JP2014129341A - Multi-kinase inhibitor, anticancer agent, anti-metastasis agent, drug resistance inhibitor, pain inhibitor, and antipruritic agent - Google Patents
Multi-kinase inhibitor, anticancer agent, anti-metastasis agent, drug resistance inhibitor, pain inhibitor, and antipruritic agent Download PDFInfo
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Abstract
Description
本発明は、マルチキナーゼ阻害剤、抗癌剤、抗転移剤、薬剤耐性抑制剤、疼痛抑制剤及び止痒薬に関する。 The present invention relates to a multikinase inhibitor, an anticancer agent, an antimetastatic agent, a drug resistance inhibitor, a pain inhibitor and an antipruritic agent.
近年、遺伝子やタンパク質の解析技術が革新的に進歩したことで、疾患に関連した生体分子が数多く同定されつつある。これにより、疾患の分類が様変わりしている。例えば、従来は癌の分類は、臓器別であったが、同一の臓器の癌であっても、関連した分子の特徴を基にした分類が可能となった。これにより、治療薬の開発手法も転換期を迎えることとなった。即ち、闇雲に有効性を示す化合物をスクリーニングすることから、疾患に特徴的な分子を標的として阻害あるいは賦活させる物質、即ち、分子標的治療薬を見出す手法が標準となっている。分子標的治療薬は、診断の段階で疾患に関連する分子を特定してから治療を開始するため、高い有効性を示すことが特徴である。 In recent years, a lot of biomolecules related to diseases have been identified due to innovative advances in gene and protein analysis techniques. This has changed the classification of diseases. For example, in the past, cancer was classified by organ, but even cancers of the same organ could be classified based on the characteristics of related molecules. As a result, the development of therapeutic drugs has reached a turning point. That is, since screening for compounds that are effective against the dark clouds, a standard method is to find a substance that inhibits or activates a molecule characteristic of a disease, that is, a molecular target therapeutic agent. Molecular targeted therapeutic agents are characterized by high efficacy because they start treatment after identifying a molecule associated with a disease at the stage of diagnosis.
疾患に関連した生体分子の同定が最も進んでいるのが癌であり、いくつかのキナーゼの異常が明らかになりつつある。キナーゼは、基質特異的なリン酸化反応を触媒する酵素で、遺伝子解析の結果からヒトには518種存在すると推定されている（非特許文献１）。これらのキナーゼは、秩序立ったリン酸化経路からなるシグナルネットワークを形成しており、細胞外からの情報に応答して、代謝、転写、細胞増殖、細胞運動、アポトーシス、あるいは細胞分化等を制御する。癌細胞では、キナーゼの異常により、細胞増殖、生存能や細胞運動に関するシグナルが活性化しており、細胞分化やアポトーシスを誘導するシグナルが抑制されている。従って、これらのキナーゼを制御する物質は、癌の分子標的治療薬として応用できる。Epidermal Growth Factor Receptor (EGFR)ファミリーのキナーゼのひとつであるHuman epidermal growth factor receptor 2 （Her2）が過剰に発現した転移性乳癌や胃癌に対する分子標的治療薬としてHer2を阻害する抗体・ハーセプチン（トラスツズマブ）が、1998年に米国で乳癌の治療薬とて認可され、2001年に日本でも承認されている。また、日本のがん死亡原因の1位である肺癌の多くはEGFRに異常があることが明らかとなり、EGFRの分子標的治療薬が開発されている。イレッサ（ゲフィチニブ）とタルセバ（エルロチニブ）は、EGFRキナーゼ阻害剤であり、EGF受容体遺伝子が変異しキナーゼ活性が亢進した非小細胞肺癌患者に著効し、30カ月程度の長期生存を可能にした。 The most advanced identification of disease-related biomolecules is cancer, and several kinase abnormalities are becoming apparent. Kinases are enzymes that catalyze a substrate-specific phosphorylation reaction, and it is estimated that 518 types exist in humans from the results of gene analysis (Non-patent Document 1). These kinases form a signal network consisting of ordered phosphorylation pathways that control metabolism, transcription, cell proliferation, cell motility, apoptosis, or cell differentiation in response to information from outside the cell. . In cancer cells, kinase abnormalities activate signals related to cell proliferation, viability, and cell motility, and suppress signals that induce cell differentiation and apoptosis. Therefore, substances that regulate these kinases can be applied as molecular target therapeutics for cancer. Herceptin (Trastuzumab), an antibody that inhibits Her2, is a molecularly targeted treatment for metastatic breast cancer and gastric cancer in which human epidermal growth factor receptor 2 (Her2), one of the epidermal growth factor receptor (EGFR) family kinases, is overexpressed. In 1998, it was approved as a treatment for breast cancer in the United States, and in Japan in 2001. In addition, many of the lung cancers that are the leading cause of cancer death in Japan have been found to have abnormalities in EGFR, and molecular target therapeutics for EGFR have been developed. Iressa (gefitinib) and Tarceva (erlotinib) are EGFR kinase inhibitors that have been effective in patients with non-small cell lung cancer whose EGF receptor gene has been mutated and kinase activity increased, enabling long-term survival of about 30 months .
一方、癌以外の疾患の分子標的治療薬の開発も進んでいる。アクテムラ(トシリズマブ)は、国産初、また世界発のIL-6阻害剤として2005年に発売された。アクテムラは、ヒト化抗ヒトIL-6受容体抗体で、抗リウマチ薬としてキャッスルマン病や関節リウマチ、全身型若年性特発性関節炎の治療に用いられている。 On the other hand, development of molecular targeted therapeutics for diseases other than cancer is also progressing. Actemra (tocilizumab) was first launched in 2005 as the first domestically produced IL-6 inhibitor in the world. Actemra is a humanized anti-human IL-6 receptor antibody and is used as an anti-rheumatic drug for the treatment of Castleman's disease, rheumatoid arthritis, and systemic juvenile idiopathic arthritis.
このように、現在、様々な疾患に関連する生体分子が明らかにされるとともに、それらの分子に対する分子標的治療薬の開発が急速に進んでおり、これまで治療法のなかった難治性疾患に対する治療が可能になるものと期待されている。 In this way, biomolecules related to various diseases have been elucidated, and the development of molecular targeted therapeutic agents for these molecules is progressing rapidly. Treatment for intractable diseases for which there has been no therapeutic method so far Is expected to be possible.
現在、癌の悪性化や薬剤耐性に関与する極めて重要な分子が次々と明らかになっているが、分子標的治療薬としての医薬品シーズの開発が立ち遅れているものも少なくない。そういった標的分子として、Mesenchymal-Epithelial Transition factor（MET）や、Aurora kinase、Fms-like tyrosine kinase 3（FLT-3）がある。また、Tyrosine kinase receptor A（TrkA）は、癌の増殖や転移のみならず、癌等の疾患にともなう難治性の疼痛やアトピー性皮膚炎の掻痒に関与していることから、TrkAを標的とした阻害剤の有効性が期待されているが、分子標的治療薬の開発には至っていない。 At present, extremely important molecules involved in cancer malignancy and drug resistance have been revealed one after another, but there are many cases where development of pharmaceutical seeds as molecular targeted therapeutic drugs has been delayed. Such target molecules include Mesenchymal-Epithelial Transition Factor (MET), Aurora kinase, and Fms-like tyrosine kinase 3 (FLT-3). Tyrosine kinase receptor A (TrkA) is targeted not only for cancer growth and metastasis, but also for intractable pain associated with diseases such as cancer and pruritus of atopic dermatitis. Inhibitors are expected to be effective, but molecular target therapeutics have not been developed.
METは、肝細胞増殖因子（HGF）の受容体で、様々な癌種において遺伝子の増幅や変異、過剰発現が報告されてきた（非特許文献２）。METはHGFの刺激で活性化され、腫瘍増殖や浸潤・転移を促進する。また、最近、非小細胞肺癌の治療薬であるイレッサやタルセバに対する耐性獲得にMETが関与していることが明らかになった。EGFRキナーゼ阻害剤であるイレッサやタルセバは、著効してもほぼ例外なく1年から数年以内に耐性を獲得し、がんが再燃することが臨床上重大な問題となっている。これらのEGFRキナーゼ阻害剤に対する耐性獲得の分子メカニズムとして、EGFR遺伝子の変異とMETの過剰発現が報告されており（非特許文献３，４）、EGFRキナーゼ阻害剤にMET阻害剤を併用することによって、耐性を克服できる可能性が高いが、現在、MET阻害剤は医薬品化されていない。2010年から非小細胞肺がん患者に対して、MET阻害剤ARQ197とタルセバ併用の第II相試験が行われ、タルセバ単剤群に比べて無増悪生存期間を有意に延長したことが報告された（非特許文献５）。しかし、2012年に第III相試験が、間質性肺炎の発症率の上昇のため中断された。このようなことから、安全性の高い新たなMET阻害剤の開発が求められている。本発明では、MET発現癌の増殖を抑制する治療薬（抗癌剤）、浸潤・転移を抑制する治療薬（抗転移剤）、及びEGFRキナーゼ阻害剤耐性非小細胞肺癌の薬剤耐性を抑制する治療薬（薬剤耐性抑制剤）として有用なMET阻害剤を提供することを目的とする。 MET is a receptor for hepatocyte growth factor (HGF), and gene amplification, mutation, and overexpression have been reported in various cancer types (Non-patent Document 2). MET is activated by stimulation of HGF and promotes tumor growth, invasion and metastasis. Recently, it has become clear that MET is involved in acquiring resistance to Iressa and Tarceva, which are treatments for non-small cell lung cancer. Irresa and Tarceva, which are EGFR kinase inhibitors, have acquired resistance within one to several years, with almost no exception, even if they are effective, and a serious clinical problem is that the cancer relapses. As a molecular mechanism for acquiring resistance to these EGFR kinase inhibitors, mutation of EGFR gene and overexpression of MET have been reported (Non-Patent Documents 3 and 4), and by using MET inhibitor in combination with EGFR kinase inhibitor Although it is likely that resistance can be overcome, MET inhibitors are not currently medicinalized. Since 2010, a phase II study of MET inhibitor ARQ197 and Tarceva has been conducted in patients with non-small cell lung cancer and reported a significant increase in progression-free survival compared to the Tarceva monotherapy group ( Non-patent document 5). However, a phase III trial was discontinued in 2012 due to an increased incidence of interstitial pneumonia. For this reason, development of a new MET inhibitor with high safety is required. In the present invention, a therapeutic agent (anticancer agent) that suppresses proliferation of MET-expressing cancer, a therapeutic agent that suppresses invasion / metastasis (antimetastatic agent), and a therapeutic agent that suppresses drug resistance of EGFR kinase inhibitor-resistant non-small cell lung cancer An object is to provide a MET inhibitor useful as a (drug resistance suppressor).
