組成物を、要すれば活性後に、神経細胞に接触させ、該神経細胞を細胞分裂誘起物質で処理することなく成熟することを特徴とする、神経細胞を成熟する方法、（１３）（９）に記載の組成物を、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することを特徴とする、非神経細胞を神経細胞へ分化し、かつ該神経細胞を成熟する方法、（１４）（１０）に記載の組成物を、要すれば活性化後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することにより神経障害疾患を治療または悪性化抑制することを特徴とする、神経障害疾患を治療または悪性化抑制する方法、（１５）移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞、（１６）（１２）に記載の方法において、癌化神経細胞混入の恐れがない神経細胞を成熟することにより得られる、移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞、（１７）（１１）または（１３）に記載の方法により得られる、移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞、（１８）要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない非神経細胞または神経細胞に接触させ、該非神経細胞の神経細胞への分化、および／または該神経細胞の成熟の有無を調べることを特徴とする、非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、そして／または神経細胞を細胞分裂誘起物質で処理することなく成熟する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法、（１９）（１８）に記載の方法において、要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない非神経細胞に接触させ、該非神経細胞の神経細胞への分化の有無を調べることを特徴とする、非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法、（２０）（１８）に記載の方法において、要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない神経細胞に接触させ、該神経細胞の成熟の有無を調べることを特徴とする、神経細胞を細胞分裂誘起物質で処理することなく成熟する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法、（２１）（１８）に記載の方法において、要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない非神経細胞に接触させ、該非神経細胞の神経細胞への分化、および該神経細胞の成熟の有無を調べることを特徴とする、非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法を提供するものである。
【００１２】
【発明の実施の形態】
本発明の組成物、すなわち、（１）要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する組成物、（２）要すれば活性後に、神経細胞に接触させ、該神経細胞を細胞分裂誘起物質で処理することなく成熟する組成物、（３）要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟する組成物、（４）要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することにより神経障害疾患を治療または悪性化抑制する組成物は非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを有効成分とすることを特徴とする。本発明の組成物の有効成分は、非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグであればよく、将来合成されるものでも差し支えない。薬学的許容範囲の毒性しかない低分子物質とは、毒性が薬学的に許容できる範囲内である低分子物質を意味する。ここで用いる毒性には、急性毒性、亜急性毒性、慢性毒性、催奇形成等本来の意味の毒性の他、薬学的許容範囲外の重篤かつ高頻度の副作用も含まれる。低分子物質とは、タンパク質や高分子のペプチドなど高分子でない物質で、抗原性がなく、血液脳関門を通過可能な程度に低分子の物質である。本発明の組成物は本発明の方法と本発明の神経細胞を得るためにも用いる。本発明の探索する方法によって得られる非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、そして／または神経細胞を細胞分裂誘起物質で処理することなく成熟する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグは本発明の組成物に属する。
【００１３】
非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する能力を有する、薬学的許容範囲の毒性しかない低分子物質としては、これに制限されるわけではないが好ましくは、３−ヒドロキシ−３−メチルグルタリル−補酵素Ａ（ＨＭＧ−ＣｏＡ）還元酵素阻害剤等が挙げられる。ＨＭＧ−ＣｏＡ還元酵素阻害剤とはコレステロール生合成の重要な律速酵素であるHMG−ＣｏＡ還元酵素によりコレステロールをはじめとする各種ステロイドやテルペン生合成の前駆体で，ケトン体代謝の中間体であるＨＭＧ−ＣｏＡをメバロン酸に変換する反応を阻害する物質である。ＨＭＧ−ＣｏＡ還元酵素阻害剤としてはＨＭＧ−ＣｏＡ還元酵素の作用を阻害する物質で薬学的許容範囲の毒性しかない低分子物質であればよい。ＨＭＧ−ＣｏＡ還元酵素阻害剤としては、これに限定されるわけではないが好ましくは、アトルバスタチン、イタバスタチン（別名ＮＫ−１０４）、コンパクチン、シンバスタチン、ジヒドロコンパクチン、ダルバスタチン、ピタバスタチン、フルバスタチン、プラバスタチン、ベルバスタチン、ベロスタチン、メバスタチン、ロスバスタチン、ロバスタチン等、およびそれらの光学または幾何異性体、およびそれらのプロドラッグ、ならびに上記化合物又はそれらのプロドラッグの薬学的に許容することのできるそれらの塩ならびにそれらの水和物等が挙げられる。その中でも、アトルバスタチン、シンバスタチン、フルバスタチン、プラバスタチンとその薬学的に許容することのできるそれらの塩ならびにそれらの水和物等は高脂血症の治療薬として長年使用されて、その安全性が確認されているという意味でより好ましく、具体的にはアトルバスタチンカルシウム水和物、シンバスタチン、フルバスタチンナトリウム、プラバスタチンナトイウム等がこれに限定されるというわけではないが特に好ましいものとして挙げられる。
【００１４】
ＨＭＧ−ＣｏＡ還元酵素阻害剤は薬学的に許容することのできる限り、どのような製法でつくられても差し支えない。これに限定するわけではないが、ＨＭＧ−ＣｏＡ還元酵素阻害剤の製法を例示すると、アトルバスタチンは米国特許第４，６８１，８９３号および特開平３−５８９６７号（ＵＳＰ５２７３９９５）に開示された方法により、イタバスタチン（別名ＮＫ−１０４）は米国特許第５，１０２，８８８号に開示された方法により、コンパクチンは米国特許第４，８０４，７７０号に開示された方法により、シンバスタチンは特開昭５６−１２２３７５号（ＵＳＰ４４４４７８４）に開示された方法により、ジヒドロコンパクチンは米国特許第４，４５０，１７１号に開示された方法により、ダルバスタチンはヨーロッパ特許出願ＥＰ７３８５１０に開示された方法により、ピタバスタチンは特開平１−２７９８６６号（ＵＳＰ５８５４２５９及びＵＳＰ５８５６３３６）に開示された方法により、フルインドスタチンはヨーロッパ特許出願公開第３６３，９３４Ａ１中に開示された方法により、フルバスタチンは特表昭６０−５０００１５号（ＵＳＰ４７３９０７３）またはヨーロッパ特許出願ＥＰ３６３９３４に開示された方法により、プラバスタチンは特開昭５７−２２４０号（ＵＳＰ４３４６２２７）に開示された方法により、ベルバスタチンは米国特許第５，０８２，８５９号に開示された方法により、ベロスタチンは米国特許第４，４４８，７８４号および米国特許第４，４５０，１７１号に開示された方法により、メバスタチンは米国特許第３，９８３，１４０号に開示された方法により、ロスバスタチンは特開平５−１７８８４１号（ＵＳＰ５２６０４４０）に開示された方法により、ロバスタチンは特開昭５７−１６３３７４号（ＵＳＰ４２３１９３８）に開示された方法により調製することができる。
【００１５】
本発明の組成物は、（１）要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する、（２）要すれば活性後に、神経細胞に接触させ、該神経細胞を細胞分裂誘起物質で処理することなく成熟する、（３）要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟する、または（４）要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することにより神経障害疾患を治療または悪性化抑制する。したがって、本発明の組成物は、該組成物を体外的に非神経細胞または未熟の神経細胞に接触させ、該細胞を分化し、かつ／または成熟することにより、移植用の神経細胞を得るために有用である。また本発明の組成物は、非神経細胞または未熟の神経細胞を体内に注入した後に該組成物を経口投与又は非経口投与することにより体内において該細胞に接触させ、体内において該細胞を分化し、かつ／または成熟することにより、または外部から非神経細胞または未熟の神経細胞を体内に注入することなく、該組成物を経口投与又は非経口投与することにより体内において内在する非神経細胞または未熟の神経細胞に接触させ、体内において該細胞を分化し、かつ／または成熟することにより神経障害疾患を治療または悪性化抑制するために有用である。すなわち、これを含有する医薬は各種組織や細胞の細胞の退行・減少又は細胞死に起因する疾患の予防及び治療薬として有用である。本発明の組成物は、低分子であることから、体外投与は勿論、経口投与又は非経口投与（筋肉内、皮下、静脈内、坐薬など）のいずれでも投与できる。体外投与の場合は本発明の組成物中にプロドラッグが含まれているときは、適宜の方法で活性化してから用いた方がより大きな効果を得るので好ましい。プロドラッグが含まれていないときはそのまま用いてよい。体内投与の場合は多くの場合体内でプロドラッグが活性化するので、プロドラッグを予め活性化しておく必要は少ない。
【００１６】
経口用製剤を調製する場合、賦形剤、さらに必要に応じて、結合剤、崩壊剤、滑沢剤、着色剤、矯味矯臭剤などを加えた後、常法により、錠剤、被服錠剤、顆粒剤、カプセル剤、溶液剤、シロップ剤、エリキシル剤、油性又は水性の懸濁液剤などとする。賦形剤としては、例えば、乳糖、コーンスターチ、白糖、ブドウ糖、ソルビット、結晶セルーロスなどが挙げられる。結合剤としては、例えば、ポリビニルアルコール、ポリビニルエーテル、エチルセルロース、メチルセルロース、アラビアゴム、トラガント、ゼラチン、シェラック、ヒドロキシプロピルセルロース、ヒドロキシプロピルスターチ、ポリビニルピロリドンなどが挙げられる。
【００１７】
崩壊剤としては、例えば、デンプン、寒天、ゼラチン未、結晶セルロース、炭酸カルシウム、炭酸水素ナトリウム、クエン酸カルシウム、デキストラン、ペクチンなどが挙げられる。滑沢剤としては、例えば、ステアリン酸マグネシウム、タルク、ポリエチレングリコール、シリカ、硬化植物油などが挙げられる。着色剤としては、医薬品に添加することが許可されているものが使用できる。矯味矯臭剤としては、ココア末、ハッカ脳、芳香酸、ハッカ油、竜脳、桂皮末などが使用できる。これらの錠剤は、顆粒剤には、糖衣、ゼラチン衣、その他必要により適宜コーティングしてもよい。
【００１８】
注射剤を調製する場合、必要により、pH調整剤、緩衝剤、安定化剤、保存剤などを添加し、常法により、皮下、筋肉内、静脈内注射剤とする。注射剤は、溶液を容器に収納後、凍結乾燥などによって、固形製剤として、用事調製の製剤としてもよい。また、一投与量を容器に収納してもよく、また、多投与量を同一の容器に収納してもよい。
【００１９】
本発明組成物の試薬としての使用濃度は通常０．０１から１０００マイクロモル、好ましくは、０．１〜１００マイクロモルで使用し、医薬としての投与量は、ヒトの場合、成人１日当たり通常０．０１〜１０００ミリグラム、好ましくは、０．１〜１００ミリグラムの範囲で、１日量を１日１回、あるいは２〜４回に分けて投与する。なお、年齢、症状により適宜増減する。
【００２０】
本発明に用いる非神経細胞とは神経細胞以外の細胞で、本発明の組成物により神経細胞に分化し得る細胞を意味する。本発明の組成物により神経細胞に分化し得る細胞としては、これに限定されるわけではないが好ましくは、ＥＳ細胞、神経幹細胞、神経前駆細胞、骨髄幹細胞、間葉系細胞、骨髄間質細胞等が挙げられる。ＥＳ細胞、神経幹細胞、神経前駆細胞、骨髄幹細胞、間葉系細胞、骨髄間質細胞等のうち、非胎児性神経幹細胞、神経前駆細胞、骨髄幹細胞、間葉系細胞、骨髄間質細胞等はＥＳ細胞、胎児由来の神経幹細胞との比較において倫理性で問題が少なく、より好ましい。非胎児性神経幹細胞、神経前駆細胞、骨髄幹細胞、間葉系細胞、骨髄間質細胞等のうち、骨髄幹細胞、間葉系細胞、骨髄間質細胞等は容易に入手し易く、移植用の神経細胞に分化させる細胞としてはより好ましく、特に自家または自家と主要組織適合抗原を実質的に同じくするまたは非常に類似する、骨髄幹細胞、間葉系細胞、骨髄間質細胞は移植用の神経細胞に分化する細胞として、または体内に注入して体内で神経細胞に分化する細胞としてより好ましい。一方、神経細胞の移植や非神経細胞等の体内注入なしに、本発明の組成物を投与して体内に内在する神経細胞の再生を行うことを目的にする場合は、内在する神経幹細胞、神経前駆細胞、骨髄幹細胞、間葉系細胞、骨髄間質細胞等が分化して神経細胞になる。したがって、目的に応じて本発明に用いる非神経細胞を選ぶことがより好ましい。
【００２１】
本発明の方法に用いる非神経細胞または神経細胞の入手先は使用目的によって限定の度合いが異なるが、適合する限りどこから入手しても良い。使用目的がヒトを対象としない場合には限定が少ないが、ヒトに対して移植する神経細胞を得る目的の場合または体内に注入して体内で分化して神経細胞にする、または体内で神経細胞を成熟する場合は、ＥＳ細胞はヒトの胎児胚より、神経幹細胞と神経前駆細胞はヒトの胎児の脳より、骨髄幹細胞、間葉系細胞、骨髄間質細胞等は移植されるヒトまたはそのヒトと主要組織適合抗原が実質的に同じ、または非常に類似するヒトの骨髄より無菌的に採取するのが好ましい。神経細胞の移植や非神経細胞等の体内注入なしに、本発明の組成物を投与して体内に内在する神経細胞の再生を行うことを目的にする場合は、用いる非神経細胞は内在非神経細胞に限られる。非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、そして／または神経細胞を細胞分裂誘起物質で処理することなく成熟する能力を有する、薬学的許容範囲の毒性しかない低分子物質および／またはそのプロドラッグを探索する方法に用いる非神経細胞または神経細胞の入手先に限定はない。
【００２２】
本発明に用いる神経細胞とは成熟した神経細胞以外の神経細胞で、本発明の組成物により成熟し得る神経細胞を意味する。本発明の組成物により成熟し得る神経細胞は、非神経細胞を本発明の方法により分化して得られる神経細胞に限定されない．非神経細胞を本発明の方法以外の方法により分化して得られる神経細胞や神経幹細胞・神経前駆細胞から自然に分化した神経細胞も用いることができる．これら神経細胞のうち、非神経細胞を細胞分裂誘起物質で処理することなく分化させることにより、移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞が好ましい。該細胞は内在する神経細胞または本発明の非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する方法で得られる。
【００２３】
本発明の神経細胞は移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞に限定される。本発明の移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞は、本発明の方法、すなわち（１）本発明の組成物を、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する方法、（２）本発明の組成物を、要すれば活性後に、神経細胞に接触させ、該神経細胞を細胞分裂誘起物質で処理することなく成熟する方法、（３）本発明の組成物を、要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟する方法により得られる。
【００２４】
本発明の神経細胞は自然に分化した神経細胞または自然に分化成熟した神経細胞に限りなく近い神経細胞である．これに限定されるわけではないが、移植用神経細胞としては最適である．本発明の神経細胞は、脳に移植の場合は中枢神経系に移植するのに適切であるように様々な方法で投与されうる。これに限定されるわけではないが、非経口投与、くも膜下腔投与、脳室内投与および黒質内投与等が挙げられる。
【００２５】
本発明の方法、すなわち、（１）非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する方法、（２）神経細胞を細胞分裂誘起物質で処理することなく成熟する方法、（３）該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟する方法、（４）該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することにより神経障害疾患を治療または悪性化抑制する方法は、本発明の組成物を、要すれば活性後に、非神経細胞または神経細胞を細胞分裂誘起物質で処理することなく、該非神経細胞または神経細胞に接触させることを特徴とする。本発明の特徴以外の操作や工程は公知の操作や工程でよく、それらは例えば前述の文献（特表２００２−５１３５４５、Ｍ．Ｄｅｚａｗａら、Ｅｕｒ．Ｊ．Ｎｅｕｒｏｓｃｉ．２００１年１４巻１７７１頁、Ｗ．Ｄｅｎｇら、Ｂｉｏｃｈｅｍ．Ｂｉｏｐｈｙｓ．Ｒｅｓ．Ｃｏｍｍｕｎ．２００１年２８２巻１４８頁、Ｄ．Ｗｏｏｄｂｕｒｙら、Ｊ．Ｎｅｕｒｏｓｃｉ．Ｒｅｓ．２０００年６１巻３６３頁、Ｊ．Ｓａｎｃｈｅｚ−Ｒａｍｏｓら、Ｅｘｐ．Ｎｅｕｒｏｌ．２０００年１６４巻２４７頁、Ｊ．Ｋｏｈｙｙａｍａら、Ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ、２００１年６８巻２３５頁、Ｈ．Ｎａｋａｔｏｍｉら，Ｃｅｌｌ、２００２年１１０巻４２９頁、特表２００１−５１７６１７）および他の文献（Ｊ．Ｋｕｎｌｉｎら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．２００１年９８巻４７１０頁）に開示されている。また、すでに本発明の組成物の項で説明したように、本発明の組成物を非神経細胞または未熟の神経細胞に接触させる方法としては、（１）本発明の組成物を体外的に非神経細胞または未熟の神経細胞に接触させる方法、（２）非神経細胞または未熟の神経細胞を体内に注入した後に本発明の組成物を経口投与又は非経口投与することにより体内において該細胞に接触させさせる方法、（３）外部から非神経細胞または未熟の神経細胞を体内に注入することなく、本発明の組成物を経口投与又は非経口投与することにより体内において内在する非神経細胞または未熟の神経細胞に接触させる方法等がある。目的に応じて適宜選択すればよい。これらの工程も本発明の特徴以外は前述の文献に開示されている。
