JP5717129B2 - Anticancer agents combined with withanolide ingredients - Google Patents

Anticancer agents combined with withanolide ingredients Download PDF

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JP5717129B2
JP5717129B2 JP2010262117A JP2010262117A JP5717129B2 JP 5717129 B2 JP5717129 B2 JP 5717129B2 JP 2010262117 A JP2010262117 A JP 2010262117A JP 2010262117 A JP2010262117 A JP 2010262117A JP 5717129 B2 JP5717129 B2 JP 5717129B2
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ワダワ レヌー
レヌー ワダワ
スニル カウル
スニル カウル
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National Institute of Advanced Industrial Science and Technology AIST
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本発明は、アシュワガンダの葉抽出物の構成成分であるウィザノン及びウィザフェリンAを有効成分として特定の割合で配合した、腫瘍細胞への殺傷能が高く、正常細胞への毒性の少ない抗癌剤に関する。   The present invention relates to an anticancer agent having a high killing ability to tumor cells and a low toxicity to normal cells, which is formulated with a specific ratio of withanone and witherferin A which are constituents of Ashwagandha leaf extract as active ingredients.

アシュワガンダ(Ashwagandha,学名Withania somnifera;ナス科)は、インドの民間医療アーユルヴェーダで最も良く用いられる薬草の一つで、種々の治療効果が知られている。
本発明者らは、従来からアシュワガンダの葉からエタノール抽出物(i-Extract)を精製して、この成分に強い抗腫瘍活性があることを示すと共に、当該エタノール抽出物(i-Extract)から各種のウィザノライド化合物を単離して、生物活性を検討する中で、成分中に含まれるウィザノン(Withanone)に、腫瘍細胞特異的にがん抑制因子p53を活性化する現象を見出し、生体内での抗腫瘍効果も確認した(特許文献1、2)。当該ウィザノンは、さらに正常細胞の寿命延長、正常細胞の酸化ストレスからの保護作用、紫外線のDNA損傷及び化学毒性からの保護作用なども報告されている(特許文献2)。
一方、同様に上記エタノール抽出物中に見出されたウィザフェリンA(WithaferinA)も腫瘍細胞殺傷能を示すが、生体内での腫瘍抑制能は見出されず、何より正常細胞に対して強い毒性を有しているという大きな欠点がある。ウィザフェリンAに対して、ウィザノンを多く含むピーク21画分と併用することが試みられたことがあるが、正常細胞毒性はかなり減少したものの、完全に毒性を失わせることはできなかった(特許文献1)。
ウィザノンは、正常細胞への毒性がきわめて低いという長所はあるが、生体内での抗腫瘍効果が穏やかなものにとどまり、単独で抗癌剤として用いるためには不十分であった。
したがって、複数のウィザノライド化合物を組み合わせた、正常細胞への毒性はなく、しかも癌細胞に対する細胞増殖抑制効果の高い抗癌剤の開発が望まれていた。
Ashwagandha (scientific name: Withania somnifera; solanaceae) is one of the most commonly used medicinal herbs in Indian folk medicine Ayurveda and is known for its various therapeutic effects.
The present inventors have conventionally purified an ethanol extract (i-Extract) from the leaves of Ashwagandha and show that this component has a strong antitumor activity. In the study of biological activity by isolating the withanolide compound, we found a phenomenon that activates the tumor suppressor p53 specifically in tumor cells in the withanone (Withanone) contained in the components. Tumor effects were also confirmed (Patent Documents 1 and 2). The withanone has also been reported to extend normal cell life, protect normal cells from oxidative stress, protect DNA from UV damage and chemical toxicity (Patent Document 2).
On the other hand, withaferin A found in the above ethanol extract also shows tumor cell killing ability, but no tumor suppressive ability is found in vivo, and above all has strong toxicity to normal cells. There is a big drawback of doing. There has been an attempt to use Witherin A in combination with the peak 21 fraction containing a large amount of Withanone, but the normal cytotoxicity was considerably reduced, but the toxicity could not be completely lost (patent) Reference 1).
With an advantage that Withanon has extremely low toxicity to normal cells, it has a mild antitumor effect in vivo and is insufficient for use alone as an anticancer agent.
Therefore, there has been a demand for the development of an anticancer agent which is a combination of a plurality of withanolide compounds, has no toxicity to normal cells, and has a high cell growth inhibitory effect on cancer cells.