Aurora kinase A及びAurora kinase Bは、細胞周期を調節するキナーゼで、これらを阻害すると細胞周期がG2/M期で停止する。Aurora kinase の発現は、種々の癌で亢進しており、予後不良と相関している（非特許文献６）。現在、Aurora kinaseに対する分子標的治療薬の開発が進められているが、医薬品として承認されたものはない。本発明では、癌の増殖を抑制する治療薬（抗癌剤）として有用なAurora kinase A 及びAurora kinase B阻害剤を提供することを目的とする。 Aurora kinase A and Aurora kinase B are kinases that regulate the cell cycle. When these are inhibited, the cell cycle stops at the G2 / M phase. The expression of Aurora kinase is increased in various cancers and correlates with poor prognosis (Non-patent Document 6). Currently, molecular targeted therapeutics for Aurora kinase are being developed, but no drugs have been approved. An object of the present invention is to provide Aurora kinase A and Aurora kinase B inhibitors useful as therapeutic agents (anticancer agents) that suppress cancer growth.
FLT-3は、急性骨髄性白血病（AML）の患者の約30%に遺伝子変異が確認され、その生命予後が不良であることが報告されている（非特許文献７）。現在、FLT-3に対する分子標的治療薬の開発が進められているが、医薬品として承認されたものはない。本発明では、AMLの増殖を抑制する治療薬（抗癌剤）として有用なFLT-3阻害剤を提供することを目的とする。
Trk Aは、乳癌の増殖と転移の促進に関与していることが報告されている（非特許文献８）。現在、Trk Aに対する分子標的治療薬の開発が進められているが、医薬品として承認されたものはない。本発明では、TrkAを発現する癌の増殖を抑制する治療薬（抗癌剤）として有用なTrk A阻害剤を提供することを目的とする。
FLT-3 has been reported to have a genetic mutation in approximately 30% of patients with acute myeloid leukemia (AML) and have a poor life prognosis (Non-patent Document 7). Currently, molecular targeted therapeutics for FLT-3 are being developed, but no drugs have been approved. An object of the present invention is to provide a FLT-3 inhibitor useful as a therapeutic agent (anticancer agent) that suppresses the proliferation of AML.
Trk A has been reported to be involved in the promotion of breast cancer growth and metastasis (Non-patent Document 8). Currently, molecular targeted therapeutics for Trk A are being developed, but no drug has been approved. An object of the present invention is to provide a Trk A inhibitor useful as a therapeutic agent (anticancer agent) that suppresses the growth of a cancer that expresses TrkA.
最近、Nerve growth factor （NGF）とその受容体であるTrkAが疼痛や掻痒に関与していることが明らかになってきた。TrkAは、約40-50%の感覚神経に発現している受容体型キナーゼであり、感覚神経の末端が分布する臓器や組織（筋肉、皮膚、血管など）においてそのリガンドであるNGFが発現・分泌されることでNGF-TrkAシグナルが活性化され、末梢の疼痛を惹起する。したがって、TrkAのリン酸化を抑制する化合物は、鎮痛作用を有することが期待される。既存の治療薬ではコントロールが困難な癌性疼痛をはじめ、自己免疫疾患、外傷、脊椎疾患、糖尿病等に伴う疼痛は、NGF-TrkAシグナルが関与していることが明らかとなっている（非特許文献９）。また、TrkA阻害剤は、疼痛のみならず、アトピー性皮膚炎の掻痒に対しても有効であると期待されている。正常な皮膚の知覚神経は、真皮内部に留まっているが、アトピー性皮膚炎では表皮細胞が産生するNGFの作用によって表皮内まで知覚神経線維が伸長することで痒みの閾値が低下し、掻破行動を誘発すると考えられており、実際、マウスを用いたモデル実験で、NGF阻害剤が皮膚炎を抑制することが報告されている(非特許文献１０)。このような背景から、TrkAを標的とした分子標的治療薬の開発が試みられているが、臨床応用までは進んでいない。本発明では、難治性疼痛の治療薬（疼痛抑制剤）及びアトピー性皮膚炎の止痒薬として有用なTrkA阻害剤を提供することを目的とする。 Recently, it has become clear that Nerve growth factor (NGF) and its receptor TrkA are involved in pain and pruritus. TrkA is a receptor-type kinase expressed in about 40-50% of sensory nerves, and its ligand NGF is expressed and secreted in organs and tissues (muscles, skin, blood vessels, etc.) where sensory nerve terminals are distributed. As a result, the NGF-TrkA signal is activated, causing peripheral pain. Therefore, a compound that suppresses phosphorylation of TrkA is expected to have an analgesic action. NGF-TrkA signaling has been shown to be involved in pain associated with autoimmune diseases, trauma, spinal diseases, diabetes, etc., including cancer pain that is difficult to control with existing therapies (non-patented) Reference 9). Further, TrkA inhibitors are expected to be effective not only for pain but also for pruritus of atopic dermatitis. Normal skin sensory nerves remain inside the dermis, but in atopic dermatitis, sensory nerve fibers extend into the epidermis due to the action of NGF produced by epidermal cells, reducing the itch threshold and scratching behavior In fact, it has been reported that NGF inhibitors suppress dermatitis in model experiments using mice (Non-patent Document 10). Against this background, attempts have been made to develop molecular targeted therapeutic drugs targeting TrkA, but clinical application has not progressed. An object of the present invention is to provide a TrkA inhibitor useful as a therapeutic agent for intractable pain (pain suppressant) and an antipruritic agent for atopic dermatitis.
初期の分子標的治療薬は、特異的なキナーゼ阻害剤を目標として開発されてきた（特許文献１、２）。しかし、最近の臨床研究から、複数のキナーゼを阻害するマルチキナーゼ阻害剤の方が、効果が高いと考えられるようになってきた。なぜなら、癌細胞では様々なキナーゼが同時に活性化しているため、それらを総合的に阻害する方が、癌に対して高い抑制効果が得られるからである。例えば、METとAurora kinase を発現している癌では、癌転移と腫瘍増殖の両方を一剤で治療できる。また、同癌患者に、癌性疼痛の症状があれば、同時に疼痛の治療も可能となる。これにより従来の細胞障害性抗癌剤では効果がほとんどなかった癌であっても、標的となる分子を発現している場合は、相加的あるいは相乗的に優れた治療効果をもたらす。また、投与する薬剤を少なくすることができるため、副作用を軽減することができる。 Early molecular targeted therapeutic agents have been developed with the goal of specific kinase inhibitors (Patent Documents 1 and 2). However, recent clinical studies have led to the belief that multikinase inhibitors that inhibit multiple kinases are more effective. This is because various kinases are simultaneously activated in cancer cells, so that comprehensive inhibition of them can provide a higher suppressive effect on cancer. For example, in cancers expressing MET and Aurora kinase, both cancer metastasis and tumor growth can be treated with a single agent. In addition, if the cancer patient has symptoms of cancer pain, the pain can be treated at the same time. As a result, even if the cancer is almost ineffective with the conventional cytotoxic anticancer agent, when the target molecule is expressed, the therapeutic effect is excellent additively or synergistically. In addition, since fewer drugs can be administered, side effects can be reduced.
本発明はかかる問題点に鑑みてなされたものであって、マルチキナーゼ阻害剤、抗癌剤、抗転移剤、薬剤耐性抑制剤、疼痛抑制剤及び止痒薬を提供することを目的とする。 This invention is made | formed in view of this problem, Comprising: It aims at providing a multikinase inhibitor, an anticancer agent, an anti-metastatic agent, a drug resistance inhibitor, a pain inhibitor, and an antidiarrheal agent.
本発明にかかるマルチキナーゼ阻害剤、抗癌剤、抗転移剤、薬剤耐性抑制剤、疼痛抑制剤及び止痒薬は、式１を備えるフラボノールを有効成分として含有する。 The multikinase inhibitor, anticancer agent, antimetastasis agent, drug resistance inhibitor, pain inhibitor and antidiarrheal agent according to the present invention contain a flavonol comprising Formula 1 as an active ingredient.
ここで、Ｒは式２又は式３である Here, R is Formula 2 or Formula 3.
式１においてＲが式２の場合は、上記フラボノールはヘルバセチンである。また、式１においてＲが式３の場合は、上記フラボノールはゴシペチンである。 In Formula 1, when R is Formula 2, the flavonol is herbacetin. Moreover, when R is Formula 3 in Formula 1, the flavonol is gosipetin.
ヘルバセチンは、配糖体として、イワベンケイ（岩弁慶、Rhodiola rosea）、アマ（亜麻、Linum usitatissimum）等から単離されている（非特許文献１１，１２）。また、ゴシペチンは、配糖体として、イワベンケイ（岩弁慶、Rhodiola rosea）、ローゼル（Hibiscus sabdariffa）等から単離されている（非特許文献１１，１３）。 Herbacetin has been isolated as a glycoside from Iwabenkei (Rhodiola rosea), flax (linum usitatissimum) and the like (Non-patent Documents 11 and 12). Moreover, gosipetin has been isolated as a glycoside from Iwabenkei (Rhodiola rosea), Roselle (Hibiscus sabdariffa) and the like (Non-Patent Documents 11 and 13).
