JP7644471B2 - Autophagy Inhibitors - Google Patents
Autophagy Inhibitors Download PDFInfo
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- JP7644471B2 JP7644471B2 JP2020157505A JP2020157505A JP7644471B2 JP 7644471 B2 JP7644471 B2 JP 7644471B2 JP 2020157505 A JP2020157505 A JP 2020157505A JP 2020157505 A JP2020157505 A JP 2020157505A JP 7644471 B2 JP7644471 B2 JP 7644471B2
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- autophagy
- cancer
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- cells
- anticancer
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Description
本発明は、オートファジー阻害剤に関する。より詳しくは、優れたオートファジー阻害活性を有する、新規化合物、オートファジー阻害剤、Atg4B阻害剤、及びこれを用いた抗癌剤の活性増強剤、並びに医薬品組成物、及び癌治療用キットに関する。 The present invention relates to an autophagy inhibitor. More specifically, the present invention relates to a novel compound, an autophagy inhibitor, an Atg4B inhibitor, and an activity enhancer for an anticancer drug using the same, as well as a pharmaceutical composition and a kit for cancer treatment, all of which have excellent autophagy inhibitory activity.
腫瘍におけるオートファジーの誘導は、異常タンパク質や異常細胞小器官の蓄積、その結果生じる酸化ストレスなどを抑制することで、腫瘍悪性化や抗癌剤耐性獲得に関与するため、オートファジー阻害剤は既存抗癌剤の有効性を高める新規抗癌剤として期待されている。オートファジー阻害剤が抗癌剤として臨床使用されている例はいまだないが、同様に既存の抗癌剤の作用増強を可能にする薬物としてオブジーボ（ニボルマブ）をはじめとする免疫療法薬が挙げられる。癌免疫療法薬の市場規模は大きく、今後も成長すると予測されている。しかしながら、癌免疫療法薬は臨床で優れた治療成績を示すが、有効な患者は約２割と言われており、その他の作用機序を介して既存の抗癌剤の作用増強を可能にする薬物の開発が望まれている。 The induction of autophagy in tumors is involved in tumor malignancy and the acquisition of anticancer drug resistance by suppressing the accumulation of abnormal proteins and abnormal organelles and the resulting oxidative stress, and therefore autophagy inhibitors are expected to be new anticancer drugs that enhance the effectiveness of existing anticancer drugs. Although there are no cases yet in which autophagy inhibitors have been used clinically as anticancer drugs, immunotherapy drugs such as Opdivo (nivolumab) are examples of drugs that can similarly enhance the effects of existing anticancer drugs. The market size of cancer immunotherapy drugs is large and is expected to continue to grow. However, although cancer immunotherapy drugs have shown excellent treatment results in clinical trials, it is said that only about 20% of patients are effective, and there is a need to develop drugs that can enhance the effects of existing anticancer drugs through other mechanisms of action.
また、オートファジーに関する研究は増加傾向にあり、2017年には5000件を超える関連論文が発表されており（例えば、非特許文献１～３等参照）、いまだ増加傾向にある。しかし、研究試薬として用いられているオートファジー阻害剤はいまだPI3キナーゼ阻害剤とリソソーム阻害剤しかなく、両者はオートファジー選択的な阻害剤ではない。オートファジーという現象を正しく理解するためにはオートファジー選択的阻害剤の開発が必要であり、試薬レベルでもその使用範囲は広いと推測される。なお、既存の医薬のいくつかにオートファジー阻害活性があることが知られている（特許文献１参照）。 Furthermore, research on autophagy is on the rise, with over 5,000 related papers published in 2017 (see, for example, Non-Patent Documents 1-3, etc.), and this trend is still on the rise. However, the only autophagy inhibitors used as research reagents are PI3 kinase inhibitors and lysosomal inhibitors, neither of which are selective inhibitors of autophagy. To properly understand the phenomenon of autophagy, the development of selective autophagy inhibitors is necessary, and it is expected that their use will be widespread even at the reagent level. It is also known that some existing pharmaceuticals have autophagy inhibitory activity (see Patent Document 1).
従来、KRAS変異型を含む一部の難治性の腫瘍がオートファジー依存的に生存・進展すると考えられ、オートファジー阻害剤によって腫瘍の封じ込めが可能になると考えられてきた。そのため、リソソーム阻害活性を介するオートファジー阻害剤として知られるクロロキンやヒドロキシクロロキンを用いた既存抗癌剤との併用療法の臨床試験が行われてきた。しかしながら、最近になってクロロキンによる抗癌活性にオートファジーが関与していないことを示す報告も発表され、癌におけるオートファジーの意義について再考する必要性が出てきた。このような事態が引き起こされてきた背景には、いまだオートファジー選択的阻害剤が開発されていないことが考えられる。したがって、オートファジー選択的阻害剤の開発はオートファジーの生理学的、病態生理学的意義の解明において最も重要な課題の一つである。 It has been thought that some intractable tumors, including KRAS mutant tumors, survive and progress in an autophagy-dependent manner, and that autophagy inhibitors can contain tumors. For this reason, clinical trials have been conducted on combination therapy with existing anticancer drugs, such as chloroquine and hydroxychloroquine, which are known to be autophagy inhibitors that inhibit lysosomes. However, a recent report has shown that autophagy is not involved in the anticancer activity of chloroquine, calling for a reconsideration of the significance of autophagy in cancer. The reason for this situation is thought to be that selective autophagy inhibitors have not yet been developed. Therefore, the development of selective autophagy inhibitors is one of the most important issues in elucidating the physiological and pathophysiological significance of autophagy.
このような実情のもと、本発明では、優れたオートファジー阻害活性を有する、オートファジー阻害剤を提供することを主目的とする。 Under these circumstances, the main objective of the present invention is to provide an autophagy inhibitor that has excellent autophagy inhibitory activity.
すなわち、本発明では、まず、下記一般式（１）で表される化合物若しくはその塩を有効成分として含有するオートファジー阻害剤、又は下記一般式（１）で表される化合物若しくはその塩を有効成分として含有するAtg4B阻害剤を含む、抗癌剤の活性増強剤を提供する。
（一般式（１）中、ｎは、２～２０の整数である。）
更に、本発明では、前記抗癌剤の活性増強剤と抗癌剤とを含む、医薬品組成物を提供する。
また、本発明では、前記抗癌剤の活性増強剤と抗癌剤とを有する、癌治療用キットも提供する。
That is , the present invention first provides an autophagy inhibitor containing, as an active ingredient, a compound represented by the following general formula (1) or a salt thereof, or an activity enhancer for an anticancer agent comprising an Atg4B inhibitor containing, as an active ingredient, a compound represented by the following general formula (1) or a salt thereof:
(In general formula (1), n is an integer from 2 to 20.)
Furthermore, the present invention provides a pharmaceutical composition comprising the anticancer agent and the activity enhancer for the anticancer agent.
The present invention also provides a kit for cancer treatment comprising the anticancer agent activity enhancer and an anticancer agent.
本発明によれば、優れたオートファジー阻害活性を有する、オートファジー阻害剤を提供することができる。
なお、ここに記載された効果は、必ずしも限定されるものではなく、本明細書中に記載されたいずれかの効果であってもよい。
According to the present invention, it is possible to provide an autophagy inhibitor having excellent autophagy inhibitory activity.
Note that the effects described here are not necessarily limited to those described herein, and may be any of the effects described in this specification.
以下、本発明を実施するための好適な形態について説明する。
以下に説明する実施形態は、本発明の代表的な実施形態の一例を示したものであり、これにより本発明の範囲が狭く解釈されることはない。
Preferred embodiments for carrying out the present invention will now be described.
The embodiment described below is an example of a typical embodiment of the present invention, and the scope of the present invention is not to be construed as being narrow.
１．上記一般式（１）で表される化合物又はその塩
本発明に係る化合物は、上記一般式（１）で表される化合物又はその塩である。
本発明に係る化合物は、後述する実施例に示す通り、前立腺癌細胞株を用いた検討において、細胞レベルで飢餓誘導性オートファジーを有意に抑制した。また、既存の抗癌剤の作用増強効果も認められた。したがって、本発明に係る化合物は、既存の抗癌剤の作用増強を可能にする癌アジュバント薬として、また、Atg4B阻害を作用点とするオートファジー阻害剤として実用化可能である。
1. Compound represented by the above general formula (1) or a salt thereof The compound according to the present invention is a compound represented by the above general formula (1) or a salt thereof.
As shown in the Examples below, the compound according to the present invention significantly suppressed starvation-induced autophagy at the cellular level in a study using a prostate cancer cell line. In addition, the compound was also found to have an effect of enhancing the action of existing anticancer drugs. Therefore, the compound according to the present invention can be put to practical use as a cancer adjuvant drug that can enhance the action of existing anticancer drugs, and as an autophagy inhibitor that inhibits Atg4B as its action site.
上記一般式（１）において、ｎは、２～２０の整数であり、好ましくは、ｎは、２、４、６、８～１２、１４、１６～１８、又は２０であり、より好ましくは、ｎは、４、６、８～１２、１４、１６～１８、又は２０であり、更に好ましくは、ｎは、６、８～１２、１７、又は１８であり、より更に好ましくは、ｎは、９、１０、１１、又は１７であり、特に好ましくは、ｎは、１０である。 In the above general formula (1), n is an integer from 2 to 20, preferably n is 2, 4, 6, 8 to 12, 14, 16 to 18, or 20, more preferably n is 4, 6, 8 to 12, 14, 16 to 18, or 20, even more preferably n is 6, 8 to 12, 17, or 18, even more preferably n is 9, 10, 11, or 17, and particularly preferably n is 10.