国際公開WO2005/082392International Publication WO2005 / 083392 特開2008−195704号公報JP 2008-195704 A

本発明は、複数のウィザノライド化合物を特定の割合で組み合わせることで、正常細胞への毒性はなく、しかも癌細胞に対する細胞増殖抑制効果の高い抗癌剤を提供することを目的とする。   An object of the present invention is to provide an anticancer agent that has no toxicity to normal cells and has a high cell growth inhibitory effect on cancer cells by combining a plurality of withanolide compounds at a specific ratio.

本発明では、アシュワガンダ葉由来のエタノール抽出物(i-Extract)に含まれる二つのウィザノライド化合物であるウィザノンとウィザフェリンAに着目し、両者の配合割合を様々に変化させた組成物を作製し、正常細胞への細胞毒性と共に、癌細胞に対する細胞増殖抑制能を解析することによって、それぞれの成分の相乗効果が現れる配合割合を見出した。
すなわち、in vitroでは、ウィザノン(下記式1)とウィザフェリンA(下記式2)とを重量比で20:1の割合で含んだ組成物の場合において、正常細胞への細胞毒性がなく、しかも、癌細胞に対する細胞増殖抑制能が強力であるという画期的な効果が奏せられることが確認できた。以下、当該組成物を「WiNA-201」という。
<式1>ウィザノン:

<式2>ウィザフェリンA:
In the present invention, paying attention to two withanolide compounds, wizzanone and witherferin A, contained in an ethanol extract (i-Extract) derived from Ashwagandha leaves, a composition in which the blending ratio of the two is varied is prepared. By analyzing the cell growth inhibitory ability against cancer cells as well as cytotoxicity against normal cells, the compounding ratios in which the synergistic effects of the respective components appear were found.
That is, in vitro, in the case of a composition containing Withanone (Formula 1 below) and Witherin A (Formula 2 below) in a weight ratio of 20: 1, there is no cytotoxicity to normal cells. It was confirmed that the epoch-making effect that the cell growth-inhibiting ability against cancer cells was strong was exhibited. Hereinafter, the composition is referred to as “WiNA-201”.
<Formula 1> Withanon:

<Formula 2> Witherin A:

そして、このように両者の配合として、正常細胞に対する毒性がなく、癌細胞に対しては細胞増殖抑制能が強力である最適な数値範囲が存在するという知見が得られたので、本発明者らは、生体内に投与した場合(in vivo)の最適割合について、両成分の溶解性、半減期を勘案し、体重あたりの最大投与量を決定し、ウィザノンとウィザフェリンAとを重量比で2:1の割合で含む場合がin vivoでの最適数値範囲であることを突き止めた。そして、生体内においては、このような重量比2:1の割合で含む組成物(「WiNA-21」)の場合に、正常細胞、組織への毒性はなく、かつ強い腫瘍細胞増殖抑制効果を得ることができ、さらに癌細胞の浸潤性抑制効果と共に、癌細胞の転移抑制作用という優れた抗癌剤機能を実証できた。
これらの知見を得たことで、本発明を完成させた。
Thus, as a combination of the two, the present inventors have found that there is an optimal numerical range that is not toxic to normal cells and has a strong cell growth inhibitory ability against cancer cells. Determines the maximum dose per body weight for the optimal ratio when administered in vivo (in vivo), taking into account the solubility and half-life of both ingredients, and witheron and witherferin A in a weight ratio of 2 : The case where the ratio of 1 was included was found to be the optimal numerical range in vivo. In vivo, in the case of a composition containing such a weight ratio of 2: 1 ("WiNA-21"), there is no toxicity to normal cells and tissues and a strong tumor cell growth inhibitory effect. Furthermore, it was possible to demonstrate an excellent anticancer agent function of cancer cell metastasis inhibitory effect as well as cancer cell invasive inhibitory effect.
Obtaining these findings completed the present invention.

すなわち、本発明は、具体的には以下の通りである。
〔1〕ウィザノン及びウィザフェリンAを有効成分とし、両成分を2:1(重量比)の割合で含む抗癌剤。
〔2〕注射剤として投与することを特徴とする、前記〔1〕に記載の抗癌剤。
ウィザノン及びウィザフェリンAを有効成分とし、両成分を2:1(重量比)の割合で含む抗癌剤。
That is, the present invention is specifically as follows.
[1] An anticancer agent comprising withanone and witherin A as active ingredients and containing both ingredients in a ratio of 2: 1 (weight ratio).
[2] The anticancer agent according to [1], which is administered as an injection.
An anticancer agent comprising Withanone and Witherin A as active ingredients and containing both ingredients in a ratio of 2: 1 (weight ratio).

