JP7388702B2 - Antitumor agents and combination drugs - Google Patents
Antitumor agents and combination drugs Download PDFInfo
- Publication number
- JP7388702B2 JP7388702B2 JP2019554284A JP2019554284A JP7388702B2 JP 7388702 B2 JP7388702 B2 JP 7388702B2 JP 2019554284 A JP2019554284 A JP 2019554284A JP 2019554284 A JP2019554284 A JP 2019554284A JP 7388702 B2 JP7388702 B2 JP 7388702B2
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- glutathione
- sulfasalazine
- inhibitor
- dyclonine
- aldehyde dehydrogenase
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Description
本発明は、抗腫瘍剤及び配合剤に関する。 The present invention relates to antitumor agents and combination agents.
がん治療においては、抗がん剤や放射線などの治療に対して抵抗性を持つ細胞が存在することが、再発や転移の原因となり、がんの治療を妨げている。このような治療抵抗性細胞として、近年がん幹細胞の存在が注目されている。がん幹細胞は、各種ストレスに対して耐性が高く、がん幹細胞を標的とした薬剤の開発ががんの根治のためには急務である。しかし、がん幹細胞を標的にした治療の開発のための、がん幹細胞のストレス耐性の分子機構の解析は端緒についたばかりである。 In cancer treatment, the presence of cells that are resistant to treatments such as anticancer drugs and radiation causes recurrence and metastasis, hindering cancer treatment. Cancer stem cells have recently attracted attention as such treatment-resistant cells. Cancer stem cells are highly resistant to various types of stress, and the development of drugs that target cancer stem cells is an urgent need for a complete cure of cancer. However, analysis of the molecular mechanisms underlying stress resistance in cancer stem cells has only just begun, in order to develop treatments that target cancer stem cells.
上皮性がん幹細胞のマーカーの一つであるＣＤ４４は、そのストレス耐性に関与する分子として知られている（Cancer Cell. 2011 Mar 8;19(3):387-400）。ＣＤ４４には、スプライスバリアントフォーム（以下、ＣＤ４４ｖ）が存在し、ＣＤ４４ｖが細胞膜上にシスチントランスポーターｘＣＴを安定して発現させる。ｘＣＴは細胞内にシスチンを取り込む機能を有し、それによって取り込まれたシスチンはグルタチオン（ＧＳＨ）の産生に用いられるために、ＣＤ４４ｖを高発現している細胞では、ＧＳＨの量が増加する。ＧＳＨは強力な抗酸化作用を持ち、細胞に生じたストレスを減少させる役割を持つために、ＣＤ４４ｖを高発現するがん幹細胞は、治療に対して抵抗性を有するとされる。 CD44, which is one of the markers of epithelial cancer stem cells, is known as a molecule involved in their stress resistance (Cancer Cell. 2011 Mar 8;19(3):387-400). A splice variant form (hereinafter referred to as CD44v) of CD44 exists, and CD44v stably expresses the cystine transporter xCT on the cell membrane. xCT has the function of taking cystine into cells, and the cystine thus taken in is used for the production of glutathione (GSH), so the amount of GSH increases in cells that highly express CD44v. Since GSH has a strong antioxidant effect and plays a role in reducing stress generated in cells, cancer stem cells that highly express CD44v are said to be resistant to treatment.
一方、潰瘍性大腸炎や関節リウマチの治療に使用されている薬剤に、スルファサラジン（Sulfasalazine）(別名：サラゾスルファピリジン、サラゾピリン、サリチルアゾスルファピリジン)がある。スルファサラジンは、スルファピリジンと５－アミノサリチル酸（５－ＡＳＡ）の酸性アゾ化合物であり、経口投与すると、腸内で腸内細菌によりスルファピリジンと５－アミノサリチル酸（５－ＡＳＡ）に分解される。前記疾患に対しては、特に５－ＡＳＡが主な有効成分とされている。 On the other hand, sulfasalazine (also known as salazosulfapyridine, salicylazosulfapyridine, salicylazosulfapyridine) is a drug used to treat ulcerative colitis and rheumatoid arthritis. Sulfasalazine is an acidic azo compound of sulfapyridine and 5-aminosalicylic acid (5-ASA), and when administered orally, it is broken down by intestinal bacteria into sulfapyridine and 5-aminosalicylic acid (5-ASA). Ru. In particular, 5-ASA is considered to be the main active ingredient for the above-mentioned diseases.
近年、分解される前の未変化体のスルファサラジンにｘＣＴ阻害作用があり、抗腫瘍剤として有効であることが明らかになった（Leukemia vol.15, pp.1633-1640, 2001）。つまり、スルファサラジンを癌細胞に添加すると、ｘＣＴによる細胞内へのシスチンの取り込みが抑制され、グルタチオン産生量が低下し、その結果、癌細胞の酸化ストレス耐性が下がり、抗腫瘍剤への感受性が上昇する。 In recent years, it has been revealed that the unchanged form of sulfasalazine before being degraded has an xCT inhibitory effect and is effective as an antitumor agent (Leukemia vol. 15, pp. 1633-1640, 2001). In other words, when sulfasalazine is added to cancer cells, the uptake of cystine into the cells by xCT is suppressed, and the amount of glutathione production is reduced, resulting in a decrease in the cancer cells' resistance to oxidative stress and an increase in their sensitivity to antitumor drugs. do.
ＣＤ４４ｖを高発現するがん幹細胞に対しても、ｘＣＴ阻害作用を有するスルファサラジンは有効に増殖を抑制することが知られている（特開２０１２－１４４４９８）。 It is known that sulfasalazine, which has an xCT inhibitory effect, effectively suppresses the proliferation of cancer stem cells that highly express CD44v (Japanese Patent Laid-Open No. 2012-144498).
本発明は、新規な抗腫瘍剤及び配合剤を提供することを課題とするものである。 An object of the present invention is to provide novel antitumor agents and combination agents.
本発明者らは、スルファサラジンは、未分化な腫瘍細胞がほとんどである腫瘍に対しては単独で抗腫瘍効果を有するが、分化形質を示す腫瘍細胞を含むような分化型腫瘍に対してはＣＤ４４ｖを高発現するがん幹細胞を減少させるものの、腫瘍全体の体積を減少させる効果がないことを見出した。そこで、そのような分化型腫瘍に対し、スルファサラジンが抗腫瘍効果を有しない腫瘍細胞に対して抗腫瘍効果をもつ薬剤を開発することによって分化型腫瘍に対する抗腫瘍剤を得ようと鋭意努力したところ、アルデヒド脱水素酵素阻害剤をスルファサラジンと併用すれば、スルファサラジン単独では効果の弱い腫瘍細胞に対し顕著な抗腫瘍効果があることを見出し、本発明の完成に至った。 The present inventors found that sulfasalazine alone has an antitumor effect on tumors that are mostly composed of undifferentiated tumor cells, but that sulfasalazine has an antitumor effect on CD44V alone against differentiated tumors that contain tumor cells that exhibit differentiated traits. They found that although the drug reduced cancer stem cells that highly expressed , it had no effect on reducing the overall tumor volume. Therefore, we made efforts to obtain an antitumor agent for such differentiated tumors by developing a drug that has an antitumor effect on tumor cells in which sulfasalazine does not have an antitumor effect. The inventors have discovered that when an aldehyde dehydrogenase inhibitor is used in combination with sulfasalazine, it has a remarkable antitumor effect on tumor cells, which is weakly effective with sulfasalazine alone, leading to the completion of the present invention.
本発明の一実施態様は、有効量のアルデヒド脱水素酵素阻害剤と同時に投与される、グルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤を有効成分として含有する抗腫瘍剤であるか、あるいは有効量のグルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤と同時に投与される、アルデヒド脱水素酵素阻害剤を有効成分として含有する抗腫瘍剤である。 One embodiment of the present invention is an antitumor agent containing as an active ingredient a glutathione concentration lowering agent or a glutathione S-transferase inhibitor, which is administered simultaneously with an effective amount of an aldehyde dehydrogenase inhibitor, or an effective amount of an aldehyde dehydrogenase inhibitor. This is an antitumor agent containing an aldehyde dehydrogenase inhibitor as an active ingredient, which is administered simultaneously with a glutathione concentration lowering agent or a glutathione S-transferase inhibitor.
本発明の他の実施態様は、アルデヒド脱水素酵素阻害剤とグルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤とを有効成分として含有する配合剤である。 Another embodiment of the present invention is a combination product containing an aldehyde dehydrogenase inhibitor and a glutathione concentration lowering agent or a glutathione S-transferase inhibitor as active ingredients.
本発明のさらなる実施態様は、上記配合剤を含有する抗腫瘍剤である。 A further embodiment of the invention is an anti-tumor agent containing the above formulation.
前記グルタチオン濃度低下剤がｘＣＴ、Thioredoxin-1（チオレドキシン-１：ＴＲＸ-１）、glutamate-cysteine ligase（ＧＣＬ）（ＥＣ６．３．２．２）（γ－グルタミルシステイン合成酵素とも呼ばれる）、グルタチオン合成酵素（ＥＣ６．３．２．３）のいずれかの活性を阻害する薬剤であってもよい。前記薬剤がxCTトランスポーターの阻害剤であってもよい。前記xCTトランスポーターの阻害剤がスルファサラジン、エラスチン、またはソラフェニブであってもよい。前記アルデヒド脱水素酵素阻害剤が下記式（Ｉ）で表される化合物またはその薬理学的に許容される塩であってもよい。 The glutathione concentration lowering agent is xCT, Thioredoxin-1 (thioredoxin-1: TRX-1), glutamate-cysteine ligase (GCL) (EC6.3.2.2) (also called γ-glutamylcysteine synthetase), glutathione synthesis It may also be a drug that inhibits the activity of any enzyme (EC6.3.2.3). The drug may be an inhibitor of xCT transporter. The xCT transporter inhibitor may be sulfasalazine, elastin, or sorafenib. The aldehyde dehydrogenase inhibitor may be a compound represented by the following formula (I) or a pharmacologically acceptable salt thereof.