【００２６】
本発明の探索方法、すなわち（１）非神経細胞を細胞分裂誘起物質で処理することなく分化させて神経細胞にする能力を有する、薬学的許容範囲の毒性しかない低分子物質を探索する方法、（２）神経細胞を細胞分裂誘起物質で処理することなく成熟する能力を有する、薬学的許容範囲の毒性しかない低分子物質を探索する方法、（３）非神経細胞を細胞分裂誘起物質で処理することなく分化成熟させて神経細胞にする能力を有する、薬学的許容範囲の毒性しかない低分子物質を探索する方法は、要すれば活性後に、薬学的許容範囲の毒性しかない低分子物質を、細胞分裂誘起物質で処理していない非神経細胞または神経細胞に接触させ、該非神経細胞の神経細胞への分化の有無、または神経細胞の成熟の有無または神経細胞への分化と該神経細胞の成熟の有無を調べることを特徴とる。すなわち、細胞分裂誘起物質で処理しない非神経細胞または神経細胞を用いることと薬学的許容範囲の毒性しかない低分子物質を探索の対象とすることが特徴で、この特徴以外の工程は前述の文献に開示されている。
【００２７】
【実施例】
以下、実施例により本発明を更に詳細に説明するが、本発明はこれらの実施例に限定されるものではない。なお、操作や工程のうち、公知部分の詳細は以下の文献にも開示されている。Ｄ．Ｓｔｏｋｅｓら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．１９９８年９５巻３９０８頁、Ｒ．Ｊ．Ｇｅｒｓｏｎら、Ａｍ．Ｊ．Ｍｅｄ．１９８９年８７巻２８Ｓ頁、Ｐ．Ａ．Ｔｏｄｄら、Ｄｒｕｇｓ、１９９０年４０巻５８３頁、Ｐ．Ｈｏｆｓｔｅｔｔｅｒら、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．２００２年９９巻２１９９頁、Ｅ．Ｚｅａ−Ｌｏｎｇａら、Ｓｔｒｏｋｅ、１９８９年２０巻８４年、メルクマニュアル１７版日本版。
【００２８】
実施例１．インビトロ分化：骨髄は、１０週齢の雌ＳＤラットの大腿骨と脛骨またはヒト骨髄吸引物から得た。ヒト骨髄を、ダルベッコ修飾イーグル最少必須培地（ＤＭＥＭ）（低濃度グルコース）（ＧＩＢＣＯ／ＢＲＬ）および１０％胎児血清（ＦＢＳ）（ＨＹＣＬＯＮＥ）、１００単位／ミリリットルペニシリン、１００マイクログラム／ミリリットルストレプトマイシン（ＳＩＧＭＡ）で１：１に希釈し、密度勾配で、５分間８００グラムで遠心分離した。上清および界面を合せ、１０％胎児血清を含む
MEMで約２０ミリリットルに希釈し、ポリエチレンイミンでコートされたプラスチック製フラスコにプレートした。ラット骨髄を、１０ミリリットルＰＢＳおよび５％アルブミンに入れた。細胞を、この培地中で洗浄し、８００ｒｐｍで５分間遠心分離した。細胞を、２ｍＭグルタミン、１０％血清（ＦＢＳ）、１００単位／ミリリットルペニシリン、１００マイクログラム／ミリリットルストレプトマイシン（ＳＩＧＭＡ）を加えたDMEM（低濃度グルコース）からなる増殖培地中に再懸濁した。細胞を、フラスコ中で１日間培養し、非接着細胞を除去した。その後、毎３日間培養し、培地を交換した。培養物が、コンフルエンシーにほとんど達した後、トリプシン（０．２５％）処理を行い、細胞を剥がした。細胞を８００ｒｐｍで５分間遠心分離して集めた。細胞を上記培地に懸濁し、上記プラスチック製フラスコに植え、上記培地で培養した。この操作を4回繰返した。この操作に通常行う上皮成長因子(EGF)または血小板由来成長因子(PDGF)のようなマイトジェン添加はしなかった。
【００２９】
骨髄間質細胞のニューロン様細胞への分化は、次の工程で行った。まだコンフルエンシーに達していない骨髄間質細胞を上記プラスチック製フラスコ底から剥がし、１０，０００細胞数／平方センチメートルの濃度で０．０５ミリグラム／ミリリットルポリ−Ｄ−リジン（ＳＩＧＭＡ）でコートした２４穴の培地プレートの１０％胎児血清（ＦＢＳ）、１００単位／ミリリットルペニシリン、１００マイクログラム／ミリリットルストレプトマイシンを加えたＤＭＥＭ（低濃度グルコース）に植えた。このとき、前もって４ミリグラムのシンバスタチンのプロドラッグを１００マイクロリットルのエタノールに溶かし、０．１ＮのＮａＯＨを加え摂氏５０度で２時間反応させ、ＨＣＬでｐＨを７．０に調整し、４ミリグラム／ミリリットルの濃度にし、摂氏4度で保存しておいた活性化シンバスタチンを２．５マイクロモル、５マイクロモル、１０マイクロモル添加して７２時間培養した。通常添加する脳由来成長因子（ＢＤＮＦ）、グリア由来神経栄養因子（ＧＤＮＥ）および神経成長因子（ＮＧＦ）などのニューロン成長因子は一切加えなかった。
【００３０】
培養した細胞を４％（ｗｔ／ｖｏｌ）パラホルムアルデヒド固定０．１モルリン酸緩衝液ｐＨ７．４で３０分固定した。神経細胞特異的エノラーゼ（ＮＳＥ）とニューロン特異的核抗原（ＮｅｕＮ）染色のため、洗浄後、細胞を０．２５％トリトンＸ−１００と５％正常のヒツジ血清（ＰＢＳＴＳ）を含有するＰＢＳで1時間室温でブロックした。次に細胞をＰＢＳＴＳで希釈した第１次抗体液中摂氏４度で一晩培養した。ＰＢＳで３回洗浄した後、室温でアビジンービオチン複合体を１時間室温で接触させた。ＰＢＳで３回洗浄した。ＭＡＰ２とタウ染色のためには、洗浄後、細胞を０．２５％トリトンＸ−１００と５％正常のヒツジ血清（ＰＢＳＴＳ）を含有するＰＢＳで1時間室温でブロックした。次に細胞をＰＢＳＴＳで希釈した第１次抗体液中摂氏４度で一晩培養した。ＰＢＳで３回洗浄した後、細胞をＰＢＳＴＳで希釈した第２次抗体液中摂氏４度で１時間暗所で培養した。洗浄後、細胞をグリセロール／ＰＢＳ（１：１）で包埋し、蛍光顕微鏡で観察した。第１次抗体はラットＮＳＥに対するウサギモノクローナル抗体を１：２００、ラットＮｅｕＮに対するマウスモノクローナル抗体を１：２００、ＭＡＰ２に対するマウスマウスモノクローナル抗体を５マイクログラム／ミリリットル、タウに対するウサギモノクローナル抗体を１：２００に希釈して用いた。第２次抗体はウサギＩｇＧに対するビオチン化ヒツジ抗体を１０マイクログラム／ミリリットル、ラットＩｇＧに対するマウスＩｇＧに対するビオチン化ウマ抗体を１０マイクログラム／ミリリットルに希釈して用いた。ＭＡＰ２はマウスＣｙ−３に対するヒツジ抗体（５マイクログラム／ミリリットル）で可視化し、タウはウサギＣｙ２に対するヒツジ抗体で可視化した。
【００３１】
ラット骨髄から得られた骨髄間質細胞を分離し、プラスチックプレート上増殖を４回繰返した。大きな平らな細胞と比較的引伸ばされた紡錘体状細胞の２種類に分かれた。これに対し、０．２５マイクロモル、５マイクロモルと１０マイクロモルの活性化したシンバスタチンが接触した骨髄間質細胞は驚くべきことに２４時間後に形態を変化した。形態変化した細胞は丸みを帯びた細胞体と屈折を示し、単純な２極性の細胞や非常に枝分かれした複雑な多極性の細胞等神経様細胞の性格を示し、シナプス様の形態形成も見られた（図１）。４８時間後には神経様細胞のシナプス形態も明確になった（図２）。活性化したシンバスタチンを接触させた神経細胞様の細胞を２４時間、４８時間、７２時間培養後に固定し、神経マーカーのＮＳＥ用に染色した。シンバスタチンを接触させなかった平板状の骨髄間質細胞は非常に低くしか染色されなかった。しかし、ＮＳＥタンパク質のレベルは検知可能であった。これに対し、活性化したシンバスタチンを接触させた神経細胞様の細胞はＮＳＥを顕著に発現させた。我々は次にＮｅｕＮの存在につぃて調べた。シンバスタチンを接触させなかった平板状の骨髄間質細胞にＮｅｕＮが検出できなかったのに対し、活性化したシンバスタチンを接触させた神経細胞様の細胞はＮｅｕＮを発現した形態を示した。活性化したシンバスタチンを接触させた神経細胞様の細胞は細胞体中にＭＡＰ２とタウを発現していた（図３と図４）。これに対し、シンバスタチンを接触させなかった平板状の骨髄間質細胞は細胞体中にＭＡＰ２とタウを発現しなかった。
【００３２】
活性化シンバスタチンを接触させたままＮＥＳ陽性、ＮｅｕＮ陽性、ＭＡＰ２陽性、タウ陽性の形態学的にも神経細胞様の細胞を培養続けた。細胞から樹状突起が複雑に伸び、周りの多くの細胞とシナプスを形成し、神経細胞が成長したことが確認された。したがって、シンバスタチン接触２４時間後に出現した神経様細胞は神経細胞そのものであることが確認された。非神経細胞から神経細胞へ２４時間以内に分化した報告は未だなく、本実施例が初めてである。
【００３３】
実施例２．実施例１においてシンバスタチンの代わりに、アトルバスタチン、イタバスタチン、コンパクチン、ジヒドロコンパクチン、ダルバスタチン、ピタバスタチン、フルインドスタチン、フルバスタチン、プラバスタチン、ベルバスタチン、ベロスタチン、メバスタチン、ロスバスタチン、ロバスタチンを別々に用いて同様にラットの骨髄間質細胞をマイトジェン処理無しに神経細胞に分化させ、その分化させた神経細胞を成熟させた。
【００３４】
実施例３．実施例１においてシンバスタチンの代わりに、アトルバスタチンとフルバスタチンの混合物を用いて同様にラットの骨髄間質細胞をマイトジェン処理無しに神経細胞に分化させ、その分化させた神経細胞を成熟させた。
【００３５】
実施例４．インビボ分化：骨髄を１０週齢の雌ＳＤラットの大腿骨と脛骨またはヒト骨髄吸引物から得た。ヒト骨髄を、ダルベッコ修飾イーグル最少必須培地（ＤＭＥＭ）（低濃度グルコース）（ＧＩＢＣＯ／ＢＲＬ）および１０％胎児血清（ＦＢＳ）（ＨＹＣＬＯＮＥ）、１００単位／ミリリットルペニシリン、１００マイクログラム／ミリリットルストレプトマイシン（ＳＩＧＭＡ）で１：１に希釈し、密度勾配で、５分間８００ｇで遠心分離した。上清および界面を合せ、１０％胎児血清を含むMEMで約２０ミリリットルに希釈し、ポリエチレンイミンでコートされたプラスチック製フラスコにプレートした。ラット骨髄を、１０ミリリットルＰＢＳおよび５％アルブミンに入れた。細胞を、この培地中で洗浄し、８００ｒｐｍで５分間遠心分離した。細胞を、２ミリモルグルタミン、１０％血清（ＦＢＳ）、１００単位／ミリリットルペニシリン、１００マイクログラム／ミリリットルストレプトマイシン（ＳＩＧＭＡ）を加えたDMEM（低濃度グルコース）からなる増殖培地中に再懸濁した。細胞を、フラスコ中で１日間培養し、非接着細胞を除去した。その後、毎３日間培養し、培地を交換した。培養物が、コンフルエンシーにほとんど達した後、トリプシン（０．２５％）処理を行い、細胞を剥がした。細胞を８００ｒｐｍで５分間遠心分離して集めた。細胞を上記培地に懸濁し、上記プラスチック製フラスコに植え、上記培地で培養した。この操作を4回繰返した。この操作に通常行う上皮成長因子(EGF)または血小板由来成長因子(PDGF)のようなマイトジェン添加はしなかった。
【００３６】
LXSNベクター（CLONTECH）を用いて発現マーカーとして緑蛍光タンパク質をコードし、ネオマイシン（Ｇ４１８）選択性マーカーとしてアミノグリコシドポスホトランスフェラーゼをコードしたレトロウイルス性のプラスミドをつくった。カルシウムリン酸沈殿を用いてフェニックスアンホトロピックパッケェジした細胞（ＡＴＣＣ）にＬＸＳＮ−ＧＦＰプラスミドを感染させた。感染後４８時間後にウイルス性の上清を集め、穴の大きさが０．４５−マイクロメートルのフィルターで濾した。使うまで摂氏マイナス８０度で保存した。ウイルスを採取した時にフェニックスアンホトロピックパッケェジした細胞のGFPの発現を解析して確認した。約１００，０００個の骨髄間質細胞を２０％加熱不活化したＦＣＳを含む完全培地の入った２１．０平方センチメートルのプレートに蒔き、一晩培養した。翌日２．５ミリリットルの２０％加熱不活化したFCSを含む完全培地中で該細胞を５００マイクロリットルのウイルス上清液と８マイクログラム／ミリリットルのポリブレン（Ｓｉｇｍａ）と混合して感染させた。ウイルス感染した日を第１日とした。第２日にウイルス感染操作を繰返した。第３日に培養液を２０％の非加熱ＦＢＳを含む新しい完全培地と交換した。第４日に細胞を２００マイクログラム／ミリリットルのＧ４１８（Ｓｉｇｍａ）を含む完全培地の入った５５．０平方センチメートルのプレートに１：３の割合で分け、２１日間選択した。ＧＦＰプラスミドが遺伝子に入り込んだ骨髄間質細胞を得た。この細胞の増殖を３回繰返して増やした。
【００３７】
雄のＳＤラットに１３７Ｃｓを９．０（９００ｒａｄｓ）の強度っで照射した。照射２４時間後にこのラットの尾っぽの静脈にＧＦＰプラスミドが遺伝子に入り込んだ骨髄間質細胞約５，０００，０００個１ミリリットルを注入した。骨髄間質細胞移植ラットを８週間飼育した。
【００３８】
骨髄間質細胞移植ラットを４％イソフルラン、６６％Ｎ２Ｏ、３０％酸素の混合ガスで麻酔し、１．５％のイソフルラン、６８．５％Ｎ２Ｏ、３０％酸素の混合ガスで麻酔を維持した。血圧、血液冲のガス分圧、血糖値を左の大腿骨動脈でモニターした。直腸温度、中大脳動脈閉塞の反対側の側頭筋温度、を常時モニターして、加熱パッドで摂氏３７．０−３７．５度に維持した。Ｅ．Ｚｅａ−Ｌｏｎｇａ等の方法に従い、縫合糸による管内中大脳動脈閉塞によってラットに虚血を起した。すなわち、左外頚動脈を６−０絹縫合糸で結び、遠位を切開し、左内頚動脈を分離し、迷走神経から離した。左内頚動脈の頭蓋外枝をその枝分れの根元のところで、６−０絹縫合糸で結んだ。円形チップの付いた３−０外科用単層ナイロン縫合糸を外頚動脈断片を通して左内頚動脈に導き、その頚動脈の枝分れ部分より２０ミリメートル過ぎたところで、９０分間結び、その後直ぐ解いた。
【００３９】
縫合糸による閉塞による中大脳動脈閉塞を起したラット１０匹に対して、即日より殺す日まで毎日２０ミリグラム／キログラムの割合でシンバスタチンを経口投与した。対照として中大脳動脈閉塞を起した別のラット１０匹に対してシンバスタチンを投与しなかった。
【００４０】
中大脳動脈閉塞後第１，３，７，１４日にこれらのラット２匹ずつをキシラジン１０ミリグラム／キログラムとケタミン８０ミリグラム／キログラムで麻酔して殺した。直ちに、生理的食塩水で還流し、続いて４％パラフォルムアルデヒドで還流した。脳組織を２ミリメートル間隔で７等分の環状ブロックに切断した。前頭面で」各切断ブロックから６マイクロメートルの厚さの切片をつくり、実施例１と同様にＧＦＰとＮｅｕＮに対する二重免疫標識した。シンバスタチンを投与したラットは中大脳動脈閉塞後第３日に殺した群からＧＦＰとＮｅｕＮの二重陽性が認められ、日数に比例して二重陽性が強くなった。シンバスタチンを投与しなかったラットは中大脳動脈閉塞後１４日になってもＧＦＰとＮｅｕＮの二重陽性は確認できなかった。このことから、移植した骨髄間質細胞が脳血管の関門を通って、脳内に浸潤し、シンバスタチンによって神経細胞へ分化したことが確認された。
【００４１】
殺さなかったラットは３ヶ月間シンバスタチンを投与した。対照ラットにシンバスタチンを投与しなかった。このラットについて高架式十字迷路試験を行った。高架式十字迷路試験とは高架式の十字形の細長い平板の一方にラットの餌を置き、他方の一つにラットを放すと、ラットは最初は試行錯誤で十字路の一方の行き止まりまで行き、そこに餌が無いことを知って、十字路にもどり、別の道を行く、そうこうしているうちにラットが餌の置いてある端にたどりつく。もう一度実験をすると記憶力のあるラットは迷うことなく、餌がおいてある道を進み餌にありつく。しかし、記憶力の悪いラットは１回目と同様に試行錯誤を繰返す。この試験によりラットの記憶力が測定できる。シンバスタチンを投与しなかったラットは餌にたどり着くまでに多くの時間を要し、記憶力が極度に低下していた。これに対し、シンバスタチンを投与したラットは餌にたどり着くまでの時間が短く、ある程度の記憶力を回復しており、シンバスタチンにより再生した神経細胞は機能性があることを示唆した。
【００４２】
実施例５．実施例４においてシンバスタチンの代わりに、アトルバスタチン、イタバスタチン、コンパクチン、ジヒドロコンパクチン、ダルバスタチン、ピタバスタチン、フルインドスタチン、フルバスタチン、プラバスタチン、ベルバスタチン、ベロスタチン、メバスタチン、ロスバスタチン、ロバスタチンを別々に投与して同様の操作をし、移植骨髄間質細胞が人工的に脳虚血を起させたラットの脳内で神経細胞へ分化し、かつ神経細胞機能的に再生させたことを確認した。
【００４３】
実施例６．実施例４においてシンバスタチンの代わりに、アトルバスタチンとフルバスタチンの混合物を投与して同様の操作をし、同様の効果を得た。
【００４４】
実施例７．アルツハイマー病に対する薬効評価。日本語で読み書きができ、自分の意志を医者に伝えることが可能であるがアルツハイマー病の可能性ありという臨床診断を受けている患者8人で、プラバスタチンナトリウム等ＨＭＧ−ＣｏＡ還元酵素阻害剤に過敏症の既往歴がなく、腎機能と肝代謝が低下している疑いがなく、かつ妊娠または妊娠している可能性のある人および授乳婦以外の人に対して、下記ミニメンタルステート試験を行う。なお、患者は重症度の指標が２５以下であることを確認し、重症度の指標が２５以上の患者はいる場合は、重症度の指標が２５以下である患者と入れ替える。また、測定前４ヶ月以内に治験薬を使用していた患者は除外する。また、シメチジン、プロプラノロール等親油性６遮断剤やクロニジン、抗コリン作動薬、および抗コリン活性を有する抗うつ剤、神経弛緩剤、推定認識力増強物質および中枢神経刺激物質、または半減期が長いベンゾジアゼピンは投与を禁止する。
【００４５】
ミニメンタルステート試験の内容：見当識（１．今の年は？１点、季節は？１点、曜日は？１点、日付は？１点、月は？１点、２．私たちが今いる県の名前は？１点、郡の名前は？１点、市／町の名前は？１点、階数は？１点、は？１点、住所／建物の名前は？１点）、記銘力（３．医師が３つの物の名前を書く秒かけて言う。医師が言った後で次に患者に３つ全部の名前を尋ねる。患者が３つ全てを正しく言えるまで答えを繰返させる。３点）、注意と計算（４．１００から続けて７を引かせる。正しい答え１つにつき１点与える。５回答えたところで終える。５点）、想起（５．質問３で覚えた３つの物の名前を尋ねる。正しい答え１つにつき１点与える。３点）、言語（６．医師が鉛筆と時計を指す。患者に医師が指した物の名前を言わせる。２点、７．患者に「いいえ、もし、そして、しかし」と言わせる。１点、８．患者に３段階の命令を与え、それに従ってもらう。「右手で紙をつまみ上げてください。紙を半分に折ってください。紙を机の上に置いてください。」３点、９．患者に次の指示を読ませ、それに従ってもらう。「目を閉じてください。」１点、１０．患者に自由に１つの文章を書いてもらう。文章には守護つと目的語１つを含み、しかも意味をなしている必要がある。得点には書字の誤りは考慮しない。１点、１１．１辺が約５センチメートルの２つの五角形が１頂点だけ重なった図形を見せ、患者に書き写させる。全ての角および角度が保たれており、しかも交わった領域が四角形をなしていれば１点をあたえる。１点）、合計３０点。
【００４６】
医師の管理下に被験者に市販の医療用プラバスタチンナトリウム１０ミリグラム錠を１日２回、６ヶ月間投与する。６ヵ月後にミニメンタルステート試験を行う。ミニメンタルステート試験によりプラバスタチンナトリウム投与による見当識、記銘力、注意と計算、想起、言語の改善を確認できる。
【００４７】
【発明の効果】
