JP2019064973A - No production inhibitor and metastasis/invasion inhibitor for solid tumor - Google Patents
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本発明は、ＮＯ産生抑制剤及び固形腫瘍に対する転移・浸潤抑制剤に関する。 The present invention relates to an NO production inhibitor and a metastasis / infiltration inhibitor for solid tumors.
ＩＫＤ-８３４４は、放線菌から単離された２８員環のマクロライド系の抗生物質であるが、殺虫剤など（例えば、特許文献１参照）や、医薬としての抗菌剤や白血病細胞に対する増殖抑制剤（例えば、特許文献２参照）としても知られている。現在まで、化学合成の方法も報告されている（例えば、特許文献２、非特許文献１〜３参照）。 IKD-8344 is a 28-membered ring macrolide antibiotic isolated from actinomycetes, but it is a growth inhibitor against insecticides (see, for example, Patent Document 1), an antibacterial agent as a medicine, and leukemia cells. Also known as an agent (see, for example, Patent Document 2). To date, methods of chemical synthesis have also been reported (see, for example, Patent Document 2 and Non-Patent Documents 1 to 3).
本発明は、新規ＮＯ産生抑制剤及び固形腫瘍に対する新規転移・浸潤抑制剤を提供することを目的とする。 An object of the present invention is to provide a novel NO production inhibitor and a novel metastasis / infiltration inhibitor for solid tumors.
本発明の一実施態様は、下記構造式（Ｉ）を有する化合物またはその薬学的に許容できる塩を有効成分として含有する、ＮＯ産生抑制剤である。 One embodiment of the present invention is an NO production inhibitor comprising a compound having the following structural formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.
このＮＯ産生抑制剤は、抗炎症剤または抗アレルギー剤として用いられてもよい。 This NO production inhibitor may be used as an antiinflammatory agent or an antiallergic agent.
本発明の他の一実施態様は、固形腫瘍に対する腫瘍転移抑制剤であって、下記構造式（Ｉ）を有する化合物またはその薬学的に許容できる塩を有効成分として含有する腫瘍転移抑制剤である。 Another embodiment of the present invention is a tumor metastasis inhibitor for solid tumors, which comprises, as an active ingredient, a compound having the following structural formula (I) or a pharmaceutically acceptable salt thereof: .
この腫瘍転移抑制剤が、卵巣癌に対する腫瘍転移抑制剤であってもよい。 The tumor metastasis inhibitor may be a tumor metastasis suppressor for ovarian cancer.
また、この腫瘍転移抑制剤は、前記固形腫瘍に対する腫瘍細胞増殖抑制効果を有する抗腫瘍剤と併用されてもよい。 In addition, this tumor metastasis inhibitor may be used in combination with an antitumor agent having a tumor cell proliferation inhibitory effect on the solid tumor.
本発明のさらなる一実施態様は、固形腫瘍に対する腫瘍浸潤抑制剤であって、下記構造式（Ｉ）を有する化合物またはその薬学的に許容できる塩を有効成分として含有する腫瘍浸潤抑制剤である。 A further embodiment of the present invention is a tumor invasion suppressant for solid tumors, which comprises, as an active ingredient, a compound having the following structural formula (I) or a pharmaceutically acceptable salt thereof.
この腫瘍浸潤抑制剤が、卵巣癌に対する腫瘍浸潤抑制剤であってもよい。 The tumor invasion inhibitor may be a tumor invasion suppressor for ovarian cancer.
また、この腫瘍浸潤抑制剤は、前記固形腫瘍に対する腫瘍細胞増殖抑制効果を有する抗腫瘍剤と併用されてもよい。 In addition, the tumor invasion inhibitor may be used in combination with an antitumor agent having a tumor cell growth inhibitory effect on the solid tumor.