（式中、Ｒ１はＣ１～６の直鎖または分岐アルキル基であり、Ｒ２及びＲ３は独立して選択されるＣ１～６の直鎖または分岐アルキル基であるか、Ｒ２とＲ３が一緒になってそれらが結合するＮをヘテロ原子とする４員環、５員環、６員環、または７員環のアザシクロアルキル基を形成し、Ｒ４は水素またはハロゲンである。）
本発明のさらなる実施態様は、Ｃ１～６の直鎖または分岐アルキル基であり、Ｒ２及びＲ３は独立して選択されるＣ１～６の直鎖または分岐アルキル基であるか、Ｒ２とＲ３が一緒になってそれらが結合するＮをヘテロ原子とする４員環、５員環、または６員環のアザシクロアルキル基を形成する。）
前記式（Ｉ）で表される化合物がジクロニン（Dyclonine）、BAS00363846、STL327701、PHAR033081、PHAR298639、またはAldi-2であってもよい。 (wherein R 1 is a C1-6 straight chain or branched alkyl group, R 2 and R 3 are independently selected C1-6 straight chain or branched alkyl groups, or R 2 and R 3 together form a 4-, 5-, 6-, or 7-membered azacycloalkyl group in which the N to which they are bonded is a heteroatom, and R 4 is hydrogen or halogen. )
A further embodiment of the invention is a C1-6 straight-chain or branched alkyl group, and R 2 and R 3 are independently selected C1-6 straight-chain or branched alkyl groups, or R 2 and R 3 together form a 4-, 5-, or 6-membered azacycloalkyl group in which the N to which they are bonded is a heteroatom. )
The compound represented by formula (I) may be Dyclonine, BAS00363846, STL327701, PHAR033081, PHAR298639, or Aldi-2.
本発明のさらなる実施態様は、アルデヒド脱水素酵素阻害剤とグルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤をin vitroで腫瘍細胞に同時に投与する工程と、前記腫瘍細胞の増殖速度または細胞生存率を測定する工程と、を含む測定方法である。 A further embodiment of the invention comprises the step of simultaneously administering an aldehyde dehydrogenase inhibitor and a glutathione concentration lowering agent or a glutathione S-transferase inhibitor to tumor cells in vitro, and determining the proliferation rate or cell viability of said tumor cells. This is a measuring method including a step of measuring.
本発明のさらなる実施態様は、グルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤と併用効果を有するアルデヒド脱水素酵素阻害剤の特定方法であって、特定のグルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤と複数のアルデヒド脱水素酵素阻害剤をin vitroで腫瘍細胞に同時に投与する工程と、前記腫瘍細胞の増殖速度または細胞生存率を測定する工程と、を含む特定方法である。 A further embodiment of the present invention is a method for identifying an aldehyde dehydrogenase inhibitor having a combined effect with a glutathione concentration lowering agent or a glutathione S-transferase inhibitor, the method comprising: and a plurality of aldehyde dehydrogenase inhibitors to tumor cells in vitro; and measuring the proliferation rate or cell viability of the tumor cells.
本発明のさらなる実施態様は、アルデヒド脱水素酵素阻害剤と併用効果を有するグルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤の特定方法であって、抗腫瘍剤である特定のアルデヒド脱水素酵素阻害剤と、複数のグルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤とをin vitroで腫瘍細胞に同時に投与する工程と、前記腫瘍細胞の増殖速度または細胞生存率を測定する工程と、を含む特定方法である。 A further embodiment of the present invention is a method for identifying a glutathione concentration lowering agent or a glutathione S-transferase inhibitor that has a combined effect with an aldehyde dehydrogenase inhibitor, the method comprising: a specific aldehyde dehydrogenase inhibitor that is an antitumor agent; and simultaneously administering a plurality of glutathione concentration lowering agents or glutathione S-transferase inhibitors to tumor cells in vitro; and measuring the proliferation rate or cell viability of the tumor cells. be.
本発明のさらなる実施態様は、アルデヒド脱水素酵素阻害剤とグルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤との併用効果を奏する腫瘍細胞の特定方法であって、アルデヒド脱水素酵素阻害剤とグルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤の特定の組み合わせをin vitroで複数の腫瘍細胞に同時に投与する工程と、前記複数の腫瘍細胞の増殖速度または細胞生存率を測定する工程と、を含む特定方法である。 A further embodiment of the present invention is a method for identifying tumor cells exhibiting a combined effect of an aldehyde dehydrogenase inhibitor and a glutathione concentration lowering agent or a glutathione S-transferase inhibitor, the method comprising: A specific method comprising simultaneously administering a specific combination of inhibitors or glutathione S-transferase inhibitors to a plurality of tumor cells in vitro, and measuring the proliferation rate or cell viability of the plurality of tumor cells. It is.
上記いずれかの特定方法または測定方法において、前記腫瘍細胞が、グルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤に耐性であってもよい。 In any of the above identification methods or measurement methods, the tumor cells may be resistant to a glutathione concentration lowering agent or a glutathione S-transferase inhibitor.
＝＝関連文献とのクロスリファレンス＝＝
本出願は、２０１７年１１月１５日付で出願した日本国特許出願２０１７－２２０２３１に基づく優先権を主張するものであり、当該基礎出願を引用することにより、本明細書に含めるものとする。==Cross reference with related literature==
This application claims priority based on Japanese Patent Application No. 2017-220231 filed on November 15, 2017, and is incorporated herein by reference to the basic application.
以下、本発明の実施の形態を、実施例を挙げながら詳細に説明する。なお、本発明の目的、特徴、利点、および、そのアイデアは、本明細書の記載により、当業者には明らかであり、本明細書の記載から、当業者であれば容易に本発明を再現できる。以下に記載された発明の実施の形態及び具体的な実施例などは、本発明の好ましい実施態様を示すものであり、例示又は説明のために示されているのであって、本発明をこれらに限定するものではない。本明細書で開示されている本発明の意図並びに範囲内で、本明細書の記載に基づき、様々な改変並びに修飾ができることは、当業者にとって明らかである。 Hereinafter, embodiments of the present invention will be described in detail by giving examples. Note that the objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of this specification, and those skilled in the art will be able to easily reproduce the present invention from the description of this specification. can. The embodiments and specific examples of the invention described below indicate preferred embodiments of the present invention and are provided for illustration or explanation, and the present invention is not limited thereto. It is not limited. It will be apparent to those skilled in the art that various changes and modifications can be made based on the description herein within the spirit and scope of the present invention disclosed herein.
なお、実施の形態及び実施例に特に説明がない場合には、標準的なプロトコール集に記載の方法、あるいはそれを修飾したり、改変したりした方法を用いる。また、市販の試薬キットや測定装置を用いる場合には、特に説明が無い場合、それらに添付のプロトコールを用いる。 Note that unless otherwise specified in the embodiments and examples, methods described in standard protocol collections or methods modified or modified therefrom are used. Furthermore, when using commercially available reagent kits or measurement devices, the protocols attached to them should be used unless otherwise specified.
＝＝抗腫瘍剤＝＝
本発明の一実施形態は、有効量のアルデヒド脱水素酵素阻害剤と同時に投与される、グルタチオン濃度低下剤を有効成分として含有する抗腫瘍剤である。ここで、有効量のアルデヒド脱水素酵素阻害剤とは、抗腫瘍活性として、グルタチオン濃度低下剤と併用効果を有する量のアルデヒド脱水素酵素阻害剤である。==Anti-tumor agent==
One embodiment of the present invention is an antitumor agent containing a glutathione concentration lowering agent as an active ingredient, which is administered simultaneously with an effective amount of an aldehyde dehydrogenase inhibitor. Here, an effective amount of an aldehyde dehydrogenase inhibitor is an amount of an aldehyde dehydrogenase inhibitor that has an antitumor activity in combination with a glutathione concentration lowering agent.
また、本発明の他の実施形態は、有効量のグルタチオン濃度低下剤と同時に投与される、アルデヒド脱水素酵素阻害剤を有効成分として含有する抗腫瘍剤である。ここで、有効量のグルタチオン濃度低下剤とは、抗腫瘍活性として、アルデヒド脱水素酵素阻害剤と併用効果を有する量のグルタチオン濃度低下剤である。 Another embodiment of the present invention is an antitumor agent containing an aldehyde dehydrogenase inhibitor as an active ingredient, which is administered simultaneously with an effective amount of a glutathione concentration lowering agent. Here, the effective amount of the glutathione concentration-lowering agent is an amount of the glutathione concentration-lowering agent that has an antitumor activity in combination with an aldehyde dehydrogenase inhibitor.
アルデヒド脱水素酵素阻害剤は、アルデヒド脱水素酵素２（アルデヒド脱水素酵素２；ＡＬＤＨ）（ＥＣ１．２．１．１０）の酵素活性を阻害する薬剤である。阻害対象とするＡＬＤＨのタイプおよびイソタイプは特に限定されず、ＡＬＤＨ１～５、及びそれらのイソタイプのいずれであってもよい。抗腫瘍剤で使用されるアルデヒド脱水素酵素阻害剤は特に限定されないがクロープロパミド（chlorpropamide）、トルブタミド（tolbutamide）、ジエチルアミノベンズアルデヒド、ジスルフィラム(tetraethylthioperoxydicarbonic diamide)、シアナミド(cyanamide)、オキシフェドリン、シトラル（3,7-dimethyl-2,6-octadienal）、コプリン（coprine）、ダイジン（daidzin）、DEAB(4-(Diethylamino)benzaldehyde)、ゴシポール（gossypol）、キヌレニン代謝物（3-hydroxykynurenine, 3-hydroxyanthranilic acid, kynurenic acid, および indol-3-ylpyruvic acid）、モリネート（Molinate）、ニトログリセリン、パージリン（N-benzyl-N-methylprop-2-yn-1-amine）及びそれらの類縁体、またはそれらの薬理学的に許容される塩が例示できる。特に、以下に示すジクロニン及びジクロニン類縁体（Ｉ）が好ましく、図７に示した化合物（BAS00363846、STL327701、PHAR033081、PHAR298639、およびAldi-2）がより好ましい。 Aldehyde dehydrogenase inhibitors are drugs that inhibit the enzymatic activity of aldehyde dehydrogenase 2 (ALDH) (EC1.2.1.10). The type and isotype of ALDH to be inhibited is not particularly limited, and may be any of ALDH 1 to 5 and their isotypes. Aldehyde dehydrogenase inhibitors used as antitumor agents are not particularly limited, but include chlorpropamide, tolbutamide, diethylaminobenzaldehyde, tetraethylthioperoxydicarbonic diamide, cyanamide, oxyphedrine, and citral ( 3,7-dimethyl-2,6-octadienal), coprine, daidzin, DEAB (4-(Diethylamino)benzaldehyde), gossypol, kynurenine metabolite (3-hydroxykynurenine, 3-hydroxyanthranilic acid) , kynurenic acid, and indol-3-ylpyruvic acid), molinate, nitroglycerin, pargyline (N-benzyl-N-methylprop-2-yn-1-amine) and their analogs, or their pharmacology Examples include legally acceptable salts. In particular, dyclonine and dyclonine analogs (I) shown below are preferred, and the compounds shown in FIG. 7 (BAS00363846, STL327701, PHAR033081, PHAR298639, and Aldi-2) are more preferred.