本発明の組成物は、（１）要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する能力、または（２）要すれば活性後に、神経細胞に接触させ、該神経細胞を細胞分裂誘起物質で処理することなく成熟する能力、または（３）要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟する能力、または（４）要すれば活性後に、非神経細胞に接触させ、該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することにより神経障害疾患を治療または悪性化抑制する能力を有する。したがって、本発明組成物を体外投与することにより、移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞をつくるために有用であり、また非神経細胞または移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞を体内に注入した後、または注入せずに、本発明組成物を体内投与することにより、アルツハイマー病、筋萎縮性側索硬化症、脳腫瘍、小脳変性症、交通性水頭症、ハンチントン病、前頭葉への照射、多発性硬化症、正常圧水頭症、パーキンソン病、ピック病、進行性多巣性白質脳症、進行性核上麻痺、拳闘家痴呆、脳外傷、外科手術、脳腫瘍、慢性硬膜下血腫、脳卒中、脳血管性痴呆、ウィルソン病細菌性心内膜炎、クロイツフェルト・ヤコブ病、ゲルストマン・シュトロイスラー・シャインカー病、HIV関連疾患、神経梅毒、結核性および真菌性髄膜炎、ウイルス性脳炎、無酸素症、B１２欠乏症、慢性的な薬物-アルコール-栄養性乱用、葉酸欠乏症、副甲状腺機能亢進症に伴う高カルシウム血症、低血糖、甲状腺機能低下症、肝性脳症、肺性脳症、尿毒素性脳症等の臓器系不全、ペラグラ等由来の神経障害疾患の治療または悪性化抑制薬として有用である。
【００４８】
本発明の方法は、（１）非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化する方法、（２）神経細胞を細胞分裂誘起物質で処理することなく成熟する方法、（３）該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟する方法、（４）該非神経細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することにより神経障害疾患を治療または悪性化抑制する方法である。したがって、本発明方法は移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞をつくる方法として有用であり、また前述の神経障害疾患を治療または悪性化抑制する方法として有用である。
【００４９】
本発明の神経細胞は移植拒絶反応を起す高分子と癌化神経細胞の混入の恐れがない神経細胞である。したがって、自然に分化した神経細胞または自然に分化成熟した神経細胞に限りなく、研究用または治療用として有用である。
【００５０】
本発明の探索方法は、（１）非神経細胞を細胞分裂誘起物質で処理することなく分化させて神経細胞にする能力を有する、薬学的許容範囲の毒性しかない低分子物質を探索する方法、（２）神経細胞を細胞分裂誘起物質で処理することなく成熟する能力を有する、薬学的許容範囲の毒性しかない低分子物質を探索する方法、（３）非神経細胞を細胞分裂誘起物質で処理することなく分化成熟させて神経細胞にする能力を有する、薬学的許容範囲の毒性しかない低分子物質を探索する方法である。これらの方法により有用な医薬品や試薬を創出する可能性があるので有用である。
【図面の簡単な説明】
【図１】５マイクロモルの活性化シンバスタチン２４時間接触による骨髄間質細胞の神経細胞への分化。普通光顕微鏡写真。倍率１００倍。スケールバーの長さ：５０マイクロメーター（実施例１）
【図２】５マイクロモルの活性化シンバスタチン４８時間接触による骨髄間質細胞の神経細胞への分化。普通光顕微鏡写真。倍率１００倍。スケールバーの長さ：５０マイクロメーター（実施例１）
【図３】５マイクロモルの活性化シンバスタチン２４時間接触による骨髄間質細胞の神経細胞への分化。ＭＡＰ２（緑色）とタウ（赤色）の二重免疫標識標本の蛍光光顕微鏡写真。倍率１００倍。スケールバーの長さ：５０マイクロメーター（実施例１）
【図４】５マイクロモルの活性化シンバスタチン４８時間接触による骨髄間質細胞の神経細胞への分化。ＭＡＰ２（緑色）とタウ（赤色）の二重免疫標識標本の蛍光顕微鏡写真。倍率１００倍。スケールバーの長さ：５０マイクロメーター（実施例１）[0001]
[Technical field to which the invention belongs]
The present invention comprises (1) a pharmaceutically acceptable low molecular weight substance and / or prodrug thereof having an ability to differentiate non-neuronal cells into neurons without being treated with a mitogenic substance, and / or a prodrug thereof as an active ingredient. A composition that contacts non-neuronal cells after activation if necessary, and differentiates the non-neuronal cells into neurons without being treated with a mitogenic substance, (2) cell division of non-neuronal cells A pharmacologically acceptable low molecular weight substance capable of differentiating into a neuron without treatment with an inducer is a 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor The composition according to (1) selected from: (3) the HMG-CoA reductase inhibiting composition is atorvastatin, itavastatin, compactin, simvastatin, dihydrocompact , Dalvastatin, pitavastatin, fluindostatin, fluvastatin, pravastatin, bervastatin, verostatin, mevastatin, rosuvastatin, lovastatin, and their optical or geometric isomers, and their prodrugs, as well as the above compounds or their prodrugs The composition according to (2), which is selected from pharmaceutically acceptable salts thereof and hydrates thereof, (4) the HMG-CoA reductase inhibitor composition is atorvastatin calcium hydrate, simvastatin, fluvial The composition according to (2) selected from statin sodium and pravastatin sodium, and (5) the composition according to (1) to (4), wherein the non-neuronal cell is selected from a myeloid non-neuronal cell and a neuronal non-neuronal cell (6) Myeloid non-neuronal cells are bone marrow stems The composition according to (5), selected from cysts, mesenchymal cells, and bone marrow stromal cells; (7) the composition according to (5), wherein the non-neuronal nervous system cells are selected from neural stem cells and neural progenitor cells; (8) The composition according to (1) to (7) is used as an active ingredient, and if necessary, after activation, it is brought into contact with a nerve cell and the nerve cell is treated with a mitogenic substance. A composition that matures completely, (9) characterized in that the composition according to (1) to (7) is used as an active ingredient, and if necessary after contact with non-neuronal cells, A composition that differentiates into a nerve cell without treatment with a mitogenic substance and matures the nerve cell, (10) The composition according to (1) to (7) is an active ingredient, and if necessary, after activation, Without contact with non-neuronal cells and without treatment of the non-neuronal cells with a mitogenic substance A composition for treating or malignantly treating a neuropathic disease, characterized in that the neuropathic disease is treated or malignant-suppressed by differentiating into a transcellular cell and maturing the neuron, (11) (1) The composition according to (7) is contacted with non-neuronal cells after activation if necessary, and the non-neuronal cells are differentiated into nerve cells without being treated with a mitogenic substance. A method for differentiating cells into nerve cells, wherein the composition according to (12) (8) is contacted with nerve cells after activation if necessary, and the nerve cells are matured without being treated with a mitogenic substance. A method of maturation of nerve cells, characterized by comprising: contacting the non-neuronal cells with the composition according to (13) or (9), if necessary after activation, and treating the non-neuronal cells with a mitogenic substance Without being differentiated into nerve cells and the god A method of differentiating non-neuronal cells into nerve cells and maturating the nerve cells, characterized by maturation of the cells, and (14) the composition according to (10), Contacting a nerve cell, differentiating the non-neuronal cell into a nerve cell without treating with a mitogenic substance, and maturating the nerve cell to treat or suppress malignant neuropathy, A method for treating or suppressing malignant transformation of a neuropathy disease, (15) a nerve cell that is not likely to be mixed with a macromolecule that causes transplant rejection and cancerous nerve cells, and (16) the method according to (12), (17) (11) or (13), a nerve cell obtained by maturation of a nerve cell that is not likely to be contaminated with a nerve cell, and that is not likely to be contaminated with a macromolecule that causes transplant rejection and a cancerous nerve cell. Obtained by the method (18) If necessary, a low molecular weight substance having a pharmaceutically acceptable tolerance can be used as a cell division-inducing substance after activation, if necessary. A non-neuronal cell or a neuronal cell is contacted, and the non-neuronal cell is examined for differentiation into the neuronal cell and / or maturation of the non-neuronal cell, and a non-neuronal cell mitogen A pharmaceutically acceptable low molecular weight substance and / or prodrug thereof capable of differentiating into a nerve cell without being treated with and / or maturing the nerve cell without being treated with a mitogenic substance. In the method described in (19) and (18), a non-neural cell that has not been treated with a mitogenic substance is treated with a low-molecular-weight substance that has only a pharmaceutically acceptable range after activation, if necessary. A non-neuronal cell having a capability of differentiating into a neuronal cell without being treated with a mitogenic agent, characterized in that the non-neuronal cell is examined for the presence or absence of differentiation into a neuron by contacting the cell In the method described in (20) and (18), a low molecular weight substance having only a pharmaceutically acceptable range is activated after activation in the method according to (20) or (18). A pharmaceutically acceptable agent having the ability to mature a neuron without treatment with a mitogen, characterized by contacting a neuron that has not been treated with a mitogen and examining the maturity of the neuron. A method for searching for a low-molecular-weight substance having only a range of toxicity and / or a prodrug thereof, (21) In the method described in (18), if necessary, after the activity, only a pharmaceutically acceptable toxicity can be obtained. A non-neural cell, wherein the non-neural cell is contacted with a non-neuronal cell that has not been treated with a mitogenic substance, and the non-neuronal cell is differentiated into a neuronal cell and examined for the presence or absence of maturation of the neuronal cell. The present invention relates to a method for searching for a low-molecular substance having a pharmaceutically acceptable toxicity and / or a prodrug thereof having the ability to differentiate into a nerve cell without treatment with a mitogenic substance and to mature the nerve cell. .