本発明によって、新規ＮＯ産生抑制剤及び固形腫瘍に対する新規転移・浸潤抑制剤を提供することができるようになった。 According to the present invention, it has become possible to provide a novel NO production inhibitor and a novel metastasis / infiltration inhibitor for solid tumors.
実施の形態及び実施例に特に説明がない場合には、M. R. Green & J. Sambrook (Ed.), Molecular cloning, a laboratory manual (4th edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2012); F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J.G. Seidman, J. A. Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd.などの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。 Unless otherwise described in the embodiments and examples, MR Green & J. Sambrook (Ed.), Molecular cloning, a laboratory manual (4th edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2012) Described in standard protocols such as FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd. Or a modified or modified method of Moreover, when using a commercially available reagent kit or measuring apparatus, unless otherwise described, the protocol attached thereto is used.
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。 The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art will readily reproduce the present invention from the descriptions of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for the purpose of illustration or explanation. 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 of the present specification within the intent and scope of the present invention disclosed herein.
＝＝ＮＯ産生抑制剤＝＝
本発明の一実施形態は、下記構造式（Ｉ）を有する化合物ＩＫＤ-８３４４またはその薬学的に許容できる塩を有効成分として含有する、ＮＯ産生抑制剤である。このＮＯ産生抑制剤は、医薬・試薬などの薬剤、化粧品、機能性食品などに有用である。医薬としては、炎症性疾患、虚血性疾患、自己免疫性疾患、アレルギー性疾患、などに対する抗炎症剤、抗アレルギー剤などとして、利用できる。
== NO production inhibitor ==
One embodiment of the present invention is an NO production inhibitor comprising a compound IKD-8344 having the following structural formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient. This NO production inhibitor is useful for medicines, reagents such as medicines, cosmetics, functional foods and the like. As a medicine, it can be used as an anti-inflammatory agent, an anti-allergic agent and the like for inflammatory diseases, ischemic diseases, autoimmune diseases, allergic diseases and the like.
＝＝転移及び／又は浸潤抑制剤＝＝
また、本発明の他の一実施形態は、下記構造式（Ｉ）を有する化合物ＩＫＤ-８３４４またはその薬学的に許容できる塩を有効成分として含有する、固形腫瘍に対する転移及び／又は浸潤抑制剤である。ここで、治療対象の固形腫瘍は特に限定されず、血液細胞やリンパ球以外の腫瘍であればよく、例えば、線維腫、脂肪腫、粘液腫、軟骨腫、骨腫、血管腫、黒色腫、筋腫、神経腫、神経膠腫、骨肉腫、筋肉腫、線維肉腫、乳頭腫、腺腫、脳腫瘍、頚癌、食道癌、舌癌、肺癌、乳癌、膵癌、胃癌、十二指腸・空腸・回腸等の小腸癌、結腸・盲腸・直腸等の大腸癌、膀胱癌、腎癌、肝癌、胆嚢癌、前立腺癌、子宮頸癌、子宮癌、卵巣癌、甲状腺癌、咽頭癌、皮膚癌などが例示できるが、好ましくは卵巣癌である。
== Metastasis and / or infiltration inhibitor ==
In addition, another embodiment of the present invention is a metastasis and / or invasion inhibitor to a solid tumor, comprising Compound IKD-8344 having the following structural formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient: is there. Here, the solid tumor to be treated is not particularly limited as long as it is a tumor other than blood cells and lymphocytes, for example, fibroma, lipoma, myxoma, chondroma, osteoma, hemangioma, melanoma, Myoma, neuroma, glioma, osteosarcoma, myosarcoma, fibrosarcoma, papilloma, adenoma, brain tumor, cervical cancer, esophageal cancer, tongue cancer, lung cancer, breast cancer, pancreatic cancer, gastric cancer, small intestine such as duodenum, jejunum and ileum Examples include cancer, colon cancer such as colon, cecum and rectum, bladder cancer, renal cancer, liver cancer, gall bladder cancer, prostate cancer, cervical cancer, uterine cancer, ovarian cancer, thyroid cancer, pharyngeal cancer, skin cancer and the like. Preferably it is ovarian cancer.
