JP2018002619A - Autophagy inhibitor - Google Patents

Autophagy inhibitor Download PDF

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JP2018002619A
JP2018002619A JP2016128769A JP2016128769A JP2018002619A JP 2018002619 A JP2018002619 A JP 2018002619A JP 2016128769 A JP2016128769 A JP 2016128769A JP 2016128769 A JP2016128769 A JP 2016128769A JP 2018002619 A JP2018002619 A JP 2018002619A
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autophagy
cancer
autophagy inhibitor
anticancer agent
cells
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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/JP2017/023683 priority patent/WO2018003829A1/en
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Abstract

PROBLEM TO BE SOLVED: To provide a new autophagy inhibitor.SOLUTION: An autophagy inhibitor contains, as an active ingredient, a component selected from albendazole, carbazochrome sulfonic acid, carvedilol, cinacalcet, clemastine, clomiphene, loratadine, pimobendan, testosterone enanthate, zonisamide and salt thereof.SELECTED DRAWING: None

Description

本発明は、オートファジー阻害剤及び抗がん剤に関する。   The present invention relates to an autophagy inhibitor and an anticancer 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.

オートファジーは正常細胞の機能に重要であるが、一部のがん細胞はオートファジーにより依存していることが示唆されている（非特許文献１）。これは、がん細胞が栄養学的に不利な状況にあることや、急速な細胞内代謝回転を必要としていることなどが理由と考えられる。実際に、米国では難治性腫瘍に対するオートファジーを標的とした臨床試験が行われている（非特許文献２〜６）。   Although autophagy is important for the function of normal cells, it has been suggested that some cancer cells depend on autophagy (Non-patent Document 1). This may be because cancer cells are in a nutritionally disadvantageous situation or require rapid intracellular turnover. In fact, clinical trials targeting autophagy for refractory tumors have been conducted in the United States (Non-Patent Documents 2 to 6).

Amaravadi et al. (2007) Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest. 117:326-36.Amaravadi et al. (2007) Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest. 117: 326-36. Rosenfeld et al. (2014) A phase I/II trial of hydroxychloroquine in conjunction with radiation therapy and concurrent and adjuvant temozolomide in patients with newly diagnosed glioblastoma multiforme. Autophagy. 2014 10:1359-68.Rosenfeld et al. (2014) A phase I / II trial of hydroxychloroquine in conjunction with radiation therapy and concurrent and adjuvant temozolomide in patients with newly diagnosed glioblastoma multiforme.Autophagy. 2014 10: 1359-68. Rangwala et al. (2014) Phase I trial of hydroxychloroquine with dose-intense temozolomide in patients with advanced solid tumors and melanoma. Autophagy. 10:1369-79.Rangwala et al. (2014) Phase I trial of hydroxychloroquine with dose-intense temozolomide in patients with advanced solid tumors and melanoma. Autophagy. 10: 1369-79. Vogl et al. Combined autophagy and proteasome inhibition: a phase 1 trial of hydroxychloroquine and bortezomib in patients with relapsed/refractory myeloma. Autophagy. 2014 10:1380-90.Vogl et al. Combined autophagy and proteasome inhibition: a phase 1 trial of hydroxychloroquine and bortezomib in patients with relapsed / refractory myeloma. Autophagy. 2014 10: 1380-90. Mahalingam et al. (2014) Combined autophagy and HDAC inhibition: a phase I safety, tolerability, pharmacokinetic, and pharmacodynamic analysis of hydroxychloroquine in combination with the HDAC inhibitor vorinostat in patients with advanced solid tumors. Autophagy. 10:1403-14.Mahalingam et al. (2014) Combined autophagy and HDAC inhibition: a phase I safety, tolerability, pharmacokinetic, and pharmacodynamic analysis of hydroxychloroquine in combination with the HDAC inhibitor vorinostat in patients with advanced solid tumors.Autophagy. 10: 1403-14. Barnard et al. (2014) Phase I clinical trial and pharmacodynamic evaluation of combination hydroxychloroquine and doxorubicin treatment in pet dogs treated for spontaneously occurring lymphoma. Autophagy. 10:1415-25.Barnard et al. (2014) Phase I clinical trial and pharmacodynamic evaluation of combination hydroxychloroquine and doxorubicin treatment in pet dogs treated for spontaneously occurring lymphoma. Autophagy. 10: 1415-25.

しかしながら、臨床試験で使用されている薬剤クロロキンおよびヒドロキシクロロキンは、オートファジー特異的ではなく、リソソームを含めた酸性コンパートメントの機能全般を抑制する。
従って、本発明の課題は、より特異性の高い新たなオートファジー阻害剤を提供することにある。 However, the drugs chloroquine and hydroxychloroquine used in clinical trials are not autophagy specific and inhibit the overall function of acidic compartments including lysosomes.
Accordingly, an object of the present invention is to provide a new autophagy inhibitor with higher specificity.