これまでに、これらの化合物について、いくつかの薬理作用が報告されている。ヘルバセチンは、抗酸化作用を有すること（非特許文献１４）、ノイラミニダーゼ阻害作用による抗インフルエンザウイルス作用を有すること（非特許文献１５）、ジメチルエーテル付加体がKA3IT細胞とNIH3T3細胞に対して細胞毒性を示し、EGF受容体によるリン酸化反応を抑制すること（非特許文献１６）、配糖体が抗酸化作用を有すること（非特許文献１７）、が知られている。また、ゴシペチンは、ノイラミニダーゼ阻害作用による抗インフルエンザウイルス作用を有すること（非特許文献１４）、低比重リポ蛋白を修飾してマクロファージに取り込まれやすくさせる作用を有すること（非特許文献１８）、アロマターゼ阻害作用を有すること（非特許文献１９）が知られている。しかしながら、これらの化合物が、MET、FLT3、Aurora kinase、TrkA等の複数のキナーゼに対する阻害作用、すなわちマルチキナーゼ阻害作用を有することは知られていない。
本発明によれば、複数のキナーゼを阻害するマルチキナーゼ阻害剤が得られる。即ち、癌細胞の増殖や転移を促す複数のチロシンキナーゼを狙い撃ち、更に疼痛に関与するキナーゼも阻害するため、従来の細胞障害性抗癌剤では効果がほとんどなかった悪性腫瘍の増殖や転移に対して優れた治療効果をもたらすと同時に、薬剤耐性の抑制や癌性疼痛の治療を行うことができる。また、本発明によれば、複数の薬剤を使用せずに、単一の薬剤により複数のキナーゼを阻害するため、癌治療において投与する薬剤を少なくすることができ、これにより患者に対する副作用を軽減することができる。また、本発明によれば、癌の悪性化や薬剤耐性に関与する極めて重要な分子にも関わらず、分子標的治療薬としての医薬品シーズの開発が立ち遅れている、Mesenchymal-Epithelial Transition factor（MET）、Aurora kinase、Fms-like tyrosine kinase 3（FLT-3）及びTyrosine kinase receptor A（TrkA）に対する分子標的治療薬が得られる。更に、TrkAが関わる疾患の治療薬、例えば、癌性疼痛を含む難治性の疼痛治療薬やアトピー性皮膚炎の止痒薬が得られる。 According to the present invention, a multikinase inhibitor that inhibits a plurality of kinases can be obtained. In other words, it aims at multiple tyrosine kinases that promote cancer cell growth and metastasis, and also inhibits kinases involved in pain, so it is excellent against the growth and metastasis of malignant tumors that were hardly effective with conventional cytotoxic anticancer agents In addition to providing therapeutic effects, it is possible to suppress drug resistance and treat cancer pain. Further, according to the present invention, since a plurality of kinases are inhibited by a single drug without using a plurality of drugs, the number of drugs administered in cancer treatment can be reduced, thereby reducing side effects on patients. can do. In addition, according to the present invention, despite the extremely important molecules involved in cancer malignancy and drug resistance, the development of pharmaceutical seeds as molecular target therapeutics has been delayed, Mesenchymal-Epithelial Transition factor (MET) , Aurora kinase, Fms-like tyrosine kinase 3 (FLT-3) and Tyrosine kinase receptor A (TrkA) are targeted molecules. Furthermore, therapeutic agents for diseases involving TrkA, such as intractable pain therapeutic agents including cancer pain, and antidiarrheal agents for atopic dermatitis can be obtained.
以下、添付の図面を参照して本発明の実施形態について具体的に説明するが、当該実施形態は本発明の原理の理解を容易にするためのものであり、本発明の範囲は、下記の実施形態に限られるものではなく、当業者が以下の実施形態の構成を適宜置換した他の実施形態も、本発明の範囲に含まれる。 Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. However, the embodiments are for facilitating understanding of the principle of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiments, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.
本発明者らは鋭意研究により、式１を基本骨格として備えるフラボノールがマルチキナーゼ阻害活性を有することを初めて見出し、この新知見に基づいて本発明を完成させた。 The inventors of the present invention have found for the first time that flavonols having Formula 1 as a basic skeleton have multikinase inhibitory activity through intensive studies, and have completed the present invention based on this new finding.
ここで、Ｒは式２又は式３である Here, R is Formula 2 or Formula 3.
式１においてＲが式２の場合は、上記フラボノールはヘルバセチンである。また、式１においてＲが式３の場合は、上記フラボノールはゴシペチンである。 In Formula 1, when R is Formula 2, the flavonol is herbacetin. Moreover, when R is Formula 3 in Formula 1, the flavonol is gosipetin.
本発明において、実施例で用いたヘルバセチン及びゴシペチンは、和光純薬工業株式会社から購入したものを用いた。 In the present invention, those purchased from Wako Pure Chemical Industries, Ltd. were used as the herbacetin and gosipetin used in the examples.
マルチキナーゼ阻害活性の一つは、MET阻害活性である。METは、肝細胞増殖因子（HGF）の受容体で、癌細胞の増殖や運動能を促進するシグナルに関与しており、METを阻害すると細胞増殖や細胞運動が抑制されることから、MET阻害活性を備えることは抗癌抗転移薬として機能することを意味する。また、METは非小細胞肺癌のイレッサやタルセバの耐性に関与していることから、MET阻害により耐性非小細胞肺癌の治療が可能となる。 One of the multikinase inhibitory activities is the MET inhibitory activity. MET is a receptor for hepatocyte growth factor (HGF) and is involved in signals that promote cancer cell growth and motility. Inhibiting MET inhibits cell growth and motility. Having activity means functioning as an anticancer antimetastatic agent. In addition, since MET is involved in the resistance of Iressa and Tarceva in non-small cell lung cancer, MET inhibition makes it possible to treat resistant non-small cell lung cancer.
また、マルチキナーゼ阻害活性の他の一つは、Aurora kinase阻害活性である。ヒトにおいてAurora family には３種類（Aurora A、B、C)が存在するが、本発明においては、Aurora kinase AとAurora kinase Bである。Aurora kinase A及びAurora kinase Bは、細胞周期を調節する分子で、これらを阻害すると細胞周期がG2/M期で停止し、癌細胞の増殖が抑制される。本発明において式１を備えるフラボノールは、Aurora kinase A及びAurora kinase Bに対するキナーゼ阻害作用を有するため、細胞周期調節剤となる。 Another one of the multikinase inhibitory activities is the Aurora kinase inhibitory activity. In humans, there are three types of Aurora family (Aurora A, B, C). In the present invention, they are Aurora kinase A and Aurora kinase B. Aurora kinase A and Aurora kinase B are molecules that regulate the cell cycle. When they are inhibited, the cell cycle stops at the G2 / M phase, and the growth of cancer cells is suppressed. In the present invention, the flavonol having Formula 1 has a kinase inhibitory action on Aurora kinase A and Aurora kinase B, and thus becomes a cell cycle regulator.
また、マルチキナーゼ阻害活性の他の一つは、FLT3阻害活性である。Fms-Like Tyrosine Kinase 3（FLT3） はclass III receptor tyrosine kinase family に属し、急性骨髄性白血病（AML）で最も高頻度に活性化変異が認められ、なかでも、FLT3-Internal Tandem Duplication（ITL）変異は、AML患者の30％に観察され、再発・難治性を示す。本発明において式１を備えるフラボノールは、FLT3に対するキナーゼ阻害活性を有するため、細胞増殖を抑制し、例えばAMLの治療薬となる。 Another multikinase inhibitory activity is FLT3 inhibitory activity. Fms-Like Tyrosine Kinase 3 (FLT3) belongs to the class III receptor tyrosine kinase family and has the most frequent activating mutation in acute myeloid leukemia (AML), including FLT3-Internal Tandem Duplication (ITL) mutation Is observed in 30% of patients with AML and is relapsed or refractory. In the present invention, the flavonol having Formula 1 has a kinase inhibitory activity against FLT3, and therefore suppresses cell proliferation and becomes a therapeutic agent for AML, for example.
また、マルチキナーゼ阻害活性の他の一つは、TrkAに対するキナーゼ阻害活性である。TrkAは、Nerve growth factor（NGF）の受容体であり、TrkAを発現する癌の増殖と転移の促進に関与している。そのため、本発明において式１を備えるフラボノールは、例えばTrkA発現乳癌の治療薬となる。 Another one of the multikinase inhibitory activities is a kinase inhibitory activity against TrkA. TrkA is a receptor for Nerve growth factor (NGF) and is involved in promoting the growth and metastasis of cancers that express TrkA. Therefore, the flavonol having Formula 1 in the present invention is, for example, a therapeutic agent for TrkA-expressing breast cancer.
さらに、TrkAは、末梢神経での疼痛や掻痒の伝達受容体として機能している。本発明において式１を備えるフラボノールは、TrkAに対するキナーゼ阻害活性を有するため、癌の骨転移による疼痛やアトピー性皮膚炎の掻痒に対し、NGF-TrkAシグナルをブロックすることで高い鎮痛作用や止痒作用が得られ、癌患者等の疼痛鎮痛薬あるいは止痒薬となる。 Furthermore, TrkA functions as a transmission receptor for pain and pruritus in peripheral nerves. In the present invention, the flavonol having Formula 1 has kinase inhibitory activity against TrkA, and thus it is highly analgesic and antipruritic by blocking NGF-TrkA signal against pain due to bone metastasis of cancer and pruritus of atopic dermatitis. The effect is obtained, and it becomes a pain analgesic or antipruritic agent for cancer patients and the like.
本発明において、上記フラボノールは配糖体として用いることも可能である。フラボノイドに結合している糖類としては、特に限定されるものではないが、例えば、グルコース、ガラクトース、ラムノース、キシロース、アラビノース、アピオース等の単糖、ルチノース、ネオヘスペリドース、ソフォロース、サンブビオース、ラミナリビオース等の二糖、ゲンチオトリオース、グルコシルルチノース、グルコシルネオヘスペリドース等の三糖、これらの糖に更に糖類が結合したもの、又はこれらの混合物が挙げられる。 In the present invention, the flavonol can also be used as a glycoside. The saccharide bound to the flavonoid is not particularly limited, but examples thereof include monosaccharides such as glucose, galactose, rhamnose, xylose, arabinose, and apiose, rutinose, neohesperidose, sofolose, sambubiose, and laminaribiose. And the like, disaccharides such as gentiotriose, glucosyl rutinose, glucosyl neohesperidose, etc., those obtained by further linking saccharides to these sugars, or mixtures thereof.