[0002]
[Prior art]
Alzheimer's disease, amyotrophic lateral sclerosis, brain tumor, cerebellar degeneration, traffic hydrocephalus, Huntington's disease, frontal lobe irradiation, multiple sclerosis, normal pressure hydrocephalus, Parkinson's disease, Pick's disease, progressive multifocal Leukoencephalopathy, progressive supranuclear palsy, fighting dementia, brain injury, surgery, brain tumor, chronic subdural hematoma, stroke, cerebrovascular dementia, Wilson's bacterial endocarditis, Creutzfeldt-Jakob disease, Gerstmann-Stroisler-Scheinker disease, HIV-related disease, neurosyphilis, tuberculosis and fungal meningitis, viral encephalitis, anoxia, B12 deficiency, chronic drug-alcohol-nutrient abuse, folate deficiency, Hypercalcemia associated with hyperparathyroidism, hypoglycemia, hypothyroidism, hepatic encephalopathy, pulmonary encephalopathy, organ system failure such as uremic encephalopathy, neurological diseases such as pellagra, fibrosis, immune response, Ductal lesions, nutrients and oxygen deprivation, infection or the like, nerve cells degeneration, reduction, and cell death, or injury, by being excluded, tissues and organs are thought to develop lose its function. Therefore, it is conceivable to replace or regenerate lost neurons by some method as one method for treating or preventing these diseases.
[0003]
Therefore, ES cells or neural stem cells have attracted attention. This is because ES cells or neural stem cells are undifferentiated cells, and when nerve cells die, they start to differentiate so as to compensate for the lost cells and may greatly contribute to the maintenance of biological functions. In recent years, attempts have been made to improve or treat the disease state by transplanting ES cells or neural stem cells to tissues that have lost their function, and specifically differentiating them into cells that express individual biological functions. However, ES cells are embryonic stem cells and have the ethical problem of using human embryos as raw materials and the problem of transplantation antigenicity. On the other hand, in the case of neural stem cells, only fetal-derived neural stem cells are difficult to obtain, and fetal-derived neural stem cells have ethical problems and transplant antigenic problems.
[0004]
Therefore, attention has been focused on non-neuronal cells other than ES cells and fetal-derived neural stem cells that can differentiate and mature into neurons. Bone marrow stromal cells (also called bone marrow stromal cells) that are readily available from the body are treated with mitogenic agents (also called mitogens) and then selected from retinoic acid, growth factors, fetal neuronal cells or combinations thereof Have been reported (National Table 2002-513545). However, this bone marrow cell-derived neuronal cell requires a mitogenic substance treatment operation in the process, and therefore when a mitogenic substance is administered into the body, it may cause abnormal growth of other cells and may cause cancer of the cell. In addition, even in the case of in vitro administration, there is a possibility that the nerve cells produced by such a treatment operation are contaminated with cancerous nerve cells, and transplanting them into humans is a problem. Therefore, low molecular differentiation promoting factors such as mercaptoethanol, dibutyryl cyclic AMP, bone morphogenetic factor (BMP), brain-derived neurotrophic factor (BDNF), fibroblast growth factor, etc. It has been reported that differentiation into nerve cells is caused by the action of a macromolecular differentiation promoting factor such as (bFGF) or a bone morphogenetic protein inhibitor nodine (Noggin) and a demethylating agent 5-azacytidine (M. Dezawa). Eur. J. Neurosci. 2001, 14: 1771, W. Deng et al., Biochem. Biophys. Res. Commun. 2001, 282, 148, D. Woodbury et al., J. Neurosci. Res. 363, J. Sanchez-Ramos et al., Exp. Neurol. 2000, 164, 247, J. Kohyayama et al., Differentiation, 2001, 68, 235), and the use of these in the prevention and treatment of the above-mentioned diseases can also be considered. Since it is a large peptide, it is easily degraded in vivo and cannot pass through the blood-brain barrier, so there is a problem that the administration method is extremely limited, and the differentiation of mercaptoethanol, dibutyryl cyclic AMP, etc. Small molecules such as promoters, bone morphogenetic protein inhibitors and demethylating agents are highly toxic and are difficult to put into practical use.
[0005]
On the other hand, with regard to neural stem cells and neural progenitor cells, which are substantially the same as the autologous or autologous major histocompatibility antigen, differentiate in vitro to nerve cells, and transfer drugs to humans rather than transplanting them. It is considered easier to replenish lost nerves by differentiating neural stem cells or neural progenitor cells present in the body by administration to differentiate them into nerve cells. Recently, a report that 40% of the lost nerve cells recovered after injecting nerve growth factor after temporarily suspending the rat cerebral artery and causing cerebral ischemia to injure hippocampal neurons. (H. Nakatomi et al., Cell, 2002, 110, 429). Although great as a research, injecting a protein called nerve growth factor into the brain is problematic.
[0006]
Therefore, (1) a non-neuronal cell having the ability to differentiate into a neuron without being treated with a mitogenic substance and having a pharmaceutically acceptable low molecular weight substance and / or prodrug thereof as an active ingredient A composition that contacts non-neuronal cells after activation, if necessary, and differentiates the non-neuronal cells into neurons without being treated with a mitogenic substance, (2) non-neuronal cells are myeloid non-neural The composition according to (1) selected from cells and non-neuronal cells, and (3) the composition according to (2), wherein the myeloid non-neuronal cells are selected from bone marrow stem cells, mesenchymal cells, and bone marrow stromal cells. (4) The composition according to (2), wherein the non-neuronal nervous system cell is selected from neural stem cells and neural progenitor cells, and (5) the composition according to (1) to (4) as an active ingredient Nerve cells characterized by activity, if necessary after activation A composition which is brought into contact and matures without treatment of the nerve cell with a mitogenic substance, (6) characterized in that the composition according to (1) to (4) is an active ingredient, (7) From (1) to (4), a composition that contacts non-neuronal cells after activation, differentiates the non-neuronal cells into neurons without being treated with a mitogenic substance, and matures the neurons. The composition as described above is used as an active ingredient, and if necessary after contact with non-neuronal cells, the non-neuronal cells are differentiated into neurons without being treated with a mitogenic substance, and the neurons are matured A composition for treating or inhibiting malignant neuropathy, characterized by treating or inhibiting neuropathy, or (8) a composition described in (1) to (4) being active if necessary Later, contact with non-neuronal cells, the non-neural cells A method of differentiating non-neuronal cells into nerve cells, characterized in that the composition according to (9) (5) is differentiated into neurons without treatment with a cell division-inducing substance. A method of maturing a neuronal cell, characterized in that the neuronal cell is brought into contact with the neuron and matured without being treated with a mitogenic agent, and the composition according to (10) (6) is required. After activation, the non-neuronal cell is converted into a neuron without contacting with the non-neuronal cell, and the non-neuronal cell is differentiated into a neuron without being treated with a mitogenic substance, and the neuron is matured. A method for differentiating and maturating the nerve cell, (11) The composition described in (7) is contacted with a non-neuronal cell, if necessary after activation, and the non-neuronal cell is treated with a mitogenic substance. Without being differentiated into nerve cells, and the nerve cells A method of treating or inhibiting malignant neuropathy by maturating a neuropathy by maturation of the disease, (12) mixing of a macromolecule causing transplantation rejection and cancerous neurons (13) In the method according to (13) or (9), a macromolecule that causes transplantation rejection and a cancerous nerve cell obtained by maturation of a nerve cell that is not likely to be mixed with a cancerous nerve cell (15) A nerve cell obtained by the method according to (14), (8), or (10), and a nerve cell that is not likely to be mixed with a macrophage that causes transplant rejection and a cancerated nerve cell, (15) ) After activation, if necessary, contacting a non-neuronal cell or neuronal cell that has not been treated with a mitogenic agent with a pharmaceutically acceptable small molecule that is not toxic, differentiation of the non-neuronal cell into a neuronal cell, and / Or Non-neuronal cells are differentiated into neurons without being treated with mitogens and / or matured without being treated with mitogens, characterized by examining whether neurons are mature A method for searching for a low-molecular-weight substance and / or its prodrug having only a pharmaceutically acceptable tolerance, and a pharmaceutically acceptable toxicity after the activity, if necessary, in the method according to (16) (15) A non-neuronal cell is made to contact a non-neuronal cell that is not treated with a mitogenic substance, and the presence or absence of differentiation of the non-neuronal cell into a neuron is examined. (17) (15) A method for searching for a low-molecular weight substance and / or a prodrug thereof having a pharmaceutically acceptable toxicity having an ability to differentiate into a nerve cell without being treated with (17) (15) In the method described in 1., after activation, a low-molecular substance having a pharmaceutically acceptable toxicity is contacted with a nerve cell that has not been treated with a cell division-inducing substance, and the presence or absence of maturation of the nerve cell is examined. (18) (15) A method for searching for a pharmaceutically acceptable low-molecular weight substance and / or prodrug thereof having the ability to mature without treating cells with a mitogenic substance, ), After activation, if necessary, a low molecular weight substance having a pharmaceutically acceptable range is contacted with a non-neuron cell that has not been treated with a mitogenic substance, and the non-neuron cell is brought into contact with the neuron. It has the ability to differentiate non-neuronal cells into neurons without treatment with mitogens and to mature the neurons, characterized by examining the differentiation and the maturity of the neurons Therefore, development of a method for searching for low molecular weight substances having only a pharmaceutically acceptable range and / or prodrugs thereof is strongly desired.
[0007]
On the other hand, HMG-CoA reductase inhibitor has a cholesterol synthesis inhibiting action, a lipid lowering action, an arteriosclerosis progression inhibiting action, an exogenous cholesterol absorption inhibiting action, and has low toxicity even after long-term administration. It is used to treat or prevent malignant hypercholesterolemia and hyperlipidemia. On the other hand, although not yet put into practical use, prevention or treatment of heart failure by a combination of a specific HMG-CoA reductase inhibitor and an angiotensin 2 receptor antagonist (JP 2002-145770 A), HMG-CoA reductase inhibitor Prevention of secondary heart attack by a combination of an angiotensin converting enzyme (ACE) inhibitor (JP-A-8-291082), TNA-a suppression by a combination of an HMG-CoA reductase inhibitor and an insulin sensitizer ( JP-A 2001-294537), prevention or treatment of arteriosclerosis with the same combination (JP-A-9-71540), differentiation of hematopoietic stem cell-derived cells into endothelial cells with an HMG-CoA reductase inhibitor statin (J. Llevadot et al. J. Clinical Investigation, 2001, 108. Vol. 399) Promotion of osteogenesis by combination of statin and polyphosphonate and treatment of atherosclerosis (Japanese Patent Laid-Open No. 2001-253827), Enhancement of bone formation by combination of HMG-CoA reductase inhibitor and prostaglandin agonist (JP 2002-179595), inhibition of bone resorption by HMG-CoA reductase inhibitors (special table 2002-506030), stimulation of bone formation by statin (G. Mundy et al., SCIENCE, 286, 1946, 1999), cerivastatin There are reports such as the effect of sodium on chronic HIV-1 (Japanese Patent Laid-Open No. 2002-29973), the treatment and prevention of anorexia due to the androgen activity antagonistic action of an HMG-CoA reductase inhibitor (Special Table 2001-513572), and the like.