本発明のWiNA-201は、in vitro条件下で、またWiNA-21はin vivo条件下において、正常細胞に対しては無毒であって、癌細胞特異的に毒性がある。いずれも、それぞれin vitro条件下、及びin vivo条件下において顕著な抗腫瘍活性を有すると共に、癌細胞の浸潤能を弱め、抗転移能を有しているので、優れた抗癌剤として期待される。   WiNA-201 of the present invention is nontoxic to normal cells under in vitro conditions and WiNA-21 under in vivo conditions, and is toxic specifically for cancer cells. Each of them is promising as an excellent anticancer agent because it has a remarkable antitumor activity under in vitro conditions and in vivo conditions, as well as weakening the invasion ability of cancer cells and having antimetastatic ability.

ウィザノン及びウィザフェリンAの各種配合割合での腫瘍細胞増殖抑制作用及び正常細胞に対する毒性作用の比較。 [30時間培養後、ウィザノン及びウィザフェリンAが20:1の場合(WiNA-201)のみがin vitro条件下で腫瘍細胞の増殖を抑制し、かつ正常細胞への毒性がないことを示す。]Comparison of tumor cell growth inhibitory effect and normal cell toxic effect at various blending ratios of Withanone and Witherin A. [After 30 hours of incubation, only with Wizanone and Withaferin A 20: 1 (WiNA-201) shows that tumor cell growth is suppressed under in vitro conditions and that there is no toxicity to normal cells. ] ヒト神経膠芽腫細胞(YKG1)の遊走抑制作用。 [細胞層に引っ掻き傷を作ったYKG1細胞に対する8時間後の周囲の細胞の遊走(=細胞層の修復)作用を比較した。i-Extract(エタノール抽出液)、ウィザフェリンA、ウィザノン、WiNA-201のうち、WiNA-201がin vitro条件下で最も効果的に遊走を遅らせた。]Inhibition of migration of human glioblastoma cells (YKG1). [Migration of the surrounding cells (= repair of cell layer) after 8 hours on YKG1 cells that had scratched the cell layer was compared. Of i-Extract (ethanol extract), Withaferin A, Withanon and WiNA-201, WiNA-201 most effectively delayed migration under in vitro conditions. ] ヒト線維肉腫細胞(HT1080)の浸潤抑制。 [Boydenチャンバーアッセイでの測定による。ウィザフェリンA、ウィザノンおよび WiNA-201の全てに於いて浸潤能の抑制が観察されたが、WiNA-201がin vitro条件下で最も強力な作用を示した。]Inhibition of human fibrosarcoma cell (HT1080) infiltration. [By measurement in Boyden chamber assay. Inhibition of invasive ability was observed in all of Witherin A, Withanone and WiNA-201, but WiNA-201 showed the strongest effect under in vitro conditions. ] ヒト線維肉腫細胞(HT1080)の浸潤抑制。 [QCMTM細胞浸潤アッセイキットによる。WiNA-201がin vitro条件下で最も強力な浸潤能抑制作用を示すことが定量的に示された。]Inhibition of human fibrosarcoma cell (HT1080) infiltration. [By QCM TM cell invasion assay kit. It was quantitatively shown that WiNA-201 exhibited the strongest invasive ability suppression effect under in vitro conditions. ] ヒト線維肉腫細胞(HT1080)とフィブロネクチンとの接着能の抑制。 [細胞接着アッセイにより測定。ウィザフェリンA、ウィザノンおよびWiNA-201の全てに於いてフィブロネクチンとの細胞接着が抑制されたが、WiNA-201がin vitro条件下で最も強力な作用を示した。]Inhibition of adhesion between human fibrosarcoma cells (HT1080) and fibronectin. [Measured by cell adhesion assay. Witherin A, withanone and WiNA-201 all inhibited cell adhesion to fibronectin, but WiNA-201 showed the most potent effect under in vitro conditions. ] マウスへの静脈注射による毒性試験。 [WiNA-21などをヌードマウスの尾静脈に注射後のマウスの体重を測定。体重から判断する限り、WiNA-21はマウスに毒性を示さなかった。]Toxicity test by intravenous injection to mice. [Weighs mice after injecting WiNA-21 etc. into the tail vein of nude mice. As judged by body weight, WiNA-21 was not toxic to mice. ] ヌードマウス腫瘍アッセイでのWiNA-21の強力な抗がん作用。 [ヌードマウス皮下移植下でのヒト線維肉腫細胞株(HT1080)の増殖を、WiNA-21注射群は比較対照群と比較し強力ながん抑制作用を示した。]