本発明において式１を基本骨格として備えるフラボノールは、上述したように、TrkAに対するキナーゼ阻害活性、MET阻害活性、及び、FLT3阻害活性を有するため、例えば、TrkA発現乳癌、MET発現肝癌、及びFLT3発現AMLの治療のために用いられる。 In the present invention, the flavonol having Formula 1 as a basic skeleton has a kinase inhibitory activity, a MET inhibitory activity, and a FLT3 inhibitory activity against TrkA as described above. For example, TrkA-expressing breast cancer, MET-expressing liver cancer, and FLT3 expression Used for the treatment of AML.
例えば乳癌細胞においてTGFβ刺激によるEMT誘導によってGEP100-Arf6-AMAP1経路が受容体型チロシンキナーゼであるMETの活性化を介して活性化されることが知られている。また例えばAurora kinase Aは、一対の中心小体(centriole)及びそれを取り巻く中心小体外周物質から構成される細胞内小器官である中心体に局在し、中心体の成熟と分離に必須の働きを行っており、乳癌等の多くのヒトの癌でAurora kinase Aの遺伝子増幅や過剰発現が報告されている。また例えばFLT3は主に幼若造血細胞の細胞膜上に発現し、血液細胞の分化・増殖と造血幹細胞の自己複製における重要なシグナル伝達機構に関与しており、上述のようにFLT3はAMLで最も高頻度に活性化変異が認められるが、乳癌においても発現が報告されている。そこで、例えば本発明にかかるマルチキナーゼ阻害剤を乳癌患者に投与することにより、高い鎮痛効果とともに癌細胞増殖を抑制することができ、総合的に極めて高い治療効果が得られうる。 For example, it is known that GEP100-Arf6-AMAP1 pathway is activated through activation of MET, which is a receptor tyrosine kinase, by induction of EMT by TGFβ stimulation in breast cancer cells. For example, Aurora kinase A is localized in the centrosome, an intracellular organelle composed of a pair of centriole and surrounding centriole, and is essential for centrosome maturation and separation. Aurora kinase A gene amplification and overexpression have been reported in many human cancers such as breast cancer. For example, FLT3 is expressed mainly on the cell membrane of immature hematopoietic cells and is involved in important signal transduction mechanisms in blood cell differentiation and proliferation and hematopoietic stem cell self-renewal. Although activating mutations are frequently observed, expression has also been reported in breast cancer. Therefore, for example, by administering the multikinase inhibitor according to the present invention to a breast cancer patient, cancer cell proliferation can be suppressed together with a high analgesic effect, and an extremely high therapeutic effect can be obtained comprehensively.
本発明にかかるマルチキナーゼ阻害剤、抗癌剤、抗転移剤、疼痛抑制剤、及び止痒薬は、例えば、錠剤、顆粒剤、細粒剤、カプセル剤等のような経口投与の製剤、経口投与に適した様々な液体製剤、又は注射剤のような非経口投与用製剤とすることが可能である。 The multikinase inhibitor, anticancer agent, antimetastasis agent, pain suppressing agent, and antipruritic agent according to the present invention are, for example, for oral administration such as tablets, granules, fine granules, capsules, etc. It can be a variety of suitable liquid preparations or preparations for parenteral administration such as injections.
経口投与の製剤の場合、式(1)を備えるフラボノールの微粉末を製剤担体と共に錠剤、顆粒剤、細粒剤、カプセル剤等の形態で製剤化して得られる。 In the case of a preparation for oral administration, it is obtained by formulating a fine powder of flavonol having the formula (1) together with a preparation carrier in the form of tablets, granules, fine granules, capsules and the like.
製剤担体としては、賦形剤、結合剤、崩壊剤、滑沢剤、及び可塑剤等を使用できる。賦形剤としては、例えば、白糖、塩化ナトリウム、マンニトール、乳糖、ブドウ糖、でんぷん、炭酸カルシウム等を使用できる。結合剤としては、例えば、水、エタノール、プロパノール、ブドウ糖液、デンプン液、ゼラチン液、等を使用できる。崩壊剤としては、例えば、カルボキシメチルセルロースカルシウム、乾燥デンプン、炭酸水素ナトリウム、等を使用できる。滑沢剤としては、例えば、精製タルク、ステアリン酸塩、ホウ酸末、ポリエチレングリコール等を使用できる。可塑剤としては、例えば、グリセリン脂肪酸エステル、ジオクチルフタレート、ジブチルフタレート、ヒマシ油等を使用できる。 As the pharmaceutical carrier, excipients, binders, disintegrants, lubricants, plasticizers and the like can be used. As the excipient, for example, sucrose, sodium chloride, mannitol, lactose, glucose, starch, calcium carbonate and the like can be used. As the binder, for example, water, ethanol, propanol, glucose solution, starch solution, gelatin solution and the like can be used. As the disintegrant, for example, carboxymethylcellulose calcium, dry starch, sodium hydrogen carbonate, and the like can be used. As the lubricant, for example, purified talc, stearate, boric acid powder, polyethylene glycol and the like can be used. As the plasticizer, for example, glycerin fatty acid ester, dioctyl phthalate, dibutyl phthalate, castor oil and the like can be used.
上記の経口投与の製剤化の場合、水溶性高分子及び界面活性剤等の分散剤を使用することも可能である。水溶性高分子としては、例えば、ヒドロキシプロピルセルロース、ポリビニルアルコール、ポリビニルピロリドン等を使用できる。界面活性剤としては、例えば、ラウリル硫酸ナトリウム、ラウリル硫酸マグネシウム等を使用できる。 In the case of the above-mentioned formulation for oral administration, it is possible to use a dispersing agent such as a water-soluble polymer and a surfactant. As the water-soluble polymer, for example, hydroxypropyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone and the like can be used. As the surfactant, for example, sodium lauryl sulfate, magnesium lauryl sulfate and the like can be used.
錠剤を調製するには、式(1)を備えるフラボノールの微粉末を上記製剤担体を用いて常法により錠剤とする。顆粒剤又は細粒剤は、式(1)を備えるフラボノールの微粉末に上記製剤担体を添加し、流動層造粒、高速攪拌造粒、攪拌流動層造粒、遠心流動造粒、押し出し造粒等で顆粒化することにより調製できる。カプセル剤は、不活性な医薬充填剤又は希釈剤と共に混合して調製し、硬ゼラチンカプセル又は軟カプセルに詰める。 To prepare a tablet, a fine powder of flavonol having the formula (1) is made into a tablet by a conventional method using the above-mentioned preparation carrier. Granules or fine granules are prepared by adding the above formulation carrier to a flavonol fine powder having the formula (1), fluidized bed granulation, high speed agitation granulation, agitated fluidized bed granulation, centrifugal fluidized granulation, extrusion granulation Etc., and can be prepared by granulating. Capsules are prepared by mixing with an inert pharmaceutical filler or diluent and packed into hard gelatin capsules or soft capsules.
経口液体製剤は、式(1)を備えるフラボノールと、甘味料（例えば、ショ糖）、保存剤（例えば、メチルパラベン、プロピルパラベン）、着色料、香料等とを混合して調製する。 An oral liquid preparation is prepared by mixing a flavonol having the formula (1) with a sweetener (for example, sucrose), a preservative (for example, methylparaben, propylparaben), a colorant, a flavor, and the like.
注射用製剤は、例えば、液剤、乳濁液、又は懸濁液の形態で調製され、血液に対して等張にされる。液体、乳濁液又は懸濁液の形態の製剤は、例えば、水性媒体、エチルアルコール、プロピレングリコール、エトキシ化イソステアリルアルコール等を用いて調製される。 Injectable preparations are prepared, for example, in the form of solutions, emulsions or suspensions and are made isotonic with blood. Formulations in the form of liquids, emulsions or suspensions are prepared using, for example, an aqueous medium, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol and the like.
注射用製剤において、当技術分野で通常使用されている添加剤を適宜用いることができ、例えば、等張化剤、安定化剤、緩衝剤、保存剤、キレート剤、抗酸化剤等を使用できる。等張化剤としては、例えば、ブドウ糖、ソルビトール、マンニトール等が挙げられる。安定化剤としては、例えば亜硫酸ナトリウム等が挙げられる。緩衝剤としては、例えば、ホウ酸緩衝剤、リン酸緩衝剤、クエン酸緩衝剤等が挙げられる。保存剤としては、例えば、パラオキシ安息香酸エステル、ベンジルアルコール等が挙げられる。キレート剤としては、例えば、エデト酸ナトリウム、クエン酸ナトリウム等が挙げられる。抗酸化剤としては、例えば、亜硫酸ナトリウム、亜硫酸水素ナトリウム等が挙げられる。 In injectable preparations, additives commonly used in the art can be used as appropriate, for example, isotonic agents, stabilizers, buffers, preservatives, chelating agents, antioxidants, and the like can be used. . Examples of the isotonic agent include glucose, sorbitol, mannitol and the like. Examples of the stabilizer include sodium sulfite. Examples of the buffer include borate buffer, phosphate buffer, and citrate buffer. Examples of the preservative include paraoxybenzoic acid ester and benzyl alcohol. Examples of chelating agents include sodium edetate and sodium citrate. Examples of the antioxidant include sodium sulfite and sodium hydrogen sulfite.
本願発明のマルチキナーゼ阻害剤、抗癌剤、抗転移剤、疼痛抑制剤、及び止痒薬の投与量としては、例えば、一回の投与につき体重1 kgあたり0.5mgから500mg、好ましくは2.5 mgから25 mgの範囲で投与量が選択できる。しかしながら、本発明の医薬組成物はこれらの投与量に制限されるものではない。 The dosage of the multikinase inhibitor, anticancer agent, antimetastatic agent, pain suppressor and antidiarrheal agent of the present invention is, for example, 0.5 mg to 500 mg per kg body weight per administration, preferably 2.5 mg to 25 The dose can be selected within the range of mg. However, the pharmaceutical composition of the present invention is not limited to these doses.
