JP6746022B1 - 腫瘍細胞におけるアスパラギン酸合成の阻害剤、腫瘍細胞のスフェロイド形成阻害剤、腫瘍細胞の転移抑制剤、解糖系阻害剤の作用増強剤、並びに腫瘍の転移の抑制および/または予防用医薬組成物 - Google Patents
腫瘍細胞におけるアスパラギン酸合成の阻害剤、腫瘍細胞のスフェロイド形成阻害剤、腫瘍細胞の転移抑制剤、解糖系阻害剤の作用増強剤、並びに腫瘍の転移の抑制および/または予防用医薬組成物 Download PDFInfo
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Abstract
Description
ここではまず、有効成分として用いられる下記化学式1Aまたは化学式1Bで表される化合物(以下、「本発明の化合物」とも称する)について説明する。なお、本発明において「化学式1Aまたは化学式1Bで表される化合物を含有する」とは、化学式1Aで表される化合物を含有する形態、化学式1Bで表される化合物を含有する形態、さらには、化学式1Aで表される化合物と化学式1Bで表される化合物とをともに含有する形態のいずれをも含む概念である。また、「置換基を有していてもよい炭化水素基」とは、炭化水素が、ヘテロ原子(酸素原子や窒素原子、硫黄原子等)を介してシクロヘキシル骨格に結合している形態を含む概念である。
続いて、上述した本発明の各形態に係る用途について、説明する。
では、有効成分は、クエン酸ナトリウムまたはリン酸二カルシウムのような1つまたは複数の製薬上許容できる担体、および/または以下のもののいずれかと混合される:デンプン、ラクトース、スクロース、グルコース、マンニトール、および/またはケイ酸のような充填剤または増量剤;例えばカルボキシメチルセルロース、アルギン酸塩、ゼラチン、ポリビニルピロリドン、スクロースおよび/またはアラビアゴムのような粘結剤;グリセロールのような保湿剤;寒天、炭酸カルシウム、バレイショまたはタピオカデンプン、アルギン酸、ある特定のケイ酸塩、および炭酸ナトリウムのような崩壊剤;パラフィンのような溶解遅延剤;4級アンモニウム化合物のような吸収促進剤;セチルアルコールおよびモノステアリン酸グリセロールのような湿潤剤;カオリンおよびベントナイト粘土のような吸収剤;タルク、ステアリン酸カルシウム、ステアリン酸マグネシウム、固体ポリエチレングリコール、ラウリル硫酸ナトリウム、およびそれらの混合物のような潤滑剤;ならびに着色剤。カプセル、錠剤および丸薬の場合、これらは緩衝剤を含んでもよい。同様の種類の固体組成物が、ラクトースまたは乳糖のような補形薬と、高分子量ポリエチレングリコール等とを用いたソフトおよびハード充填ゼラチンカプセル内の充填剤としても使用可能である。
ペタシン、S−ペタシン、ネオペタシンおよびネオ−S−ペタシンの4種の化合物について、以下の手法により、マウスまたはイヌ由来の悪性黒色腫細胞に対する増殖抑制作用を評価した。
マウス悪性黒色腫肺転移モデルを用いて、ネオペタシンの腫瘍細胞に対する転移抑制効果を調べた。具体的には、C57BL/6マウス(6週齢、メス)を日本SLC(浜松)より購入した。次いで、B16F10細胞を尾静脈より0.25×106細胞/100μL PBSの量で静脈投与した。そして、細胞を播種させた日からネオペタシン(50mg/kg)を2日ごと1回(合計5回)腹腔内投与した。投与開始から10日後に解剖を行って肺を摘出して、肺表面に認められる転移巣の数を計測した。なお、グラフパッドプリズムソフトウェアシステム(グラフパッドソフトウェア社)を用い、ステューデントT検定を用いてP値を算出した。
解糖系阻害剤である2−デオキシグルコース(2-Deoxyglucose;2−DG)は、グルコースと競合してGLUT1を介してがん細胞に取り込まれ、がん細胞の解糖系を阻害することによりがん細胞の増殖速度を低下させることが知られている。本試験例では、このような2−DGの腫瘍細胞に対する制がん作用に対してネオペタシンが及ぼす影響を調べた。
B16F10細胞について、ネオペタシンの存在下でスフェロイド形成アッセイを行い、ネオペタシンが当該細胞のスフェロイド形成能に及ぼす影響を調べた。
ネオペタシンが腫瘍細胞の遊走作用に及ぼす影響を調べる目的で、スクラッチ創傷治癒アッセイを行った。
ネオペタシンが腫瘍細胞に対して増殖抑制活性や転移抑制活性を示す作用機序を明らかにすることを目的として、ネオペタシンがB16F10細胞のミトコンドリアに与える影響を評価した。ネオペタシン処置により影響を受けるシグナル伝達経路を、マイクロアレイを用いたKEGG Pathway解析により調べた。具体的には、A2058細胞を0.5×105細胞/mLとなるように6ウェルプレートに播種し、その24時間後に、ネオペタシン(終濃度は3μM)を培地中に懸濁して8時間インキュベートした。このようにしてネオペタシン(3μM)で8時間処置したA2058細胞をセルスクレイパーで回収した。なお、陰性対照区にはDMSOを投与して同様の実験を行った。次いで、SurePrint G3 Human CGH マイクロアレイ 8x60K(Agilent、カリフォルニア州、アメリカ合衆国)を使用してデータを収集し、各サンプルについて得られた生データを75パーセンタイルの値で正規化(global normalization)した。そして、Gene Set Enrichment Analysis(GSEA、ブロード研究所、マサチューセッツ工科大学、マサチューセッツ州,アメリカ合衆国)を用いてKEGG,Reactome,GOに登録されているpathwayを検索した。