なお、薬理学的に許容される塩とは、そ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。 The pharmacologically acceptable salt is not limited as long as it forms a salt with the compound, but specifically, for example, hydrochloride, sulfate, nitrate, hydrobromide, hydrogen iodide Addition salts of inorganic acids such as acid salts, perchlorates and phosphates, addition salts of organic acids such as oxalate, maleate, fumarate and succinate, methanesulfonate, ethanesulfonic acid There may be mentioned salts, addition salts of sulfonic acids such as benzenesulfonate, p-toluenesulfonate, camphorsulfonate and the like, addition salts of amino acids and the like. Furthermore, it is needless to say that those compounds or their pharmacologically acceptable salts include not only anhydrate but also hydrates and crystalline polymorphs.
この抗腫瘍剤は、固形腫瘍細胞の増殖を抑制したり、生存率を低下させたりするのではなく、固形腫瘍細胞の転移及び／又は浸潤を抑制することで、その効力を発揮する。従って、この抗腫瘍剤を、上記のような固形腫瘍に対する増殖抑制効果を有する抗腫瘍剤（細胞増殖抑制剤）と併用することによって、相乗効果が得られる。併用する抗腫瘍剤は、固形腫瘍に対する増殖抑制効果を有するものであれば特に限定されず、例えばシクロホスファミド、イホスファミド、メルファラン、ブスルファン、チオテパなどのナイトロジェンマスタード類や、ニムスチン、ラニムスチン、ダカルバシン、プロカルバシン、テモゾロマイド、カルムスチン、ストレプトゾトシン、ベンダムスチンなどのニトロソウレア類などのアルキル化薬；シスプラチン、カルボプラチン、オキサリプラチン、ネダプラチンなどの白金製剤；フルオロウラシルなどのピリミジン代謝阻害薬や、６−メルカプトプリンなどのプリン代謝阻害薬や、プリンアナログ、ピリミジンアナログなどのヌクレオチドアナログなどの代謝拮抗薬；アントラサイクリン系、エピポドフィロトキシン系、キノロン系などのトポイソメラーゼ阻害薬などが挙げられる。 This antitumor agent exerts its efficacy by suppressing the metastasis and / or invasion of solid tumor cells, rather than suppressing the growth of solid tumor cells or reducing the survival rate. Therefore, a synergistic effect is obtained by using this antitumor agent in combination with an antitumor agent (cytostatic agent) having a growth inhibitory effect on solid tumors as described above. The antitumor agent to be used in combination is not particularly limited as long as it has a growth inhibitory effect on solid tumors, and examples thereof include nitrogen mustards such as cyclophosphamide, ifosfamide, melphalan, busulfan, thiotepa, nimustine, lanimustine, Alkylating agents such as dacarbacin, procarbacin, temozolomide, carmustine, streptozotocin, bendamustine and other nitrosoureas; platinum preparations such as cisplatin, carboplatin, oxaliplatin and nedaplatin; pyrimidine metabolism inhibitors such as fluorouracil, and 6-mercaptopurine Antimetabolites such as purine metabolism inhibitors, purine analogues, nucleotide analogues such as pyrimidine analogues; Anthracyclines, epipodophyllotoxins, quinolones Such as topoisomerase inhibitors and the like.
これらの医薬の形状、投与形態、投与量は特に限定されず、患者の症状、体重、年齢、性別等に従って、最適な形状、投与形態、投与量を容易に決定できる。 The form, dosage form and dosage of these pharmaceuticals are not particularly limited, and the optimum form, dosage form and dosage can be easily determined according to the patient's condition, body weight, age, sex and the like.
（実施例１）ＩＫＤ-８３４４は腫瘍細胞に対し、ＮＯ産生抑制効果を有する。 Example 1 IKD-8344 has an effect of suppressing NO production on tumor cells.