（式中、Ｒ１はＣ１～６の直鎖または分岐アルキル基であり、Ｒ２及びＲ３は独立して選択されるＣ１～６の直鎖または分岐アルキル基であるか、Ｒ２とＲ３が一緒になってそれらが結合するＮをヘテロ原子とする４員環、５員環、６員環、または７員環のアザシクロアルキル基を形成し、Ｒ４は水素またはハロゲンである。Ｒ１はＣ４～５の直鎖または分岐アルキル基であることが好ましく、Ｒ２及びＲ３はＣ２のアルキル基か、またはＲ２とＲ３が一緒になってそれらが結合するＮをヘテロ原子とする６員環のアザシクロアルキル基であることが好ましい。なお、Ｒ１はＣ４の直鎖アルキル基であり、Ｒ２とＲ３が一緒になってそれらが結合するＮをヘテロ原子とする６員環のアザシクロアルキル基である化合物はジクロニンである。ハロゲンは、Ｆ、Ｃｌ、Ｉ、Ｂｒ、Ｉが好ましい。）
なお、薬理学的に許容される塩とは、それらの化合物と塩を形成するものであれば限定されないが、具体的には例えば塩酸塩、硫酸塩、硝酸塩、臭化水素酸塩、ヨウ化水素酸塩、過塩素酸塩、リン酸塩などの無機酸の付加塩、シュウ酸塩、マレイン酸塩、フマル酸塩、コハク酸塩などの有機酸の付加塩、メタンスルホン酸塩、エタンスルホン酸塩、ベンゼンスルホン酸塩、p-トルエンスルホン酸塩、カンファースルホン酸塩などのスルホン酸の付加塩、アミノ酸の付加塩などを挙げることができ、好ましくは塩酸塩、シュウ酸塩、マレイン酸塩、メタンスルホン酸塩である。さらに、それらの化合物またはその薬理学的に許容される塩は、無水物のみならず水和物や結晶多形も含まれることは言うまでもない。 (wherein R 1 is a C1-6 straight chain or branched alkyl group, R 2 and R 3 are independently selected C1-6 straight chain or branched alkyl groups, or R 2 and R 3 together form a 4-, 5-, 6-, or 7-membered azacycloalkyl group in which the N to which they are bonded is a heteroatom, and R 4 is hydrogen or halogen. Preferably, R 1 is a C4-5 linear or branched alkyl group, and R 2 and R 3 are C2 alkyl groups, or R 2 and R 3 together represent the N to which they are bonded to a heteroatom. It is preferable that it is a 6-membered ring azacycloalkyl group. Note that R 1 is a C4 straight chain alkyl group, and R 2 and R 3 are taken together and the N to which they are bonded is a hetero atom. The compound that is a 6-membered azacycloalkyl group is dyclonine.The halogen is preferably F, Cl, I, Br, or I.)
Note that pharmacologically acceptable salts are not limited as long as they form salts with these compounds, but specific examples include hydrochlorides, sulfates, nitrates, hydrobromides, and iodides. Addition salts of inorganic acids such as hydrates, perchlorates, phosphates, addition salts of organic acids such as oxalates, maleates, fumarates, succinates, methanesulfonates, ethanesulfones Examples include addition salts of sulfonic acids such as acid salts, benzenesulfonates, p-toluenesulfonates, and camphorsulfonates, and addition salts of amino acids, with preference given to hydrochlorides, oxalates, and maleates. , methanesulfonate. Furthermore, it goes without saying that these compounds or their pharmacologically acceptable salts include not only anhydrous forms but also hydrates and crystalline polymorphs.
グルタチオン濃度低下剤は、細胞内におけるグルタチオン濃度を低下させる薬剤である。これらの抗腫瘍剤で使用されるグルタチオン濃度低下剤は限定されないが、ｘＣＴが細胞内に取り込んだシスチンからグルタチオンが産生される経路を阻害する薬剤が好ましく、ｘＣＴ、Thioredoxin-1（チオレドキシン-１：ＴＲＸ-１）、glutamate-cysteine ligase（ＧＣＬ）（ＥＣ６．３．２．２）（γ－グルタミルシステイン合成酵素とも呼ばれる）、グルタチオン合成酵素（ＥＣ６．３．２．３）のいずれかの活性を阻害する薬剤であることがより好ましく、ｘＣＴ阻害剤がより好ましい。ｘＣＴ阻害剤は特に限定されないが、スルファサラジン、エラスチン、ソラフェニブ、または抗ｘＣＴ抗体であることが好ましい。 A glutathione concentration lowering agent is a drug that lowers the glutathione concentration within cells. The glutathione concentration-lowering agents used in these antitumor agents are not limited, but drugs that inhibit the pathway in which glutathione is produced from cystine taken into cells by xCT are preferred, such as xCT, Thioredoxin-1 (thioredoxin-1: TRX-1), glutamate-cysteine ligase (GCL) (EC6.3.2.2) (also called γ-glutamylcysteine synthetase), or glutathione synthase (EC6.3.2.3). A drug that inhibits xCT is more preferable, and an xCT inhibitor is more preferable. The xCT inhibitor is not particularly limited, but is preferably sulfasalazine, elastin, sorafenib, or an anti-xCT antibody.
グルタチオンＳ-トランスフェラーゼ阻害剤は、グルタチオンＳ-トランスフェラーゼ（ＥＣ２．５．１．１８）の酵素活性を阻害する薬剤であって、特に、ＨＮＥ（４－ＨＮＥ；４-ヒドロキシ-２-ノネナール）をＨＮＥ－ＧＳＨに変換する活性を阻害する薬剤である。グルタチオンＳ-トランスフェラーゼ阻害剤は特に限定されないが、グルタチオンアナログ（例えば、WO95/08563、WO96/40205、WO99/54346など）、ケトプロフェン、インドメタシン、エタクリン酸、ピロプロスト、抗ＧＳＴ抗体、ＧＳＴのドミナントネガティブ変異体等が挙げられる。 Glutathione S-transferase inhibitors are drugs that inhibit the enzymatic activity of glutathione S-transferase (EC2.5.1.18), and in particular, inhibit HNE (4-HNE; 4-hydroxy-2-nonenal). -A drug that inhibits the activity of converting to GSH. Glutathione S-transferase inhibitors are not particularly limited, but include glutathione analogs (for example, WO95/08563, WO96/40205, WO99/54346, etc.), ketoprofen, indomethacin, ethacrynic acid, piroprost, anti-GST antibodies, dominant negative mutants of GST etc.
ここで、二つの薬剤を「同時に投与する」とは、時間的に同時に投与することのみならず、一方の薬剤の効果が残っている間に、他方の薬剤を投与する限り、時間的に前後してそれぞれ単独で投与することも意味するものとする。二つの薬剤を同時に投与する場合、一方のみを含有する薬剤を２種類同時に投与してもよいが、配合剤として二つの薬剤を一つの剤形にして投与してもよい。 Here, "administering two drugs at the same time" does not only mean administering them at the same time, but also as long as the other drug is administered before or after the other drug while the effect of one drug remains. It shall also mean that each is administered alone. When two drugs are administered simultaneously, two drugs containing only one of them may be administered at the same time, or the two drugs may be administered in one dosage form as a combination drug.