実施例１ ヒト乳癌由来MDA-MB-231細胞を用いたHGFによって誘導される運動能に対するヘルバセチンの効果
癌細胞の運動能と転移能は高い相関関係があり、癌細胞の運動能を抑制することによって転移を抑制することができる。
Example 1 Effect of herbacetin on the motility induced by HGF using human breast cancer-derived MDA-MB-231 cells The motility and metastasis of cancer cells are highly correlated and suppress the motility of cancer cells Can suppress metastasis.
１）MET陽性のヒト乳癌由来MDA-MB-231細胞を用い、HGFにより誘導される細胞運動能に対するヘルバセチンの抑制効果を、トランスウェルでの移動細胞数を指標に解析した。また、比較対照として、ヘルバセチンの構造に類似したフラボノイドである、式４に示されるケンフェロール、式５に示されるアピゲニン、及び式６に示されるイソスクテラレインを用いた。 1) Using MET-positive human breast cancer-derived MDA-MB-231 cells, the inhibitory effect of herbacetin on cell motility induced by HGF was analyzed using the number of migrating cells in the transwell as an index. As a comparative control, kaempferol represented by the formula 4, apigenin represented by the formula 5, and isoscutellarein represented by the formula 6, which are flavonoids similar to the structure of herbacetin, were used.
下部ウェルは、24ウェルプレートを用い、50 ng/mL HGF添加培地を添加した。無添加培地あるいは5 μg/mLヘルバセチン、ケンフェロール、アピゲニン、あるいは、イソスクテラレイン添加培地に懸濁したMDA-MB-231細胞をトランスウェルに添加し、24ウェルプレートに載せた。CO2インキュベーターで24時間培養した後、トランスウェルから下部ウェルに移動した細胞数を位相差顕微鏡下で計数した。その結果、HGFにより誘導される細胞運動は、ヘルバセチンの添加により最も強く（約90%）阻害された（図１（ａ））。 For the lower well, a 24-well plate was used, and a medium supplemented with 50 ng / mL HGF was added. MDA-MB-231 cells suspended in an additive-free medium or 5 μg / mL herbacetin, kaempferol, apigenin, or an isoscutellarein-added medium were added to a transwell and placed on a 24-well plate. After culturing for 24 hours in a CO 2 incubator, the number of cells migrated from the transwell to the lower well was counted under a phase contrast microscope. As a result, cell motility induced by HGF was inhibited most strongly (about 90%) by the addition of herbacetin (FIG. 1 (a)).
２）上記の実験条件における、細胞の生存能に対するヘルバセチン及び各種フラボノイドの効果を解析した。MDA-MB-231細胞を50 ng/mL HGF添加培地 (Control) あるいは、50 ng/mL HGF及び5μg/mLヘルバセチン、ケンフェロール、アピゲニン、あるいは、イソスクテラレイン添加培地に懸濁し、96ウェルプレートに播種した。CO2インキュベーターで24時間培養した後、WSTアッセイ溶液10 μLを添加して、2時間インキュベートした。その後、マイクロプレートリーダーを用いて450 nmの吸光度を測定した。その結果、アピゲニンの添加で細胞生存能の低下が観察されたが、ヘルバセチンや他のフラボノイドの添加は影響を与えなかった（図１（ｂ））。従って、アピゲニンによる運動能の低下は，細胞生存能の低下によると考えられた。 2) The effects of herbacetin and various flavonoids on cell viability under the above experimental conditions were analyzed. Suspend MDA-MB-231 cells in 50 ng / mL HGF-supplemented medium (Control) or 50 ng / mL HGF and 5 μg / mL herbacetin, kaempferol, apigenin, or isosterrain-supplemented medium. Sowing. After culturing for 24 hours in a CO 2 incubator, 10 μL of WST assay solution was added and incubated for 2 hours. Thereafter, the absorbance at 450 nm was measured using a microplate reader. As a result, a decrease in cell viability was observed with the addition of apigenin, but the addition of herbacetin and other flavonoids had no effect (FIG. 1 (b)). Therefore, the decrease in motility due to apigenin was thought to be due to the decrease in cell viability.
３）種々の濃度のヘルバセチンを用いて、HGFにより誘導される細胞運動に対する効果を解析した結果、ヘルバセチンの添加濃度依存的に運動能が低下することが明らかとなり、そのIC50値は、523 ng/mL（1.7 μM）であった（図２）。 3) As a result of analyzing the effect on cell motility induced by HGF using various concentrations of herbacetin, it was found that the motility decreased depending on the concentration of added herbacetin, and its IC 50 value was 523 ng. / mL (1.7 μM) (FIG. 2).
以上の結果から、ヘルバセチンはMDA-MB-231細胞の生存能に影響を与えない濃度で、HGFによって誘導される細胞運動を抑制することが明らかになった。
実施例２ HGF-METシグナルに対するヘルバセチンの効果
HGFにより誘導されるMETのチロシンリン酸化に対するヘルバセチンの効果をMDA-MB-231細胞を用いて解析した。また、比較対照として、ヘルバセチンの構造に類似したフラボノイドである、ケンフェロール、アピゲニン、及びイソスクテラレインを用いた。
From the above results, it was clarified that herbacetin suppresses cell movement induced by HGF at a concentration that does not affect the viability of MDA-MB-231 cells.
Example 2 Effect of herbacetin on HGF-MET signal
The effect of herbacetin on the tyrosine phosphorylation of MET induced by HGF was analyzed using MDA-MB-231 cells. As a comparative control, kaempferol, apigenin, and isoscutellarein, which are flavonoids similar to the structure of herbacetin, were used.
１）MDA-MB-231細胞を10%血清添加培地で48時間培養した。無添加培地で24時間培養した後、2.5 μg/mLヘルバセチン、ケンフェロール、アピゲニン、あるいは、イソスクテラレイン及び50 ng/mL HGF添加培地で5分間インキュベートし、PBSで洗浄した後、タンパク質抽出用細胞溶解液で細胞を溶解した。細胞の溶解液についてSDS-PAGEを行い、抗リン酸化MET抗体を用いたウエスタンブロッティングで検出した。その結果、HGFによって誘導されるMETのチロシンリン酸化は、ヘルバセチンで抑制されたが、他のフラボノイドでは抑制されなかった（図３）。図中、P-Metはリン酸化Metを示す。 1) MDA-MB-231 cells were cultured in a medium supplemented with 10% serum for 48 hours. Incubate for 24 hours in non-added medium, then incubate with 2.5 μg / mL herbacetin, kaempferol, apigenin, or isucterralein and 50 ng / mL HGF-added medium for 5 minutes, wash with PBS, and for protein extraction Cells were lysed with cell lysate. The cell lysate was subjected to SDS-PAGE and detected by Western blotting using an anti-phosphorylated MET antibody. As a result, tyrosine phosphorylation of MET induced by HGF was suppressed by herbacetin, but not by other flavonoids (FIG. 3). In the figure, P-Met indicates phosphorylated Met.
２）各種濃度のヘルバセチン（0〜5 μg/mL）について、METのチロシンリン酸化の抑制効果を同様の方法で解析した結果、ヘルバセチンの添加濃度依存的に抑制されることが明らかとなった（図４（ａ），図４（ｂ））。
実施例３ ヘルバセチンと各種フラボノイドのMETチロシンキナーゼ活性阻害効果の解析
１）METキナーゼドメインの組換えタンパク質と合成基質ペプチドを用いたキナーゼ阻害アッセイを行った。20 mM HEPES，0.01% Triton X-100，2 mM DTT，25 μM ATP，5 mM MgCl2，pH7.5に0.94 nM 組換えMETキナーゼドメイン，1 μM Srctide及び0.001〜30 μM ヘルバセチンを添加し、室温で1時間反応させた。基質ペプチドと生成したリン酸化ペプチド量をLab Chip 3000（Caliper Lifesciences）を用いて測定した。統計解析ソフトウエア（GraphPad Prism 5）を用いてヘルバセチンの添加濃度に対するリン酸化ペプチドの生成阻害率のプロットに対する4パラメーターロジスティック曲線の近似式を求め、そのパラメーターよりIC50値を算出した。その結果、ヘルバセチンは，濃度依存的にMETのチロシンキナーゼ活性を阻害することが明らかとなり、そのIC50値は、1.284 μMであった（図５）。
2) As a result of analyzing the inhibitory effect of MET on tyrosine phosphorylation in the same way for various concentrations of herbacetin (0-5 μg / mL), it was revealed that it was suppressed depending on the concentration of herbacetin added ( FIG. 4 (a), FIG. 4 (b)).
Example 3 Analysis of MET tyrosine kinase activity inhibitory effect of herbacetin and various flavonoids 1) Kinase inhibition assay using recombinant protein of MET kinase domain and synthetic substrate peptide was performed. 20 mM HEPES, 0.01% Triton X-100, 2 mM DTT, 25 μM ATP, 5 mM MgCl 2 , pH 7.5 with 0.94 nM recombinant MET kinase domain, 1 μM Srctide and 0.001-30 μM herbacetin, room temperature For 1 hour. The amount of the phosphopeptide generated with the substrate peptide was measured using Lab Chip 3000 (Caliper Lifesciences). Using a statistical analysis software (GraphPad Prism 5), an approximate expression of a 4-parameter logistic curve with respect to a plot of the inhibition rate of phosphorylated peptide production against the added concentration of herbacetin was determined, and an IC 50 value was calculated from the parameters. As a result, it was revealed that herbacetin inhibited the tyrosine kinase activity of MET in a concentration-dependent manner, and its IC 50 value was 1.284 μM (FIG. 5).