[0008]
In addition, regarding a HMG-CoA reductase inhibitor, a composition for increasing the level of ApoE is administered to a neurodegenerative disease patient as a therapeutic method for the neurodegenerative disease based on the fact that the neurodegenerative disease patient has low apoliboprotein (ApoE). And a method for searching for an ApoE-elevating compound that measures the presence or absence of an increase in ApoE in a neuron cell (Special Table 2001-517617), total cholesterol in a human brain suffering from Alzheimer's disease ( In order to administer a plasma triglyceride level-lowering agent to humans suffering from Alzheimer's disease based on the fact that the concentration of TC) and LDL is higher than that of a healthy person, or to reduce LDL-C level and increase HDL level, Co-administering a cholesterol level-lowering agent if desired to lower plasma triglyceride levels And the administration of a composition that decreases blood cholesterol level to an individual with an elevated level of cholesterol at risk for Alzheimer's disease. There is a report of a method for reducing production (Japanese Patent Publication No. 2002-507564). These reports indicate that neurodegenerative diseases such as Alzheimer's disease can be prevented or treated by applying effects such as an increase in ApoE, a decrease in cholesterol level, and a decrease in LDL level used to treat hyperlipidemia of HMG-CoA reductase inhibitors. I'm just thinking. It does not directly look at the development of tissues and organs that lose their function due to the degeneration, decrease, cell death, injury, or exclusion of neurons due to neurodegenerative diseases such as Alzheimer's disease. No matter how much ApoE is increased, cholesterol level is decreased, and LDL level is decreased, degenerated, decreased, cell death, or injury, excluded neurons are not regenerated. There is a strong demand for the development of a composition containing an active ingredient of a low molecular weight substance capable of regenerating nerves and having only a pharmaceutically acceptable toxicity.
[0009]
[Problems to be solved by the invention]
The object of the present invention is to effectively use (1) a low molecular weight substance having pharmacologically acceptable toxicity and / or a prodrug thereof capable of differentiating non-neuronal cells into nerve cells without being treated with a mitogenic substance. (2) a composition comprising: a component that is brought into contact with a non-neuronal cell after activation, if necessary, and that differentiates the non-neuronal cell into a neuron without being treated with a mitogenic substance; The composition according to (1), which is selected from myeloid non-neuronal cells and neural non-neuronal cells, (3) the myeloid non-neuronal cells are selected from bone marrow stem cells, mesenchymal cells, and bone marrow stromal cells (2) (4) the composition according to (2), (5) the composition according to (1) to (4), wherein the non-neuronal nervous system cell is selected from neural stem cells and neural progenitor cells Ingredients, if necessary after activation, A composition that is brought into contact with a transcellular cell and matures without treatment of the nerve cell with a mitogenic agent, (6) characterized in that the composition according to (1) to (4) is an active ingredient, (7) From (1) to (7) (1), wherein after activation, a composition that contacts non-neuronal cells, differentiates the non-neuronal cells into neurons without being treated with a mitogenic substance, and matures the neurons The composition described in 4) is used as an active ingredient, and if necessary, after activation, it is brought into contact with a non-neuronal cell, the non-neuronal cell is differentiated into a neuron without being treated with a mitogenic substance, and the neuron is matured A composition for treating or inhibiting malignant neuropathy disease, comprising treating the neuropathy disease or suppressing malignant transformation, and (8) the composition described in (1) to (4), After activation, it is contacted with non-neuronal cells, A method for differentiating non-neuronal cells into neurons, characterized by differentiating neurons into neurons without treatment with a mitogenic substance, (9) If the composition according to (5) is required A method for maturating a nerve cell, comprising contacting the nerve cell after the activity and maturating the nerve cell without being treated with a mitogenic agent, (10) the composition according to (6), If necessary, after activation, the non-neuronal cell is brought into contact with the non-neuronal cell, the non-neuronal cell is differentiated into a neuron without being treated with a mitogenic substance, and the neuron is matured. A method for differentiating into a cell and maturating the neuron, (11) The composition according to (7) is contacted with a non-neuron after activation if necessary, and the non-neuron is treated with a mitogenic substance. Differentiating into neurons without treatment, and A method for treating or inhibiting malignant diseases, characterized by treating or inhibiting malignant diseases by maturation of neurons, (12) a macromolecule and cancerous neurons that cause transplant rejection (13) In the method according to (9), a macromolecule that causes transplant rejection and canceration obtained by maturation of a nerve cell that is not likely to be contaminated with cancerous neurons A nerve cell that is not likely to be mixed with a nerve cell, a nerve cell that is obtained by the method according to (14), (8), or (10), and is not likely to be mixed with a macromolecule that causes transplant rejection and cancerous nerve cells, (15) After activation, if necessary, a low molecular weight substance having only a pharmaceutically acceptable range is brought into contact with a non-neuronal cell or a nerve cell that has not been treated with a cell division-inducing substance, and the non-neuronal cell is differentiated into a nerve cell. ,and Or non-neuronal cells are differentiated into nerve cells without being treated with a mitogen and / or nerve cells are not treated with a mitogen, characterized by examining the presence or absence of maturation of the nerve cells A method for searching for a pharmaceutically acceptable low molecular weight substance having the ability to mature and / or a prodrug thereof, (16) In the method according to (15), if necessary after the activity, the pharmaceutically acceptable range A non-neuronal cell is divided by contacting a non-neuronal cell that is not toxic with a non-neuronal cell that has not been treated with a mitogenic substance, and examining whether the non-neuronal cell has differentiated into a neuron. A method for searching for a pharmaceutically acceptable small molecule and / or prodrug thereof having an ability to differentiate into a nerve cell without treatment with an inducer (17) In the method according to (15), if necessary, after activation, a low molecular weight substance having a pharmaceutically acceptable range is contacted with a nerve cell not treated with a cell division inducing substance, and whether or not the nerve cell is matured A method of searching for a pharmaceutically acceptable low-molecular substance and / or prodrug thereof having the ability to mature nerve cells without being treated with a mitogenic substance, characterized in that ) In the method according to (15), if necessary, after activation, a low-molecular substance having a pharmaceutically acceptable toxicity is brought into contact with a non-neuron cell that has not been treated with a mitogenic substance, and the nerve of the non-neuron cell is contacted. Differentiating non-neuronal cells into neurons without treatment with a mitogenic agent, and maturating the neurons characterized by examining the differentiation into cells and the presence or absence of maturation of the neurons It is an object of the present invention to provide a method for searching for a low-molecular substance having a capability and having only a pharmaceutically acceptable toxicity and / or a prodrug thereof.
[0010]
[Means for Solving the Problems]
In light of such circumstances, the present inventors have conducted various searches for low-molecular substances that have the ability to differentiate non-neuronal cells into nerve cells without being treated with cell division-inducing substances and have only a pharmaceutically acceptable toxicity. The composition containing an HMG-CoA reductase inhibitor as an active ingredient has been found to have an action of differentiating non-neuronal cells into neurons without treating them with mitogenic substances and an action of maturing the neurons. As a result of further various studies, (1) a pharmacologically acceptable low molecular weight substance and / or prodrug thereof having the ability to differentiate non-neuronal cells into neurons without being treated with mitogenic substances is effective. A composition that is made into a component, is contacted with a non-neuronal cell after activation if necessary, and differentiates the non-neuronal cell into a neuron without being treated with a mitogenic substance, (2 A pharmaceutically acceptable low molecular weight substance capable of differentiating non-neuronal cells into neurons without treatment with mitogenic substances is 3-hydroxy-3-methylglutaryl-coenzyme A (HMG- CoA) a composition according to (1) selected from reductase inhibitors; (3) an HMG-CoA reductase inhibitor composition is atorvastatin, itavastatin, compactin, simvastatin, dihydrocompactin, dalvastatin, pitavastatin, fluiddostatin , Fluvastatin, pravastatin, bervastatin, verostatin, mevastatin, rosuvastatin, lovastatin, and optical or geometric isomers thereof, and their prodrugs, and pharmaceutically acceptable of the above compounds or their prodrugs If salt And (4) the HMG-CoA reductase inhibitor composition is selected from atorvastatin calcium hydrate, simvastatin, fluvastatin sodium, and pravastatin sodium (2). (5) The composition according to (1) to (4), wherein the non-neuronal cell is selected from myeloid non-neuronal cells and neural non-neuronal cells, and (6) the myeloid non-neuronal cell The composition according to (5), which is selected from bone marrow stem cells, mesenchymal cells, and bone marrow stromal cells, and (7) the composition according to (5), wherein the nervous system non-neuronal cells are selected from neural stem cells and neural progenitor cells. (8) The composition according to (1) to (7) is used as an active ingredient, and if necessary, after activation, it is brought into contact with a nerve cell, and the nerve cell is treated with a mitogenic substance. A composition that matures without (9) The composition according to (1) to (7) is used as an active ingredient, and if necessary, after activation, it is brought into contact with non-neuronal cells, and the non-neuronal cells are treated with a mitogenic substance. A composition that differentiates into a nerve cell and matures the nerve cell, and (10) the composition according to (1) to (7) is used as an active ingredient and, if necessary, after contact with non-neuronal cell Treating a neuropathic disease characterized by treating or suppressing malignant neuropathy by differentiating the non-neuronal cell into a neuronal cell without treating with a mitogenic substance and maturing the neuronal cell Alternatively, a composition for suppressing malignant transformation, or a composition according to (11) (1) to (7), if necessary, after contact with non-neuronal cells, and treating the non-neuronal cells with a mitogenic substance It is characterized by differentiation into nerve cells A method for differentiating non-neuronal cells into neurons, (12) without contacting the neurons after activation, if necessary, after treating the composition, and treating the neurons with a mitogenic agent A method of maturating a nerve cell, characterized by maturation, the composition according to (13) (9), if necessary, after contact with a non-neuron cell, and then activating the non-neuron cell with a mitogenic substance A method for differentiating non-neuronal cells into nerve cells and maturating the nerve cells, characterized by differentiating into nerve cells without treatment and maturating the nerve cells, (14) (10) A neuropathy disorder by contacting a non-neuronal cell after activation, if necessary, differentiating the non-neuronal cell into a neuron without treatment with a mitogenic substance, and maturing the neuron To treat or suppress malignant transformation A method for treating or suppressing malignant neuropathy, (15) a nerve cell that is not likely to be mixed with a macromolecule that causes transplant rejection and a cancerous nerve cell, (16) the method according to (12) And (17) (11) or (11), which is obtained by maturation of a nerve cell that is not likely to be contaminated with cancerous nerve cells, and is not likely to be contaminated with a polymer that causes transplant rejection and cancerous nerve cells. 13) a nerve cell which is obtained by the method described in 13) and which is not likely to be mixed with a macromolecule causing transplantation rejection and a cancerous nerve cell; and (18) if necessary, a small molecule having only a pharmaceutically acceptable toxicity after activation. A substance is contacted with a non-neuronal cell or a neuron that has not been treated with a mitogenic substance, and the differentiation of the non-neuronal cell into a neuron and / or maturation of the neuron is characterized. Fine non-neuronal cells A pharmaceutically acceptable low molecular weight substance capable of differentiating into a neuronal cell without treatment with a mitogenic agent and / or maturing the neuronal cell without treatment with a mitogenic agent and / or A method for searching for a prodrug thereof, (19) In the method described in (18), a non-neural nerve that has not been treated with a mitogenic substance, if necessary, after activation, a low molecular weight substance having a pharmaceutically acceptable toxicity A pharmaceutically acceptable range having the capability of differentiating non-neuronal cells into neurons without being treated with a mitogenic substance, characterized by contacting cells and examining the presence or absence of differentiation of the non-neuronal cells into neurons A method of searching for a low-molecular-weight substance and / or prodrug thereof having only toxicity, and in the method according to (20) (18), if necessary, after the activity, a pharmaceutically acceptable toxicity A neuron is matured without being treated with a mitogenic agent, characterized by contacting a low molecular weight substance with a nerve cell that has not been treated with a mitogenic substance and examining the maturity of the nerve cell. A method for searching for a low-molecular-weight substance having ability and having only a pharmaceutically acceptable toxicity and / or a prodrug thereof, (21) In the method described in (18), if necessary, after the activity, a pharmaceutically acceptable toxicity A non-neuronal cell that is not treated with a mitogenic substance is contacted with a low-molecular substance that is only a non-mitotic agent, and the non-neuronal cell is differentiated into a neuron and the presence or absence of maturation of the neuron is characterized. A pharmacologically acceptable low molecular weight substance and / or its ability to differentiate into a neuronal cell without treatment with a mitogenic substance and to mature the neuronal cell. Developed a method for searching a prodrug, the present invention has been completed.