そこで、本発明者らは、新たなオートファジー阻害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１０種の成分に強いオートファジー阻害作用があることから抗がん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 inventors have screened components that have already been used as pharmaceuticals in order to search for new autophagy inhibitors. As a result, specific 10 types of components have a strong autophagy inhibitory action. The present invention was completed by finding it useful as an anticancer 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〕アルベンダゾール、カルバゾクロムスルホン酸、カルベジロール、シナカルセト、クレマスチン、クロミフェン、ロラタジン、ピモベンダン、テストステロンエナント酸エステル、ゾニサミド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阻害剤。
〔２〕アルベンダゾール、カルバゾクロムスルホン酸、カルベジロール、シナカルセト、クレマスチン、クロミフェン、ロラタジン、ピモベンダン、テストステロンエナント酸エステル、ゾニサミド及びこれらの塩から選ばれる成分を有効成分とする抗がん剤。 [1] An autophagy inhibitor comprising as an active ingredient an ingredient selected from albendazole, carbazochrome sulfonic acid, carvedilol, cinacalcet, clemastine, clomiphene, loratadine, pimobendan, testosterone enanthate, zonisamide, and salts thereof.
[2] An anticancer agent comprising as an active ingredient an ingredient selected from albendazole, carbazochrome sulfonic acid, carvedilol, cinacalcet, clemastine, clomiphene, loratadine, pimobendan, testosterone enanthate, zonisamide, and salts thereof.

本発明のオートファジー阻害剤及び抗がん剤の有効成分は、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阻害作用を有し、がん細胞の生存にオートファジーが重要な役割を果たしているがんの治療に有効である。   Since the active ingredient of the autophagy inhibitor and anticancer agent of the present invention is an ingredient already used as a pharmaceutical, safety has been confirmed and it has a strong autophagy inhibitory action, and cancer cells Autophagy plays an important role in the survival of cancer.

薬剤処理による細胞増殖への影響を示す図である。It is a figure which shows the influence on the cell growth by a chemical | medical agent process. 薬剤処理を行った細胞の顕微鏡画像である。It is a microscope image of the cell which performed the chemical | medical agent process.

本発明のオートファジー阻害剤及び抗がん剤の有効成分は、アルベンダゾール、カルバゾクロムスルホン酸、カルベジロール、シナカルセト、クレマスチン、クロミフェン、ロラタジン、ピモベンダン、テストステロンエナント酸エステル、ゾニサミド及びこれらの塩から選ばれる成分である。   The active ingredient of the autophagy inhibitor and anticancer agent of the present invention is an ingredient selected from albendazole, carbazochrome sulfonic acid, carvedilol, cinacalcet, clemastine, clomiphene, loratadine, pimobendan, testosterone enanthate, zonisamide and salts thereof It is.

アルベンダゾールは、種々の寄生虫の駆除薬として知られており、寄生虫病の治療薬として用いられている。   Albendazole is known as an antiparasitic agent for various parasites and is used as a therapeutic agent for parasitic diseases.

カルバゾクロムスルホン酸は、血管強化作用を有し、止血剤として用いられている。カルバゾクロムスルホン酸の塩としては、ナトリウム塩等が挙げられる。   Carbazochrome sulfonic acid has a blood vessel strengthening action and is used as a hemostatic agent. Examples of the salt of carbazochrome sulfonic acid include sodium salt.

カルベジロールは、αβ遮断薬の一つであり、血管拡張作用を有し、高血圧、心不全等の治療に用いられている。   Carvedilol is one of αβ blockers, has a vasodilatory effect, and is used for the treatment of hypertension, heart failure and the like.

シナカルセトは、カルシウム受容体作動薬であり、副甲状腺ホルモンの分泌を抑え、二次性副甲状腺機能亢進症又は副甲状腺がん等に伴う高カルシウム血症の治療に用いられている。シナカルセトの塩としては、塩酸塩等が挙げられる。   Cinacalcet is a calcium receptor agonist, suppresses the secretion of parathyroid hormone, and is used to treat hypercalcemia associated with secondary hyperparathyroidism or parathyroid cancer. Examples of cinacalcet salts include hydrochloride.

クレマスチンは、抗ヒスタミン薬の一つであり、アレルギー性疾患の治療に用いられている。クレマスチンの塩としては、フマル酸塩等が挙げられる。   Clemastine is one of antihistamines and is used for the treatment of allergic diseases. Examples of the salt of clemastine include fumarate.

