JP2017214302A - Autophagy inducer - Google Patents

Autophagy inducer Download PDF

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JP2017214302A
JP2017214302A JP2016107609A JP2016107609A JP2017214302A JP 2017214302 A JP2017214302 A JP 2017214302A JP 2016107609 A JP2016107609 A JP 2016107609A JP 2016107609 A JP2016107609 A JP 2016107609A JP 2017214302 A JP2017214302 A JP 2017214302A
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autophagy
gfp
cells
rfp
cloperastine
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昇 水島
Noboru Mizushima
昇 水島
剛志 貝塚
Takeshi Kaizuka
剛志 貝塚
祐太郎 濱
Yutaro Hama
祐太郎 濱
雄一郎 豊田
Yuichiro Toyoda
雄一郎 豊田
水島　徹
Toru Mizushima
徹 水島
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LTT Bio Pharma Co Ltd
University of Tokyo NUC
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LTT Bio Pharma Co Ltd
University of Tokyo NUC
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Priority to PCT/JP2017/019848 priority patent/WO2017209027A1/en
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    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
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    • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
    • A61K31/569—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone substituted in position 17 alpha, e.g. ethisterone
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Abstract

PROBLEM TO BE SOLVED: To provide a novel autophagy inducer.SOLUTION: An autophagy inducer contains, as an active ingredient, a component selected from adefovir dipivoxil, azidothymidine, benproperine, cloperastine, enalapril, homochlorcyclizine, lomerizine, methyltestosterone, norethisterone, oxaprozin, prulifloxacin, tipepidine and salts thereof.SELECTED DRAWING: None

Description

本発明は、オートファジー誘導剤及び神経変性疾患治療剤に関する。   The present invention relates to an autophagy-inducing agent and a neurodegenerative disease therapeutic agent.

オートファジーは、細胞が持っている、細胞内のタンパク質を分解するための仕組みの一つであり、自食作用ともよばれる。オートファジーではまず細胞質の一部が隔離膜によって取り囲まれてオートファゴソームが形成され、次にオートファゴソームの外膜がリソソーム膜と融合しオートリソソームとなり、内容物が分解される。オートファジーはタンパク質だけではなくミトコンドリアなどのオルガネラをも分解できる大規模なシステムで、酵母から高等動植物に至るまで非常によく保存されている。オートファジーでは約１μｍの領域がランダムに包み込まれるため、オートファジーは原則として非選択的な分解システムであると考えられてきたが、最近になって一部の基質は選択的に取り込まれることもわかってきた。   Autophagy is one of the mechanisms that cells have to break down intracellular proteins and is also called autophagy. In autophagy, a part of the cytoplasm is first surrounded by an isolation membrane to form an autophagosome, and then the outer membrane of the autophagosome is fused with the lysosomal membrane to become an autolysosome and the contents are degraded. Autophagy is a large-scale system capable of degrading not only proteins but also organelles such as mitochondria, and is well preserved from yeast to higher animals and plants. Autophagy has been considered to be a non-selective degradation system in principle, since the region of about 1 μm is randomly wrapped in autophagy, but recently, some substrates may be selectively incorporated. I understand.