抗腫瘍剤の投与対象は、脊椎動物であれば特に限定されないが、ヒトのがん患者であることが好ましい。治療対象の腫瘍は特に限定されないが、グルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤に対して耐性を有する腫瘍細胞を含む腫瘍が好ましい。この腫瘍細胞は、アルデヒド脱水素酵素が高発現していてもよい。グルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤は、ｘＣＴ阻害剤であることが好ましく、スルファサラジンであることがより好ましい。耐性を有する腫瘍細胞とは、in vivoでは、患者に通常治療濃度で通常治療日数投与したときに生き残る腫瘍細胞であって、in vitroでは、８０％以上の種類の細胞株において生存率が５０％以下である濃度で生存率が９０％以上の腫瘍細胞をいうものとする。例えば、スルファサラジン耐性腫瘍細胞とは、in vivoでは、患者にＡＵＣ０－２４が５０～３００μｇ・ｈ／ｍＬで約２週間投与したときに生き残る腫瘍細胞であって、in vitroでは、２００μＭで生存率が９０％以上の腫瘍細胞をいうものとする。スルファサラジン耐性腫瘍細胞は、ＣＤ４４ｖ発現も低レベルであるか、陰性であることが好ましい。アルデヒド脱水素酵素が過剰発現している腫瘍細胞とは、ＡＬＤＨ１Ａ１、ＡＬＤＨ２、ＡＬＤＨ１Ｂ１，ＡＬＤＨ３Ａ１のいずれかの遺伝子発現が、ＯＳＣ１９細胞に比較して３倍以上、好ましくは１０倍以上高いレベルで発現している細胞をいうものとする。この治療対象の腫瘍には、ＣＤ４４ｖが発現している腫瘍細胞が混入していてもよい。ＣＤ４４ｖが発現している腫瘍細胞に対しては、スルファサラジンが効果的に抗腫瘍作用を有するからである。ＣＤ４４ｖが発現している腫瘍細胞とは、ＣＤ４４ｖ発現が検出できる細胞であればよいが、高発現している細胞が好ましく、その場合の高発現とは、卵巣腫瘍細胞の平均レベルと同じ程度か高ければよいが、２倍以上高いことが好ましく、４倍以上高いことがより好ましく、１０倍以上高いことがさらに好ましい。The subject to whom the antitumor agent is administered is not particularly limited as long as it is a vertebrate, but it is preferably a human cancer patient. The tumor to be treated is not particularly limited, but tumors containing tumor cells that are resistant to glutathione concentration lowering agents or glutathione S-transferase inhibitors are preferred. The tumor cells may highly express aldehyde dehydrogenase. The glutathione concentration lowering agent or glutathione S-transferase inhibitor is preferably an xCT inhibitor, more preferably sulfasalazine. Resistant tumor cells are tumor cells that survive in vivo when administered to patients at normal therapeutic concentrations for normal treatment days, and that in vitro have a survival rate of 50% in more than 80% of cell lines. Tumor cells with a survival rate of 90% or more at a concentration below. For example, sulfasalazine-resistant tumor cells are tumor cells that survive in vivo when administered to a patient for about 2 weeks at an AUC 0-24 of 50 to 300 μg/h/mL, and in vitro, survival rate is 200 μM. 90% or more of tumor cells. Preferably, the sulfasalazine-resistant tumor cells also express low levels of CD44v or are negative. Tumor cells overexpressing aldehyde dehydrogenase are those in which the gene expression of ALDH1A1, ALDH2, ALDH1B1, or ALDH3A1 is expressed at a level that is 3 times or more, preferably 10 times or more higher than that of OSC19 cells. This refers to the cells that are present. The tumor to be treated may be contaminated with tumor cells expressing CD44v. This is because sulfasalazine has an effective antitumor effect on tumor cells expressing CD44v. Tumor cells expressing CD44v may be any cells in which CD44v expression can be detected, but cells with high expression are preferable, and in this case, high expression means approximately the same level as the average level of ovarian tumor cells. It may be as high as possible, but preferably twice or more, more preferably four times or more, and even more preferably ten times or more.
腫瘍の種類は特に限定されないが、固形がんであることが好ましく、大腸腺癌、胃腺癌、乳腺癌、肺腺癌、膵腺癌、頭頸部の扁平上皮癌、卵巣腫瘍、精巣腫瘍を例示することができる。 The type of tumor is not particularly limited, but it is preferably a solid cancer, examples of which include colon adenocarcinoma, gastric adenocarcinoma, mammary adenocarcinoma, lung adenocarcinoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, ovarian tumor, and testicular tumor. be able to.
抗腫瘍剤は、通常の方法により、錠剤、粉剤、顆粒剤、散剤、カプセル剤、液剤、乳剤、懸濁剤などに剤形化されてもよい。その際、当業者に知られた薬学的に許容できる添加剤、例えば賦形剤や担体を用いて製造される。 The antitumor agent may be formulated into tablets, powders, granules, powders, capsules, solutions, emulsions, suspensions, etc. by conventional methods. In this case, it is manufactured using pharmaceutically acceptable additives such as excipients and carriers known to those skilled in the art.
抗腫瘍剤は、有効量の範囲内で、投与対象に対して適した方法で投与すればよい。有効量は、剤形の種類、投与方法、投与対象の年齢や体重、投与対象の病状等を考慮して、最終的には医師または獣医師の判断により適宜決定することができる。例えば、化合物の投与量は１日当たり、０．１ｍｇ／ｋｇ以上であることが好ましく、１ｍｇ／ｋｇ以上であることがより好ましく、１０ｍｇ／ｋｇ以上であることがさらに好ましく、１０００ｍｇ／ｋｇ以下であることが好ましく、３００ｍｇ／ｋｇ以下であることがより好ましく、１００ｍｇ／ｋｇ以下であることがさらに好ましい。投与方法は、特に限定されず、例えば、経口投与してもよいし、腹腔内や静脈内への注射や点滴により非経口投与してもよいし、注射等によりがん内に直接投与してもよい。 The antitumor agent may be administered within an effective amount by a method suitable for the subject. The effective amount can be determined as appropriate based on the final judgment of a physician or veterinarian, taking into consideration the type of dosage form, administration method, age and weight of the subject, medical condition of the subject, and the like. For example, the dosage of the compound is preferably 0.1 mg/kg or more, more preferably 1 mg/kg or more, even more preferably 10 mg/kg or more, and 1000 mg/kg or less per day. The amount is preferably 300 mg/kg or less, more preferably 100 mg/kg or less. The method of administration is not particularly limited, and for example, it may be administered orally, parenterally by intraperitoneal or intravenous injection or infusion, or directly administered into the cancer by injection, etc. Good too.
＝＝腫瘍細胞の増殖速度または細胞生存率の測定方法＝＝
本発明の一実施形態は、アルデヒド脱水素酵素阻害剤と、グルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤とをin vitroで腫瘍細胞に同時に投与する工程と、薬剤を投与した腫瘍細胞の増殖速度または細胞生存率を測定する工程と、を含む測定方法である。本節におけるアルデヒド脱水素酵素阻害剤、グルタチオン濃度低下剤、グルタチオンＳ-トランスフェラーゼ阻害剤は、「抗腫瘍剤」の節で詳述したものに準じる。==Method for measuring proliferation rate or cell viability of tumor cells==
One embodiment of the present invention provides a step of simultaneously administering an aldehyde dehydrogenase inhibitor and a glutathione concentration lowering agent or a glutathione S-transferase inhibitor to tumor cells in vitro, and the growth rate of the tumor cells to which the drugs have been administered. or a step of measuring cell viability. The aldehyde dehydrogenase inhibitors, glutathione concentration lowering agents, and glutathione S-transferase inhibitors in this section are the same as those detailed in the "Antitumor Agents" section.
アルデヒド脱水素酵素阻害剤と、グルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤とは、抗腫瘍活性について併用効果を有しているので、この測定方法によって、併用効果の高い薬剤の組み合わせを見出したり、ある薬剤の組み合わせに対し、特に有効な腫瘍細胞を見出したりすることができる。 Since aldehyde dehydrogenase inhibitors and glutathione concentration lowering agents or glutathione S-transferase inhibitors have a combined effect on antitumor activity, this measurement method can be used to find combinations of drugs that have a high combined effect. It is possible to discover tumor cells that are particularly effective against certain drug combinations.
具体的には、特定のグルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤と複数のアルデヒド脱水素酵素阻害剤をin vitroで腫瘍細胞に同時に投与し、薬剤を投与した腫瘍細胞の増殖速度または細胞生存率を測定することによって、特定のグルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤と併用効果を有するアルデヒド脱水素酵素阻害剤を特定することができる。また、特定のアルデヒド脱水素酵素阻害剤と複数のグルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤をin vitroで腫瘍細胞に同時に投与し、薬剤を投与した腫瘍細胞の増殖速度または細胞生存率を測定することによって、特定のアルデヒド脱水素酵素阻害剤と併用効果を有するグルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤を特定することができる。あるいは、アルデヒド脱水素酵素阻害剤とグルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤の特定の組み合わせをin vitroで複数の腫瘍細胞に同時に投与し、薬剤を投与した複数の腫瘍細胞の増殖速度または細胞生存率を測定することにより、アルデヒド脱水素酵素阻害剤とグルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤との併用効果を奏する腫瘍細胞を特定することができる。 Specifically, a specific glutathione concentration lowering agent or a glutathione S-transferase inhibitor and multiple aldehyde dehydrogenase inhibitors were simultaneously administered to tumor cells in vitro, and the proliferation rate or cell survival of drug-treated tumor cells was determined. By measuring the rate, it is possible to identify aldehyde dehydrogenase inhibitors that have a combined effect with a particular glutathione concentration lowering agent or glutathione S-transferase inhibitor. In addition, a specific aldehyde dehydrogenase inhibitor and multiple glutathione concentration-lowering agents or glutathione S-transferase inhibitors are simultaneously administered to tumor cells in vitro, and the proliferation rate or cell survival rate of drug-treated tumor cells is measured. By doing so, it is possible to specify a glutathione concentration lowering agent or a glutathione S-transferase inhibitor that has a combined effect with a specific aldehyde dehydrogenase inhibitor. Alternatively, certain combinations of aldehyde dehydrogenase inhibitors and glutathione concentration-lowering agents or glutathione S-transferase inhibitors can be administered simultaneously to multiple tumor cells in vitro, and the proliferation rate or cell By measuring the survival rate, it is possible to identify tumor cells that exhibit the effect of the combined use of an aldehyde dehydrogenase inhibitor and a glutathione concentration lowering agent or a glutathione S-transferase inhibitor.
（実験例１）スルファサラジンとジクロニンとの併用効果
（目的）本実験例では、スルファサラジン耐性細胞の生存率低下に関し、ｘＣＴ阻害効果のあるスルファサラジンとジクロニンとが併用効果を奏することを示す。(Experimental Example 1) Effect of combined use of sulfasalazine and dyclonine (Purpose) This experimental example shows that sulfasalazine and dyclonine, which have xCT inhibitory effects, have a combined effect on reducing the survival rate of sulfasalazine-resistant cells.