[0011]
That is, the present invention is effective in (1) a low-molecular substance having a capability of differentiating non-neuronal cells into neurons without being treated with a mitogenic substance and having only a pharmaceutically acceptable toxicity and / or a prodrug thereof. A composition that is made into a component and, if necessary, after activation, is brought into contact with a non-neuronal cell, and the non-neuronal cell is differentiated into a neuron without being treated with a mitogenic substance, (2) A pharmacologically acceptable low molecular weight substance capable of differentiating into nerve cells without treatment with a cell division inducer is 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase The composition according to (1) selected from inhibitors, and (3) an HMG-CoA reductase inhibitor composition is atorvastatin, itavastatin, compactin, simvastatin, dihydride Compactin, dalvastatin, pitavastatin, fluindostatin, fluvastatin, pravastatin, bervastatin, verostatin, mevastatin, rosuvastatin, lovastatin, and their optical or geometric isomers, and their prodrugs, as well as the compounds or their prodrugs The composition according to (2), which is selected from pharmaceutically acceptable salts thereof and hydrates thereof, (4) the HMG-CoA reductase inhibitor composition is atorvastatin calcium hydrate, simvastatin, fluvial The composition according to (2) selected from statin sodium and pravastatin sodium, and (5) the composition according to (1) to (4), wherein the non-neuronal cell is selected from a myeloid non-neuronal cell and a neuronal non-neuronal cell (6) Myeloid non-neural Is selected from bone marrow stem cells, mesenchymal cells, and bone marrow stromal cells. (7) The composition according to (5), wherein the non-neuronal cells are selected from neural stem cells and neural progenitor cells. (8) characterized in that the composition according to (1) to (7) is used as an active ingredient, and if necessary, after activation, the nerve cell is contacted and treated with a cell division-inducing substance. A composition that matures without being treated, (9) characterized in that the composition according to (1) to (7) is used as an active ingredient, and if necessary after contact with non-neuronal cells, A composition that differentiates into a nerve cell without treatment with a mitogenic substance and matures the nerve cell, (10) the composition according to (1) to (7) as an active ingredient, and if necessary, active Later, contact the non-neuronal cell and treat the non-neuronal cell with a mitogen A composition for treating or malignantly treating a neuropathic disease, characterized in that the neuropathic disease is treated without maturation and matured, and the neuropathy is matured to suppress malignant disease, or (11) ( The composition according to 1) to (7) is contacted with a non-neuronal cell, if necessary after activation, and the non-neuronal cell is differentiated into a neuron without being treated with a mitogenic substance, A method of differentiating non-neuronal cells into nerve cells, (12) After the activation, if necessary, the composition according to (8) is brought into contact with the nerve cells and matured without being treated with a mitogenic substance. A method for maturation of nerve cells, wherein the composition according to (13) and (9) is contacted with non-neuronal cells after activation, if necessary, and the non-neuronal cells are treated with a mitogenic substance. Without being differentiated into nerve cells And a method for differentiating non-neuronal cells into neurons and maturating the neurons, wherein the composition according to (14) and (10) is activated if necessary It is characterized in that it is brought into contact with a non-neuronal cell, the non-neuronal cell is differentiated into a neuron without being treated with a mitogenic substance, and the neuron is matured to treat or suppress malignant diseases. A method for treating or inhibiting malignant neuropathy, (15) a nerve cell that is not likely to be mixed with a macrophage that causes transplant rejection and a cancerous nerve cell, (16) in the method according to (12) A neuron which is obtained by maturation of a nerve cell which is not likely to be contaminated with cancerous nerve cells, and which is not likely to be contaminated with a macromolecule causing a transplant rejection and cancerous nerve cells, (17) (11) or (13) ) A neuron that is not likely to be contaminated with a macromolecule that causes transplant rejection and a cancerous neuron, and (18) if necessary, a low-molecular substance having a pharmacologically acceptable toxicity after cell activation. Non-neuronal cells or cells that are not treated with a substance are contacted, and the non-neuronal cells are examined for differentiation into non-neuronal cells and / or maturation of the non-neuronal cells. A pharmaceutically acceptable low molecular weight substance and / or its progenitor that has the ability to differentiate into neurons without treatment with an inducer and / or mature neurons without treatment with a mitogen. In the method of searching for a drug, (19) or (18), if necessary, a low molecular weight substance having a pharmaceutically acceptable toxicity is not treated with a cell division inducing substance after activation. A pharmaceutical product having the ability to differentiate into a non-neuronal cell without treatment with a mitogenic substance, wherein the non-neuronal cell is contacted with a non-neuronal cell and examined for the differentiation of the non-neuronal cell into a neuron A method for searching for a low molecular weight substance having only a toxic tolerance and / or a prodrug thereof, (20) In the method described in (18), if necessary, a low molecular weight substance having a pharmaceutically acceptable tolerance after activation Having the ability to mature without treatment with a mitogen, characterized by contacting a nerve cell that has not been treated with a mitogen and examining the maturity of the nerve cell, A method for searching for a low molecular weight substance having only a pharmaceutically acceptable toxicity and / or a prodrug thereof, (21) In the method described in (18), if necessary, after the activity, the pharmaceutically acceptable range Contacting a non-neuronal cell that is not treated with a mitogenic substance with a low-molecular substance that is only toxic, and examining the differentiation of the non-neuronal cell into a neuron and the maturation of the neuron, Search for a pharmaceutically acceptable low molecular weight substance and / or prodrug thereof that has the ability to differentiate non-neuronal cells into neurons without treatment with mitogenic substances and to mature the neurons. A method is provided.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
The composition of the present invention, that is, (1) a composition that, after activation, if necessary, contacts non-neuronal cells and differentiates the non-neuronal cells into neurons without being treated with a mitogenic substance, (2) A composition that is brought into contact with a nerve cell after activation and matures without treatment of the nerve cell with a mitogenic substance, and (3) if necessary, after contact with a non-neuron cell, the non-neuron cell is cell-divided. A composition that differentiates into a nerve cell without being treated with an inducer and matures the nerve cell, (4) if necessary, after activation, is contacted with a non-neuron cell, and the non-neuron cell is treated with a cell division inducer A composition for treating or malignantly treating a neuropathy disease by maturating the nerve cell and maturating the nerve cell has the ability to differentiate a non-neuron cell into a nerve cell without treatment with a mitogen. Have Characterized in that the biological tolerance of toxicity only low molecular substances and / or active ingredient prodrugs thereof. The active ingredient of the composition of the present invention is a pharmaceutically acceptable small molecule substance and / or prodrug thereof having the ability to differentiate non-neuronal cells into neurons without being treated with mitogenic substances. It only needs to be present and may be synthesized in the future. A low molecular weight substance having only a pharmaceutically acceptable toxicity means a low molecular weight substance having a toxicity within a pharmaceutically acceptable range. Toxicity used here includes acute toxicity, subacute toxicity, chronic toxicity, toxicity inherent in nature such as teratogenesis, as well as severe and frequent side effects outside the pharmaceutically acceptable range. The low molecular weight substance is a non-high molecular weight substance such as a protein or a high molecular weight peptide, and is a low molecular weight substance that is not antigenic and can pass through the blood brain barrier. The composition of the present invention is also used to obtain the method of the present invention and the nerve cell of the present invention. A pharmaceutical having the ability to differentiate non-neuronal cells obtained by the searching method of the present invention into neurons without being treated with mitogens and / or to mature neurons without being treated with mitogens Low molecular weight substances and / or prodrugs thereof which have only acceptable tolerances belong to the compositions of the present invention.
[0013]
As a low molecular weight substance having only a pharmaceutically acceptable toxicity and capable of differentiating non-neuronal cells into nerve cells without being treated with a mitogenic substance, preferably, but not limited to, 3- And hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors. HMG-CoA reductase inhibitor is a precursor of biosynthesis of various steroids and terpenes including cholesterol by HMG-CoA reductase, which is an important rate-limiting enzyme of cholesterol biosynthesis, and is an intermediate of ketone body metabolism. -A substance that inhibits the reaction of converting CoA to mevalonic acid. The HMG-CoA reductase inhibitor may be any low-molecular substance that inhibits the action of HMG-CoA reductase and has only a pharmaceutically acceptable toxicity. The HMG-CoA reductase inhibitor is preferably, but not limited to, atorvastatin, itavastatin (also known as NK-104), compactin, simvastatin, dihydrocompactin, dalvastatin, pitavastatin, fluvastatin, pravastatin, Bervastatin, verostatin, mevastatin, rosuvastatin, lovastatin, etc., and optical or geometric isomers thereof, and prodrugs thereof, and pharmaceutically acceptable salts of the above compounds or their prodrugs and water thereof Japanese products are listed. Among them, atorvastatin, simvastatin, fluvastatin, pravastatin and their pharmaceutically acceptable salts and hydrates thereof have been used for many years as a therapeutic agent for hyperlipidemia, and its safety has been confirmed. More specifically, atorvastatin calcium hydrate, simvastatin, fluvastatin sodium, pravastatin sodium and the like are not particularly limited, but are particularly preferable.
[0014]
The HMG-CoA reductase inhibitor can be produced by any method as long as it is pharmaceutically acceptable. Although it is not necessarily limited to this, exemplifying the production method of the HMG-CoA reductase inhibitor, atorvastatin is obtained by the method disclosed in U.S. Pat. No. 4,681,893 and JP-A-3-58967 (USP 527939). Itavastatin (also known as NK-104) is produced by the method disclosed in US Pat. No. 5,102,888, compactin is produced by the method disclosed in US Pat. No. 4,804,770, and simvastatin is produced by No. 122375 (US Pat. No. 4,444,784), dihydrocompactin according to the method disclosed in US Pat. No. 4,450,171, dalbastatin according to the method disclosed in European Patent Application EP 728510, and pitavastatin according to JP -279866 (USP 5854259 and SP5856336) is disclosed in European Patent Application Publication No. 363,934A1 and fluvastatin is disclosed in Japanese Patent Publication No. 60-500015 (USP 4739073) or European Patent Application EP363934. Pravastatin according to the method disclosed in JP-A-57-2240 (USP 4346227), velvastatin according to the method disclosed in US Pat. No. 5,082,859, and verostatin as US Pat. No. 4,448,859. 784 and U.S. Pat. No. 4,450,171, mevastatin is disclosed in U.S. Pat. No. 3,983,140, and rosuvastatin is disclosed in JP-A-5-178841 (USP 5260440). Is The method, lovastatin may be prepared by the method disclosed in JP 57-163374 (USP4231938).
[0015]
The composition of the present invention is (1) contacted with non-neuronal cells after activation if necessary, and differentiates the non-neuronal cells into neurons without being treated with a mitogenic substance, (2) after activation if necessary Contact with a neuron and mature without treatment with a mitogenic substance, (3) contact with a non-neuron after activation if necessary, and treat the non-neuron with a mitogen (4) after activation, if necessary, contact with a non-neuronal cell, and separate the non-neuronal cell into a neuronal cell without treatment with a mitogenic substance. And maturating the nerve cells to treat or malignantly inhibit neuropathy diseases. Therefore, the composition of the present invention provides a neuron for transplantation by bringing the composition into contact with a non-neuronal cell or an immature neuron cell in vitro, and differentiating and / or maturing the cell. Useful for. In addition, the composition of the present invention allows non-neuronal cells or immature nerve cells to be injected into the body, and then the composition is orally or parenterally brought into contact with the cells in the body to differentiate the cells in the body. And / or matured or without externally injecting non-neuronal cells or immature nerve cells into the body, or by administering the composition orally or parenterally, It is useful for treating a neuropathy disease or suppressing malignant transformation by bringing it into contact with other nerve cells, differentiating the cells in the body, and / or maturing. That is, a medicine containing this is useful as a prophylactic and therapeutic drug for diseases caused by cell regression / reduction or cell death of various tissues and cells. Since the composition of the present invention has a low molecular weight, it can be administered by oral administration or parenteral administration (intramuscular, subcutaneous, intravenous, suppository, etc.) as well as in vitro administration. In the case of in vitro administration, when a prodrug is contained in the composition of the present invention, it is preferable to use it after activating it by an appropriate method because it provides a greater effect. When a prodrug is not included, it can be used as it is. In many cases, the prodrug is activated in the body in the case of in vivo administration, so that it is not necessary to activate the prodrug in advance.
[0016]
When preparing an oral preparation, after adding an excipient and, if necessary, a binder, a disintegrating agent, a lubricant, a coloring agent, a corrigent, etc., a tablet, a coated tablet, a granule by a conventional method. Preparations, capsules, solutions, syrups, elixirs, oily or aqueous suspensions, and the like. Examples of the excipient include lactose, corn starch, sucrose, glucose, sorbit, crystal cellulose and the like. Examples of the binder include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl starch, and polyvinylpyrrolidone.
[0017]
Examples of the disintegrant include starch, agar, gelatin not yet, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextran, and pectin. Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, hydrogenated vegetable oil, and the like. As the colorant, those permitted to be added to pharmaceuticals can be used. As a flavoring agent, cocoa powder, mint brain, aromatic acid, mint oil, dragon brain, cinnamon powder and the like can be used. These tablets may be coated with granules, sugar coats, gelatin coats, etc. as necessary.
[0018]
When preparing injections, pH adjusters, buffers, stabilizers, preservatives and the like are added as necessary, and subcutaneous, intramuscular and intravenous injections are prepared by conventional methods. An injection may be a preparation prepared for daily use as a solid preparation by lyophilization after storing the solution in a container. One dose may be stored in a container, and multiple doses may be stored in the same container.
[0019]
The concentration of the composition of the present invention used as a reagent is usually 0.01 to 1000 micromolar, preferably 0.1 to 100 micromolar. The dosage as a pharmaceutical is usually 0 per day for an adult. The daily dose is administered in the range of 0.01 to 1000 milligrams, preferably 0.1 to 100 milligrams, once a day, or divided into 2 to 4 times. The dosage may be adjusted according to age and symptoms.
[0020]
The non-neuronal cell used in the present invention means a cell other than a neuronal cell, which can be differentiated into a neuronal cell by the composition of the present invention. The cells that can be differentiated into nerve cells by the composition of the present invention are not limited thereto, but preferably ES cells, neural stem cells, neural progenitor cells, bone marrow stem cells, mesenchymal cells, bone marrow stromal cells Etc. Among ES cells, neural stem cells, neural progenitor cells, bone marrow stem cells, mesenchymal cells, bone marrow stromal cells, etc., non-fetal neural stem cells, neural progenitor cells, bone marrow stem cells, mesenchymal cells, bone marrow stromal cells, etc. There are few problems with ethics in comparison with ES cells and embryonic neural stem cells, which is more preferable. Among non-fetal neural stem cells, neural progenitor cells, bone marrow stem cells, mesenchymal cells, bone marrow stromal cells, etc., bone marrow stem cells, mesenchymal cells, bone marrow stromal cells, etc. are easily available, and nerves for transplantation Bone marrow stem cells, mesenchymal cells, and bone marrow stromal cells that are substantially the same as or very similar to autologous or autologous major histocompatibility antigens are particularly preferred as cells for transplantation. It is more preferable as a cell that differentiates or as a cell that is injected into the body and differentiates into a nerve cell in the body. On the other hand, in the case where the composition of the present invention is administered without regeneration of nerve cells, such as transplantation of non-neuronal cells or in vivo injection, in order to regenerate the nerve cells in the body, Progenitor cells, bone marrow stem cells, mesenchymal cells, bone marrow stromal cells, etc. are differentiated into nerve cells. Therefore, it is more preferable to select non-neuronal cells used in the present invention according to the purpose.
[0021]
Although the degree of limitation varies depending on the purpose of use, the source of non-neuronal cells or nerve cells used in the method of the present invention may be obtained from any place as long as it fits. There are few limitations when the purpose of use is not intended for humans, but for the purpose of obtaining nerve cells to be transplanted into humans, or by injecting into the body to differentiate into nerve cells, or nerve cells in the body ES cells are transplanted from human fetal embryos, neural stem cells and neural progenitor cells are transplanted from human fetal brain, bone marrow stem cells, mesenchymal cells, bone marrow stromal cells, etc. And aseptically from human bone marrow where the major histocompatibility antigens are substantially the same or very similar. In the case where the composition of the present invention is administered to regenerate nerve cells existing in the body without transplanting nerve cells or in vivo injection of non-neuron cells, the non-neuron cells to be used are endogenous non-neuronal cells. Limited to cells. A pharmaceutically acceptable, small molecule that has the ability to differentiate non-neuronal cells into neurons without treatment with mitogens and / or mature neurons without treatment with mitogens There is no limitation on the source of non-neuronal cells or neurons used in the method for searching for substances and / or prodrugs thereof.