塩とは、具体的には、例えば「薬学的に許容される塩」であり、これは、親化合物（塩フリーの化合物）の生物学的有効性を備えており、生物学的に無毒性か、若しくは生物学的に毒性の低い無機又は有機の酸又は塩基の付加塩をいう。前記塩としては、例えば、塩酸、硫酸等との無機酸付加塩；ギ酸、酢酸、トリフルオロ酢酸、酒石酸等との有機酸付加塩；ナトリウム、カリウム等とのアルカリ金属塩；カルシウム、マグネシウム等とのアルカリ土類金属塩；メチルアミン、エチルアミン、ジエタノールアミン等との有機アミン塩等が挙げられる。 Specifically, the salt is, for example, a "pharmaceutical acceptable salt", which refers to an addition salt of an inorganic or organic acid or base that has the biological effectiveness of the parent compound (salt-free compound) and is biologically non-toxic or has low biological toxicity. Examples of the salt include inorganic acid addition salts with hydrochloric acid, sulfuric acid, etc.; organic acid addition salts with formic acid, acetic acid, trifluoroacetic acid, tartaric acid, etc.; alkali metal salts with sodium, potassium, etc.; alkaline earth metal salts with calcium, magnesium, etc.; organic amine salts with methylamine, ethylamine, diethanolamine, etc.
２．オートファジー阻害剤、Atg4B阻害剤
本発明に係るオートファジー阻害剤は、上記一般式（１）で表される化合物又はその塩を有効成分として含有する。本発明に係るオートファジー剤は、上記の一般式（１）で表される化合物又はその塩を含有しており、且つ、本発明の効果を損なわない限り特に制限されず、後述する薬学的に許容される担体や、既知又は未知の他の有効成分などを含有していてもよい。
2. Autophagy Inhibitors, Atg4B Inhibitors The autophagy inhibitors according to the present invention contain the compound represented by the above general formula (1) or a salt thereof as an active ingredient. The autophagy agent according to the present invention contains the compound represented by the above general formula (1) or a salt thereof, and is not particularly limited as long as it does not impair the effects of the present invention, and may contain a pharma- ceutically acceptable carrier described below, other known or unknown active ingredients, and the like.
本明細書において、「オートファジー阻害剤」とは、オートファジーを阻害ないし抑制する剤をいう。 As used herein, "autophagy inhibitor" refers to an agent that inhibits or suppresses autophagy.
本発明に係るオートファジー阻害剤は、オートファジーが関与する各種疾患のための予防又は治療剤として使用することができる。また、研究用試薬として使用できるほか、診断薬等にも使用できる。 The autophagy inhibitor according to the present invention can be used as a preventive or therapeutic agent for various diseases in which autophagy is involved. It can also be used as a research reagent and as a diagnostic agent, etc.
本発明に係るAtg4B阻害剤は、上記一般式（１）で表される化合物又はその塩を有効成分として含有する。本発明に係るAtg4B阻害剤は、上記の一般式（１）で表される化合物又はその塩を含有しており、且つ、本発明の効果を損なわない限り特に制限されず、後述する薬学的に許容される担体や、既知又は未知の他の有効成分などを含有していてもよい。 The Atg4B inhibitor according to the present invention contains a compound represented by the above general formula (1) or a salt thereof as an active ingredient. The Atg4B inhibitor according to the present invention contains a compound represented by the above general formula (1) or a salt thereof, and is not particularly limited as long as it does not impair the effects of the present invention, and may contain a pharma- ceutically acceptable carrier described below, other known or unknown active ingredients, and the like.
本明細書において、「Atg4B阻害剤」とは、Atg4Bの活性を阻害ないし抑制する剤をいう。
Atg4Bは、Autophagin-1とも称され、オートファゴソーム膜の構成成分であるLC3の成熟化に必須なシステインプロテアーゼである。Atg4Bが正常に機能しないと、オートファジーが完結しないことが知られる。したがって、Atg4Bの阻害は合理的なオートファジー阻害の作用点であると考えられている。
As used herein, the term "Atg4B inhibitor" refers to an agent that inhibits or suppresses the activity of Atg4B.
Atg4B, also known as Autophagin-1, is a cysteine protease essential for the maturation of LC3, a component of the autophagosomal membrane. It is known that autophagy is not completed unless Atg4B functions normally. Therefore, inhibition of Atg4B is thought to be a rational point of action for inhibiting autophagy.
本発明に係るAtg4B阻害剤は、Atg4Bが関与する各種疾患のための予防又は治療剤として使用することができる。また、研究用試薬として使用できるほか、診断薬等にも使用できる。 The Atg4B inhibitor according to the present invention can be used as a preventive or therapeutic agent for various diseases in which Atg4B is involved. It can also be used as a research reagent and as a diagnostic agent, etc.
本発明に係るオートファジー阻害剤及びAtg4B阻害剤の投与対象は、通常、ヒトであるが、本発明では、ヒト以外の哺乳動物、例えばイヌ、ネコ等のペット動物、ウシ、ヒツジ、ブタ等の家畜も含む。 The subjects for administration of the autophagy inhibitors and Atg4B inhibitors according to the present invention are usually humans, but the present invention also includes mammals other than humans, such as pet animals such as dogs and cats, and livestock such as cows, sheep, and pigs.
本発明に係るオートファジー阻害剤及びAtg4B阻害剤の投与形態は、固体製剤及び液体製剤のいずれの形態でもよく、例えば注射剤、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。 The administration form of the autophagy inhibitor and Atg4B inhibitor according to the present invention may be either a solid formulation or a liquid formulation, and examples of such formulations include injections, oral preparations (tablets, granules, powders, capsules), ointments, creams, patches, suppositories, etc.
これらの医薬品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、トラ癌ト、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が、これらに限定されるものではない。 To make these pharmaceutical compositions, they can be formulated with a pharma- ceutically acceptable carrier. Examples of such carriers include, but are not limited to, lactose, glucose, D-mannitol, starch, crystalline cellulose, calcium carbonate, kaolin, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, ethanol, carboxymethylcellulose, carboxymethylcellulose calcium salt, magnesium stearate, talc, acetylcellulose, sucrose, titanium oxide, benzoic acid, paraoxybenzoic acid esters, sodium dehydroacetate, gum arabic, tragacanth, methylcellulose, egg yolk, surfactants, sucrose, simple syrup, citric acid, distilled water, ethanol, glycerin, propylene glycol, macrogol, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate, glucose, sodium chloride, phenol, thimerosal, paraoxybenzoic acid esters, and sodium hydrogen sulfite.
前記医薬品組成物中における有効成分の含有量は、製剤の形によって大きく変動するため特に限定されるものではないが、例えば医薬品組成物全量に対して０．００１～１００質量％とすることができる。 The content of the active ingredient in the pharmaceutical composition is not particularly limited because it varies greatly depending on the form of the formulation, but can be, for example, 0.001 to 100% by mass of the total amount of the pharmaceutical composition.
３．抗癌剤の活性増強剤
本発明に係る活性増強剤は、前述したオートファジー阻害剤又はAtg4B阻害剤を含む。本発明に係る活性増強剤は、前述したオートファジー阻害剤又はAtg4B阻害剤を含んでおり、且つ、本発明の効果を損なわない限り特に制限されず、前述した薬学的に許容される担体や、既知又は未知の他の有効成分などを含有していてもよい。
3. Activity enhancer for anticancer agent The activity enhancer according to the present invention includes the above-mentioned autophagy inhibitor or Atg4B inhibitor. The activity enhancer according to the present invention is not particularly limited as long as it includes the above-mentioned autophagy inhibitor or Atg4B inhibitor and does not impair the effects of the present invention, and may contain the above-mentioned pharma- ceutically acceptable carrier, other known or unknown active ingredients, and the like.
一般に、オートファジーは生体防御的に誘導される。そのため、抗癌剤処理された細胞はアポトーシスを惹起して細胞死に向かう一方で、自身を保護するためにオートファジーも誘導する。このオートファジーの誘導は、抗癌剤の感受性の低下の原因になることも知られ、オートファジー阻害剤を抗癌剤と併用することで抗癌剤の作用増強につながると考えられている。 Generally, autophagy is induced as a defense mechanism for the body. Therefore, while cells treated with anticancer drugs undergo apoptosis and head toward cell death, they also induce autophagy to protect themselves. This induction of autophagy is also known to cause a decrease in sensitivity to anticancer drugs, and it is thought that using autophagy inhibitors in combination with anticancer drugs will enhance the effects of the anticancer drugs.
癌は世界的に主な死亡原因であり、癌の予防及び根治を目指した新規抗癌剤の開発が急務とされている。近年、治療奏効率の優れた抗癌剤が上市されているが、その大半は抗体医薬である。抗体医薬は癌の原因タンパクに直接作用するために高い効果が得られる反面、大量生産が困難などの原因によるその高い薬価が問題となり、金銭上の理由から治療を選択できない患者も多く存在する。そのため、大量生産が容易であり、比較的安価である低分子医薬の開発が望まれる。これに対し、本発明に係る活性増強剤は、化学合成も容易であり、大量生産にも耐えられることが想定されるため、癌治療への適応が得られれば、患者の治療機会の向上、ひいては国民医療費の削減に寄与できると考えられる。 Cancer is the leading cause of death worldwide, and there is an urgent need to develop new anticancer drugs aimed at preventing and curing cancer. In recent years, anticancer drugs with excellent therapeutic success rates have been launched on the market, but the majority of these are antibody drugs. Although antibody drugs are highly effective because they act directly on the proteins that cause cancer, their high drug prices due to factors such as the difficulty of mass production are a problem, and many patients are unable to choose treatment for financial reasons. Therefore, the development of small molecule drugs that are easy to mass-produce and relatively inexpensive is desired. In contrast, the activity enhancer of the present invention is easy to chemically synthesize and is expected to withstand mass production, so if it is adapted to cancer treatment, it is believed that it will contribute to improving patients' treatment opportunities and ultimately reducing national medical expenses.