オートファジーは日常的に一定のレベルでおこっており、細胞内構成成分の品質管理機構として重要である。オートファジー能を欠損させたマウスでは反射異常、運動機能障害などをともなう神経変性を呈する（非特許文献１）。またヒトのパーキンソン病や、鉄沈着を伴う神経変性疾患（ＳＥＮＤＡ）においてもオートファジー関連遺伝子の変異が発見されている（非特許文献２、３）。これらの事実を踏まえ、オートファジーによる細胞内浄化を亢進させることで、異常タンパク質蓄積を特徴とする疾患を治療することが動物実験で試みられている（非特許文献４〜８）。ヒトでは、肝細胞内に変異タンパク質が蓄積するα１アンチトリプシン欠損症で、オートファジー活性化効果のあるカルバマゼピンによって症状が軽減したという報告がある（非特許文献９）。 Autophagy occurs at a certain level on a daily basis, and is important as a quality control mechanism for intracellular components. Mice deficient in autophagy exhibit neurodegeneration with reflex abnormalities, motor dysfunction, etc. (Non-patent Document 1). Autophagy-related gene mutations have also been found in human Parkinson's disease and neurodegenerative diseases (SENDA) with iron deposition (Non-patent Documents 2 and 3). Based on these facts, animal experiments have attempted to treat diseases characterized by abnormal protein accumulation by enhancing intracellular purification by autophagy (Non-Patent Documents 4 to 8). In humans, there is a report that symptoms are reduced by carbamazepine having an autophagy activation effect in α 1 antitrypsin deficiency in which mutant proteins accumulate in hepatocytes (Non-patent Document 9).

Hara, T. et al. (2006) Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature, 441: 885-889.Hara, T. et al. (2006) Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice.Nature, 441: 885-889. Narendra, D. et al. (2008) Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J Cell Biol, 183: 795-803.Narendra, D. et al. (2008) Parkin is recruited selectively to impaired mitochondria and promotes their autophagy.J Cell Biol, 183: 795-803. Saitsu, H. et al. De novo mutations in the autophagy gene WDR45 cause static encephalopathy of childhood with neurodegeneration in adulthood. Nat. Genet., 45: 445-449.Saitsu, H. et al. De novo mutations in the autophagy gene WDR45 cause static encephalopathy of childhood with neurodegeneration in adulthood. Nat. Genet., 45: 445-449. Ravikumar et al. (2004) Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat. Genet., 36, 585-595.Ravikumar et al. (2004) Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat. Genet., 36, 585-595. Williams et al. (2008) Novel targets for Huntington's disease in an mTOR-independent autophagy pathway. Nat Chem Biol. 4:295-305.Williams et al. (2008) Novel targets for Huntington's disease in an mTOR-independent autophagy pathway. Nat Chem Biol. 4: 295-305. Caccamo A et al. (2010) Molecular interplay between mammalian target of rapamycin (mTOR), amyloid-beta, and Tau: effects on cognitive impairments. J Biol Chem. 285:13107-20.Caccamo A et al. (2010) Molecular interplay between mammalian target of rapamycin (mTOR), amyloid-beta, and Tau: effects on cognitive impairments.J Biol Chem. 285: 13107-20. Wang IF et al. (2012) Autophagy activators rescue and alleviate pathogenesis of a mouse model with proteinopathies of the TAR DNA-binding protein 43. Proc Natl Acad Sci U S A. 109:15024-9.Wang IF et al. (2012) Autophagy activators rescue and alleviate pathogenesis of a mouse model with proteinopathies of the TAR DNA-binding protein 43. Proc Natl Acad Sci U S A. 109: 15024-9. Cortes et al. (2012) Rapamycin delays disease onset and prevents PrP plaque deposition in a mouse model of Gerstmann-Straussler-Scheinker disease. J Neurosci. 32:12396-405.Cortes et al. (2012) Rapamycin delays disease onset and prevents PrP plaque deposition in a mouse model of Gerstmann-Straussler-Scheinker disease. J Neurosci. 32: 12396-405. Hidvegi T et al. (2010) An autophagy-enhancing drug promotes degradation of mutant alpha1-antitrypsin Z and reduces hepatic fibrosis. Science. 329:229-32.Hidvegi T et al. (2010) An autophagy-enhancing drug promotes degradation of mutant alpha1-antitrypsin Z and reduces hepatic fibrosis. Science. 329: 229-32.

このようにオートファジーの誘導を促進させるというアプローチは、現在有効性の高い薬剤が存在しない神経変性疾患の治療に有効であることが考えられる。
従って、本発明の課題は、新たなオートファジー誘導剤を提供することにある。 Thus, the approach of promoting the induction of autophagy is considered to be effective for the treatment of neurodegenerative diseases for which there are no drugs that are currently highly effective.
Accordingly, an object of the present invention is to provide a new autophagy inducer.