[0022]
The nerve cell used in the present invention means a nerve cell other than a mature nerve cell, which can be matured by the composition of the present invention. The nerve cells that can be matured by the composition of the present invention are not limited to the nerve cells obtained by differentiating non-neuronal cells by the method of the present invention. Neurons obtained by differentiating non-neuronal cells by methods other than the method of the present invention, and neurons naturally differentiated from neural stem cells / progenitor cells can also be used. Among these nerve cells, non-neuronal cells are preferably differentiated without being treated with a mitogenic substance, so that nerve cells that are free from the risk of contamination of macromolecules that cause transplant rejection and cancerated neurons are preferred. The cell can be obtained by a method of differentiating an endogenous nerve cell or a non-neuronal cell of the present invention into a nerve cell without treatment with a cell division inducer.
[0023]
The nerve cells of the present invention are limited to nerve cells that are not likely to be mixed with macromolecules that cause transplant rejection and cancerous neurons. The nerve cells which are not likely to be mixed with the macromolecule causing the transplant rejection of the present invention and the cancerous nerve cells are prepared by the method of the present invention, that is, (1) the composition of the present invention, if necessary, after the activation. A method of differentiating the non-neuronal cell into a neuronal cell without treatment with a mitogenic substance, (2) contacting the neuronal cell after activation of the composition of the present invention, if necessary, A method of maturation without treatment with a cell division inducer; (3) a composition of the present invention is contacted with non-neuronal cells after activation, if necessary, and the non-neural cells are treated without treatment with a cell division inducer. It is obtained by a method of differentiating into cells and maturing the nerve cells.
[0024]
The nerve cell of the present invention is a nerve cell that is as close as possible to a naturally differentiated nerve cell or a naturally differentiated and mature nerve cell. Although not limited to this, it is optimal as a nerve cell for transplantation. The nerve cells of the present invention can be administered in a variety of ways to be suitable for transplantation into the central nervous system when transplanted into the brain. Examples include, but are not limited to, parenteral administration, subarachnoid space administration, intraventricular administration, and intrastitial administration.
[0025]
The method of the present invention, that is, (1) a method of differentiating non-neuronal cells into neurons without being treated with a mitogenic substance, (2) a method of maturing neurons without being treated with a mitogenic substance, ( 3) A method of differentiating the non-neuronal cell into a neuronal cell without treatment with a mitogenic substance and maturating the neuronal cell, (4) Dissemination of the non-neuronal cell into a neuronal cell without treatment with a mitogenic substance. The method of treating or suppressing malignant transformation of a neuropathic disease by maturation and maturation of the nerve cell comprises treating the non-neuron cell or nerve cell with a mitogenic substance after activation of the composition of the present invention, if necessary. Without contacting the non-neuronal cell or the neuron. The operations and processes other than the features of the present invention may be known operations and processes, for example, the above-mentioned literature (Special Table 2002-513545, M. Dezawa et al., Eur. J. Neurosci. 2001 14: 1771, W Deng et al., Biochem. Biophys.Res. Commun. 2001, 282, 148, D. Woodbury et al., J. Neurosci. Res. 2000, 61, 363, J. Sanchez-Ramos et al., Exp. 164, 247, J. Kohyayama et al., Differentiation, 2001, 68, 235, H. Nakatomi, et al., Cell, 110, 429, pp. 2001-517617, and other documents (J. Kunlin et al., Proc . atl.Acad.Sci.U.S.A. are disclosed in 98, pp. 4710, 2001). In addition, as already explained in the section of the composition of the present invention, as a method of bringing the composition of the present invention into contact with non-neuronal cells or immature neurons, (1) the composition of the present invention is non-extracorporeally. A method of contacting a nerve cell or an immature nerve cell; (2) contacting the cell in the body by orally or parenterally administering the composition of the present invention after injecting the non-neuron cell or immature nerve cell into the body; (3) Non-neuronal cells or immature cells existing in the body by orally or parenterally administering the composition of the present invention without injecting non-neuronal cells or immature nerve cells from the outside into the body There are methods such as contacting with nerve cells. What is necessary is just to select suitably according to the objective. These steps are also disclosed in the above-mentioned document except for the features of the present invention.
[0026]
The search method of the present invention, that is, (1) a method for searching for a low molecular weight substance having only a pharmacologically acceptable toxicity, which has the ability to differentiate a non-neuronal cell into a neuron without being treated with a mitogenic substance, (2) A method for searching for a low-molecular-weight substance having a pharmacologically acceptable toxicity that has the ability to mature without treating neuronal cells with mitogenic substances, and (3) treating non-neuronal cells with mitogenic substances. A method of searching for a low molecular weight substance having only a pharmaceutically acceptable range that has the ability to differentiate and mature into a nerve cell without performing a pharmacologically acceptable low molecular weight substance after activation is necessary. Contact with non-neuronal cells or neurons that have not been treated with a mitogenic substance, whether or not the non-neuronal cells have differentiated into neurons, or whether or not neurons have matured, or differentiation into nerve cells and the god Take wherein the check for cell maturation. In other words, it is characterized by the use of non-neuronal cells or neurons that are not treated with mitogenic substances and low molecular substances that have only a pharmaceutically acceptable toxicity, and the processes other than this feature are described above. Is disclosed.
[0027]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples. Of the operations and processes, details of known parts are also disclosed in the following documents. D. Stokes et al., Proc. Natl. Acad. Sci. U. S. A. 1998, 95, 3908, R.A. J. et al. Gerson et al., Am. J. et al. Med. 1989, 87, 28S, p. A. Todd et al., Drugs, 1990, 40, 583, p. Hofsetter et al., Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 2199; Zea-Longa et al., Stroke, 1989, 20 volumes, 84 years, Merck Manual 17th edition, Japanese version.
[0028]
Example 1. In vitro differentiation: Bone marrow was obtained from femoral and tibia or human bone marrow aspirates from 10-week-old female SD rats. Human bone marrow is treated with Dulbecco's modified Eagle minimal essential medium (DMEM) (low concentration glucose) (GIBCO / BRL) and 10% fetal serum (FBS) (HYCLONE), 100 units / milliliter penicillin, 100 micrograms / milliliter streptomycin (SIGMA) Diluted 1: 1 with a density gradient and centrifuged at 800 grams for 5 minutes. Combine supernatant and interface and contain 10% fetal serum
Dilute to about 20 milliliters with MEM and plate into plastic flasks coated with polyethyleneimine. Rat bone marrow was placed in 10 ml PBS and 5% albumin. Cells were washed in this medium and centrifuged at 800 rpm for 5 minutes. Cells were resuspended in growth medium consisting of DMEM (low concentration glucose) supplemented with 2 mM glutamine, 10% serum (FBS), 100 units / milliliter penicillin, 100 micrograms / milliliter streptomycin (SIGMA). Cells were cultured for 1 day in flasks to remove non-adherent cells. Thereafter, the cells were cultured every 3 days, and the medium was changed. After the culture almost reached confluency, it was treated with trypsin (0.25%) to detach the cells. Cells were collected by centrifugation at 800 rpm for 5 minutes. The cells were suspended in the medium, planted in the plastic flask, and cultured in the medium. This operation was repeated 4 times. Mitogens such as epidermal growth factor (EGF) or platelet-derived growth factor (PDGF), which are usually used in this procedure, were not added.
[0029]
Differentiation of bone marrow stromal cells into neuron-like cells was performed in the following steps. Bone marrow stromal cells that have not yet reached confluence are detached from the bottom of the plastic flask and coated with 0.05 mg / ml poly-D-lysine (SIGMA) at a concentration of 10,000 cells / square centimeter. Medium plates were planted in DMEM (low concentration glucose) supplemented with 10% fetal serum (FBS), 100 units / ml penicillin, 100 microgram / ml streptomycin. At this time, 4 milligrams of simvastatin prodrug was dissolved in 100 microliters of ethanol in advance, 0.1N NaOH was added and reacted at 50 degrees Celsius for 2 hours, pH was adjusted to 7.0 with HCL, and 4 milligrams / gram. Activated simvastatin at a concentration of milliliters and stored at 4 degrees Celsius was added at 2.5 micromolar, 5 micromolar and 10 micromolar and cultured for 72 hours. Normally added neuron growth factors such as brain-derived growth factor (BDNF), glial-derived neurotrophic factor (GDNE) and nerve growth factor (NGF) were not added.
[0030]
The cultured cells were fixed with 4% (wt / vol) paraformaldehyde-fixed 0.1 molar phosphate buffer pH 7.4 for 30 minutes. After washing for neuron-specific enolase (NSE) and neuron-specific nuclear antigen (NeuN) staining, the cells were washed with PBS containing 0.25% Triton X-100 and 5% normal sheep serum (PBSTS). Blocked at room temperature for hours. The cells were then cultured overnight at 4 degrees Celsius in a primary antibody solution diluted with PBSTS. After washing 3 times with PBS, the avidin-biotin complex was contacted for 1 hour at room temperature. Washed 3 times with PBS. For MAP2 and tau staining, after washing, cells were blocked with PBS containing 0.25% Triton X-100 and 5% normal sheep serum (PBSTS) for 1 hour at room temperature. The cells were then cultured overnight at 4 degrees Celsius in a primary antibody solution diluted with PBSTS. After washing 3 times with PBS, the cells were cultured in a secondary antibody solution diluted with PBSTS at 4 degrees Celsius for 1 hour in the dark. After washing, the cells were embedded with glycerol / PBS (1: 1) and observed with a fluorescence microscope. Primary antibodies were rabbit monoclonal antibody against rat NSE 1: 200, mouse monoclonal antibody against rat NeuN 1: 200, mouse mouse monoclonal antibody against MAP2 at 5 microgram / milliliter, and rabbit monoclonal antibody against tau at 1: 200. Used diluted. As the secondary antibody, a biotinylated sheep antibody against rabbit IgG was diluted to 10 microgram / ml, and a biotinylated horse antibody against mouse IgG against rat IgG was diluted to 10 microgram / ml. MAP2 was visualized with a sheep antibody against mouse Cy-3 (5 microgram / milliliter) and tau was visualized with a sheep antibody against rabbit Cy2.
[0031]
Bone marrow stromal cells obtained from rat bone marrow were separated and expanded on a plastic plate 4 times. They were divided into two types: large flat cells and relatively elongated spindle-like cells. In contrast, bone marrow stromal cells contacted with 0.25 micromolar, 5 micromolar and 10 micromolar activated simvastatin surprisingly changed morphology after 24 hours. Reshaped cells exhibit refraction and rounded cell bodies, show characteristics of neuronal cells such as simple bipolar cells and highly branched multipolar cells, and also have synaptic morphogenesis (FIG. 1). After 48 hours, the synaptic morphology of the neuron-like cells became clear (Fig. 2). Neuron-like cells contacted with activated simvastatin were fixed after 24 hours, 48 hours, and 72 hours of culture, and stained for the NSE neuronal marker. Platelet bone marrow stromal cells that were not contacted with simvastatin stained very low. However, the level of NSE protein was detectable. On the other hand, the neuron-like cells contacted with activated simvastatin expressed NSE significantly. We next examined the existence of NeuN. NeuN could not be detected in flat bone marrow stromal cells not contacted with simvastatin, whereas neuron-like cells contacted with activated simvastatin showed a form of expressing NeuN. Neuron-like cells contacted with activated simvastatin expressed MAP2 and tau in the cell body (FIGS. 3 and 4). In contrast, plate-like bone marrow stromal cells not contacted with simvastatin did not express MAP2 and tau in the cell body.
[0032]
NES-positive, NeuN-positive, MAP2-positive, and tau-positive morphologically neuron-like cells were kept in culture while being in contact with activated simvastatin. It was confirmed that the dendrites extended from the cells in a complex manner, formed synapses with many surrounding cells, and the neurons grew. Therefore, it was confirmed that the nerve-like cells that appeared 24 hours after contact with simvastatin were the nerve cells themselves. There is no report of differentiation from non-neuronal cells to neurons within 24 hours, and this example is the first.
[0033]
Example 2 In Example 1, instead of simvastatin, atorvastatin, itavastatin, compactin, dihydrocompactin, dalvastatin, pitavastatin, fluindostatin, fluvastatin, pravastatin, velvastatin, verostatin, mevastatin, rosuvastatin, and lovastatin are used in the same manner. Bone marrow stromal cells were differentiated into neurons without mitogen treatment, and the differentiated neurons were matured.
[0034]
Example 3 FIG. In Example 1, instead of simvastatin, a mixture of atorvastatin and fluvastatin was used to similarly differentiate rat bone marrow stromal cells into neurons without mitogen treatment, and the differentiated neurons were matured.
[0035]
Example 4 In Vivo Differentiation: Bone marrow was obtained from femur and tibia or human bone marrow aspirates from 10 week old female SD rats. Human bone marrow is treated with Dulbecco's modified Eagle minimal essential medium (DMEM) (low concentration glucose) (GIBCO / BRL) and 10% fetal serum (FBS) (HYCLONE), 100 units / milliliter penicillin, 100 micrograms / milliliter streptomycin (SIGMA) Diluted 1: 1 with a density gradient and centrifuged at 800 g for 5 minutes. The supernatant and interface were combined, diluted to approximately 20 milliliters with MEM containing 10% fetal serum, and plated into plastic flasks coated with polyethyleneimine. Rat bone marrow was placed in 10 ml PBS and 5% albumin. Cells were washed in this medium and centrifuged at 800 rpm for 5 minutes. Cells were resuspended in growth medium consisting of DMEM (low concentration glucose) supplemented with 2 mM glutamine, 10% serum (FBS), 100 units / milliliter penicillin, 100 microgram / milliliter streptomycin (SIGMA). Cells were cultured for 1 day in flasks to remove non-adherent cells. Thereafter, the cells were cultured every 3 days, and the medium was changed. After the culture almost reached confluency, it was treated with trypsin (0.25%) to detach the cells. Cells were collected by centrifugation at 800 rpm for 5 minutes. The cells were suspended in the medium, planted in the plastic flask, and cultured in the medium. This operation was repeated 4 times. Mitogens such as epidermal growth factor (EGF) or platelet-derived growth factor (PDGF), which are usually used in this procedure, were not added.
[0036]
Using LXSN vector (CLONTECH), a retroviral plasmid encoding green fluorescent protein as an expression marker and aminoglycoside phosphorotransferase as a neomycin (G418) selection marker was prepared. LXSN-GFP plasmids were infected with Phoenix amphotropic packaged cells (ATCC) using calcium phosphate precipitation. Viral supernatant was collected 48 hours after infection and filtered through a filter with a hole size of 0.45-micrometer. Stored at minus 80 degrees Celsius until use. When the virus was collected, the expression of GFP in the cells that were packaged in the Phoenix amphotropic package was analyzed and confirmed. About 100,000 bone marrow stromal cells were plated on 21.0 square centimeter plates with complete medium containing 20% heat-inactivated FCS and cultured overnight. The next day, the cells were infected by mixing 500 microliters of virus supernatant and 8 micrograms / milliliter of polybrene (Sigma) in complete medium containing 2.5 milliliters of 20% heat-inactivated FCS. The day of virus infection was defined as the first day. On the second day, the virus infection operation was repeated. On day 3, the culture medium was replaced with fresh complete medium containing 20% unheated FBS. On day 4, cells were split at a ratio of 1: 3 into 55.0 square centimeter plates with complete medium containing 200 micrograms / milliliter of G418 (Sigma) and selected for 21 days. Bone marrow stromal cells in which the GFP plasmid was incorporated into the gene were obtained. The proliferation of the cells was increased three times.