本明細書において、「癌」は広義に解釈され、「悪性腫瘍」と互換的に使用される。また、病理学的に診断が確定される前の段階、すなわち、腫瘍としての良性、悪性のどちらかが確定される前には、良性腫瘍、良性悪性境界病変、悪性腫瘍を総括的に含む場合もあり得る。本発明に係る活性増強剤の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。本発明では、特に、前立腺癌が好ましく、その中でも特に、去勢抵抗性前立腺癌（CRPC）が好ましい。 In this specification, "cancer" is interpreted in a broad sense and is used interchangeably with "malignant tumor." In addition, before a pathological diagnosis is confirmed, i.e., before a tumor is confirmed to be benign or malignant, it may collectively include benign tumors, benign/malignant borderline lesions, and malignant tumors. The cancers that are the target of the activity enhancer of the present invention are not particularly limited, and examples thereof include head and neck cancer, esophageal cancer, gastric cancer, duodenal cancer, colon cancer, rectal cancer, liver cancer, gallbladder/bile duct cancer, biliary tract cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, kidney cancer, bladder cancer, prostate cancer, testicular tumor, bone and soft tissue sarcoma, blood cancer, multiple myeloma, skin cancer, brain tumor, mesothelioma, blood cancer, and the like. In the present invention, prostate cancer is particularly preferred, and castration-resistant prostate cancer (CRPC) is particularly preferred.
ホルモン依存性癌の治療にはホルモン療法が有効であるが、先天的に若しくは治療の過程でホルモン感受性が消失した去勢抵抗性前立腺癌（CRPC）についてはホルモン療法では全く効果がないため、新規抗癌剤の開発が急務とされている。本発明に係る活性増強剤は、後述する実施例に示す通り、CRPC治療薬の活性を増強するため、非常に有用である。 Hormonal therapy is effective in treating hormone-dependent cancers, but it is completely ineffective for castration-resistant prostate cancer (CRPC), in which hormone sensitivity is lost congenitally or during the course of treatment, and so there is an urgent need to develop new anticancer drugs. As shown in the examples below, the activity enhancer of the present invention is extremely useful because it enhances the activity of CRPC therapeutic drugs.
本明細書において、「抗癌剤」とは、標的の疾病ないし病態である、癌に対する治療的又は予防的効果を示す薬剤のことをいう。なお、治療的効果には、癌に特徴的な症状又は随伴症状を緩和すること（軽症化）、症状の悪化を阻止ないし遅延すること等も含まれる。本発明に係る活性増強剤の対象となる抗癌剤は特に限定されないが、例えば、フルタミド、ニルタミド、ビカルタミド、エンザルタミド、アパルタミド、ダロルタミド、酢酸シプロテロン、酢酸メゲストロール、酢酸クロルマジノン、スピロノラクトン、カンレノン、ドロスピレノン、ケトコナゾール、トピルタミド（フルリジル）、シメチジン等の抗アンドロゲン剤；アビラテロン等のＣＹＰ１７リアーゼ阻害剤；ドセタキセル、カバジタキセル等のタキサン系抗癌剤等が挙げられる。本発明では、特に、去勢抵抗性前立腺癌（CRPC）治療薬が好ましく、その中でも特に、アビラテロン、アパルタミド、エンザルタミド、カバジタキセルが好ましい。 In this specification, the term "anticancer drug" refers to a drug that exhibits a therapeutic or preventive effect against cancer, which is a target disease or pathology. The therapeutic effect also includes alleviating (alleviating) symptoms characteristic of cancer or associated symptoms, and preventing or delaying the worsening of symptoms. The anticancer drug that is the target of the activity enhancer of the present invention is not particularly limited, and examples thereof include antiandrogens such as flutamide, nilutamide, bicalutamide, enzalutamide, apalutamide, darolutamide, cyproterone acetate, megestrol acetate, chlormadinone acetate, spironolactone, canrenone, drospirenone, ketoconazole, topirutamide (fluridil), and cimetidine; CYP17 lyase inhibitors such as abiraterone; and taxane anticancer drugs such as docetaxel and cabazitaxel. In the present invention, castration-resistant prostate cancer (CRPC) therapeutic agents are particularly preferred, and among these, abiraterone, apalutamide, enzalutamide, and cabazitaxel are particularly preferred.
４．医薬品組成物
本発明に係る医薬品組成物は、前述した活性増強剤と抗癌剤とを含む。本発明に係る医薬品組成物は、前述した活性増強剤及び抗癌剤を含んでおり、且つ、本発明の効果を損なわない限り特に制限されず、前述した薬学的に許容される担体や、既知又は未知の他の有効成分などを含有していてもよい。
4. Pharmaceutical Composition The pharmaceutical composition according to the present invention contains the above-mentioned activity enhancer and anticancer agent. The pharmaceutical composition according to the present invention is not particularly limited as long as it contains the above-mentioned activity enhancer and anticancer agent and does not impair the effects of the present invention, and may contain the above-mentioned pharma- ceutical acceptable carriers, other known or unknown active ingredients, and the like.
本発明に係る医薬品組成物は、前述した活性増強剤と、例えば前述した抗癌剤のうち１種と組み合わせた癌治療用の医薬品組成物として調製され、対象に投与し得る。なお、特定の癌に対して複数の抗癌剤の臨床的使用が認められている場合、それらに本発明に係る活性増強剤を組み合わせた抗癌組成物を調製し、用いてもよい。また、異なる２種以上の癌に対して、複数の抗癌剤の投与が行われる場合も、それらに本発明に係る活性増強剤を組み合わせて用いることもできる。 The pharmaceutical composition according to the present invention can be prepared as a pharmaceutical composition for cancer treatment in which the aforementioned activity enhancer is combined with, for example, one of the aforementioned anticancer agents, and administered to a subject. When multiple anticancer agents are approved for clinical use against a particular cancer, an anticancer composition in which they are combined with the activity enhancer according to the present invention may be prepared and used. In addition, when multiple anticancer agents are administered against two or more different types of cancer, they can also be combined with the activity enhancer according to the present invention.
本発明に係る活性増強剤を抗癌剤とともに用いる場合、他の癌治療法と組み合わせて実施することが可能であり、それぞれの治療法を単独で実施したときに比べてより効果的な治療効果が期待できる。例えば、外科的切除と組み合わせて実施する場合は、手術の前後を問わず化学療法を継続することが可能である。また、放射線療法と組み合わせて治療することも可能である。 When the activity enhancer of the present invention is used together with an anticancer drug, it can be combined with other cancer treatments, and more effective therapeutic effects can be expected than when each treatment is performed alone. For example, when combined with surgical resection, chemotherapy can be continued regardless of whether it is before or after surgery. It is also possible to combine it with radiation therapy.
抗癌剤と組み合わせる本発明に係る活性増強剤の投与量は、患者の年齢、症状等により適宜選択することができ、抗癌剤の抗癌活性を増強する量であれば、特に制限はない。実際には、抗癌剤の所定の投与量に従って、広範囲に変動し得る。 The dosage of the activity enhancer of the present invention to be combined with an anticancer drug can be appropriately selected depending on the age, symptoms, etc. of the patient, and is not particularly limited as long as it is an amount that enhances the anticancer activity of the anticancer drug. In practice, it can vary over a wide range depending on the prescribed dosage of the anticancer drug.
本発明に係る活性増強剤と抗癌剤とは、両者を混和後、単一の混合製剤（抗癌組成物）として対象に投与することが好ましい。ただし、それぞれ別の投与経路（例えば、活性増強剤は静脈内投与、抗癌剤は経口投与など）で投与しても同様の治療効果が期待できる。 The activity enhancer and anticancer agent of the present invention are preferably mixed together and then administered to a subject as a single mixed preparation (anticancer composition). However, the same therapeutic effect can be expected even if each is administered via a different administration route (e.g., intravenous administration of the activity enhancer and oral administration of the anticancer agent).
本発明に係る活性増強剤と抗癌剤とを別々に投与する場合、投与する抗癌剤の種類、対象の病状等に合わせてそれぞれの投与量等を決定することが可能である。本発明に係る活性増強剤は、抗癌剤の投与の前、或いは投与の後のいずれでも投与できるが、その投与間隔はあまり長くないのが好ましい。 When the activity enhancer of the present invention and the anticancer agent are administered separately, it is possible to determine the dosage of each agent according to the type of anticancer agent to be administered and the condition of the subject. The activity enhancer of the present invention can be administered either before or after the administration of the anticancer agent, but it is preferable that the administration interval is not too long.
５．癌治療用キット
本発明に係る癌治療用キットは、前述した活性増強剤と抗癌剤とを有する。本発明に係る癌治療用キットは、前述した活性増強剤と抗癌剤とを有しており、且つ、本発明の効果を損なわない限り特に制限されず、その他のものを有していてもよい。
5. Cancer Treatment Kit The cancer treatment kit according to the present invention comprises the above-mentioned activity enhancer and an anticancer agent. The cancer treatment kit according to the present invention comprises the above-mentioned activity enhancer and an anticancer agent, and is not particularly limited as long as it does not impair the effects of the present invention, and may also comprise other substances.
本発明に係る癌治療用キットは、例えば本発明に係る活性増強剤を含む第一の収容部と、抗癌剤を含む第二の収容部とを備えるものとすることができる。当該キットにおける「収容部」とは、それぞれの薬剤が混合せずに、独立して存在するために有効な形態であれば特に制限はなく、例えば容器や個別包装形態などであってもよく、一のシート状で独立して区分けされた領域としての形態であってもよい。 The cancer treatment kit according to the present invention may, for example, comprise a first storage section containing the activity enhancer according to the present invention, and a second storage section containing an anticancer drug. The "storage section" in the kit is not particularly limited as long as it is in an effective form in which the respective drugs exist independently without being mixed, and may be, for example, a container or an individually packaged form, or may be in the form of a single sheet-like, independently divided region.