本実施例では、マウス単球由来の白血病細胞株であるＲＡＷ２６４．７を用い、ＩＫＤ-８３４４がＮＯ産生抑制効果を有することを示す。 In this example, it is shown that IKD-8344 has an effect of suppressing NO production using RAW264.7 which is a leukemia cell line derived from mouse monocytes.
（生存率の測定）１０％ＦＢＳ含有ＲＰＭＩ培地で培養したＲＡＷ２６４．７細胞の懸濁液（１ｘ１０４個／ｍＬ）を１００μＬ／ウエルで９６穴プレートに播種し、３７°Ｃ、５％ＣＯ２存在下で、２４時間培養した。そしてＩＫＤ-８３４４（終濃度０．００１、０．０１、０．１、１、１０μｇ／ｍＬ）を添加し、さらに２４時間培養した。その後、各ウエルに１０μＬのＭＴＴ溶液（Cayman社 Cat No 10009591）を添加し、２時間培養後、培地を吸引除去し、１００μＬ／ウエルでＤＭＳＯを添加した。こうしてformazanを細胞から溶出させ、５７０ｎｍの吸光度をマイクロプレートリーダー(Bio-Rad社)で測定した。各条件ごとに３連で実験を行い、その平均値を求めてグラフにプロットした（図１の折れ線グラフ）。なお、コントロールとして、ＩＫＤ-８３４４非存在下（図ではＣｏｎｔ）以外は同様の条件で実験した細胞を用いた。 (Measurement of viability) A suspension (1 × 10 4 cells / mL) of RAW 264.7 cells cultured in RPMI medium containing 10% FBS is seeded at 100 μL / well in a 96-well plate at 37 ° C., 5% CO 2 The cells were cultured in the presence for 24 hours. Then, IKD-8344 (final concentration 0.001, 0.01, 0.1, 1, 10 μg / mL) was added, and culture was further continued for 24 hours. Thereafter, 10 μL of MTT solution (Cayman Cat No 10009591) was added to each well, and after 2 hours of culture, the medium was aspirated and removed, and DMSO was added at 100 μL / well. Thus, formazan was eluted from the cells, and the absorbance at 570 nm was measured with a microplate reader (Bio-Rad). The experiment was performed in triplicate for each condition, and the average value was determined and plotted on a graph (line graph in FIG. 1). As a control, cells tested under the same conditions except in the absence of IKD-8344 (Cont in the figure) were used.
その結果、細胞生存率は、ＩＫＤ-８３４４が０．１μｇ／ｍＬまでは有意な低下は観察されず、１μｇ／ｍＬ以上で若干の低下傾向にあった。 As a result, no significant decrease was observed in the cell viability up to 0.1 μg / mL of IKD-8344, and a slight tendency tended to decrease at 1 μg / mL or more.