２）ヘルバセチン及び比較対照として、ヘルバセチンの構造に類似したフラボノイドである、アピゲニン、ケンフェロール、及びケルセチンについて、METキナーゼ活性に対する阻害効果を比較するために、METのキナーゼドメインの組換えタンパク質と合成基質ペプチドを用い、消費ATP量の低下を指標としたキナーゼ阻害アッセイを行った。40 mM Tris-HCl pH7.5，2 mM DTT，7 mM MgCl2，10 μM ATP に4 nM 組換えMETキナーゼドメイン，0.2 μg/mL Poly (Glu4,Tyr1)及び10 μM フラボノイドを添加し、室温で1時間反応させ、ADP-Gloアッセイキット（Promega）を用いて消費ATP量を求めた。フラボノイド無添加における消費ATP量を100%とし、各フラボノイド添加による消費ATPの減少量から阻害率を求めた（表1）。 2) In order to compare the inhibitory effect on MET kinase activity of apigenin, kaempferol, and quercetin, which are flavonoids similar to the structure of herbacetin, as a comparative control, the recombinant protein and synthetic substrate of MET kinase domain A kinase inhibition assay was performed using peptides as indicators of a decrease in the amount of ATP consumed. 40 mM Tris-HCl pH 7.5, 2 mM DTT, 7 mM MgCl 2 , 10 μM ATP, 4 nM recombinant MET kinase domain, 0.2 μg / mL Poly (Glu4, Tyr1) and 10 μM flavonoids are added at room temperature. The reaction was carried out for 1 hour, and the amount of ATP consumed was determined using an ADP-Glo assay kit (Promega). The amount of ATP consumed without the addition of flavonoids was taken as 100%, and the inhibition rate was determined from the amount of ATP consumed with the addition of each flavonoid (Table 1).
その結果、添加濃度10 μMにおける各フラボノイドのMETチロシンキナーゼ活性の阻害率は、ヘルバセチンが最も高い53.7%を示した。なお、上記1)のIC50値は1.284 μMであったが、本実験では10 μMで53.7%の阻害率であった。これはヘルバセチンの阻害効果が、5μM以上からプラトーになること（図５）によると考えられる。 As a result, the inhibition rate of MET tyrosine kinase activity of each flavonoid at an added concentration of 10 μM was 53.7%, which was highest for herbacetin. The IC 50 value of 1) above was 1.284 μM. In this experiment, the inhibition rate was 53.7% at 10 μM. This is considered to be due to the fact that the inhibitory effect of herbacetin becomes a plateau from 5 μM or more (FIG. 5).
以上の結果から、METのキナーゼ活性は、ケルセチン等の比較対照としたフラボノイドよりもヘルバセチンによって最も強く阻害されることが明らかとなった。 From the above results, it became clear that the kinase activity of MET is most strongly inhibited by herbacetin than the flavonoid used as a comparative control such as quercetin.
以上の結果から、ヘルバセチンはMETのチロシンキナーゼを阻害することによってHGFによって誘導される運動能を抑制することが明らかになった。HGFは血清中に存在する運動能誘導因子である。また、MET発現癌細胞が自ら分泌したHGFによるオートクリン作用や、癌細胞の周辺の間充織が分泌したHGFによるパラクリン作用によって運動能を高め、浸潤や転移を促進することが報告されている。 These results indicate that herbacetin suppresses the motility induced by HGF by inhibiting the tyrosine kinase of MET. HGF is a motility inducer present in serum. In addition, autocrine action by HGF secreted by MET-expressing cancer cells itself and paracrine action by HGF secreted by the mesenchyme around the cancer cells have been reported to enhance motility and promote invasion and metastasis. .
従って、ヘルバセチンは、HGF-METシグナルによる癌細胞の運動能を抑制する作用を有することから、MET発現癌の転移抑制剤となることが示された。 Accordingly, herbacetin has been shown to be an inhibitor of metastasis of MET-expressing cancer because it has an action of suppressing the motility of cancer cells by HGF-MET signal.
更に、本実施例からヘルバセチンは、METキナーゼ活性の阻害作用を有することが明らかとなり、MET過剰発現によって生じる癌の転移及び増悪化やMET発現が原因の耐性非小細胞肺癌の治療に用いることができる。
実施例４ ヘルバセチンの網羅的キナーゼ阻害活性の解析
ヘルバセチンのキナーゼ阻害活性の特異性を解明するために、受容体型チロシンキナーゼと細胞周期に関連したキナーゼ等、50種のキナーゼについて網羅的な解析を行った。
Furthermore, it is clear from this example that herbacetin has an inhibitory effect on MET kinase activity, and it can be used for the treatment of resistant non-small cell lung cancer caused by metastasis and exacerbation of cancer caused by MET overexpression and MET expression. it can.
Example 4 Analysis of Comprehensive Kinase Inhibitory Activity of Herbacetin In order to elucidate the specificity of the kinase inhibitory activity of herbacetin, a comprehensive analysis was performed on 50 kinases including receptor tyrosine kinases and cell cycle related kinases. It was.
各キナーゼの組換えタンパク質と基質となる合成ペプチドを用い、リン酸化ペプチドの生成量の低下を指標としたMETキナーゼ阻害アッセイを行った。20 mM HEPES，0.01% Triton X-100，2 mM DTT，25 μM ATP，5 mM MgCl2，pH7.5に0.94 nM 組換えキナーゼドメインと基質となる1 μM合成ペプチド、及び1 μM ヘルバセチンを添加し、室温で1時間反応させた。基質ペプチドと生成したリン酸化ペプチド量をLab Chip 3000（Caliper Life sciences）を用いて測定した。各キナーゼについて、ヘルバセチン添加によるリン酸化ペプチドの生成量の減少量から阻害率を求めたところ、特にFLT3、Aurora kinase A、Aurora kinase B、Trk Aのキナーゼ活性が、低濃度のヘルバセチンによって抑制された（表2）。 Using a recombinant protein of each kinase and a synthetic peptide as a substrate, a MET kinase inhibition assay was performed using a decrease in the amount of phosphorylated peptide as an index. Add 20 mM HEPES, 0.01% Triton X-100, 2 mM DTT, 25 μM ATP, 5 mM MgCl 2 , pH 7.5, 0.94 nM recombinant kinase domain, 1 μM synthetic peptide as substrate, and 1 μM herbacetin. And allowed to react at room temperature for 1 hour. The amount of the substrate peptide and the generated phosphorylated peptide was measured using Lab Chip 3000 (Caliper Life sciences). For each kinase, the inhibition rate was calculated from the decrease in the amount of phosphorylated peptide produced by the addition of herbacetin. In particular, the kinase activity of FLT3, Aurora kinase A, Aurora kinase B, and Trk A was suppressed by low concentrations of herbacetin. (Table 2).
以上の結果から、ヘルバセチンは、METキナーゼ以外にもFLT3、Aurora kinase A、Aurora kinase B、Trk A等、様々なキナーゼを阻害するマルチキナーゼ阻害作用を有することが明らかとなった。 From the above results, it was revealed that herbacetin has a multikinase inhibitory action that inhibits various kinases such as FLT3, Aurora kinase A, Aurora kinase B, and Trk A in addition to MET kinase.
キナーゼの活性亢進は、癌の浸潤・転移のみならず、増殖に関わっていることが知られている。しかしながら、例えば、肝臓癌と診断されたものであっても、その性質は千差万別であるため、増殖亢進の要となるキナーゼは、個々の肝臓癌で異なっている。そのため、今や、キナーゼ阻害剤が、どのキナーゼをどの程度阻害するのかを明確にし、治療対象となる癌の性質を分子レベルで診断することが標準的な治療戦略となっている。例えば、FLT3は、一部の急性骨髄性白血病細胞で活性が亢進していることが明らかとなっているためFLT3阻害剤が増殖抑制剤として治療薬となっているが、FLT3活性が亢進していない急性骨髄性白血病細胞に対してはFLT3阻害剤が無効であることが知られている。即ち、作用する標的キナーゼを明確にすることで、初めて有用な用途を示すことが可能となる。従って、ヘルバセチンは、FLT3、Aurora kinase A、Aurora kinase Bが亢進した癌の増殖阻害剤となることが示された。
実施例５ ヒト急性骨髄性白血病細胞由来MV4-11細胞の増殖に対するヘルバセチンの効果
FLT3が恒常的に活性化しているヒト急性骨髄性白血病細胞由来MV4-11細胞を10%血清添加培地で懸濁し、48ウェルプレートに播種し、ヘルバセチンを最終濃度0-40 μg/mLとなるように添加し、3日間培養した後、WSTアッセイで生細胞数を定量した。その結果、MV4-11細胞の細胞数は、ヘルバセチンの添加濃度に依存して減少しており、そのIC50値は17.7 μMであった（図６）。
Kinase activity enhancement is known to be involved not only in cancer invasion and metastasis, but also in proliferation. However, for example, even if it is diagnosed as liver cancer, its properties are different, and the kinases that are important for promoting growth are different in each liver cancer. Therefore, it is now a standard treatment strategy to clarify which kinases and how much kinase inhibitors inhibit and to diagnose the nature of the cancer to be treated at the molecular level. For example, FLT3 has been shown to have increased activity in some acute myeloid leukemia cells, so FLT3 inhibitors have become therapeutic agents as growth inhibitors, but FLT3 activity has increased. It is known that FLT3 inhibitors are ineffective against non-acute myeloid leukemia cells. That is, it becomes possible to show a useful application for the first time by clarifying the target kinase that acts. Accordingly, it was shown that herbacetin is a cancer growth inhibitor with enhanced FLT3, Aurora kinase A, and Aurora kinase B.
Example 5 Effect of herbacetin on proliferation of MV4-11 cells derived from human acute myeloid leukemia cells
MV4-11 cells derived from human acute myeloid leukemia cells in which FLT3 is constantly activated are suspended in a medium supplemented with 10% serum and seeded in a 48-well plate, so that the final concentration of herbacetin is 0-40 μg / mL. After culturing for 3 days, the number of viable cells was quantified by WST assay. As a result, the number of MV4-11 cells decreased depending on the addition concentration of herbacetin, and its IC 50 value was 17.7 μM (FIG. 6).
以上の結果から、ヘルバセチンは、FLT3が恒常的に活性化しているヒト急性骨髄性白血病細胞の増殖を抑制することが明らかになった。 From the above results, it was revealed that herbacetin suppresses the growth of human acute myeloid leukemia cells in which FLT3 is constantly activated.