本発明に係る癌治療用キットは、それぞれの薬剤を単独で投与してもよく、必要時にそれぞれの収容部から本発明に係る活性増強剤と抗癌剤とを取り出して混合し、製剤（抗癌組成物）を調製してもよい。 In the cancer treatment kit according to the present invention, each drug may be administered alone, or when necessary, the activity enhancer according to the present invention and the anticancer drug may be taken out of their respective containers and mixed to prepare a formulation (anticancer composition).
以下、実施例に基づいて本発明を更に詳細に説明する。
なお、以下に説明する実施例は、本発明の代表的な実施例の一例を示したものであり、これにより本発明の範囲が狭く解釈されることはない。
The present invention will now be described in further detail with reference to examples.
The embodiment described below is merely a representative example of the present invention, and the scope of the present invention should not be construed as being narrow.
＜実験例１＞化合物の合成
Ar雰囲気下、アルキルベンゼン (1a-n, 0.252 mmol) のCH2Cl2 (1.5 mL) 溶液に室温にて、無水マレイン酸 (25 mg, 0.252 mmol)、塩化アルミニウム (67 mg, 0.504 mmol) を順次加え、室温にて1時間撹拌を行った。氷冷下、水(1.5 mL) を加え、CH2Cl2 層を分離後、水層を CH2Cl2 (1.5 mL) を用いて3回抽出し、先のCH2Cl2 層と合わせた。合わせた CH2Cl2層は無水硫酸ナトリウムを用いて乾燥後、ロータリーエバポレーターを用いて溶媒を留去し、得られた残渣をシリカゲルカラムクロマトグラフィー (SiO2: 7g, n-hexane : acetone = 3 : 1 ～ 1 : 1) で精製することにより、白色結晶であるAUP誘導体を得た。 Under Ar atmosphere, maleic anhydride (25 mg, 0.252 mmol) and aluminum chloride (67 mg, 0.504 mmol) were added to a solution of alkylbenzene (1a-n, 0.252 mmol) in CH 2 Cl 2 (1.5 mL) at room temperature, and the mixture was stirred at room temperature for 1 hour. Water (1.5 mL) was added under ice cooling, and the CH 2 Cl 2 layer was separated. The aqueous layer was extracted three times with CH 2 Cl 2 (1.5 mL) and combined with the previous CH 2 Cl 2 layer. The combined CH 2 Cl 2 layer was dried over anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator. The resulting residue was purified by silica gel column chromatography (SiO 2 : 7 g, n-hexane : acetone = 3 : 1 to 1 : 1) to obtain the AUP derivative as white crystals.
(E)-4-Oxo-4-(4-propylphenyl)but-2-enoic acid (AUP06)
1H-NMR (400 MHz, CDCl3) δ: 0.96 (3H, t, J = 7.2 Hz), 1.68 (2H, sext, J = 7.2 Hz), 2.67 (2H, t, J= 7.2 Hz), 6.88 (1H, d, J = 16.0 Hz), 7.32 (2H, d, J = 8.4 Hz), 7.93 (2H, d, J = 8.4 Hz), 7.99 (1H, d, J = 16.0 Hz)
(E)-4-Oxo-4-(4-propylphenyl)but-2-enoic acid (AUP06)
1 H-NMR (400 MHz, CDCl 3 ) δ: 0.96 (3H, t, J = 7.2 Hz), 1.68 (2H, sext, J = 7.2 Hz), 2.67 (2H, t, J= 7.2 Hz), 6.88 (1H, d, J = 16.0 Hz), 7.32 (2H, d, J = 8.4 Hz), 7.93 (2H, d, J = 8.4 Hz), 7.99 (1H, d, J = 16.0 Hz)
(E)-4-Oxo-4-(4-pentylphenyl)but-2-enoic acid (AUP07)
1H-NMR (400 MHz, CDCl3) δ: 0.90 (3H, t, J = 7.6 Hz), 1.25-1.38 (4H, m), 1.65 (2H, quin, J = 7.6 Hz), 2.69 (2H, t, J = 7.6 Hz), 6.88 (1H, d, J = 15.2 Hz), 7.32 (2H, d, J = 8.0 Hz), 7.93 (2H, d, J = 8.0 Hz), 7.99 (1H, d, J= 15.2 Hz)
(E)-4-Oxo-4-(4-pentylphenyl)but-2-enoic acid (AUP07)
1 H-NMR (400 MHz, CDCl 3 ) δ: 0.90 (3H, t, J = 7.6 Hz), 1.25-1.38 (4H, m), 1.65 (2H, quin, J = 7.6 Hz), 2.69 (2H, t, J = 7.6 Hz), 6.88 (1H, d, J = 15.2 Hz), 7.32 (2H, d, J = 8.0 Hz), 7.93 (2H, d, J = 8.0 Hz), 7.99 (1H, d, J= 15.2 Hz)
(E)-4-(4-Heptylphenyl)-4-oxobut-2-enoic acid (AUP08)
1H-NMR (400 MHz, CDCl3) δ: 0.89 (3H, t, J = 7.6 Hz), 1.24-1.38 (8H, m), 1.66 (2H, quin, J = 7.6 Hz), 2.69 (2H, t, J = 7.6 Hz), 6.90 (1H, d, J = 16.0 Hz), 7.34 (2H, d, J = 8.0 Hz), 7.94 (2H, d, J = 8.0 Hz), 8.01 (1H, d, J= 16.0 Hz)
(E)-4-(4-Heptylphenyl)-4-oxobut-2-enoic acid (AUP08)
1 H-NMR (400 MHz, CDCl 3 ) δ: 0.89 (3H, t, J = 7.6 Hz), 1.24-1.38 (8H, m), 1.66 (2H, quin, J = 7.6 Hz), 2.69 (2H, t, J = 7.6 Hz), 6.90 (1H, d, J = 16.0 Hz), 7.34 (2H, d, J = 8.0 Hz), 7.94 (2H, d, J = 8.0 Hz), 8.01 (1H, d, J= 16.0 Hz)
(E)-4-(4-Nonylphenyl)-4-oxobut-2-enoic acid (AUP09)
1H-NMR (400 MHz, CDCl3) δ: 0.88 (3H, t, J = 7.2 Hz), 1.20-1.36 (12H, m), 1.64 (2H, quin, J = 7.2 Hz), 2.69 (2H, t, J = 7.2 Hz), 6.88 (1H, d, J = 15.6 Hz), 7.32 (2H, d, J = 8.4 Hz), 7.93 (2H, d, J = 8.4 Hz), 7.98 (1H, d, J= 15.6 Hz)
(E)-4-(4-Nonylphenyl)-4-oxobut-2-enoic acid (AUP09)
1 H-NMR (400 MHz, CDCl 3 ) δ: 0.88 (3H, t, J = 7.2 Hz), 1.20-1.36 (12H, m), 1.64 (2H, quin, J = 7.2 Hz), 2.69 (2H, t, J = 7.2 Hz), 6.88 (1H, d, J = 15.6 Hz), 7.32 (2H, d, J = 8.4 Hz), 7.93 (2H, d, J = 8.4 Hz), 7.98 (1H, d, J= 15.6 Hz)
(E)-4-(4-Decylphenyl)-4-oxobut-2-enoic acid (AUP15)
1H-NMR (400 MHz, CDCl3) δ: 0.88 (3H, t, J = 7.6 Hz), 1.21-1.35 (14H, m), 1.64 (2H, quin, J = 7.6 Hz), 2.69 (2H, t, J = 7.6 Hz), 6.88 (1H, d, J = 15.2 Hz), 7.32 (2H, d, J = 8.4 Hz), 7.93 (2H, d, J = 8.4 Hz), 8.00 (1H, d, J= 15.2 Hz)
(E)-4-(4-Decylphenyl)-4-oxobut-2-enoic acid (AUP15)
1 H-NMR (400 MHz, CDCl 3 ) δ: 0.88 (3H, t, J = 7.6 Hz), 1.21-1.35 (14H, m), 1.64 (2H, quin, J = 7.6 Hz), 2.69 (2H, t, J = 7.6 Hz), 6.88 (1H, d, J = 15.2 Hz), 7.32 (2H, d, J = 8.4 Hz), 7.93 (2H, d, J = 8.4 Hz), 8.00 (1H, d, J= 15.2 Hz)
(E)-4-Oxo-4-(4-undecylphenyl)but-2-enoic acid (AUP01)
1H-NMR (400 MHz, CDCl3) δ: 0.88 (3H, t, J = 7.2 Hz), 1.20-1.35 (16H, m), 1.64 (2H, quin, J = 7.2 Hz), 2.69 (2H, t, J = 7.2 Hz), 6.88 (1H, d, J = 15.2 Hz), 7.32 (2H, d, J = 8.4 Hz), 7.93 (2H, d, J = 8.4 Hz), 8.00 (1H, d, J= 15.2 Hz)
(E)-4-Oxo-4-(4-undecylphenyl)but-2-enoic acid (AUP01)
1 H-NMR (400 MHz, CDCl 3 ) δ: 0.88 (3H, t, J = 7.2 Hz), 1.20-1.35 (16H, m), 1.64 (2H, quin, J = 7.2 Hz), 2.69 (2H, t, J = 7.2 Hz), 6.88 (1H, d, J = 15.2 Hz), 7.32 (2H, d, J = 8.4 Hz), 7.93 (2H, d, J = 8.4 Hz), 8.00 (1H, d, J= 15.2 Hz)