そこで本発明者は、新たなオートファジー誘導剤を探索すべく既に医薬として用いられている成分を対象としてスクリーニングしてきたところ、特定の１２種の成分に強いオートファジー誘導作用があることから神経変性疾患治療剤として有用であることを見出し、本発明を完成した。   Therefore, the present inventor has screened components already used as pharmaceuticals in order to search for new autophagy-inducing agents, and since 12 specific components have a strong autophagy-inducing action, neurodegeneration has occurred. The present invention was completed by finding it useful as a disease therapeutic agent.

すなわち、本発明は、次の〔１〕〜〔２〕を提供するものである。   That is, the present invention provides the following [1] to [2].

〔１〕アデフォビル ピボキシル、アジドチミジン、ベンプロペリン、クロペラスチン、エナラプリル、ホモクロルシクリジン、ロメリジン、メチルテストステロン、ノルエチステロン、オキサプロジン、プルリフロキサシン、チペピジン及びこれらの塩から選ばれる成分を有効成分とするオートファジー誘導剤。
〔２〕アデフォビル ピボキシル、アジドチミジン、ベンプロペリン、クロペラスチン、エナラプリル、ホモクロルシクリジン、ロメリジン、メチルテストステロン、ノルエチステロン、オキサプロジン、プルリフロキサシン、チペピジン及びこれらの塩から選ばれる成分を有効成分とする神経変性疾患治療剤。 [1] Autophagy induction containing as an active ingredient an ingredient selected from adefovir pivoxil, azidothymidine, benproperin, cloperastine, enalapril, homochlorcyclidine, lomerizine, methyltestosterone, norethisterone, oxaprozin, pullrifloxacin, tipipedin and salts thereof Agent.
[2] Neurodegenerative diseases comprising as an active ingredient an ingredient selected from adefovir pivoxil, azidothymidine, benproperin, cloperastine, enalapril, homochlorcyclidine, lomerizine, methyltestosterone, norethisterone, oxaprozin, pullrifloxacin, tipipedin and salts thereof Therapeutic agent.

本発明のオートファジー誘導剤及び神経変性疾患治療剤の有効成分は、既に医薬として使用されている成分であるから、安全性は確認されており、かつ強いオートファジー誘導作用を有し、アミロイドβに代表されるペプチド凝集体等を原因とする神経変性疾患の治療に有効である。   Since the active ingredients of the autophagy-inducing agent and neurodegenerative disease therapeutic agent of the present invention are components already used as pharmaceuticals, safety has been confirmed and they have a strong autophagy-inducing action, and amyloid β It is effective in the treatment of neurodegenerative diseases caused by peptide aggregates represented by

ＧＦＰ−ＬＣ３ドット形成の誘導を示す（溶媒のみ）。Shows induction of GFP-LC3 dot formation (solvent only). ＧＦＰ−ＬＣ３ドット形成の誘導を示す（クロペラスチン塩酸塩）。Shows the induction of GFP-LC3 dot formation (cloperastine hydrochloride). ＧＦＰ−ＬＣ３ドット形成の誘導を示す（アデフォビルピボキシル）。Shows induction of GFP-LC3 dot formation (Adefovir pivoxil). 薬剤処理による凝集体陽性細胞数の減少を示す。ＧＦＰシグナルの蛍光顕微鏡像。明るい小さな輝点がＰｏｌｙＱ凝集体を示す。The reduction | decrease in the number of aggregate positive cells by a chemical | medical agent process is shown. Fluorescent microscope image of GFP signal. Bright small bright spots indicate PolyQ aggregates. 薬剤処理による凝集体陽性細胞数の減少を示す。凝集体陽性率の定量結果。The reduction | decrease in the number of aggregate positive cells by a chemical | medical agent process is shown. Quantitative result of aggregate positive rate.