クロミフェンは、選択的エストロゲン受容体調節薬（ＳＥＲＭ）であり、排卵誘発薬として用いられている。クロミフェンの塩としては、クエン酸塩等が挙げられる。   Clomiphene is a selective estrogen receptor modulator (SERM) and is used as an ovulation inducer. Examples of the salt of clomiphene include citrate.

ロラタジンは、抗ヒスタミン薬の一つであり、アレルギー性疾患の治療に用いられている。   Loratadine is one of antihistamines and is used for the treatment of allergic diseases.

ピモベンダンは、ホスホジエステラーゼ阻害作用及び心筋のＣａ２＋感受性増加作用を有し、心不全の治療に用いられている。 Pimobendan has a phosphodiesterase inhibitory action and a myocardial Ca 2+ sensitivity increasing action, and is used for the treatment of heart failure.

テストステロンエナント酸エステルは、男性ホルモン薬であり、男子性腺機能不全、造精機能障害による男子不妊症等の治療に用いられている。   Testosterone enanthate is a male hormone drug and is used to treat male infertility due to male gonad dysfunction and spermatogenic dysfunction.

ゾニサミドは、神経作用薬であり、てんかん治療薬又はパーキンソン病治療薬として用いられている。   Zonisamide is a neuroactive agent and is used as a therapeutic agent for epilepsy or a therapeutic agent for Parkinson's disease.

前記本発明のオートファジー阻害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薬として用いられているが、オートファジー阻害作用を有すること、また抗がん剤として有用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 inhibitor of the present invention has already been used as a therapeutic agent for various diseases, but has an autophagy inhibitory action and is useful as an anticancer agent. Not known at all.

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阻害作用を有するため、抗がん剤として有用である。対象となる癌は特に制限はされないが、例えば、頭頚部癌、食道癌、胃癌、十二指腸癌、結腸癌、直腸癌、肝臓癌、胆嚢・胆管癌、胆道癌、膵臓癌、肺癌、乳癌、卵巣癌、子宮頚癌、子宮体癌、腎癌、膀胱癌、前立腺癌、精巣腫瘍、骨・軟部肉腫、血液癌、多発性骨髄腫、皮膚癌、脳腫瘍、中皮腫、血液癌等が挙げられる。中でも脳腫瘍、膵癌、多発性骨髄腫などの癌が好ましい。本発明のオートファジー阻害剤は、がん細胞の生存にオートファジーが重要な役割を果たしているがんにおいて、がん細胞の増殖を阻害することにより、がんを治療することができる。   As shown in the below-mentioned Examples, the component has an excellent autophagy inhibitory action, and thus is useful as an anticancer agent. The target cancer is not particularly limited, but for example, head and neck cancer, esophageal cancer, stomach cancer, duodenal cancer, colon cancer, rectal cancer, liver cancer, gallbladder / bile duct cancer, biliary tract cancer, pancreatic cancer, lung cancer, breast cancer, ovary Cancer, cervical cancer, endometrial cancer, renal cancer, bladder cancer, prostate cancer, testicular tumor, bone / soft tissue sarcoma, blood cancer, multiple myeloma, skin cancer, brain tumor, mesothelioma, blood cancer, etc. . Of these, cancers such as brain tumor, pancreatic cancer and multiple myeloma are preferred. The autophagy inhibitor of the present invention can treat cancer by inhibiting the growth of cancer cells in cancer in which autophagy plays an important role in the survival of cancer cells.

本発明のオートファジー阻害剤及び抗がん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 dosage form of the autophagy inhibitor and anticancer agent of the present invention include injections, oral preparations (tablets, granules, powders, capsules), ointments, creams, patches, suppositories and the like. . 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阻害剤及び抗がん剤の投与量は、投与する患者の症状、年齢、投与方法等によって異なるが、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 dose of the autophagy inhibitor and anticancer agent of the present invention varies depending on the symptoms, age, administration method, etc. 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.