(E)-4-(4-Dodecylphenyl)-4-oxobut-2-enoic acid (AUP16)
1H-NMR (400 MHz, CDCl3) δ: 0.88 (3H, t, J = 6.8 Hz), 1.20-1.34 (18H, m), 1.63 (2H, quin, J = 6.8 Hz), 2.68 (2H, t, J = 6.8 Hz), 6.86 (1H, d, J = 15.2 Hz), 7.32 (2H, d, J = 8.4 Hz), 7.93 (2H, d, J = 8.4 Hz), 7.98 (1H, d, J= 15.2 Hz)
(E)-4-(4-Dodecylphenyl)-4-oxobut-2-enoic acid (AUP16)
1 H-NMR (400 MHz, CDCl 3 ) δ: 0.88 (3H, t, J = 6.8 Hz), 1.20-1.34 (18H, m), 1.63 (2H, quin, J = 6.8 Hz), 2.68 (2H, t, J = 6.8 Hz), 6.86 (1H, d, J = 15.2 Hz), 7.32 (2H, d, J = 8.4 Hz), 7.93 (2H, d, J = 8.4 Hz), 7.98 (1H, d, J= 15.2 Hz)
(E)-4-Oxo-4-(4-tridecylphenyl)but-2-enoic acid (AUP02)
1H-NMR (400 MHz, CDCl3) δ: 0.88 (3H, t, J = 7.2 Hz), 1.20-1.35 (20H, m), 1.60-1.68 (2H, m), 2.68 (2H, t, J = 7.2 Hz), 6.88 (1H, d, J = 16.0 Hz), 7.32 (2H, d, J = 8.0 Hz), 7.93 (2H, d, J = 8.0 Hz), 7.98 (1H, d, J = 16.0 Hz)
(E)-4-Oxo-4-(4-tridecylphenyl)but-2-enoic acid (AUP02)
1 H-NMR (400 MHz, CDCl 3 ) δ: 0.88 (3H, t, J = 7.2 Hz), 1.20-1.35 (20H, m), 1.60-1.68 (2H, m), 2.68 (2H, t, J = 7.2 Hz), 6.88 (1H, d, J = 16.0 Hz), 7.32 (2H, d, J = 8.0 Hz), 7.93 (2H, d, J = 8.0 Hz), 7.98 (1H, d, J = 16.0 Hz)
(E)-4-Oxo-4-(4-pentadecylphenyl)but-2-enoic acid (AUP03)
1H-NMR (400 MHz, CDCl3) δ: 0.88 (3H, t, J = 6.8 Hz), 1.20-1.35 (24H, m), 1.60-1.66 (2H, m), 2.68 (2H, t, J = 6.8 Hz), 6.88 (1H, d, J = 15.2 Hz), 7.32 (2H, d, J = 8.0 Hz), 7.93 (2H, d, J = 8.0 Hz), 7.98 (1H, d, J = 15.2 Hz)
(E)-4-Oxo-4-(4-pentadecylphenyl)but-2-enoic acid (AUP03)
1 H-NMR (400 MHz, CDCl 3 ) δ: 0.88 (3H, t, J = 6.8 Hz), 1.20-1.35 (24H, m), 1.60-1.66 (2H, m), 2.68 (2H, t, J = 6.8 Hz), 6.88 (1H, d, J = 15.2 Hz), 7.32 (2H, d, J = 8.0 Hz), 7.93 (2H, d, J = 8.0 Hz), 7.98 (1H, d, J = 15.2 Hz)
(E)-4-(4-Heptadecylphenyl)-4-oxobut-2-enoic acid (AUP04)
1H-NMR (400 MHz, CDCl3) δ: 0.86 (3H, t, J = 6.8 Hz), 1.20-1.34 (28H, m), 1.60-1.67 (2H, m), 2.68 (2H, t, J = 6.8 Hz), 6.88 (1H, d, J = 15.2 Hz), 7.32 (2H, d, J = 8.0 Hz), 7.93 (2H, d, J = 8.0 Hz), 7.98 (1H, d, J = 15.2 Hz)
(E)-4-(4-Heptadecylphenyl)-4-oxobut-2-enoic acid (AUP04)
1 H-NMR (400 MHz, CDCl 3 ) δ: 0.86 (3H, t, J = 6.8 Hz), 1.20-1.34 (28H, m), 1.60-1.67 (2H, m), 2.68 (2H, t, J = 6.8 Hz), 6.88 (1H, d, J = 15.2 Hz), 7.32 (2H, d, J = 8.0 Hz), 7.93 (2H, d, J = 8.0 Hz), 7.98 (1H, d, J = 15.2 Hz)
(E)-4-(4-Octadecylphenyl)-4-oxobut-2-enoic acid (17)
1H-NMR (400 MHz, CDCl3) δ: 0.86 (3H, t, J = 6.8 Hz), 1.21-1.34 (30H, m), 1.60-1.67 (2H, m), 2.68 (2H, t, J = 6.8 Hz), 6.88 (1H, d, J = 15.2 Hz), 7.32 (2H, d, J = 8.0 Hz), 7.93 (2H, d, J = 8.0 Hz), 7.99 (1H, d, J = 15.2 Hz)
(E)-4-(4-Octadecylphenyl)-4-oxobut-2-enoic acid (17)
1 H-NMR (400 MHz, CDCl 3 ) δ: 0.86 (3H, t, J = 6.8 Hz), 1.21-1.34 (30H, m), 1.60-1.67 (2H, m), 2.68 (2H, t, J = 6.8 Hz), 6.88 (1H, d, J = 15.2 Hz), 7.32 (2H, d, J = 8.0 Hz), 7.93 (2H, d, J = 8.0 Hz), 7.99 (1H, d, J = 15.2 Hz)
(E)-4-(4-Nonadecylphenyl)-4-oxobut-2-enoic acid (AUP05)
1H-NMR (400 MHz, CDCl3) δ: 0.88 (3H, t, J = 6.8 Hz), 1.19-1.33 (32H, m), 1.59-1.66 (2H, m), 2.68 (2H, t, J = 6.8 Hz), 6.88 (1H, d, J = 15.2 Hz), 7.32 (2H, d, J = 8.4 Hz), 7.93 (2H, d, J = 8.4 Hz), 7.98 (1H, d, J = 15.2 Hz)
(E)-4-(4-Nonadecylphenyl)-4-oxobut-2-enoic acid (AUP05)
1 H-NMR (400 MHz, CDCl 3 ) δ: 0.88 (3H, t, J = 6.8 Hz), 1.19-1.33 (32H, m), 1.59-1.66 (2H, m), 2.68 (2H, t, J = 6.8 Hz), 6.88 (1H, d, J = 15.2 Hz), 7.32 (2H, d, J = 8.4 Hz), 7.93 (2H, d, J = 8.4 Hz), 7.98 (1H, d, J = 15.2 Hz)
(E)-4-(4-Henicosylphenyl)-4-oxobut-2-enoic acid (AUP10)
1H-NMR (400 MHz, CDCl3) δ: 0.88 (3H, t, J = 7.6 Hz), 1.20-1.34 (36H, m), 1.60-1.68 (2H, m), 2.68 (2H, t, J = 7.6 Hz), 6.88 (1H, d, J = 15.2 Hz), 7.32 (2H, d, J = 8.8 Hz), 7.93 (2H, d, J = 8.8 Hz), 7.99 (1H, d, J = 15.2 Hz)
(E)-4-(4-Henicosylphenyl)-4-oxobut-2-enoic acid (AUP10)
1 H-NMR (400 MHz, CDCl 3 ) δ: 0.88 (3H, t, J = 7.6 Hz), 1.20-1.34 (36H, m), 1.60-1.68 (2H, m), 2.68 (2H, t, J = 7.6 Hz), 6.88 (1H, d, J = 15.2 Hz), 7.32 (2H, d, J = 8.8 Hz), 7.93 (2H, d, J = 8.8 Hz), 7.99 (1H, d, J = 15.2 Hz)
＜実験例２＞各化合物のAtg4B阻害活性評価、及びPLA2阻害活性評価 <Experimental Example 2> Evaluation of Atg4B inhibitory activity and PLA2 inhibitory activity of each compound
［方法：Atg4B阻害活性評価］
精製したLC3のC末にグルタチオン-S-トランスフェラーゼを融合させたLC3-GSTとAtg4B、阻害剤を混合し、37 ℃ 、3時間インキュベート後にSDS-PAGEを行い染色し、LC3-GSTからLC3とGSTへの変換量を観察した。各バンドはImage Jを用いて定量解析した。対照群としてdimethylsulfoxide (DMSO) を添加したサンプルを調製した。変換量、変換率 (%) は以下の式により算出した。変換率 (%) は対照群を100%として算出した。
変換量 = (GST + LC3) / (LC3-GST + GST + LC3)
変換率 (%) = (阻害剤添加時の変換量) / (DMSO添加時の変換量) ×100
[Method: Evaluation of Atg4B inhibitory activity]
LC3-GST, which is made by fusing glutathione-S-transferase to the C-terminus of purified LC3, was mixed with Atg4B and an inhibitor, and incubated at 37°C for 3 hours. SDS-PAGE was then performed and stained to observe the amount of conversion from LC3-GST to LC3 and GST. Each band was quantitatively analyzed using Image J. As a control, a sample was prepared to which dimethylsulfoxide (DMSO) was added. The amount of conversion and conversion rate (%) were calculated using the following formula. The conversion rate (%) was calculated assuming the control group to be 100%.