本発明のオートファジー誘導剤及び神経変性疾患治療剤の有効成分は、アデフォビル ピボキシル、アジドチミジン、ベンプロペリン、クロペラスチン、エナラプリル、ホモクロルシクリジン、ロメリジン、メチルテストステロン、ノルエチステロン、オキサプロジン、プルリフロキサシン、チペピジン及びこれらの塩から選ばれる成分である。   The active ingredients of the autophagy-inducing agent and neurodegenerative disease therapeutic agent of the present invention include adefovir pivoxil, azidothymidine, benproperin, cloperastine, enalapril, homochlorcyclidine, lomerizine, methyltestosterone, norethisterone, oxaprozin, pullrifloxacin, tipipedin and It is a component selected from these salts.

アデフォビル ピボキシルは、Ｂ型慢性肝炎の治療に用いられる逆転写阻害剤である。アジドチミジンは、核酸系逆転写酵素阻害剤の一種で、ＨＩＶの治療薬として用いられる成分である。ベンプロペリンは、咳中枢に作用する鎮咳剤である。ベンプロペリンの塩としては、リン酸塩が挙げられる。クロペラスチンは、咳中枢に直接作用し、鎮咳剤として用いられている。クロペラスチンの塩としては、塩酸塩等が挙げられる。   Adefovir pivoxil is a reverse transcription inhibitor used in the treatment of chronic hepatitis B. Azidothymidine is a kind of nucleic acid reverse transcriptase inhibitor and is a component used as a therapeutic agent for HIV. Benproperin is an antitussive agent that acts on the cough center. Examples of the salt of benproperin include phosphate. Cloperastine acts directly on the cough center and is used as an antitussive. Examples of the salt of cloperastine include hydrochloride.

エナラプリルは、アンジオテンシン変換酵素阻害剤として知られており、高血圧症治療薬として用いられている。エナラプリルの塩としては、マレイン酸塩が挙げられる。   Enalapril is known as an angiotensin converting enzyme inhibitor and is used as a therapeutic agent for hypertension. The salt of enalapril includes maleate.

ホモクロルシクリジンは、ヒスタミンＨ1受容体遮断薬であり、皮膚疾患に伴うそう痒、じん麻疹、アレルギー性鼻炎等の治療に用いられている。ホモクロルシクリジンの塩としては、塩酸塩等が挙げられる。ロメリジンは、カルシウム拮抗薬であり、片頭痛治療薬として用いられている。ロメリジンの塩としては、塩酸塩等が挙げられる。 Homochlorcyclidine is a histamine H 1 receptor blocker and is used to treat pruritus, urticaria, allergic rhinitis and the like associated with skin diseases. Examples of the salt of homochlorocyclidine include hydrochloride. Lomeridine is a calcium antagonist and is used as a migraine treatment. Examples of the salt of romeridine include hydrochloride.

メチルテストステロンは、男性ホルモン薬（アンドロゲン）であり、男子性腺機能不全、造精機能障害による男子不妊症等に用いられる。ノルエチステロンは、別名ノルエチンドロンであり、黄体ホルモン薬であって、無月経、月経周期異常等の治療に用いられている。   Methyltestosterone is a male hormone drug (androgen) and is used for male infertility due to male gonad dysfunction and spermatogenic dysfunction. Norethisterone, also known as norethindrone, is a luteinizing hormone drug used for the treatment of amenorrhea, menstrual cycle abnormalities and the like.

オキサプロジンは、非ステロイド抗炎症薬の一つであり、消炎鎮痛薬として用いられている。   Oxaprozin is one of non-steroidal anti-inflammatory drugs and is used as an anti-inflammatory analgesic.