（方法）９６ウエルプレートに、スルファサラジン耐性細胞株である口腔扁平上皮がん細胞株ＨＳＣ－４を２０００個／ウエルで播種し、培養を開始した。培地は、ＤＭＥＭを用いた。２４時間後、５０μＭジクロニンまたは等量のＤＭＳＯと、０μＭ（無添加）、５０μＭ、１００μＭ、２００μＭ、または４００μＭのスルファサラジンを含有した培地に交換し、４８時間、培養を続けた。その後、Celltiter-Glo（Promega社）を用いて細胞生存率を測定し、コントロール（ＤＭＳＯ添加、スルファサラジン無添加）の細胞生存数を１００％として、各場合の細胞生存率を算出した。図１にスルファサラジンの各濃度に対する生存率を表すグラフを作成した。 (Method) The oral squamous cell carcinoma cell line HSC-4, which is a sulfasalazine-resistant cell line, was seeded at 2000 cells/well in a 96-well plate, and culture was started. DMEM was used as the medium. After 24 hours, the medium was replaced with a medium containing 50 μM dyclonine or an equivalent amount of DMSO and 0 μM (no addition), 50 μM, 100 μM, 200 μM, or 400 μM sulfasalazine, and culture was continued for 48 hours. Thereafter, the cell survival rate was measured using Celltiter-Glo (Promega), and the cell survival rate in each case was calculated by setting the cell survival number of the control (DMSO added, sulfasalazine not added) as 100%. A graph showing the survival rate for each concentration of sulfasalazine was created in FIG.
（結果）ＨＳＣ４は、スルファサラジン耐性細胞株であって、スルファサラジン単独では細胞生存率にほとんど影響がない。また、ジクロニン単独（ジクロニン添加、スルファサラジン無添加）でも、８０％の生存率を示す。しかしながら、ジクロニンとスルファサラジンを両方添加すると、スルファサラジンが１００μＭ以上で、生存率が１０％以下となる。 (Results) HSC4 is a sulfasalazine-resistant cell line, and sulfasalazine alone has little effect on cell viability. Furthermore, dyclonine alone (with addition of dyclonine and without addition of sulfasalazine) shows a survival rate of 80%. However, when both dyclonine and sulfasalazine are added, the survival rate becomes 10% or less when sulfasalazine is 100 μM or more.
このように、スルファサラジン耐性細胞の生存率低下に関し、スルファサラジンとジクロニンとが併用効果を奏する。 Thus, sulfasalazine and dyclonine have a combined effect on reducing the survival rate of sulfasalazine-resistant cells.
（実験例２）ｘＣＴノックダウンによるジクロニン感受性の変化
（目的）本実験例では、ｘＣＴ阻害効果のあるスルファサラジンの代わりにｘＣＴをノックダウンしても、同様なジクロニンとの併用効果が得られることを示すことによって、スルファサラジンとジクロニンとが併用効果を有するのは、スルファサラジンのｘＣＴ阻害効果を介していることを示す。(Experimental Example 2) Change in dyclonine sensitivity due to xCT knockdown (Purpose) In this experimental example, we demonstrated that even if xCT is knocked down instead of sulfasalazine, which has an xCT inhibitory effect, a similar combined effect with dyclonine can be obtained. This shows that the combined effect of sulfasalazine and dyclonine is mediated by the xCT inhibitory effect of sulfasalazine.
（方法）９６ウエルプレートにスルファサラジン耐性細胞株である口腔扁平上皮がん細胞株ＨＳＣ－４細胞を３０００個／ウエルで播種し、非サイレンシングコントロール（スクランブル(Sense: UUCUCCGAACGUGUCACGUtt（配列番号１）, Antisense: ACGUGACACGUUCGGAGAAtt（配列番号２）)）ｓｉＲＮＡまたはｘＣＴ特異的ｓｉＲＮＡ（xCT siRNA#1 Sense: AGAAAUCUGGAGGUCAUUAtt（配列番号３）, Antisense:AGAAAUCUGGAGGUCAUUAtt（配列番号４）, xCT siRNA#2 Sense: CCAGAACAUUACAAAUAAUtt（配列番号５）, Antisense: AUUAUUUGUAAUGUUCUGGtt（配列番号６））をLipofectamine RNAiMAX（ThermoFisher Scientific社）を用いてリポフェクトし、培養を開始した。培地はＤＭＥＭを用いた。２４時間後、５０μＭジクロニン（溶媒はＤＭＳＯ）または等量のＤＭＳＯを含有する培地に交換し、４８時間培養を続けた。その後、Celltiter-Glo（Promega社）を用いて細胞生存率を測定し、コントロール（非サイレンシングコントロール、ＤＭＳＯ添加）の細胞生存率を１００％として、それぞれの細胞生存率を算出した。図２に結果を示す。 (Method) Oral squamous cell carcinoma cell line HSC-4 cells, which are sulfasalazine-resistant cell lines, were seeded at 3000 cells/well in a 96-well plate, and non-silencing control (scrambled (Sense: UUCUCCGAACGUGUCACGUtt (SEQ ID NO: 1), Antisense : ACGUGACACGUUCGGAGAAtt (SEQ ID NO: 2))) siRNA or xCT-specific siRNA (xCT siRNA#1 Sense: AGAAAUCUGGAGGUCAUUAtt (SEQ ID NO: 3), Antisense: AGAAAUCUGGAGGUCAUUAtt (SEQ ID NO: 4), xCT siRNA#2 Sense: CCAGAACAUUACAAAUAAUtt (SEQ ID NO: 5) , Antisense: AUUAUUUGUAAUGUUCUGGtt (SEQ ID NO: 6)) was lipofected using Lipofectamine RNAiMAX (ThermoFisher Scientific), and culture was started. DMEM was used as the medium. After 24 hours, the medium was replaced with a medium containing 50 μM dyclonine (solvent: DMSO) or an equivalent amount of DMSO, and culture was continued for 48 hours. Thereafter, cell viability was measured using Celltiter-Glo (Promega), and each cell viability was calculated by setting the cell viability of the control (non-silencing control, DMSO added) as 100%. The results are shown in Figure 2.
（結果）ＨＳＣ－４は、５０μＭのジクロニン単独では約６０％の細胞生存率を有するのに対し、ｘＣＴをノックダウンした場合、５０μＭのジクロニン存在下で、約１０～２０％の細胞生存率しか有しない。 (Results) HSC-4 has a cell viability of about 60% in the presence of 50 μM dyclonine alone, whereas when xCT is knocked down, the cell viability is only about 10-20% in the presence of 50 μM dyclonine. I don't have it.
このように、スルファサラジンとジクロニンとが併用効果を有するのは、スルファサラジンのｘＣＴ阻害効果を介している。 Thus, the reason why sulfasalazine and dyclonine have a combined effect is through the xCT inhibitory effect of sulfasalazine.
（実験例３）各種癌細胞株におけるスルファサラジン、エラスチン、またはＢＳＯとジクロニンとの併用効果
（目的）本実験例では、様々な腫瘍細胞株において、スルファサラジン、ｘＣＴの特異的阻害剤であるエラスチン、またはグルタチオン合成阻害剤であるＢＳＯとジクロニンとが併用効果を奏することを示すのと同時に、ｘＣＴの阻害がグルタチオン合成阻害を介していることを示す。(Experimental Example 3) Effect of combination of sulfasalazine, elastin, or BSO with dyclonine on various cancer cell lines (Purpose) In this experimental example, sulfasalazine, elastin, a specific inhibitor of xCT, or This study shows that BSO and dyclonine, which are glutathione synthesis inhibitors, have a combined effect, and at the same time shows that inhibition of xCT is mediated by inhibition of glutathione synthesis.
（方法）９６ウエルプレートに、図３に示す細胞株を３０００個／ウエルで播種し、培養を開始した。培地は、ＤＭＥＭを用いた。２４時間後、５０μＭジクロニンまたは等量のＤＭＳＯと、０μＭ（無添加）または４００μＭのスルファサラジン、０μＭ（無添加）または５μＭのエラスチン、０μＭ（無添加）または１００μＭのＢＳＯを含有した培地に交換し、４８時間、培養を続けた。その後、Celltiter-Glo（Promega社）を用いて細胞生存率を測定し、コントロール（ＤＭＳＯ添加、ジクロニン無添加）の細胞生存数を１００％として、それぞれの細胞生存率を算出した。図３に各場合の細胞生存率を図示した。 (Method) The cell line shown in FIG. 3 was seeded at 3000 cells/well in a 96-well plate, and culture was started. DMEM was used as the medium. After 24 hours, the medium was changed to a medium containing 50 μM dyclonine or an equivalent amount of DMSO, 0 μM (no addition) or 400 μM sulfasalazine, 0 μM (no addition) or 5 μM elastin, 0 μM (no addition) or 100 μM BSO, Culture was continued for 48 hours. Thereafter, the cell survival rate was measured using Celltiter-Glo (Promega), and each cell survival rate was calculated by setting the cell survival number of the control (DMSO added, dyclonine not added) as 100%. FIG. 3 illustrates the cell survival rate in each case.
（結果）細胞によって大小はあるが、スルファサラジン、エラスチン、またはＢＳＯとジクロニンのいずれの組み合わせにおいても同様な併用効果が観察された。 (Results) A similar combined effect was observed for any combination of sulfasalazine, elastin, or BSO and dyclonine, although the magnitude differed depending on the cells.
このように、スルファサラジンやエラスチンによるｘＣＴの阻害は、グルタチオンの合成阻害によって、その抗腫瘍効果を発揮する。従って、スルファサラジンやエラスチンの代わりにグルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤を用いることができる。 Thus, inhibition of xCT by sulfasalazine or elastin exerts its antitumor effect by inhibiting glutathione synthesis. Therefore, a glutathione concentration lowering agent or a glutathione S-transferase inhibitor can be used in place of sulfasalazine or elastin.
（実験例４）スルファサラジンとジクロニンのin vivoにおける併用効果
（目的）本実験例では、スルファサラジンとジクロニンの併用効果が、in vivoでも観察されることを示す。(Experimental Example 4) Effect of combination of sulfasalazine and dyclonine in vivo (Purpose) This experimental example shows that the combined effect of sulfasalazine and dyclonine is also observed in vivo.