Conversion rate = (GST + LC3) / (LC3-GST + GST + LC3)
Conversion rate (%) = (amount converted when inhibitor was added) / (amount converted when DMSO was added) × 100
［方法：PLA2阻害活性評価］
EnzChek Phospholipase A2 Assay Kit（@Thermofisher）を用いて、提供された実験プロトコールに基づきPLA2阻害活性を評価した。DMSOを添加したサンプルを対照群とし、各種濃度の化合物を添加したサンプルの対照群の活性に対する阻害%から50%阻害濃度を算出した。
[Method: PLA2 inhibitory activity evaluation]
The PLA 2 inhibitory activity was evaluated using the EnzChek Phospholipase A 2 Assay Kit (@Thermofisher) according to the experimental protocol provided. The 50% inhibitory concentration was calculated from the inhibition percentage of the control group activity of samples containing various concentrations of compounds, using a sample containing DMSO as the control group.
［結果及び考察］
Atg4Bに対する50%阻害濃度はアルキル鎖長が長くなるにつれて低下し、アルキル鎖長が11の時に最も小さくなり、アルキル鎖長が11のAUP01が最も強力にAtg4Bを阻害した。更に炭素数が増えるにつれ活性は低下するが、アルキル鎖長が18、19のときにまた活性は上昇した。これは、アルキル鎖長の増加に伴う酵素との相互作用の上昇によって阻害活性が上昇したが、アルキル鎖長15、17と長くなるにつれ脂溶性の亢進による溶解度の低下によって活性が低下し、更にアルキル鎖長18、19では新たな相互作用の獲得によって阻害活性が増強したと考えられた。
[Results and Discussion]
The 50% inhibitory concentration for Atg4B decreased with increasing alkyl chain length, being the smallest when the alkyl chain length was 11, and AUP01 with an alkyl chain length of 11 most potently inhibited Atg4B. Furthermore, the activity decreased with an increase in the number of carbon atoms, but the activity increased again when the alkyl chain length was 18 or 19. This is thought to be because the inhibitory activity increased due to increased interaction with the enzyme with increasing alkyl chain length, but as the alkyl chain length increased to 15 and 17, the activity decreased due to decreased solubility due to increased lipid solubility, and further, at alkyl chain lengths of 18 and 19, the inhibitory activity was enhanced by the acquisition of new interactions.
また、アルキル鎖長18、19ではPLA2に対する50%阻害濃度が、それぞれ0.049±0.013 μM、0.35±0.13 μMである一方で、AUP01はPLA2に対する50%阻害濃度が、3.5±0.3 μMであった。したがって、AUP01は、これらと比較してAtg4Bへの選択性が高いことが示唆された。 In addition, the 50% inhibitory concentrations for PLA2 were 0.049±0.013 μM and 0.35±0.13 μM for alkyl chain lengths 18 and 19, respectively, while the 50% inhibitory concentration for PLA2 for AUP01 was 3.5±0.3 μM. This suggests that AUP01 has higher selectivity for Atg4B than these.
＜実験例３＞AUP01のオートファジー阻害活性評価 <Experimental Example 3> Evaluation of autophagy inhibitory activity of AUP01
［方法］
前立腺癌LNCaP細胞細胞は37 ℃、5% CO2条件下の炭酸ガスインキュベーター内で培養した。増殖培地として5% (v/v) FBS、100 U/ml penicillin-G potassium、100 μg/ml streptomycin sulfate及び10 mM [4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid] HEPES緩衝液 (pH 7.0) を含むRPMI1640を用いた。両細胞を1 × 106cells/dishで6 cm dishに播種し、24時間後にFBS濃度を2%に変えた増殖培地に置換し、化合物AUP01若しくは化合物17を添加した。2時間後にアミノ酸枯渇培地に置換し、更に3時間培養後に細胞をDPBSで回収し、以下のようにウエスタンブロットとDAPGreen染色に供した。
[method]
LNCaP cells were cultured in a carbon dioxide gas incubator at 37°C and 5% CO2 . RPMI1640 containing 5% (v/v) FBS, 100 U/ml penicillin-G potassium, 100 μg/ml streptomycin sulfate, and 10 mM [4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid] HEPES buffer (pH 7.0) was used as the growth medium. Both cells were seeded at 1 × 106 cells/dish on a 6 cm dish, and after 24 hours, the growth medium was replaced with one containing 2% FBS, and compound AUP01 or compound 17 was added. After 2 hours, the medium was replaced with amino acid-depleted medium, and after another 3 hours of culture, the cells were harvested with DPBS and subjected to Western blotting and DAPGreen staining as follows.
［方法：ウエスタンブロット（図２Ａ）］
各種試薬で処理した細胞をDPBSで3回洗浄後、セルスクレイパーを用いて細胞を剥離した。回収した細胞はDulbecco’s phosphate buffered saline (DPBS) で洗浄後、8 M Urea、10 mM Tris (hydroxymethyl) aminomethane、50 mM Na2H2PO4を含むUrea bufferにより懸濁してソニケーションにより細胞膜を破壊した。細胞破砕液を遠心分離 (15,000 x g、15分間、4 ℃) し、その上清を細胞抽出液とした。12.5% ポリアクリルアミドゲルを用いたSDS-PAGEにより分離後、ゲル上のタンパク質を電気的にpolyvinylidene difluoride (PVDF) 膜に転写した。PVDF膜は、1% BSAを用いてブロッキング後、p62とβ-actinに対する一次抗体とhorseradish peroxidase標識二次抗体と順次反応させた。抗体反応性タンパク質はECL enhanced chemiluminescence detection kit (GE healthcare) を用いた化学発光法により検出した。バンドの濃さはImage J（NIH）を用いて解析した。
[Method: Western blot (Figure 2A)]
After the cells were treated with each reagent, they were washed three times with DPBS and then scraped off using a cell scraper. The collected cells were washed with Dulbecco's phosphate buffered saline (DPBS) and suspended in urea buffer containing 8 M urea, 10 mM Tris (hydroxymethyl) aminomethane, and 50 mM Na 2 H 2 PO 4, and the cell membrane was disrupted by sonication. The cell lysate was centrifuged (15,000 xg, 15 min, 4°C), and the supernatant was used as the cell extract. After separation by SDS-PAGE using 12.5% polyacrylamide gel, the proteins on the gel were electrically transferred to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked with 1% BSA and reacted sequentially with primary antibodies against p62 and β-actin and horseradish peroxidase-conjugated secondary antibodies. Antibody-reactive proteins were detected by chemiluminescence using an ECL enhanced chemiluminescence detection kit (GE Healthcare). Band intensity was analyzed using Image J (NIH).
［方法：DAPGreen染色（図２Ｂ）］
増殖培地に懸濁した細胞を24-well multiplate中に2 × 104 cells/500 μLずつ播種し、37 ℃、5% CO2条件下で24時間培養後、DAPGreenを添加し、30分間インキュベートした。FBS濃度を2%に変えた増殖培地に置換し、化合物AUP01を添加した。2時間後にアミノ酸枯渇培地に置換し、更に3時間培養後に細胞をDPBSで2回洗浄後、余分な水分を除去し、スライドグラス上にマウント剤 (fluoromount-G) を用いてカバーグラスを固定した。カバーグラスを共焦点レーザー顕微鏡LSM700 (Carl Zeiss) にセットし、蛍光観察を行った。倒立型蛍光顕微鏡の40倍油浸対物レンズを用いて、画像を取り込んだ。
[Method: DAPGreen staining (Figure 2B)]
Cells suspended in growth medium were seeded in a 24-well multiplate at 2 × 104 cells/500 μL each, and after 24 hours of culture at 37 °C and 5 % CO2, DAPGreen was added and incubated for 30 minutes. The growth medium was replaced with one containing 2% FBS, and compound AUP01 was added. After 2 hours, the medium was replaced with amino acid-depleted medium, and after further culture for 3 hours, the cells were washed twice with DPBS, excess water was removed, and a cover glass was fixed on a slide glass using a mounting agent (fluoromount-G). The cover glass was placed on a confocal laser microscope LSM700 (Carl Zeiss) and fluorescence observation was performed. Images were captured using a 40x oil immersion objective lens of an inverted fluorescence microscope.
［結果及び考察］
3時間のアミノ酸枯渇培地での処理によって、p62の分解とオートファゴソームの生成に起因するDAPGreenのドット状の蛍光の増加が認められ、オートファジーが誘導されていることが示された。AUP01の前処理によってp62発現量は濃度依存的に増加し、リード化合物17よりもその効果は強力であった。またアミノ酸枯渇培地で誘導されたDAPGreen蛍光も減少した。以上のことから、AUP01は培養細胞レベルでオートファジーを阻害することが示された。
[Results and Discussion]
Treatment with amino acid-depleted medium for 3 hours increased DAPGreen dot-like fluorescence due to the degradation of p62 and the generation of autophagosomes, indicating the induction of autophagy. Pretreatment with AUP01 increased p62 expression in a concentration-dependent manner, and its effect was stronger than that of lead compound 17. DAPGreen fluorescence induced by amino acid-depleted medium was also reduced. These results indicate that AUP01 inhibits autophagy at the cultured cell level.