プルリフロキサシンは、キノロン系合成抗菌剤として用いられている。チペピジンは、鎮咳去痰薬として用いられており、その塩としてはヒベンズ酸塩が挙げられる。   Plurifloxacin is used as a quinolone synthetic antibacterial agent. Tipepidine is used as an antitussive expectorant, and its salt includes hibenzate.

前記本発明のオートファジー誘導剤の有効成分として用いられる成分は、既に種々の疾患の治療剤として用いられているが、オートファジー誘導作用を有すること、また神経変性疾患に有効であることは全く知られていない。   The component used as an active ingredient of the autophagy-inducing agent of the present invention has already been used as a therapeutic agent for various diseases, but it has no autophagy-inducing action and is effective for neurodegenerative diseases. unknown.

後記実施例に示すように前記成分は、優れたオートファジー誘導作用を有するため、アミロイドに代表されるタンパク質の異常に基づく神経変性疾患の治療剤として有用である。神経変性疾患とそのタンパク質としては、アルツハイマー病（アミロイドβ、タウ蛋白質）、パーキンソン病（αシヌクレイン）、糖尿病（アミリン）、全身性アミロイド−シス（トランスサイレチン）、ハンチントン病（ハンチンチン）等が挙げられる。本発明のオートファジー誘導剤は、これらの神経変性疾患における異常タンパク質をオートファジー誘導作用により減少させることにより、神経変性疾患を治療することができる。   As will be described later in Examples, the component has an excellent autophagy-inducing action, and thus is useful as a therapeutic agent for neurodegenerative diseases based on protein abnormalities such as amyloid. Neurodegenerative diseases and their proteins include Alzheimer's disease (amyloid β, tau protein), Parkinson's disease (α synuclein), diabetes (amylin), systemic amyloid-cis (transthyretin), Huntington's disease (huntingtin), etc. Can be mentioned. The autophagy-inducing agent of the present invention can treat neurodegenerative diseases by reducing abnormal proteins in these neurodegenerative diseases by autophagy-inducing action.

本発明のオートファジー誘導剤及び神経変性疾患治療剤の投与形態としては、注射剤、経口剤（錠剤、顆粒剤、散剤、カプセル剤）、軟膏剤、クリーム剤、貼付剤、坐剤等が挙げられる。このうち、経口剤が特に好ましい。これらの医薬組成物の形態とするには、薬学的に許容される担体とともに製剤化することができる。そのような担体としては、例えば、乳糖、ブドウ糖、Ｄ−マンニトール、澱粉、結晶セルロース、炭酸カルシウム、カオリン、デンプン、ゼラチン、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルピロリドン、エタノール、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウム塩、ステアリン酸マグネシウム、タルク、アセチルセルロース、白糖、酸化チタン、安息香酸、パラオキシ安息香酸エステル、デヒドロ酢酸ナトリウム、アラビアゴム、トラガント、メチルセルロース、卵黄、界面活性剤、白糖、単シロップ、クエン酸、蒸留水、エタノール、グリセリン、プロピレングリコール、マクロゴール、リン酸−水素ナトリウム、リン酸二水素ナトリウム、リン酸ナトリウム、ブドウ糖、塩化ナトリウム、フェノール、チメロサール、パラオキシ安息香酸エステル、亜硫酸水素ナトリウム等が挙げられる。   Examples of the administration form of the autophagy-inducing agent and neurodegenerative disease therapeutic agent of the present invention include injections, oral agents (tablets, granules, powders, capsules), ointments, creams, patches, suppositories and the like. It is done. Of these, oral preparations are particularly preferred. These pharmaceutical compositions can be formulated with a pharmaceutically acceptable carrier. Examples of such carriers include lactose, glucose, D-mannitol, starch, crystalline cellulose, calcium carbonate, kaolin, starch, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, ethanol, carboxymethylcellulose, carboxymethylcellulose calcium. Salt, magnesium stearate, talc, acetylcellulose, saccharose, titanium oxide, benzoic acid, p-hydroxybenzoate, sodium dehydroacetate, gum arabic, tragacanth, methylcellulose, egg yolk, surfactant, sucrose, simple syrup, citric acid, distillation Water, ethanol, glycerin, propylene glycol, macrogol, sodium phosphate-hydrogen, sodium dihydrogen phosphate, sodium phosphate, grape , Sodium chloride, phenol, thimerosal, p-hydroxybenzoic acid esters, and sodium hydrogen sulfite.