（ＮＯ産生量の測定）次に、１０％ＦＢＳ含有ＲＰＭＩ培地で培養したＲＡＷ２６４．７細胞の懸濁液（３ｘ１０６個／ｍＬ）を１００μＬ／ウエルで９６穴プレートに播種し、ＬＰＳ（終濃度１０ｎｇ／ｍＬ）及びＩＫＤ-８３４４（終濃度０．００１、０．０１、０．１、１、１０μｇ／ｍＬ）を添加し、３７°Ｃ、５％ＣＯ２存在下で、２４時間培養した。その後、５０μＬの上清を回収して５０μＬのGriess液（０．１％Ｎ-１-ナフチルエチレンジアミン 二塩酸，ｌ％スルファニルアミド，２．５％Ｈ３ＰＯ４）を加え、亜硝酸塩量を熱量測定法で測定した。各条件ごとに３連で実験を行い、その平均値を求めてグラフを作成した（図１の棒グラフ）。なお、コントロールとして、ＩＫＤ-８３４４非存在下でＬＰＳで刺激しない細胞（図ではＣｏｎｔ）、及びＩＫＤ-８３４４非存在下でＬＰＳで刺激した細胞（図ではＬＰＳ）を用いた。 (Measurement of NO production amount) Next, a suspension (3 x 10 6 cells / mL) of RAW 264.7 cells cultured in RPMI medium containing 10% FBS is seeded at 100 μL / well in a 96-well plate to give LPS (final concentration 10 ng / mL) and IKD-8344 (final concentration 0.001, 0.01, 0.1, 1, 10 μg / mL) were added, and cultured at 37 ° C. in the presence of 5% CO 2 for 24 hours. After that, 50 μL of the supernatant was collected, and 50 μL of Griess solution (0.1% N-1-naphthylethylenediamine dihydrochloride, 1% sulfanylamide, 2.5% H 3 PO 4 ) was added to heat the amount of nitrite. It measured by the measuring method. The experiment was performed in triplicate for each condition, and the average value was determined to create a graph (bar graph in FIG. 1). As controls, cells not stimulated with LPS in the absence of IKD-8344 (Cont in the figure) and cells stimulated with LPS in the absence of IKD-8344 (LPS in the figure) were used.
その結果、ＩＫＤ-８３４４が増加するにつれて、細胞の生存率とともにＮＯ産生量が低下するが、ＮＯ産生量低下率は細胞生存率の低下率より顕著に大きい。例えば、細胞生存率がほぼ低下しない０．１μｇ／ｍＬのＩＫＤ-８３４４の場合、ＮＯ産生量は約３分の１に低下する。また、１０μｇ／ｍＬのＩＫＤ-８３４４の場合、コントロールに比べ、生存率は半分程度に低下するが、ＮＯ産生量は１０分の１以下にまで低下する。従って、この濃度では、ＩＫＤ-８３４４は、ＮＯ産生量を細胞あたり５分の１程度に抑制する効果を有する。 As a result, as IKD-8344 increases, NO production decreases with cell viability, but NO production reduction rate is significantly larger than cell viability reduction rate. For example, in the case of 0.1 μg / mL of IKD-8344, in which the cell viability is hardly reduced, the NO production amount is reduced to about one third. Further, in the case of 10 μg / mL IKD-8344, the survival rate is reduced to about half compared to the control, but the NO production amount is reduced to 1/10 or less. Therefore, at this concentration, IKD-8344 has the effect of suppressing the NO production amount to about one-fifth per cell.
このように、ＩＫＤ-８３４４は、ＬＰＳで刺激した腫瘍細胞に対し、ＮＯ産生量を抑制する効果を有する。炎症巣においては、好中球やマクロファージはＮＯと同時に活性酸素の一種であるＯ２ −を産生する。ＮＯは速やかにＯ２ −と反応し、ＮＯより反応性の高いＯＮＯＯ−を生成し、この結果、炎症、アレルギー、糖尿病、動脈硬化などが引き起こされる。従って、ＩＫＤ-８３４４は、抗炎症剤、抗アレルギー剤などとして有用である。 Thus, IKD-8344 has the effect of suppressing NO production on LPS-stimulated tumor cells. In inflammatory foci, neutrophils and macrophages O 2 is one type of NO at the same time as the active oxygen - to produce. NO is rapidly reacts with the O 2 - and, ONOO more reactive NO - generate, as a result, inflammation, allergy, diabetes, arteriosclerosis is caused. Therefore, IKD-8344 is useful as an antiinflammatory agent, an antiallergic agent and the like.
（実施例２）ＩＫＤ-８３４４は固形腫瘍細胞に対し、細胞増殖抑制効果を有しない Example 2 IKD-8344 has no cytostatic effect on solid tumor cells
本実施例では、ヒト卵巣癌細胞株ＥＳ-２を用い、ＩＫＤ-８３４４が固形腫瘍細胞に対して細胞生存率抑制効果を有しないことを示す。 In this example, human ovarian cancer cell line ES-2 is used, and it is shown that IKD-8344 has no cell survival inhibitory effect on solid tumor cells.