さらに、本発明のオートファジー阻害剤及び抗がん剤は、単独での使用に限らず、少なくとも１つの他の抗がん剤と、同時又は異なるときに、同一又は異なる頻度で、及び同一又は異なる投与方法で、併用投与することが可能である。本発明のオートファジー阻害剤及び抗がん剤並びに併用される他の抗がん剤の投与量は、併用する薬剤、投与する患者の症状、年齢、投与方法等に応じて薬剤ごとに適宜決定される。多剤併用療法における本発明のオートファジー阻害剤及び抗がん剤の投与量は前記と同様である。
ここで、本発明のオートファジー阻害剤及び抗がん剤と併用できる抗がん剤としては、通常がん治療に使用される抗がん剤が挙げられる。これらのオートファジー阻害剤及び抗がん剤並びに他の抗がん剤は、同時に投与してもよいし、別個に投与してもよい。また、投与ルートが相違してもよい。 Furthermore, the autophagy inhibitor and the anticancer agent of the present invention are not limited to use alone, but at the same or different frequency and at the same or different frequency with at least one other anticancer agent at the same time or Different administration methods can be used in combination. The dosage of the autophagy inhibitor and anticancer agent of the present invention and other anticancer agents to be used in combination is appropriately determined for each drug according to the drug to be used in combination, the symptom of the patient to be administered, age, administration method, etc. Is done. The dosage of the autophagy inhibitor and anticancer agent of the present invention in the multi-drug combination therapy is the same as described above.
Here, examples of the anticancer agent that can be used in combination with the autophagy inhibitor and the anticancer agent of the present invention include anticancer agents that are usually used for cancer treatment. These autophagy inhibitors and anticancer agents and other anticancer agents may be administered simultaneously or separately. Moreover, the administration route may be different.

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阻害活性の評価 Example 1
Evaluation of autophagy inhibitory 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、オートファジーが阻害されるとＧＦＰ／ＲＦＰ比が上昇する。この細胞を９６穴プレートに３０，０００個ずつ播種し、翌日薬剤（１０ μＭ）または溶媒のみを含む飢餓培地（１％ウシ胎仔血清と０．１Xアミノ酸を含むＤＭＥＭ培地）に交換した（１薬剤につき３ｗｅｌｌずつ使用）。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 inhibited, the GFP / RFP ratio increases. 30,000 cells were seeded in 96-well plates, and the following day, the medium was replaced with a starvation medium (DMEM medium containing 1% fetal bovine serum and 0.1 × amino acid) containing only the drug (10 μM) or solvent. Per well). 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１．５倍以上上昇したため、これらの薬剤にオートファジー阻害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 increased 1.5 times or more, indicating that these drugs have an autophagy inhibitory effect.

実施例２
薬効評価
〔方法〕
上記の１０種類の薬剤がヒト子宮頚がん由来のＨｅＬａ細胞の増殖に及ぼす影響を検証した。ＨｅＬａ細胞を４０万個ずつ播種し、各薬剤（終濃度１０ μＭ）または溶媒（ジメチルスルホキシド、ＤＭＳＯ）を添加して ２４時間および４８時間培養後、生細胞（トリパンブルー陰性の細胞）の数をカウントした。また、４８時間培養後の位相差顕微鏡画像を取得した。結果を図１及び図２に示す。 Example 2
Efficacy evaluation [method]
The effect of the above 10 kinds of drugs on the proliferation of HeLa cells derived from human cervical cancer was examined. After inoculating 400,000 HeLa cells, adding each drug (final concentration 10 μM) or solvent (dimethyl sulfoxide, DMSO) and culturing for 24 and 48 hours, the number of viable cells (trypan blue negative cells) was determined. I counted. Moreover, the phase-contrast microscope image after 48-hour culture | cultivation was acquired. The results are shown in FIGS.

〔結果〕
いずれの薬剤においても、溶媒のみの処理と比較して顕著な細胞増殖抑制が認められた（図１）。また、薬剤処理による細胞死（培養ディッシュからの剥離）が認められた（図２）。よって、これらの薬剤は抗がん作用を有していることが示唆された。 〔result〕
In any of the drugs, significant cell growth inhibition was observed as compared with the treatment with the solvent alone (FIG. 1). In addition, cell death (detachment from the culture dish) due to the drug treatment was observed (FIG. 2). Therefore, it was suggested that these drugs have an anticancer effect.

Claims (2)

アルベンダゾール、カルバゾクロムスルホン酸、カルベジロール、シナカルセト、クレマスチン、クロミフェン、ロラタジン、ピモベンダン、テストステロンエナント酸エステル、ゾニサミド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阻害剤。 An autophagy inhibitor comprising as an active ingredient an ingredient selected from albendazole, carbazochrome sulfonic acid, carvedilol, cinacalcet, clemastine, clomiphene, loratadine, pimobendan, testosterone enanthate, zonisamide, and salts thereof. アルベンダゾール、カルバゾクロムスルホン酸、カルベジロール、シナカルセト、クレマスチン、クロミフェン、ロラタジン、ピモベンダン、テストステロンエナント酸エステル、ゾニサミド及びこれらの塩から選ばれる成分を有効成分とする抗がん剤。 An anticancer agent comprising as an active ingredient an ingredient selected from albendazole, carbazochrome sulfonic acid, carvedilol, cinacalcet, clemastine, clomiphene, loratadine, pimobendan, testosterone enanthate, zonisamide, and salts thereof.
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