さらに、本発明の医薬組成物製剤中における本発明の有効成分の含有量は、製剤の形によって大きく変動し、特に限定されるものではないが、通常は、組成物全量に対して０．０１〜１００質量％、好ましくは１〜１００質量％である。   Further, the content of the active ingredient of the present invention in the pharmaceutical composition preparation of the present invention varies greatly depending on the form of the preparation and is not particularly limited, but is usually 0.01% with respect to the total amount of the composition. -100 mass%, preferably 1-100 mass%.

本発明のオートファジー誘導剤及び神経変性疾患治療剤の投与量は、投与する患者の症状、年齢、投与方法によって異なるが、前記有効成分量として、成人に対して１日あたり１〜１０００ｍｇであるのが好ましい。またこの投与量は１日に１〜４回、好ましくは２〜４回に分けて投与することもできる。   The dosage of the autophagy-inducing agent and therapeutic agent for neurodegenerative diseases of the present invention varies depending on the symptoms, age, and administration method of the patient to be administered, but the amount of the active ingredient is 1-1000 mg per day for an adult. Is preferred. In addition, this dose can be administered 1 to 4 times a day, preferably 2 to 4 times a day.

次に実施例を挙げて本発明を更に詳細に説明する。   EXAMPLES Next, an Example is given and this invention is demonstrated still in detail.

実施例１
ＧＦＰ−ＬＣ３−ＲＦＰ−ＬＣ３ΔＧプローブを用いたオートファジー活性の評価 Example 1
Evaluation of autophagy activity using GFP-LC3-RFP-LC3ΔG probe

〔方法〕
ＧＦＰ−ＬＣ３−ＲＦＰ−ＬＣ３ΔＧを安定発現するＨｅＬａ細胞を用いた。この細胞が発現するＧＦＰ−ＬＣ３−ＲＦＰ−ＬＣ３ΔＧは先頭（アミノ末端）より、緑色蛍光タンパク質（ＧＦＰ）、ｍｉｃｒｏｔｕｂｕｌｅ−ａｓｓｏｃｉａｔｅｄ ｐｒｏｔｅｉｎ ｌｉｇｈｔ ｃｈａｉｎ ３（ＬＣ３）、赤色蛍光タンパク質（ＲＦＰ）、ＬＣ３のカルボキシ末端グリシンの欠損体（ＬＣ３ΔＧ）が連結したものである。ＧＦＰ−ＬＣ３−ＲＦＰ−ＬＣ３ΔＧは細胞内で合成された後に直ちにＧＦＰ−ＬＣ３とＲＦＰ−ＬＣ３ΔＧに切断分離される。ＧＦＰ−ＬＣ３はオートファジーによって分解されるが、ＲＦＰ−ＬＣ３ΔＧは分解されずに細胞内にとどまる。従って、オートファジーが誘導されるとＧＦＰ／ＲＦＰ比が低下する。この細胞を９６穴プレートに３０，０００個ずつ播種し、翌日薬剤または溶媒のみを含む培地に交換し（１薬剤につき３ｗｅｌｌずつ使用）した。２４時間後、各ｗｅｌｌの細胞を１０分間４％パラフォルムアルデヒド溶液で固定した。マイクロプレートリーダー（ＥｎＳｐｉｒｅ，ＰｅｒｋｉｎＥｌｍｅｒ Ｊａｐａｎ）で各ウェルのＧＦＰとＲＦＰの蛍光量を測定しＧＦＰ／ＲＦＰ比を算出した。コントロール（溶媒で処理した細胞）のＧＦＰ／ＲＦＰ比との比率を表１に記す。 〔Method〕
HeLa cells stably expressing GFP-LC3-RFP-LC3ΔG were used. GFP-LC3-RFP-LC3ΔG expressed by this cell is from the head (amino terminus) to green fluorescent protein (GFP), microtube-associated protein light chain 3 (LC3), red fluorescent protein (RFP), carboxy-terminal glycine of LC3 Deficient body (LC3ΔG) is linked. GFP-LC3-RFP-LC3ΔG is immediately cleaved and separated into GFP-LC3 and RFP-LC3ΔG after being synthesized in the cell. GFP-LC3 is degraded by autophagy, but RFP-LC3ΔG remains in the cell without being degraded. Therefore, when autophagy is induced, the GFP / RFP ratio decreases. 30,000 of these cells were seeded in 96-well plates, and the following day, the medium was replaced with a medium containing only the drug or solvent (3 wells were used per drug). After 24 hours, the cells in each well were fixed with a 4% paraformaldehyde solution for 10 minutes. The GFP / RFP ratio was calculated by measuring the amount of fluorescence of GFP and RFP in each well with a microplate reader (EnSpire, PerkinElmer Japan). The ratio of the control (cells treated with the solvent) to the GFP / RFP ratio is shown in Table 1.