＜実験例４＞アビラテロンによるアポトーシスとオートファジーの誘導に関する評価 <Experimental Example 4> Evaluation of induction of apoptosis and autophagy by abiraterone
［方法：生細胞数測定］
増殖培地に懸濁したLNCaP細胞を96-well multiplate中に2 × 104 cells/200 μLずつ播種し、CO2インキュベーター内で一晩培養した。抗生物質と2% FBSを含む培地に交換し、培地中に試料を添加して更に24時間培養した。対照群としてdimethylsulfoxide (DMSO) を添加した細胞を調製した。次に、血清不含及びフェノールレッド不含の培地に交換し、40 μM resazurinを添加し、37℃で2‐4時間培養した後、マイクロプレートリーダーModel680 (Bio Rad) を用いて波長570 nm 及び600 nmの吸光度を測定した。細胞生存率 (%) は以下の式により算出した。
細胞生存率 (%) = (S-A) / (B-A) × 100
S : 試料及び細胞を添加したwellの吸光度
A : 培地のみを添加したwellの吸光度
B : DMSO及び細胞を添加したwellの吸光度
[Method: Viable cell count measurement]
LNCaP cells suspended in growth medium were seeded at 2 × 104 cells/200 μL in a 96-well multiplate and cultured overnight in a CO2 incubator. The medium was replaced with one containing antibiotics and 2% FBS, and the samples were added to the medium and cultured for another 24 hours. As a control, cells were prepared with the addition of dimethylsulfoxide (DMSO). Next, the medium was replaced with one without serum or phenol red, 40 μM resazurin was added, and the cells were cultured at 37°C for 2-4 hours. The absorbance at wavelengths of 570 nm and 600 nm was measured using a microplate reader Model 680 (Bio Rad). The cell viability (%) was calculated using the following formula:
Cell viability (%) = (SA) / (BA) × 100
S: Absorbance of the well to which the sample and cells were added
A: Absorbance of the well to which only medium was added
B: Absorbance of wells containing DMSO and cells
［方法：ウエスタンブロット］
前立腺癌LNCaP細胞細胞は37 ℃、5% CO2条件下の炭酸ガスインキュベーター内で培養した。増殖培地として5% (v/v) FBS、100 U/ml penicillin-G potassium、100 μg/ml streptomycin sulfate及び10 mM [4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid] HEPES緩衝液 (pH 7.0) を含むRPMI1640を用いた。両細胞を1 × 106 cells/dishで6 cm dishに播種し、24時間後にFBS濃度を2%に変えた10 μM アビラテロン含有培地に置換し、更に24時間培養後に細胞をDPBSで回収し、以下のようにウエスタンブロットに供した。各種試薬で処理した細胞をDPBSで3回洗浄後、セルスクレイパーを用いて細胞を剥離した。回収した細胞はDulbecco’s phosphate buffered saline (DPBS) で洗浄後、8 M Urea、10 mM Tris (hydroxymethyl) aminomethane、50 mM Na2H2PO4を含むUrea bufferにより懸濁してソニケーションにより細胞膜を破壊した。細胞破砕液を遠心分離 (15,000 x g、15分間、4 ℃) し、その上清を細胞抽出液とした。12.5% ポリアクリルアミドゲルを用いたSDS-PAGEにより分離後、ゲル上のタンパク質を電気的にpolyvinylidene difluoride (PVDF) 膜に転写した。PVDF膜は、1% BSAを用いてブロッキング後、PARP、p62、p-p62 (Ser403)、LC3とβ-actinに対する一次抗体とhorseradish peroxidase標識二次抗体と順次反応させた。抗体反応性タンパク質はECL enhanced chemiluminescence detection kit (GE healthcare) を用いた化学発光法により検出した。バンドの濃さはImage J（NIH）を用いて解析した。
[Method: Western blot]
LNCaP cells were cultured in a carbon dioxide incubator at 37°C and 5% CO2 . RPMI1640 containing 5% (v/v) FBS, 100 U/ml penicillin-G potassium, 100 μg/ml streptomycin sulfate, and 10 mM [4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid] HEPES buffer (pH 7.0) was used as the growth medium. Both types of cells were seeded on a 6 cm dish at 1 × 106 cells/dish, and after 24 hours, the medium was replaced with 10 μM abiraterone-containing medium with 2% FBS concentration. After another 24 hours of culture, the cells were harvested with DPBS and subjected to Western blotting as follows. The cells treated with various reagents were washed three times with DPBS and detached using a cell scraper. The collected cells were washed with Dulbecco's phosphate buffered saline (DPBS), suspended in urea buffer containing 8 M urea, 10 mM Tris (hydroxymethyl) aminomethane, and 50 mM Na 2 H 2 PO 4, and the cell membrane was disrupted by sonication. The cell lysate was centrifuged (15,000 xg, 15 min, 4°C), and the supernatant was used as the cell extract. After separation by SDS-PAGE using 12.5% polyacrylamide gel, the proteins on the gel were electrically transferred to a polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was blocked with 1% BSA and reacted with primary antibodies against PARP, p62, p-p62 (Ser403), LC3, and β-actin, and horseradish peroxidase-conjugated secondary antibodies in sequence. Antibody-reactive proteins were detected by chemiluminescence using an ECL enhanced chemiluminescence detection kit (GE Healthcare). Band intensity was analyzed using Image J (NIH).
［結果及び考察］
去勢抵抗性前立腺癌（CRPC）治療薬として用いられるアビラテロンはLNCaP細胞に対して用量依存的に切断型PARPを生成し、アポトーシス性細胞死が誘導されていることが示された。また、同時にp-p62とLC3-IIの生成が認められたことから、オートファジーも誘導されていることが示された。
[Results and Discussion]
Abiraterone, a drug used to treat castration-resistant prostate cancer (CRPC), was shown to induce apoptotic cell death by producing cleaved PARP in a dose-dependent manner in LNCaP cells, and also induced autophagy by producing p-p62 and LC3-II.
＜実験例５＞アビラテロンとAUP01の併用効果 <Experimental Example 5> Effect of combined use of abiraterone and AUP01
［方法：DAPGreen染色（図４Ａ）］
増殖培地に懸濁した細胞を24-well multiplate中に2 × 104 cells/500 μLずつ播種し、37 ℃、5% CO2条件下で24時間培養後、DAPGreenを添加し、30分間インキュベートした。FBS濃度を2%に変えた増殖培地に置換し、化合物AUP01を添加した。2時間後に10 μM アビラテロン含有培地に置換し、更に24時間培養後に細胞をDPBSで2回洗浄後、余分な水分を除去し、スライドグラス上にマウント剤 (fluoromount-G) を用いてカバーグラスを固定した。カバーグラスを共焦点レーザー顕微鏡LSM700 (Carl Zeiss) にセットし、蛍光観察を行った。倒立型蛍光顕微鏡の40倍油浸対物レンズを用いて、画像を取り込んだ。
[Method: DAPGreen staining (Figure 4A)]
Cells suspended in growth medium were seeded in a 24-well multiplate at 2 × 104 cells/500 μL each, and after 24 hours of culture at 37 °C and 5 % CO2, DAPGreen was added and incubated for 30 minutes. The growth medium was replaced with one containing 2% FBS, and compound AUP01 was added. After 2 hours, the medium was replaced with one containing 10 μM abiraterone, and after further culture for 24 hours, the cells were washed twice with DPBS, excess water was removed, and a cover glass was fixed on a slide glass using a mounting agent (fluoromount-G). The cover glass was set on a confocal laser microscope LSM700 (Carl Zeiss) and fluorescence observation was performed. Images were captured using a 40x oil immersion objective lens of an inverted fluorescence microscope.
［方法：免疫蛍光染色（図４Ｂ）］
増殖培地に懸濁した細胞を24-well multiplate中に2 × 104 cells/500 μLずつ播種し、37 ℃、5% CO2条件下で24時間培養後、FBS濃度を2%に変えた増殖培地に置換し、化合物AUP01を添加した。2時間後に10 μM アビラテロン含有培地に置換し、更に24時間培養後に細胞をDPBSで2回洗浄した。4% paraformaldehyde phosphate buffer solution 300 μLを加え、10分間固定し、0.1% Triton X-100、100 mM glycineを含むDPBS 300 μLを加え、10分間静置した。0.1% Tween 20、1% BSAを含むDPBS 300 μLを加え、1時間ブロッキングし、DPBSで2回洗浄後、DPBSに300 : 1 に希釈した一次抗体 (抗Caspase-3抗体) 液中に、4 ℃で一晩インキュベートした。PBSで2回洗浄後、DPBSに500 : 1 で希釈したAlexa Fluoro-488標識したウサギ二次抗体液中に、室温で1時間遮光してインキュベートした。PBSで2回洗浄後、余分な水分を除去し、スライドグラス上にマウント剤 (DAPI fluoromount-G) を用いてカバーグラスを固定した。蛍光免疫染色した細胞を、共焦点レーザー顕微鏡LSM700 (Carl Zeiss) にセットし、蛍光観察を行った。倒立型蛍光顕微鏡の40倍油浸対物レンズを用いて、画像を取り込んだ。
[Method: Immunofluorescence staining (Figure 4B)]
The cells suspended in growth medium were seeded in a 24-well multiplate at 2 × 104 cells/500 μL each, and after 24 hours of incubation at 37°C and 5 % CO2, the growth medium was replaced with one containing 2% FBS, and compound AUP01 was added. After 2 hours, the medium was replaced with one containing 10 μM abiraterone, and after another 24 hours of incubation, the cells were washed twice with DPBS. 300 μL of 4% paraformaldehyde phosphate buffer solution was added, and the cells were fixed for 10 minutes, and 300 μL of DPBS containing 0.1% Triton X-100 and 100 mM glycine was added and left to stand for 10 minutes. 300 μL of DPBS containing 0.1% Tween 20 and 1% BSA was added, and the cells were blocked for 1 hour, washed twice with DPBS, and then incubated overnight at 4°C in a solution of primary antibody (anti-Caspase-3 antibody) diluted 300:1 in DPBS. After washing twice with PBS, the cells were incubated in a solution of Alexa Fluoro-488-labeled rabbit secondary antibody diluted 500:1 in DPBS at room temperature for 1 hour in the dark. After washing twice with PBS, excess water was removed and a cover glass was fixed on a slide glass using a mounting agent (DAPI fluoromount-G). The fluorescently immunostained cells were placed on a confocal laser microscope LSM700 (Carl Zeiss) and observed for fluorescence. Images were captured using a 40x oil immersion objective lens on an inverted fluorescence microscope.