１０％ＦＢＳ含有ＤＭＥＭ培地を用い、各濃度のＩＫＤ-８３４４存在下で培養したＥＳ-２に対し、実施例１と同様にして細胞生存率を測定した。ＩＫＤ-８３４４非存在下での細胞生存率を１００％としてグラフ化したものを図２に示した。 The cell viability was measured in the same manner as in Example 1 for ES-2 cultured in the presence of IKD-8344 at various concentrations using a DMEM medium containing 10% FBS. A graph depicting cell viability in the absence of IKD-8344 as 100% is shown in FIG.
ＩＫＤ-８３４４については、ヒト卵巣癌細胞株ＥＳ-２に対し、高濃度の３μg／mLでも８０％の生存率を維持し、有意な細胞増殖抑制効果は観察されなかった。これに対し、マウス白血病細胞に対しては、０．００１μg／mLの濃度で、５８．０％の増殖抑制率を示している（特開平０２-９３８２号公開公報参照）。 With regard to IKD-8344, the survival rate was maintained at 80% even at a high concentration of 3 μg / mL against human ovarian cancer cell line ES-2, and no significant cytostatic effect was observed. On the other hand, for mouse leukemia cells, a growth inhibition rate of 58.0% is shown at a concentration of 0.001 μg / mL (see Japanese Patent Laid-Open No. 02-9382).
このように、固形腫瘍に対しては、ＩＫＤ-８３４４は顕著な増殖抑制効果を有しない。 Thus, for solid tumors, IKD-8344 has no significant growth inhibitory effect.
（実施例３）ＩＫＤ-８３４４は固形腫瘍細胞に対して浸潤抑制効果を有する。 Example 3 IKD-8344 has an anti-invasion effect on solid tumor cells.
本実施例では、ヒト卵巣癌細胞株ＥＳ-２を用い、ＩＫＤ-８３４４が固形腫瘍細胞に対して浸潤抑制効果を有することを示す。 In this example, using human ovarian cancer cell line ES-2, it is shown that IKD-8344 has an invasion suppression effect on solid tumor cells.
各濃度のＩＫＤ-８３４４存在下で培養したＥＳ-２に対し、invasion assayによって浸潤を調べた。具体的には、BD Matrigel Basement Membrane Matrix membrane (Corning Inc., Corning, NY)を用いたボイデンチャンバー法を適用した。まず、ＥＳ-２細胞を各濃度のＩＫＤ-８３４４を含有し、５００μＬのＦＢＳを含有しないＤＭＥＭに懸濁し、上層に入れた。下層には上層と同じ濃度のＩＫＤ-８３４４を含有した７５０μＬの１０％ＦＢＳ含有ＤＭＥＭを満たした。３７°Ｃ、５％ＣＯ２存在下で、２４時間培養後、membraneを固定して、下の層に浸潤した細胞を残し、浸潤せずに上の層に残った細胞を綿でこすって除いた。そして、Diff-Quick solution (Sysmex, Kobe, Japan)を用いて染色し、浸潤細胞数を計測した。ＩＫＤ-８３４４非存在下での浸潤を１００％としてグラフ化したものを図３に示した。 Invasion was examined by invasion assay for ES-2 cultured in the presence of each concentration of IKD-8344. Specifically, the Boyden chamber method using BD Matrigel Basement Membrane Matrix membrane (Corning Inc., Corning, NY) was applied. First, ES-2 cells were suspended in DMEM containing 500 μL of FBS containing each concentration of IKD-8344 and placed in the upper layer. The lower layer was filled with 750 μL of 10% FBS-containing DMEM containing the same concentration of IKD-8344 as the upper layer. After culturing for 24 hours at 37 ° C. in the presence of 5% CO 2 , the membrane is fixed to leave the infiltrated cells in the lower layer, and remove the non-invasive cells remaining in the upper layer by rubbing with cotton. The Then, the cells were stained using Diff-Quick solution (Sysmex, Kobe, Japan) to measure the number of invading cells. A graph depicting infiltration in the absence of IKD-8344 as 100% is shown in FIG.