従って、ヘルバセチンは、FLT3活性が亢進しているヒト急性骨髄性白血病細胞の増殖抑制剤として有用であることが示された。
実施例６ ヒト肝癌由来HuH-7細胞のAurora kinase B及び細胞周期に対するヘルバセチンの効果
１）Aurora kinase B陽性であるヒト肝癌由来HuH-7細胞を用いて、ヘルバセチンのAurora kinase Bに対する効果として、Aurora kinase Bの発現量とAurora kinase Bの基質であるHistone-H3のリン酸化について解析した。10%血清添加培地に懸濁したHuH-7細胞をディッシュに播種し、24時間培養した。0-20 μg/mLヘルバセチン及び10%血清添加培地で6時間培養し、PBSで洗浄した後、タンパク質抽出用細胞溶解液で細胞を溶解した。細胞の溶解液についてSDS-PAGEを行い、抗リン酸化Histone-H3抗体、抗Aurora kinase B抗体を用いたウエスタンブロッティングで検出した。その結果、ヘルバセチンの添加濃度依存的にHistone-H3のリン酸化は抑制されたが、Aurora kinase Bの発現量は変化しなかった（図７（ａ）、図７（ｂ））。
Therefore, it was shown that herbacetin is useful as a growth inhibitor of human acute myeloid leukemia cells with enhanced FLT3 activity.
Example 6 Effect of herbacetin on Aurora kinase B and cell cycle of human hepatoma-derived HuH-7 cells 1) As a result of Aurora kinase B-positive human hepatoma-derived HuH-7 cells, The expression level of kinase B and the phosphorylation of Histone-H3 which is a substrate of Aurora kinase B were analyzed. HuH-7 cells suspended in a medium supplemented with 10% serum were seeded in a dish and cultured for 24 hours. The cells were cultured in a medium supplemented with 0-20 μg / mL herbacetin and 10% serum for 6 hours, washed with PBS, and then lysed with a cell lysate for protein extraction. The cell lysate was subjected to SDS-PAGE and detected by Western blotting using anti-phosphorylated Histone-H3 antibody and anti-Aurora kinase B antibody. As a result, the phosphorylation of Histone-H3 was suppressed depending on the addition concentration of herbacetin, but the expression level of Aurora kinase B did not change (FIGS. 7A and 7B).
２）Aurora kinase Bは、Histone-H3をリン酸化することでM期の染色体凝集を制御している。従って、Aurora kinase Bが阻害されると細胞周期はG2-M期で停止することが知られている。そこで、HuH-7細胞の細胞周期に対するヘルバセチンの効果を解析した。10% 血清培地に懸濁したHuH-7細胞をディッシュに播種し、24時間培養した後、0〜20 μg/mL ヘルバセチン及び10%血清添加培地で2日間培養した。PBSで洗浄した後、0.02% EDTA含有PBSで解離させ、ヨウ化プロピジウムで染色した後、フローサイトメーターで細胞あたりのDNA量を測定した。その結果、ヘルバセチンの添加濃度依存的にG2-M期の細胞が増加した（図８）。 2) Aurora kinase B regulates M-phase chromosome aggregation by phosphorylating Histone-H3. Therefore, it is known that when Aurora kinase B is inhibited, the cell cycle stops at the G2-M phase. Therefore, the effect of herbacetin on the cell cycle of HuH-7 cells was analyzed. HuH-7 cells suspended in 10% serum medium were seeded in a dish, cultured for 24 hours, and then cultured for 2 days in a medium supplemented with 0-20 μg / mL herbacetin and 10% serum. After washing with PBS, it was dissociated with PBS containing 0.02% EDTA, stained with propidium iodide, and then the amount of DNA per cell was measured with a flow cytometer. As a result, the number of cells in the G2-M phase increased depending on the addition concentration of herbacetin (FIG. 8).
以上の結果から、ヘルバセチンは、Aurora kinase Bの活性を阻害し、HuH-7細胞の増殖をG2-M期で停止させる作用を有することが明らかになった。
実施例７ ヒト肝癌由来HuH-7細胞株の増殖に対するヘルバセチンの効果
HuH-7細胞の増殖に対するヘルバセチンの効果を解析するため、10%血清添加培地に懸濁したHuH-7細胞を48ウェルプレートに播種し、24時間培養した。0〜40 μg/mLヘルバセチン添加培地で3日間培養した後、WSTアッセイで生細胞数を定量した。その結果、HuH-7細胞の増殖は、ヘルバセチンの添加濃度依存的に抑制された（図９）。
From the above results, it was revealed that herbacetin has an action of inhibiting the activity of Aurora kinase B and stopping the proliferation of HuH-7 cells in the G2-M phase.
Example 7 Effect of herbacetin on proliferation of human hepatoma-derived HuH-7 cell line
In order to analyze the effect of herbacetin on the proliferation of HuH-7 cells, HuH-7 cells suspended in a medium supplemented with 10% serum were seeded in a 48-well plate and cultured for 24 hours. After culturing in a medium supplemented with 0-40 μg / mL herbacetin for 3 days, the number of viable cells was quantified by WST assay. As a result, the proliferation of HuH-7 cells was suppressed depending on the addition concentration of herbacetin (FIG. 9).
以上の結果から、ヘルバセチンは、Aurora kinase Bを阻害することによって、細胞周期の停止により、HuH-7細胞の増殖を抑制することが明らかになった。 From the above results, it was revealed that herbacetin inhibits the growth of HuH-7 cells by inhibiting Aurora kinase B and thereby arresting the cell cycle.
従って、ヘルバセチンは、Aurora kinaseの活性が亢進している癌の増殖抑制剤として有用であることが示された。
実施例８ ヒト肝癌由来HuH-7細胞移植ヌードマウスの腫瘍増殖に対するヘルバセチンの効果
腫瘍増殖に対するヘルバセチンの効果を解析した。Balb/cヌードマウス（チャールス・リバー）にHuH-7細胞を皮下移植し、1週間後、腫瘍形成を確認し、コントロール群とヘルバセチン投与群、各6匹ずつに分けた。1日1回、コントロール群は滅菌水を経口投与し、ヘルバセチン投与群は、35 mg/kgのヘルバセチンを経口投与した。経時的に腫瘍サイズとマウスの体重を測定し、20日目にマウスを過麻酔下に屠殺して、腫瘍を回収した。腫瘍重量は、ヘルバセチン投与群はコントロール群と比較して有意に低下していた（図１０（ａ）、図１１（ａ））が、マウスの体重は実験期間を通して、コントロール群及びヘルバセチン投与群で有意差はなかった（図１０（ｂ）、図１１（ｂ））。
Therefore, it was shown that herbacetin is useful as a cancer growth inhibitor whose Aurora kinase activity is enhanced.
Example 8 Effect of herbacetin on tumor growth in nude mice transplanted with human liver cancer-derived HuH-7 cells The effect of herbacetin on tumor growth was analyzed. HuB-7 cells were subcutaneously transplanted into Balb / c nude mice (Charles River), and after 1 week, tumor formation was confirmed, and each group was divided into a control group and a herbacetin-administered group. Once a day, the control group was orally administered sterile water, and the herbacetin administration group was orally administered 35 mg / kg herbacetin. The tumor size and the body weight of the mice were measured over time, and the mice were sacrificed on the 20th day under hyperanaesthesia, and the tumors were collected. The tumor weight was significantly decreased in the herbacetin-administered group compared to the control group (FIGS. 10 (a) and 11 (a)), but the body weight of the mice was the same in the control group and the herbacetin-administered group throughout the experiment period. There was no significant difference (FIG. 10 (b), FIG. 11 (b)).
以上の結果から、ヘルバセチンは経口投与によって、ヌードマウスにおける腫瘍増殖を抑制することが明らかになった。また、マウスの体重に影響を与えなかったことから、ヘルバセチンは毒性がないか、あるいは毒性が極めて低いことが示唆された。
実施例９ ラット副腎髄質クロム親和性細胞腫PC12細胞のNGFによって誘導されるTrkAリン酸化及び神経突起伸長に対するヘルバセチンの抑制効果
NGFにより誘導されるTrkAのチロシンリン酸化及び神経突起伸長に対するヘルバセチンの効果についてPC12細胞を用いて解析した。
From the above results, it was revealed that herbacetin suppressed tumor growth in nude mice by oral administration. In addition, since it did not affect the body weight of mice, it was suggested that herbacetin is not toxic or very low in toxicity.
Example 9 Inhibitory effect of herbacetin on NGF-induced TrkA phosphorylation and neurite outgrowth of rat adrenal medullary pheochromocytoma PC12 cells
The effect of herbacetin on TGFA tyrosine phosphorylation and neurite outgrowth induced by NGF was analyzed using PC12 cells.
１）PC12細胞を0〜20 μg/mLヘルバセチン及び100 ng/mL NGF添加無血清培地で30分間インキュベートし、PBSで洗浄した後、タンパク質抽出用細胞溶解液で細胞を溶解した。細胞の溶解液についてSDS-PAGEを行い、抗リン酸化TrkA抗体、抗TrkA抗体及び抗GAPDH抗体を用いたウエスタンブロッティングで検出した。その結果、NGFによって誘導されるTrkAのチロシンリン酸化は、ヘルバセチンの添加濃度依存的に有意に抑制された（図１２（ａ）、図１２（ｂ））。 1) PC12 cells were incubated for 30 minutes in a serum-free medium supplemented with 0 to 20 μg / mL herbacetin and 100 ng / mL NGF, washed with PBS, and then lysed with a cell lysate for protein extraction. The cell lysate was subjected to SDS-PAGE and detected by Western blotting using anti-phosphorylated TrkA antibody, anti-TrkA antibody and anti-GAPDH antibody. As a result, the tyrosine phosphorylation of TrkA induced by NGF was significantly suppressed depending on the addition concentration of herbacetin (FIGS. 12 (a) and 12 (b)).