〔結果〕
上記の薬剤で２４時間処理した細胞ではＧＦＰ／ＲＦＰ比が０．８５以下に低下したため、これらの薬剤にオートファジー誘導効果があることが示された。 〔result〕
In cells treated with the above drugs for 24 hours, the GFP / RFP ratio decreased to 0.85 or less, indicating that these drugs have an autophagy-inducing effect.

実施例２
オートファゴソーム形成の可視化による評価
〔方法〕
実施例１と同様にＧＦＰ−ＬＣ３−ＲＦＰ−ＬＣ３ΔＧを安定発現するＨｅＬａ細胞を用いた。この細胞が産生するＧＦＰ−ＬＣ３はオートファゴソームに結合するため、それらは蛍光顕微鏡で輝点として観察される。この細胞を各薬剤で２時間、２４時間処理し、それぞれ４％パラフォルムアルデヒド溶液で１０分間固定した後、蛍光顕微鏡（ＩＸ８１，ＯＬＹＭＰＵＳ）で観察した（図１〜図３）。観察されたＧＦＰ−ＬＣ３の輝点の数を−、±、＋、＋＋、＋＋＋の５段階で評価した（表２）。 Example 2
Visualization of autophagosome formation [method]
As in Example 1, HeLa cells stably expressing GFP-LC3-RFP-LC3ΔG were used. Since GFP-LC3 produced by these cells binds to autophagosomes, they are observed as bright spots with a fluorescence microscope. The cells were treated with each drug for 2 hours and 24 hours, fixed with a 4% paraformaldehyde solution for 10 minutes, and then observed with a fluorescence microscope (IX81, OLYMPUS) (FIGS. 1 to 3). The number of observed bright spots of GFP-LC3 was evaluated in five stages:-, ±, +, ++, and ++ (Table 2).

〔結果〕
薬剤で２４時間処理した細胞ではＧＦＰ−ＬＣ３輝点が増加したため、これらの薬剤にオートファジー誘導効果があることが示された。 〔result〕
In cells treated with drugs for 24 hours, GFP-LC3 bright spots increased, indicating that these drugs have an autophagy-inducing effect.