（方法）スルファサラジン耐性細胞株である口腔扁平上皮がん細胞株ＨＳＣ－２細胞の１×１０６個をヌードマウス皮下に移植し、移植後４日目より生理食塩水、スルファサラジン単独、ジクロニン単独、スルファサラジンとジクロニン両方を１日１回、各薬剤をスルファサラジン４００ｍｇ／ｋｇ、ジクロニン５ｍｇ／ｋｇの投与量で腹腔内投与し、２２日目まで続けた。３～４日おきに、腫瘍の短径、長径を測定し、以下の式により腫瘍体積を算出し、結果を図４にグラフ化した。(Method) 1 x 10 6 cells of the oral squamous cell carcinoma cell line HSC-2, which is a sulfasalazine-resistant cell line, were transplanted subcutaneously into nude mice, and from the 4th day after transplantation, saline, sulfasalazine alone, dyclonine alone, Both sulfasalazine and dyclonine were administered intraperitoneally once a day at a dose of 400 mg/kg of each drug and 5 mg/kg of dyclonine until day 22. The short axis and long axis of the tumor were measured every 3 to 4 days, and the tumor volume was calculated using the following formula, and the results are graphed in FIG. 4.
腫瘍体積＝（長径×（短径）２）／２
なお、腫瘍体積の統計解析は、２２日目にｔ検定により行った。Tumor volume = (major axis x (minor axis) 2 )/2
In addition, statistical analysis of tumor volume was performed by t-test on the 22nd day.
（結果）図４で示すように、各薬剤単独では、３５％程度の腫瘍体積の減少であったが、両方投与すると、７０％程度体積が減少した。 (Results) As shown in FIG. 4, when each drug was administered alone, the tumor volume decreased by about 35%, but when both were administered, the volume decreased by about 70%.
このように、スルファサラジンとジクロニンとの併用投与によって、スルファサラジン耐性腫瘍の増殖を低下させることができる。 Thus, co-administration of sulfasalazine and dyclonine can reduce the growth of sulfasalazine-resistant tumors.
（実験例５）ジクロニンによるＡＬＤＨの阻害
（目的）本実験例では、ジクロニンがＡＬＤＨの阻害活性を有することを示す。(Experimental Example 5) Inhibition of ALDH by Dyclonine (Purpose) This experimental example shows that dyclonine has ALDH inhibitory activity.
（方法）１０ｃｍ細胞培養デイシュに口腔届平上皮がん細胞株ＨＳＣ－４細胞を８×１０５個／ディッシュで播種し、培養を開始した。培地は、ＤＭＥＭを用いた。２４時間後、５０μＭのジクロニン（溶媒はＤＭＳＯ）を含む培地に交換し、２４時間培養した。その後、細胞を回収し、ALDEFLUOR kit（STEMCELL Technologies社）により、N，N－diethylaminobenzaldehyde（ＤＥＡＢ）存在下でのＡＬＤＨ活性を有する細胞を染色し、ＦＡＣＳで解析した（図ではDyclonine）。コントロールとして、ＤＥＡＢを添加せずALDEFLUOR kitで染色しなかったもの（図ではUnstained）、ジクロニンを含まない等量のＤＭＳＯを含む培地に交換し、ALDEFLUOR kitで染色したもの（図ではNon-treatment）についての実験結果を示す。なお、陽性細胞の計測については、DEAB存在下でALDEFLUOR kitによる染色を行ったDMSO処理サンプル（図ではDEAB）について陽性細胞がほぼ０％となるようなゲートを作製し、各場合の陽性率を計算した。(Method) Orally delivered squamous cell carcinoma cell line HSC-4 cells were seeded at 8×10 5 cells/dish in a 10 cm cell culture dish, and culture was started. DMEM was used as the medium. After 24 hours, the medium was replaced with a medium containing 50 μM dyclonine (solvent: DMSO), and cultured for 24 hours. Thereafter, the cells were collected, and cells having ALDH activity in the presence of N,N-diethylaminobenzaldehyde (DEAB) were stained using the ALDEFLUOR kit (STEMCELL Technologies) and analyzed by FACS (Dyclonine in the figure). As controls, DEAB was not added and the medium was not stained with the ALDEFLUOR kit (Unstained in the figure), and the medium was replaced with a medium containing an equal amount of DMSO without dyclonine and stained with the ALDEFLUOR kit (Non-treatment in the figure). The experimental results are shown below. Regarding the measurement of positive cells, we created a gate so that the number of positive cells was approximately 0% for the DMSO-treated sample (DEAB in the figure) stained with the ALDEFLUOR kit in the presence of DEAB, and calculated the positive rate in each case. I calculated it.
（結果）図５に示すように、ＤＭＳＯ処理細胞においてはＡＬＤＨ活性の高い細胞群が２５％程度存在しているが、ジクロニン処理細胞および既知のＡＬＤＨ阻害剤であるＤＥＡＢ処理細胞においてはＡＬＤＨ活性が高い細胞群は１％程度まで抑制されている。 (Results) As shown in Figure 5, approximately 25% of cells treated with DMSO have high ALDH activity, but cells treated with dyclonine and cells treated with DEAB, a known ALDH inhibitor, have no ALDH activity. High cell groups are suppressed to about 1%.
このように、ジクロニンはＡＬＤＨの阻害活性を有する。 Thus, dyclonine has ALDH inhibitory activity.
（実験例６）スルファサラジンとジクロニン併用によるＨＮＥの蓄積
（目的）本実験例では、スルファサラジンとジクロニンの併用によって、腫瘍細胞内でのＨＮＥのレベル、及びＨＮＥを蓄積する細胞の頻度が著しく増加することを示す。(Experiment Example 6) Accumulation of HNE by combination of sulfasalazine and dyclonine (Purpose) In this example, the combination of sulfasalazine and dyclonine significantly increases the level of HNE in tumor cells and the frequency of cells that accumulate HNE. shows.
（方法）実験例１と同様に、５０μＭジクロニンまたは等量のＤＭＳＯと、０μＭ（無添加）または４００μＭのスルファサラジンとを含有した培地でＨＳＣ－４細胞を培養し、処理後の細胞を４％ＰＦＡ－ＰＢＳにて固定した。さらに、０．２％ＴｒｉｔｏｎＸ１００－ＰＢＳにて細胞膜透過処理を行った後、３％ＢＳＡ－ＰＢＳによるブロッキングを行った。その後、１次抗体として抗ＨＮＥ抗体、２次抗体としてAlexafluor488標識抗マウスＩｇＧ抗体を用いて蛍光染色した。ポジティブコントロールとして、５０μＭＨＮＥで３０分インキュベートした細胞を用い、同様に抗体染色を行った。蛍光顕微鏡での観察像を図６に示す。 (Method) As in Experimental Example 1, HSC-4 cells were cultured in a medium containing 50 μM dyclonine or an equivalent amount of DMSO and 0 μM (no addition) or 400 μM sulfasalazine, and the cells after treatment were treated with 4% PFA. -Fixed with PBS. Furthermore, after cell membrane permeabilization was performed with 0.2% TritonX100-PBS, blocking was performed with 3% BSA-PBS. Thereafter, fluorescent staining was performed using an anti-HNE antibody as a primary antibody and an Alexafluor488-labeled anti-mouse IgG antibody as a secondary antibody. As a positive control, antibody staining was performed in the same manner using cells incubated with 50 μM HNE for 30 minutes. Figure 6 shows an image observed with a fluorescence microscope.
（結果）ジクロニンまたはスルファサラジンをそれぞれ単独で処理した場合、低頻度で細胞内ＨＮＥ濃度の増加が観察されるが、スルファサラジンとジクロニンの併用によって、高頻度で高濃度の細胞内ＨＮＥの蓄積が観察された。 (Results) When treated with dyclonine or sulfasalazine alone, an increase in intracellular HNE concentration was observed at low frequencies, but when sulfasalazine and dyclonine were treated together, accumulation of high concentrations of intracellular HNE was observed at high frequency. Ta.
このように、ｘＣＴ阻害剤とＡＬＤＨ阻害剤を併用することで、高頻度で高濃度の細胞内ＨＮＥの蓄積が観察されるようになる。この理由として、以下の理論に拘泥するものではないが、図１１に示すように、細胞内では、ＨＮＥを分解する経路が複数存在し、その中でＧＳＴを介する経路とＡＬＤＨを介する分解経路の二つを同時に阻害することにより、細胞内でＨＮＥが蓄積するものと考えられる。そして、ＨＮＥには細胞毒性があるので、腫瘍細胞が増殖できなくなると考えられる。 Thus, by using an xCT inhibitor and an ALDH inhibitor in combination, accumulation of intracellular HNE at high concentrations can be observed at high frequency. The reason for this is, without being bound by the following theory, that as shown in Figure 11, there are multiple pathways for degrading HNE within cells, and among these, the GST-mediated pathway and the ALDH-mediated degradation pathway. It is thought that by simultaneously inhibiting the two, HNE accumulates within cells. Since HNE is cytotoxic, it is thought that tumor cells cannot proliferate.
（実験例７）スルファサラジンまたはＢＳＯとジクロニン類縁体（ジクロニン骨格を有するもの）との併用効果
（目的）ジクロニン骨格を有する下記ジクロニン類縁体（Ｉ）は、スルファサラジンまたはＢＳＯとの併用効果を有することを示す。(Experiment Example 7) Effect of combined use of sulfasalazine or BSO and dyclonine analog (having a dyclonine skeleton) (Purpose) The following dyclonine analog (I) having a dyclonine skeleton has a combined effect with sulfasalazine or BSO. show.