２）PC12細胞をI型コラーゲンコーティング24穴プレートに播種し、0〜10 μg/mLヘルバセチン及び100 ng/mL NGF添加した1%ウマ血清-0.5%ウシ胎児血清添加培地で48時間インキュベートした。NGFによって誘導された神経突起の伸長は、位相差顕微鏡下で1視野あたり100個以上の細胞を画像解析ソフト（Image J）で解析し、6視野の平均値を求めた。その結果、NGF添加PC12細胞は、全体の約30%の細胞が神経突起を伸長し（図１４（ｂ））、それら細胞の神経突起の平均長は、37 μmであった（図１４（ａ））。一方、ヘルバセチンの添加によって、神経突起の長さ、及び神経突起を有する細胞の割合が有意に減少した（図１４（ａ）、図１４（ｂ））。なお、NGFを添加しなかったPC12細胞は、神経突起の伸張が観察されなかった（図１３）。 2) PC12 cells were seeded in a type I collagen-coated 24-well plate and incubated in a medium supplemented with 1% horse serum-0.5% fetal calf serum supplemented with 0 to 10 μg / mL herbacetin and 100 ng / mL NGF for 48 hours. NGF-induced neurite outgrowth was analyzed with image analysis software (Image J) for more than 100 cells per visual field under a phase contrast microscope, and the average of 6 visual fields was determined. As a result, about 30% of the NGF-added PC12 cells had neurites extended (FIG. 14 (b)), and the average length of the neurites of these cells was 37 μm (FIG. 14 (a)). )). On the other hand, the length of neurites and the ratio of cells having neurites were significantly reduced by the addition of herbacetin (FIGS. 14 (a) and 14 (b)). In addition, no extension of neurites was observed in PC12 cells to which NGF was not added (FIG. 13).
以上の結果から、ヘルバセチンは、NGFにより誘導される神経突起の伸長を抑制する作用を有することが明らかとなり、鎮痛作用や止痒作用を有することが示唆された。
実施例１０ マウスのホルマリン誘発性疼痛に対するヘルバセチンの疼痛抑制効果
ヘルバセチンがin vivoで疼痛抑制効果を有するのかどうかを明らかにするために、ホルマリン試験を行った。ヘルバセチン（50, 100, 150 mg/kg）をマウス（n=8）の腹腔内に投与し90分後に、2.5%ホルマリン溶液20 μLを左後肢足裏皮下に投与した。足を舐める、噛むなどの疼痛関連行動を経時的に測定した。ホルマリン試験は2相性の反応を示す。第1相目（0-5 分間）の反応は、ホルマリンよる知覚神経主末へ直接刺激による痛みを反映し、第2相目（15-45 分間）の反応は、ホルマリン組織侵害で生じた炎症による痛みを反映する。モルヒネのような中枢性鎮痛薬は、第1相目及び第２相の両方を抑制し、非ステロイド性抗炎症薬（NSAIDs）は、第2相のみを抑制する。ヘルバセチンは、第1相目の反応については抑制傾向を示したが有意差はなかった。一方、第2相目の反応については濃度依存的に抑制し、100〜150 mg/kgでは、有意に疼痛行動を抑制した（図１５）。
From the above results, it was revealed that herbacetin has an action of suppressing neurite outgrowth induced by NGF, suggesting that it has analgesic action and antipruritic action.
Example 10 Pain Suppressing Effect of Herbacetin on Formalin-Induced Pain in Mice In order to clarify whether herbacetin has a pain suppressing effect in vivo, a formalin test was performed. Herbacetin (50, 100, 150 mg / kg) was intraperitoneally administered to mice (n = 8), and 90 minutes later, 20 μL of 2.5% formalin solution was subcutaneously administered to the left hind foot sole. Pain-related behaviors such as licking and biting were measured over time. The formalin test shows a biphasic response. Phase 1 response (0-5 minutes) reflects pain from direct stimulation to the main sensory nerve endings of formalin, and Phase 2 response (15-45 minutes) is inflammation caused by formalin invasion. Reflects the pain caused by Central analgesics such as morphine suppress both phase 1 and phase 2, and nonsteroidal anti-inflammatory drugs (NSAIDs) suppress only phase 2. Herbacetin showed a tendency to suppress the first-phase reaction, but there was no significant difference. On the other hand, the reaction in the second phase was suppressed in a concentration-dependent manner, and pain behavior was significantly suppressed at 100 to 150 mg / kg (FIG. 15).
これらの結果から、ヘルバセチンは、NGF-TrkA阻害を介して、鎮痛作用を示すことが明らかになった。 From these results, it was revealed that herbacetin exhibits analgesic action through NGF-TrkA inhibition.
以上の実施例より、ヘルバセチンはマルチキナーゼ阻害剤として、MET発現癌の転移及び増悪化の治療、耐性非小細胞肺癌の治療、Aurora kinase及びFLT3活性亢進癌の増殖抑制剤、及び難治性疼痛に対する鎮痛剤として有用であることが示された。
実施例１１ ヘルバセチンと類似構造を有するゴシペチンのキナーゼ阻害特性の解析
式７に示されるゴシペチンは、ヘルバセチンと極めて構造が類似しているフラボノールである。ヘルバセチンで見出されたマルチキナーゼ阻害作用をゴシペチンも有しているのかを解析した。
From the above examples, herbacetin as a multikinase inhibitor, treatment of metastasis and exacerbation of MET-expressing cancer, treatment of resistant non-small cell lung cancer, growth inhibitor of cancer with enhanced Aurora kinase and FLT3 activity, and refractory pain It has been shown to be useful as an analgesic.
Example 11 Analysis of Kinase Inhibitory Properties of Gosipetin Having a Structure Similar to Herbacetin Gosipetin shown in Formula 7 is a flavonol that is very similar in structure to herbacetin. We analyzed whether gosipetin also has the multikinase inhibitory action found in herbacetin.
MET、FLT3、Aurora kinase A、Aurora kinase B、Trk Aについて、各キナーゼドメインの組換えタンパク質と合成基質ペプチドを用いたキナーゼ阻害アッセイを行った。20 mM HEPES，0.01% Triton X-100，2 mM DTT，25〜100 μM ATP，5 mM MgCl2，pH7.5に0.94 nM 組換えキナーゼドメイン，1 μM合成基質ペプチド及び0.001〜30 μM ヘルバセチンを添加し、室温で1時間反応させた。ただし、合成基質ペプチドは、MET及びFLT3では、Srctide、Aurora kinase A及びAurora kinase Bでは、Kemptide、Trk Aでは、CSKtideを用いた。また、ATP濃度は、MET、Aurora kinase A及びAurora kinase Bでは、25 μM、FLT3では、100 μM、Trk Aでは、75 μMで行った。基質ペプチドと生成したリン酸化ペプチド量をLab Chip 3000（Caliper Lifesciences）を用いて測定した。統計解析ソフトウエア（GraphPad Prism 5）を用いてヘルバセチンあるいはゴシペチンの添加濃度に対するリン酸化ペプチドの生成阻害率のプロットに対する4パラメーターロジスティック曲線の近似式を求め、そのパラメーターよりIC50値を算出した（表3）。 For MET, FLT3, Aurora kinase A, Aurora kinase B, and Trk A, kinase inhibition assays using recombinant proteins of each kinase domain and synthetic substrate peptides were performed. 20 mM HEPES, 0.01% Triton X-100, 2 mM DTT, 25-100 μM ATP, 5 mM MgCl 2 , pH 7.5 with 0.94 nM recombinant kinase domain, 1 μM synthetic substrate peptide and 0.001-30 μM herbacetin And allowed to react at room temperature for 1 hour. However, the synthetic substrate peptides used were Srctide for MET and FLT3, Kemptide for Aurora kinase A and Aurora kinase B, and CSKtide for Trk A. The ATP concentration was 25 μM for MET, Aurora kinase A and Aurora kinase B, 100 μM for FLT3, and 75 μM for Trk A. The amount of the phosphopeptide generated with the substrate peptide was measured using Lab Chip 3000 (Caliper Lifesciences). Using statistical analysis software (GraphPad Prism 5), an approximate expression of a 4-parameter logistic curve was plotted against the plot of inhibition rate of phosphorylated peptide against the addition concentration of herbacetin or gosipetin, and the IC 50 value was calculated from the parameters (Table). 3).
その結果、ゴシペチンもヘルバセチンと同様にマルチキナーゼ阻害作用を有することが明らかになった。 As a result, it has been clarified that gosipetin has a multikinase inhibitory action as well as herbacetin.
以上の結果から、ヘルバセチンは、MET、Aurora kinase A、Aurora kinase B、FLT3、Trk A等のキナーゼを標的とした，マルチキナーゼ阻害作用を有すること、ゴシペチンについても、ヘルバセチンと同等のマルチキナーゼ阻害作用を有することが明らかとなった。 Based on the above results, herbacetin has multikinase inhibitory activity targeting kinases such as MET, Aurora kinase A, Aurora kinase B, FLT3, and Trk A, and gosipetin also has multikinase inhibitory activity equivalent to herbacetin. It became clear to have.
以上の実施例から、式１を備えるフラボノールは、上記の実施例で示したヘルバセチンと同等の用途に有用であることが示された。 From the above examples, it was shown that the flavonol having the formula 1 is useful for the same use as the herbacetin shown in the above examples.
癌の治療、疼痛の治療及び掻痒の治療に利用できる。 It can be used for cancer treatment, pain treatment and pruritus treatment.
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| KR20190092314A (en) | 2018-01-30 | 2019-08-07 | 각코우호우징 기타사토켄큐쇼 | Extraction fraction containing high molecular mass condensed tannin obtained from ephedra extract or ephedrine alkaloids-free ephedra extract, method for producing same, and use of same |
| KR102108414B1 (en) * | 2018-11-21 | 2020-05-11 | 중앙대학교 산학협력단 | Use for regulating cancer cell differentiation by regulating AURKA-mediated KDM6B activity |
| WO2021177519A1 (en) * | 2019-08-30 | 2021-09-10 | (주)프론트바이오 | Pharmaceutical composition for preventing or treating cancer containing, as active ingredient, complex of biguanide-based compound and flavone, hydroxyflavone, flavanone, flavone derivative, hydroxyflavone derivative, or flavanone derivative |
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