実施例３
神経変性疾患関連凝集体の除去効果
（方法）
ドキシサイクリン（ＤＯＸ）依存的にＧＦＰ−Ｑ８１を発現するＨｅＬａ細胞を用いた。ＧＦＰ−Ｑ８１は、緑色蛍光タンパク質（ＧＦＰ）に８１個のグルタミンが連なった配列（ＰｏｌｙＱ）を付加したものである。ＰｏｌｙＱは細胞内で自己相互作用して凝集体を形成することが知られている。ＰｏｌｙＱ凝集体は複数のヒト神経変性疾患の病態に関連することが知られており、オートファジーの選択的基質となることも報告されている。この細胞をカバーガラス上に播種し、それと同時にＤＯＸを添加して凝集体の誘導を開始した。７２時間後、溶媒（ＤＭＳＯ）またはオートファジー誘導剤（１０μＭ）を含む培地に交換した。更に２４時間後、各細胞を１０分間４％パラフォルムアルデヒド溶液で固定し、核を染色した後に、蛍光顕微鏡で観察した。核染色像から画像内の総細胞数を、ＧＦＰ像から凝集体陽性の細胞数を定量し、総細胞数に対する凝集体陽性細胞の割合を計算した。結果を図４、図５及び表３に示す。 Example 3
Removal effect of neurodegenerative disease-related aggregates (method)
HeLa cells expressing GFP-Q81 in a doxycycline (DOX) dependent manner were used. GFP-Q81 is obtained by adding a sequence (PolyQ) in which 81 glutamines are linked to green fluorescent protein (GFP). PolyQ is known to self-interact in cells to form aggregates. PolyQ aggregates are known to be associated with the pathology of multiple human neurodegenerative diseases and have been reported to be selective substrates for autophagy. The cells were seeded on a cover glass, and at the same time, the induction of aggregates was started by adding DOX. After 72 hours, the medium was replaced with a medium containing a solvent (DMSO) or an autophagy inducer (10 μM). After an additional 24 hours, each cell was fixed with a 4% paraformaldehyde solution for 10 minutes, stained with nuclei, and then observed with a fluorescence microscope. The total number of cells in the image was quantified from the nuclear staining image, and the number of aggregate-positive cells from the GFP image, and the ratio of aggregate-positive cells to the total number of cells was calculated. The results are shown in FIGS. 4 and 5 and Table 3.

図４、５及び表３より、上記の薬剤はＤＭＳＯ処理時に比して凝集体陽性細胞率が低下したため（表３、図４、５）、これらの薬剤に神経変性疾患関連凝集体の除去効果があることが示唆された。   4 and 5 and Table 3, since the above-mentioned drugs had a decrease in the aggregate positive cell rate as compared to DMSO treatment (Tables 3, 4 and 5), these drugs have the effect of removing neurodegenerative disease-related aggregates. It was suggested that there is.

Claims (2)

アデフォビル ピボキシル、アジドチミジン、ベンプロペリン、クロペラスチン、エナラプリル、ホモクロルシクリジン、ロメリジン、メチルテストステロン、ノルエチステロン、オキサプロジン、プルリフロキサシン、チペピジン及びこれらの塩から選ばれる成分を有効成分とするオートファジー誘導剤。   An autophagy inducer comprising as an active ingredient an ingredient selected from adefovir pivoxil, azidothymidine, benproperin, cloperastine, enalapril, homochlorcyclidine, lomerizine, methyltestosterone, norethisterone, oxaprozin, pullrifloxacin, tipipedin and salts thereof. アデフォビル ピボキシル、アジドチミジン、ベンプロペリン、クロペラスチン、エナラプリル、ホモクロルシクリジン、ロメリジン、メチルテストステロン、ノルエチステロン、オキサプロジン、プルリフロキサシン、チペピジン及びこれらの塩から選ばれる成分を有効成分とする神経変性疾患治療剤。   A therapeutic agent for neurodegenerative diseases comprising as an active ingredient an ingredient selected from adefovir pivoxil, azidothymidine, benproperin, cloperastine, enalapril, homochlorcyclidine, lomelidine, methyltestosterone, norethisterone, oxaprozin, pullrifloxacin, tipipedin and salts thereof.
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