［方法：ウエスタンブロット（図４ＣＤ）］
前立腺癌LNCaP細胞細胞は37 ℃、5% CO2条件下の炭酸ガスインキュベーター内で培養した。増殖培地として5% (v/v) FBS、100 U/ml penicillin-G potassium、100 μg/ml streptomycin sulfate及び10 mM [4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid] HEPES緩衝液 (pH 7.0) を含むRPMI1640を用いた。両細胞を1 × 106 cells/dishで6 cm dishに播種し、FBS濃度を2%に変えた増殖培地に置換し、化合物AUP01若しくはウォルトマニン（Wo）を添加した。2時間後に10 μM アビラテロン含有培地に置換し、更に24時間培養後に細胞をDPBSで2回洗浄し、以下のようにウエスタンブロットに供した。各種試薬で処理した細胞をDPBSで3回洗浄後、セルスクレイパーを用いて細胞を剥離した。回収した細胞はDulbecco’s phosphate buffered saline (DPBS) で洗浄後、8 M Urea、10 mM Tris (hydroxymethyl) aminomethane、50 mM Na2H2PO4を含むUrea bufferにより懸濁してソニケーションにより細胞膜を破壊した。細胞破砕液を遠心分離 (15,000 x g、15分間、4 ℃) し、その上清を細胞抽出液とした。12.5% ポリアクリルアミドゲルを用いたSDS-PAGEにより分離後、ゲル上のタンパク質を電気的にpolyvinylidene difluoride (PVDF) 膜に転写した。PVDF膜は、1% BSAを用いてブロッキング後、PARPとβ-actinに対する一次抗体とhorseradish peroxidase標識二次抗体と順次反応させた。抗体反応性タンパク質はECL enhanced chemiluminescence detection kit (GE healthcare) を用いた化学発光法により検出した。バンドの濃さはImage J（NIH）を用いて解析した。
[Method: Western blot (Fig. 4C-D)]
LNCaP cells were cultured in a carbon dioxide gas incubator at 37°C and 5% CO2 . RPMI1640 containing 5% (v/v) FBS, 100 U/ml penicillin-G potassium, 100 μg/ml streptomycin sulfate, and 10 mM [4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid] HEPES buffer (pH 7.0) was used as the growth medium. Both types of cells were seeded at 1 × 106 cells/dish on a 6 cm dish, and the growth medium was replaced with one containing 2% FBS, and the compound AUP01 or wortmannin (Wo) was added. After 2 hours, the medium was replaced with 10 μM abiraterone-containing medium, and after further culture for 24 hours, the cells were washed twice with DPBS and subjected to Western blotting as follows. The cells treated with various reagents were washed three times with DPBS and detached using a cell scraper. The collected cells were washed with Dulbecco's phosphate buffered saline (DPBS) and suspended in urea buffer containing 8 M urea, 10 mM Tris (hydroxymethyl) aminomethane, and 50 mM Na2H2PO4 , and the cell membrane was disrupted by sonication. The cell lysate was centrifuged (15,000 xg, 15 min, 4°C), and the supernatant was used as the cell extract. After separation by SDS-PAGE using 12.5% polyacrylamide gel, the proteins on the gel were electrically transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking with 1% BSA, the PVDF membrane was reacted sequentially with primary antibodies against PARP and β-actin and horseradish peroxidase-labeled secondary antibodies. Antibody-reactive proteins were detected by chemiluminescence using an ECL enhanced chemiluminescence detection kit (GE Healthcare). Band intensity was analyzed using Image J (NIH).
［方法：生細胞数測定（図４Ｄ）］
増殖培地に懸濁したLNCaP細胞を96-well multiplate中に2 × 104 cells/200 μLずつ播種し、CO2インキュベーター内で一晩培養した。抗生物質と2% FBSを含む培地に交換し、AUP01（1 μM; △, 2 μM; ▲）添加2時間後に、5, 10 μM アビラテロンを添加して更に24時間培養した。対照群としてdimethylsulfoxide (DMSO) を添加した細胞を調製した。次に、血清不含及びフェノールレッド不含の培地に交換し、40 μM resazurinを添加し、37 ℃で2‐4時間培養した後、マイクロプレートリーダーModel680 (Bio Rad) を用いて波長570 nm 及び600 nmの吸光度を測定した。
[Method: Viable cell count (FIG. 4D)]
LNCaP cells suspended in growth medium were seeded at 2 × 104 cells/200 μL in a 96-well multiplate and cultured overnight in a CO2 incubator. The medium was replaced with one containing antibiotics and 2% FBS, and AUP01 (1 μM; △, 2 μM; ▲) was added 2 hours later, 5 and 10 μM abiraterone was added, and the cells were cultured for another 24 hours. As a control, cells were prepared by adding dimethylsulfoxide (DMSO). Next, the medium was replaced with serum-free and phenol red-free medium, and 40 μM resazurin was added. After 2-4 hours of culture at 37°C, the absorbance at wavelengths of 570 nm and 600 nm was measured using a microplate reader Model 680 (Bio Rad).
［結果及び考察］
アビラテロン処理によって誘導されたオートファジーはAUP01の前処理によって抑制された。また、アポトーシス性細胞死はAUP01の前処理によって増強された。よって、AUP01はアビラテロン処理時に誘導されたオートファジーを阻害することによってアポトーシスを増強したと考えられた。
[Results and Discussion]
Autophagy induced by abiraterone treatment was suppressed by pretreatment with AUP01, and apoptotic cell death was enhanced by pretreatment with AUP01. Thus, AUP01 enhanced apoptosis by inhibiting autophagy induced by abiraterone treatment.
＜実験例６＞AUP01によるCRPC治療薬の抗癌活性増強効果 <Experimental Example 6> AUP01 enhances the anticancer activity of CRPC drugs
［方法：生細胞数測定］
増殖培地に懸濁したLNCaP細胞を96-well multiplate中に2 × 104 cells/200 μLずつ播種し、CO2インキュベーター内で一晩培養した。抗生物質と2% FBSを含む培地に交換し、5 μM AUP01添加2時間後に、アパルタミド（50, 100 μM）、カバジタキセル（10, 20 μM）若しくはエンザルタミド（20, 50 μM）を添加して更に24時間培養した。対照群としてdimethylsulfoxide (DMSO) を添加した細胞を調製した。次に、血清不含及びフェノールレッド不含の培地に交換し、40 μM resazurinを添加し、37℃で2‐4時間培養した後、マイクロプレートリーダーModel680 (Bio Rad) を用いて波長570 nm 及び600 nmの吸光度を測定した。
[Method: Viable cell count measurement]
LNCaP cells suspended in growth medium were seeded at 2 × 104 cells/200 μL in a 96-well multiplate and cultured overnight in a CO2 incubator. The medium was replaced with antibiotics and 2% FBS, and 5 μM AUP01 was added 2 hours later. Apalutamide (50, 100 μM), cabazitaxel (10, 20 μM), or enzalutamide (20, 50 μM) were added and cultured for another 24 hours. As a control, cells were prepared with dimethylsulfoxide (DMSO). Next, the medium was replaced with serum-free and phenol red-free medium, and 40 μM resazurin was added. After 2-4 hours of culture at 37°C, the absorbance at wavelengths of 570 nm and 600 nm was measured using a microplate reader Model 680 (Bio Rad).
［結果及び考察］
３種の去勢抵抗性前立腺癌（CRPC）治療薬アパルタミド、カバジタキセル、エンザルタミドに対してもAUP01は有意に抗癌活性を増強させた。
[Results and Discussion]
AUP01 also significantly enhanced the anticancer activity of three castration-resistant prostate cancer (CRPC) drugs: apalutamide, cabazitaxel, and enzalutamide.
本発明によれば、優れたオートファジー阻害活性を有する、新規化合物、オートファジー阻害剤、Atg4B阻害剤、及びこれを用いた抗癌剤の活性増強剤、並びに医薬品組成物、及び癌治療用キットを提供することができる。本発明に係るオートファジー阻害剤等は、化学合成も容易であり、大量生産に適する。したがって、本発明に係るオートファジー阻害剤等を癌治療などに応用することで、患者の治療機会の向上、ひいては国民医療費の削減に寄与できると考えられる。 According to the present invention, it is possible to provide a novel compound, an autophagy inhibitor, an Atg4B inhibitor, and an activity enhancer of an anticancer drug using the same, as well as a pharmaceutical composition and a cancer treatment kit, all of which have excellent autophagy inhibitory activity. The autophagy inhibitors and the like according to the present invention can be easily chemically synthesized and are suitable for mass production. Therefore, it is believed that application of the autophagy inhibitors and the like according to the present invention to cancer treatment and the like can improve treatment opportunities for patients and ultimately contribute to reducing national medical expenses.
Claims (3)
（一般式（１）中、ｎは、２～２０の整数である。） An autophagy inhibitor containing a compound represented by the following general formula (1) or a salt thereof as an active ingredient , or an activity enhancer for an anticancer agent comprising an Atg4B inhibitor containing a compound represented by the following general formula (1) or a salt thereof as an active ingredient :
(In general formula (1), n is an integer from 2 to 20.)
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Non-Patent Citations (3)
| Title |
|---|
| KEITH BOWDEN et al.，"Structure-Activity Relations", Advances in Chemistry，1974年08月01日，p.130-140，DOI: 10.1021/BA-1972-0114.CH009 |
| NUHN P et al.，"[Synthesis of substituted benzoylacrylic acids as potential antagonists of phospholipase A2]."，Die Pharmazie，1999年02月，Vol. 54, No. 2，p.93-98 |
| 遠藤智史、外６名，がんアジュバント薬を指向したシステインプロテアーゼAtg4B阻害剤の創製，日本病態プロテアーゼ学会学術集会プログラム抄録集，24th，2019年，p.41 |
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