[0037]
Male SD rats were irradiated with 137Cs at an intensity of 9.0 (900 rads). 24 hours after irradiation, about 5,000,000 bone marrow stromal cells into which the GFP plasmid had entered the gene were injected into the tail vein of the rat. Bone marrow stromal cell transplanted rats were raised for 8 weeks.
[0038]
Bone marrow stromal cell transplanted rats were anesthetized with a mixed gas of 4% isoflurane, 66% N2O, and 30% oxygen, and anesthesia was maintained with a mixed gas of 1.5% isoflurane, 68.5% N2O, and 30% oxygen. Blood pressure, gas partial pressure of blood clot, and blood glucose level were monitored at the left femoral artery. Rectal temperature, temporal muscle temperature opposite the middle cerebral artery occlusion, was constantly monitored and maintained at 37.0-37.5 degrees Celsius with a heating pad. E. In accordance with the method of Zea-Longa et al., Rats were ischemic by intraocular middle cerebral artery occlusion with sutures. That is, the left external carotid artery was tied with 6-0 silk suture, the distal incision was made, the left internal carotid artery was isolated, and separated from the vagus nerve. The extracranial branch of the left internal carotid artery was tied with 6-0 silk suture at the base of the branch. A 3-0 surgical single layer nylon suture with a circular tip was guided through the external carotid artery fragment to the left internal carotid artery, tied 20 minutes past the carotid bifurcation, and then unwound immediately.
[0039]
Ten rats with middle cerebral artery occlusion due to occlusion with sutures were orally administered simvastatin at a rate of 20 mg / kg daily from the same day until the day of killing. As a control, simvastatin was not administered to another 10 rats with middle cerebral artery occlusion.
[0040]
On days 1, 3, 7, and 14 after occlusion of the middle cerebral artery, two of these rats were anesthetized with 10 mg / kg of xylazine and 80 mg / kg of ketamine and killed. Immediately refluxed with physiological saline followed by 4% paraformaldehyde. Brain tissue was cut into 7 equal circular blocks at 2 millimeter intervals. At the frontal plane, a section of 6 micrometers thickness was made from each cutting block and double immunolabeled for GFP and NeuN as in Example 1. In rats administered with simvastatin, double positivity of GFP and NeuN was observed from the group killed on the third day after middle cerebral artery occlusion, and double positivity became stronger in proportion to the number of days. In rats that did not receive simvastatin, double GFP and NeuN could not be confirmed even 14 days after the middle cerebral artery occlusion. From this, it was confirmed that the transplanted bone marrow stromal cells infiltrated into the brain through the cerebrovascular barrier and differentiated into neurons by simvastatin.
[0041]
Rats that were not killed received simvastatin for 3 months. Control rats did not receive simvastatin. This rat was subjected to an elevated plus maze test. The Elevated Cross Maze Test is where a rat's food is placed on one of the elevated cross-shaped slender plates and the rat is released on one of the other, and the rat first goes to a dead end on one of the crossroads by trial and error. Knowing that there is no food, go back to the crossroads and go another way, while the rat reaches the end where the food is placed. When the experiment is repeated, the memory-rich rat does not hesitate and goes on the path where the food is placed, and then comes to the food. However, rats with poor memory repeat trial and error as in the first time. This test can measure the memory ability of rats. Rats that did not receive simvastatin took much time to reach the diet and their memory was extremely reduced. In contrast, rats administered with simvastatin had a short time to reach the food and recovered some memory, suggesting that neurons regenerated with simvastatin are functional.
[0042]
Embodiment 5 FIG. In Example 4, instead of simvastatin, atorvastatin, itavastatin, compactin, dihydrocompactin, dalvastatin, pitavastatin, fluindostatin, fluvastatin, pravastatin, velvastatin, verostatin, mevastatin, rosuvastatin, and lovastatin are administered separately. By performing the operation, it was confirmed that the transplanted bone marrow stromal cells were differentiated into nerve cells and regenerated functionally in the nerve cells in the brain of the rat in which cerebral ischemia was artificially induced.
[0043]
Example 6 In Example 4, instead of simvastatin, a mixture of atorvastatin and fluvastatin was administered and the same operation was performed, and the same effect was obtained.
[0044]
Example 7 Efficacy evaluation for Alzheimer's disease. Eight patients who can read and write in Japanese and can communicate their will to the doctor but have a clinical diagnosis of possible Alzheimer's disease and are hypersensitive to HMG-CoA reductase inhibitors such as pravastatin sodium The following mini-mental state test is conducted for non-lactating and non-lactating persons who have no history of illness, are suspected of having impaired renal function and hepatic metabolism, and who are or may be pregnant . The patient confirms that the severity index is 25 or less. If there is a patient whose severity index is 25 or more, the patient is replaced with a patient whose severity index is 25 or less. Also exclude patients who were using the study drug within 4 months prior to the measurement. In addition, lipophilic 6-blockers such as cimetidine and propranolol, clonidine, anticholinergics, antidepressants with anticholinergic activity, neuroleptics, putative cognitive enhancers and central nervous stimulators, or benzodiazepines with a long half-life Prohibits administration.
[0045]
Contents of the mini-mental state examination: Orientation (1. Current year? 1 point, season? 1 point, day of the week? 1 point, date? 1 point, month? 1 point, 2. We are now The name of the prefecture is 1 point, the name of the county is 1 point, the name of the city / town is 1 point, the floor is 1 point, 1 point, 1 point, the address / building name is 1 point) Ingenuity (3. The doctor will write the names of the three things in seconds. After the doctor says, ask the patient for all three names. Repeat the answer until the patient says all three correctly. .3 points), attention and calculation (4.100, then continue to subtract 7. Give 1 point for each correct answer. Finish after 5 answers. 5 points), Recall (5. Ask for the name of the object, give one point for each correct answer, 3 points), language (6. Doctor points to pencil and watch. Tell the patient the name of the object the doctor points to. 2 points, 7. Ask the patient to say “No, if, but,” 1 point, 8. Give the patient a three-step command and obey it, “Pick up the paper with your right hand. Fold the paper in half.Place the paper on the desk. ”3 points, 9. Ask the patient to read and follow the instructions:“ Close your eyes. ”1 point, 10. Ask the patient to write one sentence freely, the sentence must contain a guardian and one object, and it must make sense, and the score does not take into account typographical errors. Show a figure with two vertices of one side approximately 5 centimeters overlapped by one vertex and let the patient transcribe it.If all corners and angles are preserved and the intersecting area is a rectangle, 1 1 point), a total of 30 points.
[0046]
Under the supervision of a doctor, a subject is administered a commercially available medical pravastatin sodium 10 mg tablet twice a day for 6 months. A mini-mental state test will be conducted after 6 months. Mini-mental state test confirms orientation, memorization, attention and calculation, recall, and language improvement with pravastatin sodium administration.
[0047]
【The invention's effect】
The composition of the present invention has (1) the ability to contact non-neuronal cells after activation, if necessary, and to differentiate the non-neuronal cells into neurons without being treated with a mitogenic substance, or (2) if necessary The ability to contact a neuron after activation and to mature the neuron without treatment with a mitogenic substance, or (3) if necessary, contact the non-neuron after activation to induce the non-neuron to undergo mitosis Ability to differentiate into neurons without treatment with substances and maturation of the neurons, or (4) contact with non-neurons, if necessary, after treatment, and treat the non-neurons with a mitogenic substance It has the ability to treat or malignantly inhibit neuropathy diseases by differentiating into nerve cells and maturing the nerve cells. Therefore, by in vitro administration of the composition of the present invention, it is useful for producing nerve cells that are not likely to be mixed with macrophages that cause transplantation rejection and cancerous neurons, and non-neuronal cells or transplantation rejection Alzheimer's disease, amyotrophic lateral sclerosis by administering the composition of the present invention into the body after injecting or without injecting nerve cells that are not likely to contaminate the macromolecules and cancerous neurons , Brain tumor, cerebellar degeneration, hydrocephalus, Huntington's disease, frontal lobe irradiation, multiple sclerosis, normal pressure hydrocephalus, Parkinson's disease, Pick's disease, progressive multifocal leukoencephalopathy, progressive supranuclear palsy, Fist dementia, brain trauma, surgery, brain tumor, chronic subdural hematoma, stroke, cerebrovascular dementia, Wilson disease bacterial endocarditis, Creutzfeldt-Jakob disease, Gerstmann-Stroisler Shy For Kerr disease, HIV-related disease, neurosyphilis, tuberculosis and fungal meningitis, viral encephalitis, anoxia, B12 deficiency, chronic drug-alcohol-nutrient abuse, folate deficiency, hyperparathyroidism It is useful as a therapeutic or malignant suppressant for hypercalcemia, hypoglycemia, hypothyroidism, hepatic encephalopathy, pulmonary encephalopathy, organ system failure such as uremic encephalopathy, neuropathy diseases derived from pellagra and the like.
[0048]
The method of the present invention comprises (1) a method of differentiating non-neuronal cells into neurons without being treated with a mitogenic substance, (2) a method of maturing neurons without being treated with a mitogenic substance, (3 ) A method of differentiating the non-neuronal cell into a nerve cell without treatment with a mitogenic substance and maturation of the nerve cell; and (4) differentiation of the non-neuronal cell into a nerve cell without treatment with a mitogenic substance. And a method of treating or inhibiting malignant neuropathy by maturating the nerve cells. Therefore, the method of the present invention is useful as a method for producing nerve cells that are not likely to be mixed with macrophages that cause transplant rejection and cancerous neurons, and is useful as a method for treating or inhibiting malignant diseases of the above-mentioned neuropathy. It is.
[0049]
The nerve cell of the present invention is a nerve cell that has no fear of contamination of a macromolecule that causes transplant rejection and cancerous nerve cells. Therefore, it is useful not only for naturally differentiated neurons or naturally differentiated and mature neurons but also for research or therapeutic purposes.
[0050]
The search method of the present invention includes (1) a method for searching for a low molecular weight substance having only a pharmaceutically acceptable toxicity, which has the ability to differentiate a non-neuronal cell into a nerve cell without treatment with a mitogenic substance, (2) A method for searching for a low-molecular-weight substance having a pharmacologically acceptable toxicity that has the ability to mature without treating neuronal cells with mitogenic substances, and (3) treating non-neuronal cells with mitogenic substances. This is a method of searching for a low molecular weight substance having a pharmaceutically acceptable toxicity that has the ability to be differentiated and matured into a neuron without being differentiated. These methods are useful because they may create useful drugs and reagents.
[Brief description of the drawings]
BRIEF DESCRIPTION OF THE FIGURES FIG. 1. Differentiation of bone marrow stromal cells into neurons upon contact with 5 micromolar activated simvastatin 24 hours. Normal light micrograph. Magnification 100 times. Scale bar length: 50 micrometers (Example 1)
FIG. 2. Differentiation of bone marrow stromal cells into neurons by contact with 5 micromolar activated simvastatin for 48 hours. Normal light micrograph. Magnification 100 times. Scale bar length: 50 micrometers (Example 1)
FIG. 3. Differentiation of bone marrow stromal cells into neurons by contact with 5 micromolar activated simvastatin for 24 hours. Fluorescence photomicrograph of a double immunolabeled specimen of MAP2 (green) and tau (red). Magnification 100 times. Scale bar length: 50 micrometers (Example 1)
FIG. 4. Differentiation of bone marrow stromal cells into neurons by contact with 5 micromolar activated simvastatin for 48 hours. Fluorescence micrograph of a double immunolabeled specimen of MAP2 (green) and tau (red). Magnification 100 times. Scale bar length: 50 micrometers (Example 1)

Claims (5)

シンバスタチンを有効成分とすることを特徴とする、該組成物中のシンバスタチンを活性化後に、骨髄間質細胞に接触させ、該骨髄間質細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化するための組成物。  Simvastatin in the composition, which is activated by simvastatin as an active ingredient, is brought into contact with bone marrow stromal cells, and the bone marrow stromal cells are differentiated into nerve cells without being treated with a mitogenic agent. Composition to do. 請求項１に記載の組成物を有効成分とすることを特徴とする、該組成物中のシンバスタチンを活性化後に、骨髄間質細胞に接触させ、該骨髄間質細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟するための組成物。The composition of claim 1 is used as an active ingredient, and after simvastatin in the composition is activated, it is brought into contact with bone marrow stromal cells, and the bone marrow stromal cells are treated with a mitogenic substance. A composition for differentiating into a nerve cell without maturation and maturing the nerve cell. 請求項１に記載の組成物を有効成分とし、該組成物中のシンバスタチンを活性化後に、骨髄間質細胞に接触させ、該骨髄間質細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することにより神経障害疾患を治療または悪性化抑制することを特徴とする、神経細胞の増加により病状を改善する神経障害疾患を治療または悪性化抑制するための組成物。The composition of claim 1 is used as an active ingredient, and after activation of simvastatin in the composition, the bone marrow stromal cells are brought into contact with the bone marrow stromal cells, and the bone marrow stromal cells are treated with neuronal cells without being treated with a mitogenic agent. A composition for treating or inhibiting malignancy of a neuropathic disease that improves a disease state by increasing the number of nerve cells, characterized by treating the neuropathy disease or suppressing malignancy by differentiating and maturating the nerve cell object. 請求項１に記載の組成物を、該組成物中のシンバスタチンを活性化後に、ヒトの生体内以外において骨髄間質細胞に接触させ、該骨髄間質細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化することを特徴とする、骨髄間質細胞を神経細胞へ分化する方法。The composition according to claim 1, after activating simvastatin in the composition, after contacting the bone marrow stromal cells outside the human body without treating the bone marrow stromal cells with a mitogenic substance. A method for differentiating bone marrow stromal cells into nerve cells, characterized by differentiating into nerve cells. 請求項２に記載の組成物を、該組成物中のシンバスタチンを活性化後に、ヒトの生体内以外において骨髄間質細胞に接触させ、該骨髄間質細胞を細胞分裂誘起物質で処理することなく神経細胞へ分化し、かつ該神経細胞を成熟することを特徴とする、骨髄間質細胞を神経細胞へ分化し、かつ該神経細胞を成熟する方法。The composition according to claim 2, after simvastatin in the composition is activated, is brought into contact with bone marrow stromal cells other than in the human body, and the bone marrow stromal cells are not treated with a mitogenic substance. A method for differentiating bone marrow stromal cells into nerve cells and maturating the nerve cells, characterized by differentiating into nerve cells and maturing the nerve cells.
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