（式中、Ｒ１はＣ１～６の直鎖または分岐アルキル基であり、Ｒ２及びＲ３は独立して選択されるＣ１～６の直鎖または分岐アルキル基であるか、Ｒ２とＲ３が一緒になってそれらが結合するＮをヘテロ原子とする４員環、５員環、６員環、または７員環のアザシクロアルキル基を形成し、Ｒ４は水素またはハロゲンである。Ｒ１はＣ４～５の直鎖または分岐アルキル基であることが好ましく、Ｒ２及びＲ３はＣ２のアルキル基か、またはＲ２とＲ３が一緒になってそれらが結合するＮをヘテロ原子とする６員環のアザシクロアルキル基であることが好ましい。なお、Ｒ１はＣ４の直鎖アルキル基であり、Ｒ２とＲ３が一緒になってそれらが結合するＮをヘテロ原子とする６員環のアザシクロアルキル基である化合物はジクロニンである。ハロゲンは、Ｆ、Ｃｌ、Ｉ、Ｂｒ、Ｉが好ましい。）
（方法）実験例１と同様に、０μＭ（無添加）、２５μＭ、５０μＭ、または１００μＭのジクロニン、又は１２．５μＭ、２５μＭ、５０μＭ、１００μＭのジクロニン類縁体BAS00363846、STL327701、PHAR033081、PHAR298639、またはAldi-2（構造式は図７Ｂ参照）と、０μＭ（無添加）または１００μＭＢＳＯまたは３００μＭスルファサラジンを含有した培地でＨＳＣ－４細胞を培養し、細胞生存率を測定して、図７Ａにグラフ化した。 (wherein R 1 is a C1-6 straight chain or branched alkyl group, R 2 and R 3 are independently selected C1-6 straight chain or branched alkyl groups, or R 2 and R 3 together form a 4-, 5-, 6-, or 7-membered azacycloalkyl group in which the N to which they are bonded is a heteroatom, and R 4 is hydrogen or halogen. Preferably, R 1 is a C4-5 linear or branched alkyl group, and R 2 and R 3 are C2 alkyl groups, or R 2 and R 3 together represent the N to which they are bonded to a heteroatom. It is preferable that it is a 6-membered ring azacycloalkyl group. Note that R 1 is a C4 straight chain alkyl group, and R 2 and R 3 are taken together and the N to which they are bonded is a hetero atom. The compound that is a 6-membered azacycloalkyl group is dyclonine.The halogen is preferably F, Cl, I, Br, or I.)
(Method) Similar to Experimental Example 1, 0 μM (no addition), 25 μM, 50 μM, or 100 μM of dyclonine, or 12.5 μM, 25 μM, 50 μM, or 100 μM of dyclonine analog BAS00363846, STL327701, PHAR033081, PHAR298639, or Aldi- HSC-4 cells were cultured in a medium containing 2 (for the structural formula, see FIG. 7B) and 0 μM (no addition), 100 μM BSO, or 300 μM sulfasalazine, and the cell viability was measured and graphed in FIG. 7A.
（結果）これらの化合物はすべてＢＳＯまたはスルファサラジンとの併用効果を奏した。 (Results) All of these compounds exhibited effects when used in combination with BSO or sulfasalazine.
このように、ジクロニン骨格を有するジクロニン類縁体（Ｉ）は、ｘＣＴ阻害剤と抗腫瘍剤として併用効果を有する。 Thus, the dyclonine analog (I) having a dyclonine skeleton has a combined effect as an xCT inhibitor and an antitumor agent.
（実験例８）ＢＳＯとジクロニン類縁体（ジクロニン骨格を有さないもの）との併用効果
（目的）ジクロニン骨格を有さないジクロニン類縁体は、ＢＳＯとの併用効果を有さないことを示す。(Experimental Example 8) Effect of combined use of BSO and dyclonine analog (without dyclonine skeleton) (Purpose) It is shown that dyclonine analog without dyclonine skeleton does not have the combined effect with BSO.
（方法）実験例１と同様に、０μＭ（無添加）、１２．５μＭ、２５μＭ、または５０μＭジクロニン、又は３．１２５μＭ、６．２５μＭ、１２．５μＭ、２５μＭ、５０μＭ、１００μＭのジクロニン類縁体（4-hydroxyacetpphenone：構造式は図８Ｂ参照）と、０μＭ（無添加）または１００μＭＢＳＯを含有した培地でＨＳＣ－４細胞を培養し、細胞生存率を測定して、図８Ａにグラフ化した。 (Method) Similar to Experimental Example 1, 0 μM (no addition), 12.5 μM, 25 μM, or 50 μM dyclonine, or 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, or 100 μM dyclonine analog (4 HSC-4 cells were cultured in a medium containing -hydroxyacetpphenone (for the structural formula, see FIG. 8B) and 0 μM (no addition) or 100 μM BSO, and the cell viability was measured and graphed in FIG. 8A.
（結果）ジクロニン骨格を有さないジクロニン類縁体はＢＳＯとの併用効果を奏しなかった。 (Results) A dyclonine analog without a dyclonine skeleton had no effect when used in combination with BSO.
このように、ｘＣＴ阻害剤との相互作用には、ジクロニン骨格が重要である。 Thus, the dyclonine skeleton is important for interaction with xCT inhibitors.
（実験例９）スルファサラジン耐性ＯＳＣ１９細胞におけるスルファサラジン、エラスチン、またはＢＳＯとジクロニンとの併用効果
（目的）ｘＣＴ阻害剤に対する耐性を獲得したがん細胞株においてジクロニンがグルタチオン合成阻害剤との併用効果を有することを示す。(Experimental Example 9) Effect of combination of sulfasalazine, elastin, or BSO with dyclonine on sulfasalazine-resistant OSC19 cells (Purpose) Dyclonine has a combined effect with glutathione synthesis inhibitor on cancer cell lines that have acquired resistance to xCT inhibitors. Show that.
（方法）スルファサラジン感受性口腔扁平上皮がん細胞株ＯＳＣ１９をスルファサラジンを含むＤＭＥＭ培地中で２ケ月間培養し、スルファサラジン耐性ＯＳＣ１９細胞を樹立した。ＯＳＣ１９細胞の親株またはＯＳＣ１９－ＳＳＺＲ細胞を９６ウエルプレートに３０００個／ウエルで播種し、２４時間培養後、図９に示した濃度のスルファサラジン、エラスチン、またはＢＳＯと、５０μＭのジクロニン（溶媒はＤＭＳＯ）または等量のＤＭＳＯを含む培地に交換し、４８時間培養した。その後、Celltiter-Glo（Promega社）により細胞生存率を測定し、コントロール（スルファサラジン、エラスチンおよびＢＳＯ無添加、ＤＭＳＯ添加）を１００％として細胞生存率を算出した。 (Method) The sulfasalazine-sensitive oral squamous cell carcinoma cell line OSC19 was cultured in a DMEM medium containing sulfasalazine for 2 months to establish sulfasalazine-resistant OSC19 cells. The parent strain of OSC19 cells or OSC19-SSZR cells were seeded at 3000 cells/well in a 96-well plate, and after culturing for 24 hours, sulfasalazine, elastin, or BSO at the concentrations shown in Figure 9 and 50 μM dyclonine (the solvent was DMSO) were added. Alternatively, the medium was replaced with a medium containing an equal amount of DMSO, and cultured for 48 hours. Thereafter, the cell viability was measured using Celltiter-Glo (Promega), and the cell viability was calculated by setting the control (no addition of sulfasalazine, elastin, and BSO, addition of DMSO) as 100%.
（結果）ジクロニンはＯＳＣ１９－ＳＳＺＲ細胞においてもスルファサラジン、エラスチンまたはＢＳＯとの併用効果を示した。 (Results) Dyclonine also showed a combined effect with sulfasalazine, elastin, or BSO in OSC19-SSZR cells.
このように、ジクロニンはｘＣＴ阻害剤に対する耐性を獲得したがん細胞においてもグルタチオン合成阻害剤との併用効果を示す。 In this way, dyclonine exhibits effects in combination with glutathione synthesis inhibitors even in cancer cells that have acquired resistance to xCT inhibitors.
（実験例１０）スルファサラジン耐性ＯＳＣ１９細胞およびＨＳＣ－４におけるＡＬＤＨ遺伝子ファミリーの発現
（目的）ｘＣＴ阻害剤に対する耐性を有するがん細胞においてＡＬＤＨ遺伝子ファミリーが高発現していることを示す。(Experimental Example 10) Expression of ALDH gene family in sulfasalazine-resistant OSC19 cells and HSC-4 (Purpose) This shows that the ALDH gene family is highly expressed in cancer cells that are resistant to xCT inhibitors.
（方法）ＨＳＣ－４細胞、ＯＳＣ１９細胞およびＯＳＣ１９－ＳＳＺＲ細胞よりメッセ
ンジャーＲＮＡを抽出し、逆転写反応を行うことにより相補的ＤＮＡを合成した。その後、得られた相補的ＤＮＡを鋳型とし、定量的ＲＴ－ＰＣＲ法によりＡＬＤＨＩＡｌ、
ＡＬＤＨＩＢｌ、ＡＬＤＨ２、ＡＬＤＨ３ＡｌおよびＲＰＳ１７の発現量を測定した。ＲＰＳ１７の発現量をリファレンスとして、ΔΔＣｔ法により各ＡＬＤＨファミリー遺伝子の発現量を定量し、図１０にグラフ化した。(Method) Messenger RNA was extracted from HSC-4 cells, OSC19 cells, and OSC19-SSZR cells, and complementary DNA was synthesized by performing a reverse transcription reaction. Then, using the obtained complementary DNA as a template, ALDHIAl,
The expression levels of ALDHIBl, ALDH2, ALDH3Al and RPS17 were measured. Using the expression level of RPS17 as a reference, the expression level of each ALDH family gene was quantified by the ΔΔCt method and graphed in FIG. 10.
（結果）ＡＬＤＨＩＡｌはＯＳＣ１９に比較して、ＯＳＣ１９－ＳＳＺＲにおいて発現が上昇していた。ＡＬＤＨＩＢｌおよびＡＬＤＨ２はＨＳＣ～４に高い発現が認められた。ＡＬＤＨ３ＡｌはＨＳＣ４およびＯＳＣ１９－ＳＳＺＲにおいて高発現であった。このように、ｘＣＴ低感受性のがん細胞株においてＡＬＤＨファミリー遺伝子の発現が高い傾向にあった。 (Results) The expression of ALDHIAl was increased in OSC19-SSZR compared to OSC19. High expression of ALDHIB1 and ALDH2 was observed in HSC-4. ALDH3Al was highly expressed in HSC4 and OSC19-SSZR. Thus, the expression of ALDH family genes tended to be high in cancer cell lines with low xCT sensitivity.