ネオペタシンで処置したB16F10細胞のメタボローム解析(アミノ酸)を行った。具体的には、試験例1と同様の手法によりネオペタシン(3μM)で処置したB16F10細胞について、処置から9時間および48時間後のそれぞれの時点で細胞を回収し、キャピラリー電気泳動−飛行時間型質量分析計(CE−TOFMS)を用いてアミノ酸濃度を測定した。なお、サンプル中のアミノ酸濃度は内部標準検量法に従って測定し、サンプル中の内部標準濃度を25μMとして算出した。また、解析にはMaster Hands ver.2.17.3.18(慶應義塾大学先端生命科学研究所)を使用した。
ネオペタシンが腫瘍細胞においてアスパラギン酸の合成を阻害する作用機序を明らかにすることを目的として、ネオペタシンがB16F10細胞のミトコンドリアに与える影響を評価した。具体的には、試験例1と同様の手法によりネオペタシン(3μM)で48時間処置したB16F10細胞を、膜電位に依存してミトコンドリアを標識するMitoTracker Orange(Thermo Fisher Scientific、マサチューセッツ州、アメリカ合衆国)を使用して染色した。なお、陽性対照区として、ネオペタシンに代えて、ミトコンドリア呼吸鎖複合体I阻害剤(ミトコンドリア毒)として知られているメトホルミンを5000μMの濃度で用いて同様の実験を行った。
ネオペタシンのミトコンドリア毒としての作用点を明らかにすることを目的として、ミトコンドリアの呼吸鎖を構成する各複合体(複合体I〜複合体V)のそれぞれの活性にネオペタシンが及ぼす影響を調べた。
ウエスタンブロット法を用いて、ネオペタシン処置細胞における様々ながん関連タンパク質の発現の変化を調べた。なお、KMeC細胞(イヌ悪性黒色腫細胞)は、東京大学大学院農学生命科学研究科獣医外科学研究室より入手した。また、KMeC細胞はRPMI1640培地を用いて、37℃のインキュベーター(空気/CO2=95/5(v/v))内で培養した。
ウエスタンブロット法を用いて、ネオペタシンまたはメトホルミンで処置したB16F10細胞における様々ながん代謝・増殖・転移関連タンパク質(AMPK、FAK、Akt、ERK1/2)の発現の経時的な変化を調べた。
上述した各試験例に示す結果をまとめると、まず、マウス高転移悪性黒色腫細胞株であるB16F10細胞を用いた腫瘍細胞肺転移試験を行ったところ、ネオペタシン投与はB16F10腫瘍細胞の肺転移を阻害することが判明した。なお、in vitroの抗腫瘍効果の特異性と一致して、ネオペタシン投与は肝臓腎臓脾臓などにほとんど毒性を示さなかった。効果発現機序の検索を進めたところ,ネオペタシンは腫瘍代謝を破綻させ、特にアスパラギン酸合成を10分の1以下にまで阻害することがわかった。ここで、アスパラギン酸はミトコンドリアで合成されるアミノ酸であり、核酸やタンパク質の合成に必須のアミノ酸である。このように、ネオペタシンは第一義的にはアミノ酸代謝を破綻させることによって作用を発現していると考えられるが、ひいてはエネルギー代謝、さらには解糖系からもペントースリン酸回路を介して核酸の合成を強く抑制する。また、FAKの発現を著しく低下させることで、細胞の運動や形態変化を障害する。以上のことから、ネオペタシンは腫瘍細胞の転移先での増殖と適応に必要な栄養素であるアスパラギン酸の合成を強力に阻害することで、腫瘍細胞の転移能を阻害しているものと考えられる。
Claims (13)
- ネオペタシンを有効成分として含有する、腫瘍細胞の転移を抑制するための、腫瘍細胞におけるアスパラギン酸合成阻害剤:
- ネオペタシンを有効成分として含有する、腫瘍細胞の転移を抑制するための、腫瘍細胞のスフェロイド形成阻害剤。
- 請求項1または2に記載の剤を含む、腫瘍細胞の転移抑制剤。
- 前記腫瘍細胞が、悪性黒色腫の細胞である、請求項3に記載の転移抑制剤。
- 前記腫瘍細胞の肺への転移を抑制する、請求項3または4に記載の転移抑制剤。
- ネオペタシンを有効成分として含有し、解糖系阻害剤と併用されて用いられる、解糖系阻害剤の作用増強剤。
- 前記解糖系阻害剤が2−デオキシ−D−グルコースである、請求項6に記載の作用増強剤。
- 請求項6または7に記載の作用増強剤を含む、腫瘍の転移の抑制および/または予防用医薬組成物。
- 解糖系阻害剤と併用されて投与される、請求項8に記載の医薬組成物。
- 前記解糖系阻害剤が2−デオキシ−D−グルコースである、請求項9に記載の医薬組成物。
- ヒト、またはペット動物に経口的に投与されて使用される、請求項8〜10のいずれか1項に記載の医薬組成物。
- ネオペタシンを有効成分として含有する、腫瘍細胞転移抑制用飲食品組成物。
- ヒト用健康食品またはペットフード組成物である、請求項12に記載の飲食品組成物。
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