ＩＫＤ-８３４４は、ヒト卵巣癌細胞株ＥＳ-２に対して、増殖抑制効果を示さない１μg／mLまたは３μg／mLで有意な浸潤抑制効果を発揮している。 IKD-8344 exerts a significant anti-invasion effect on human ovarian cancer cell line ES-2 at 1 μg / mL or 3 μg / mL showing no growth inhibitory effect.
このように、固形腫瘍に対しては、ＩＫＤ-８３４４は浸潤抑制剤として有用である。 Thus, for solid tumors, IKD-8344 is useful as an invasion control agent.
（実施例４）ＩＫＤ-８３４４は固形腫瘍細胞に対して腫瘍転移抑制効果を有する。 Example 4 IKD-8344 has a tumor metastasis suppressive effect on solid tumor cells.
本実施例では、ヒト卵巣癌細胞株ＥＳ-２を用い、ＩＫＤ-８３４４が固形腫瘍細胞に対して細胞移動抑制効果を有することを示す。 In this example, using human ovarian cancer cell line ES-2, it is shown that IKD-8344 has a cell migration inhibitory effect on solid tumor cells.
具体的には、wound healing assayによって細胞移動（migration）を調べた。まず、１０％ＦＢＳ含有ＤＭＥＭを用いて、ＥＳ-２細胞を２４穴プレートにコンフルエントになるまで培養し、ピペットマンのチップで長方形状（約１ｍｍｘ１５ｍｍ）に細胞を除去し、各濃度のＩＫＤ-８３４４を添加して２４時間培養後に写真撮影を行い、培養後の長方形の短径幅を測定した。ＩＫＤ-８３４４非存在下での長方形の幅を１００％としてグラフ化したものを図４に示した。 Specifically, cell migration was examined by wound healing assay. First, culture the ES-2 cells in a 24-well plate to confluence using DMEM containing 10% FBS, remove cells in a rectangular shape (about 1 mm x 15 mm) with a pipetman tip, and use IKD-8344 at each concentration After the culture for 24 hours after the addition, the photograph was taken and the width of the minor axis of the rectangle after the culture was measured. FIG. 4 is a graph plotting the width of the rectangle in the absence of IKD-8344 as 100%.
ＩＫＤ-８３４４は、ヒト卵巣癌細胞株ＥＳ-２に対して、増殖抑制効果を示さない０．３μｇ／ｍＬ〜３μｇ／ｍＬの濃度で有意な腫瘍細胞移動抑制効果を発揮している。 IKD-8344 exerts a significant tumor cell migration inhibitory effect on human ovarian cancer cell line ES-2 at a concentration of 0.3 μg / mL to 3 μg / mL showing no growth inhibitory effect.
このように、固形腫瘍に対しては、ＩＫＤ-８３４４は腫瘍転移抑制剤として有用である。
Thus, for solid tumors, IKD-8344 is useful as a tumor metastasis inhibitor.
Claims (8)
下記構造式（Ｉ）を有する化合物またはその薬学的に許容できる塩を有効成分として含有する腫瘍転移抑制剤。
A tumor metastasis inhibitor for solid tumors,
A tumor metastasis inhibitor comprising a compound having the following structural formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.
下記構造式（Ｉ）を有する化合物またはその薬学的に許容できる塩を有効成分として含有する腫瘍浸潤抑制剤。
Tumor invasion inhibitor for solid tumors,
A tumor invasion inhibitor comprising a compound having the following structural formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.
An NO production inhibitor comprising a compound having the following structural formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.
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