このように、ＡＬＤＨファミリー遺伝子の発現が高いがん細胞では、ＡＬＤＨファミリー遺伝子によりＨＮＥを分解しているために、ｘＣＴ阻害剤によってＧＳＴへの分解を抑制しても、ＨＮＥによる毒性は作用せず、ｘＣＴ阻害剤に耐性を得る（図１１参照）。このような細胞にＡＬＤＨ阻害剤を投与すると、ｘＣＴ阻害剤に対する感受性が上がるので、ＡＬＤＨ阻害剤及びグルタチオン濃度低下剤またはグルタチオンＳ-トランスフェラーゼ阻害剤を含有する抗腫瘍剤は、ＡＬＤＨファミリー遺伝子の発現が高いがん細胞にも効果的に作用する。 Thus, in cancer cells with high expression of ALDH family genes, HNE is degraded by ALDH family genes, so even if xCT inhibitors inhibit the degradation to GST, the toxicity of HNE does not occur. , acquires resistance to xCT inhibitors (see Figure 11). Administration of ALDH inhibitors to such cells increases their sensitivity to xCT inhibitors, so antitumor agents containing ALDH inhibitors and glutathione concentration-lowering agents or glutathione S-transferase inhibitors may inhibit the expression of ALDH family genes. It also acts effectively on cancer cells with high levels of cancer.
本発明によって、新規な抗腫瘍剤及び配合剤を提供することができるようになった。 The present invention has made it possible to provide novel antitumor agents and combination agents.
Claims (9)
前記アルデヒド脱水素酵素阻害剤は、下記式（Ｉ）で表される化合物、またはその薬理学的に許容される塩であり、
（式中、Ｒ１はＣ１～６の直鎖または分岐アルキル基であり、Ｒ２及びＲ３は独立して選択されるＣ１～６の直鎖または分岐アルキル基であるか、Ｒ２とＲ３が一緒になってそれらが結合するＮをヘテロ原子とする４員環、５員環、６員環、または７員環のアザシクロアルキル基を形成し、Ｒ４は水素またはハロゲンである。）
前記グルタチオン濃度低下剤はスルファサラジン、エラスチン、ソラフェニブ、または抗ｘＣＴ抗体であり、
前記グルタチオンＳ-トランスフェラーゼ阻害剤は、グルタチオンアナログ、ケトプロフェン、インドメタシン、エタクリン酸、ピロプロスト、抗ＧＳＴ抗体、またはＧＳＴのドミナントネガティブ変異体である、抗腫瘍剤。 An antitumor agent containing a glutathione concentration lowering agent or a glutathione S-transferase inhibitor as an active ingredient, which is administered simultaneously with an effective amount of an aldehyde dehydrogenase inhibitor,
The aldehyde dehydrogenase inhibitor is a compound represented by the following formula (I) or a pharmacologically acceptable salt thereof,
(wherein R 1 is a C1-6 straight chain or branched alkyl group, R 2 and R 3 are independently selected C1-6 straight chain or branched alkyl groups, or R 2 and R 3 together form a 4-, 5-, 6-, or 7-membered azacycloalkyl group in which the N to which they are bonded is a heteroatom, and R 4 is hydrogen or halogen. )
The glutathione concentration lowering agent is sulfasalazine, elastin, sorafenib, or an anti-xCT antibody,
The glutathione S-transferase inhibitor is an anti-tumor agent, wherein the glutathione S-transferase inhibitor is a glutathione analog, ketoprofen, indomethacin, ethacrynic acid, piroprost, an anti-GST antibody, or a dominant negative mutant of GST.
前記アルデヒド脱水素酵素阻害剤は、下記式（Ｉ）で表される化合物またはその薬理学的に許容される塩であり、
（式中、Ｒ１はＣ１～６の直鎖または分岐アルキル基であり、Ｒ２及びＲ３は独立して選択されるＣ１～６の直鎖または分岐アルキル基であるか、Ｒ２とＲ３が一緒になってそれらが結合するＮをヘテロ原子とする４員環、５員環、６員環、または７員環のアザシクロアルキル基を形成し、Ｒ４は水素またはハロゲンである。）
前記グルタチオン濃度低下剤はスルファサラジン、エラスチン、ソラフェニブ、または抗ｘＣＴ抗体であり、
前記グルタチオンＳ-トランスフェラーゼ阻害剤は、グルタチオンアナログ、ケトプロフェン、インドメタシン、エタクリン酸、ピロプロスト、抗ＧＳＴ抗体、またはＧＳＴのドミナントネガティブ変異体である、抗腫瘍剤。 An antitumor agent containing an aldehyde dehydrogenase inhibitor as an active ingredient, which is administered simultaneously with an effective amount of a glutathione concentration lowering agent or a glutathione S-transferase inhibitor,
The aldehyde dehydrogenase inhibitor is a compound represented by the following formula (I) or a pharmacologically acceptable salt thereof,
(wherein R 1 is a C1-6 straight chain or branched alkyl group, R 2 and R 3 are independently selected C1-6 straight chain or branched alkyl groups, or R 2 and R 3 together form a 4-, 5-, 6-, or 7-membered azacycloalkyl group in which the N to which they are bonded is a heteroatom, and R 4 is hydrogen or halogen. )
The glutathione concentration lowering agent is sulfasalazine, elastin, sorafenib, or an anti-xCT antibody,
The glutathione S-transferase inhibitor is an anti-tumor agent, wherein the glutathione S-transferase inhibitor is a glutathione analog, ketoprofen, indomethacin, ethacrynic acid, piroprost, an anti-GST antibody, or a dominant negative mutant of GST.
前記アルデヒド脱水素酵素阻害剤は、下記式（Ｉ）で表される化合物またはその薬理学的に許容される塩であり、
（式中、Ｒ１はＣ１～６の直鎖または分岐アルキル基であり、Ｒ２及びＲ３は独立して選択されるＣ１～６の直鎖または分岐アルキル基であるか、Ｒ２とＲ３が一緒になってそれらが結合するＮをヘテロ原子とする４員環、５員環、６員環、または７員環のアザシクロアルキル基を形成し、Ｒ４は水素またはハロゲンである。）
前記グルタチオン濃度低下剤はスルファサラジン、エラスチン、ソラフェニブ、または抗ｘＣＴ抗体であり、
前記グルタチオンＳ-トランスフェラーゼ阻害剤は、グルタチオンアナログ、ケトプロフェン、インドメタシン、エタクリン酸、ピロプロスト、抗ＧＳＴ抗体、またはＧＳＴのドミナントネガティブ変異体である、抗腫瘍剤。 An antitumor agent comprising a combination drug containing an aldehyde dehydrogenase inhibitor and a glutathione concentration lowering agent or a glutathione S-transferase inhibitor as active ingredients,
The aldehyde dehydrogenase inhibitor is a compound represented by the following formula (I) or a pharmacologically acceptable salt thereof,
(wherein R 1 is a C1-6 straight chain or branched alkyl group, R 2 and R 3 are independently selected C1-6 straight chain or branched alkyl groups, or R 2 and R 3 together form a 4-, 5-, 6-, or 7-membered azacycloalkyl group in which the N to which they are bonded is a heteroatom, and R 4 is hydrogen or halogen. )
The glutathione concentration lowering agent is sulfasalazine, elastin, sorafenib, or an anti-xCT antibody,
The glutathione S-transferase inhibitor is an anti-tumor agent , wherein the glutathione S-transferase inhibitor is a glutathione analog, ketoprofen, indomethacin, ethacrynic acid, piroprost, an anti-GST antibody, or a dominant negative mutant of GST.
前記腫瘍細胞の増殖速度または細胞生存率を測定する工程と、を含み、
前記アルデヒド脱水素酵素阻害剤は、下記式（Ｉ）で表される化合物、またはその薬理学的に許容される塩であり、
（式中、Ｒ１はＣ１～６の直鎖または分岐アルキル基であり、Ｒ２及びＲ３は独立して選択されるＣ１～６の直鎖または分岐アルキル基であるか、Ｒ２とＲ３が一緒になってそれらが結合するＮをヘテロ原子とする４員環、５員環、６員環、または７員環のアザシクロアルキル基を形成し、Ｒ４は水素またはハロゲンである。）
前記グルタチオン濃度低下剤はスルファサラジン、エラスチン、ソラフェニブ、または抗ｘＣＴ抗体であり、
前記グルタチオンＳ-トランスフェラーゼ阻害剤は、グルタチオンアナログ、ケトプロフェン、インドメタシン、エタクリン酸、ピロプロスト、抗ＧＳＴ抗体、またはＧＳＴのドミナントネガティブ変異体である、測定方法。 simultaneously administering an aldehyde dehydrogenase inhibitor and a glutathione concentration lowering agent or a glutathione S-transferase inhibitor to tumor cells in vitro;
measuring the proliferation rate or cell viability of the tumor cells,
The aldehyde dehydrogenase inhibitor is a compound represented by the following formula (I) or a pharmacologically acceptable salt thereof,
(wherein R 1 is a C1-6 straight chain or branched alkyl group, R 2 and R 3 are independently selected C1-6 straight chain or branched alkyl groups, or R 2 and R 3 together form a 4-, 5-, 6-, or 7-membered azacycloalkyl group in which the N to which they are bonded is a heteroatom, and R 4 is hydrogen or halogen. )
The glutathione concentration lowering agent is sulfasalazine, elastin, sorafenib, or an anti-xCT antibody,
The measuring method, wherein the glutathione S-transferase inhibitor is a glutathione analog, ketoprofen, indomethacin, ethacrynic acid, piroprost, an anti-GST antibody, or a dominant negative mutant of GST.
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