The potent anticancer effect of WiNA-21 in the nude mouse tumor assay. [The growth of a human fibrosarcoma cell line (HT1080) under subcutaneous transplantation of nude mice showed a strong cancer suppressive effect in the WiNA-21 injection group compared to the control group. ] ヌードマウス腫瘍アッセイでの腫瘍の体積変化。 [WiNA-21注射群及び比較対照群マウスの4匹それぞれの2か所ずつの腫瘍体積を平均値化し、腫瘍の体積変化を検討した。]Tumor volume change in nude mouse tumor assay. [Two tumor volumes in each of four mice in the WiNA-21 injection group and the comparative control group mice were averaged, and changes in tumor volume were examined. ] ヒト線維肉腫細胞に対するin vivo条件下での浸潤抑制作用。 [ヌードマウスに静脈注射後8日目のヒト線維肉腫細胞株に対してWiNA-21を腹腔投与した場合とリン酸緩衝液・生理食塩水を用いた場合(コントロール)の5週間後の肺組織へのがん細胞の転移を観察。WiNA-21注射群には腫瘍の転移は殆ど観察されない。]Inhibition of human fibrosarcoma cells under in vivo conditions. [Lung tissue 5 weeks after WiNA-21 was administered intraperitoneally to human fibrosarcoma cell line 8 days after intravenous injection in nude mice and when phosphate buffer / saline was used (control) Observe cancer cell metastasis to Little tumor metastasis is observed in the WiNA-21 injection group. ] WiNA-21(in vivo)ならびにWiNA-201(in vitro)は細胞接着タンパク質NCAM-140を増加させる。 [A:ヒト神経芽腫細胞株を、ウィザフェリンA、ウィザノンあるいは WiNA-201存在下で48時間培養後、各種NCAMの発現量の確認。B:HT1080細胞株を用いたin vivo腫瘍形成実験。ヌードマウス皮下で作らせたHT1080細胞の腫瘍中でのWiNA-21を用いた各種NCAM発現量の比較。C:HT1080細胞培養液中のWiNA-201を用いたNCAM-140とPSA-NCAMの発現量の比較。]WiNA-21 (in vivo) as well as WiNA-201 (in vitro) increases the cell adhesion protein NCAM-140. [A: Human neuroblastoma cell line was cultured for 48 hours in the presence of Witherin A, Withanone or WiNA-201, and the expression levels of various NCAMs were confirmed. B: In vivo tumor formation experiment using HT1080 cell line. Comparison of various NCAM expression levels using WiNA-21 in HT1080 cell tumors made subcutaneously in nude mice. C: Comparison of expression levels of NCAM-140 and PSA-NCAM using WiNA-201 in HT1080 cell culture. ] WiNA-201によるNCAMの強力な発現誘導とPSA-NCAMの発現抑制。 [WiNA-201をHT1080細胞培養液に添加しNCAMおよびPSA-NCAMの発現を免疫染色により検討した。WiNA-201投与によってNCAMが上昇したのに対し、PSA-NCAMは減少したことを示す。]Strong expression induction of NCAM and suppression of PSA-NCAM expression by WiNA-201. [WiNA-201 was added to HT1080 cell culture medium and the expression of NCAM and PSA-NCAM was examined by immunostaining. It shows that PSA-NCAM decreased while WiCAM-201 administration increased NCAM. ] WiNA-21(in vivo)ならびにWiNA-201(in vitro)によるマトリクスメタロプロテアーゼMMP2の発現抑制。WiNA-201、WiNA-21の投与によるMMP2タンパク質の優位な減少が観察された。 [A:YKG1細胞株の培養液中にウィザフェリンA、ウィザノンあるいはWiNA-201を添加して培養し、MMP2タンパク質の発現量をウエスタンブロットにより検討。B:HT1080細胞株の培養液中でのMMP2タンパク質の発現量。C:ヌードマウス皮下のHT1080由来腫瘍でのMMP2タンパク質の発現量。]Inhibition of matrix metalloproteinase MMP2 expression by WiNA-21 (in vivo) and WiNA-201 (in vitro). A significant decrease in MMP2 protein was observed with the administration of WiNA-201 and WiNA-21. [A: Witherferin A, withanone or WiNA-201 was added to the culture medium of the YKG1 cell line and cultured, and the expression level of MMP2 protein was examined by Western blot. B: MMP2 protein expression level in the culture medium of HT1080 cell line. C: MMP2 protein expression level in HT1080-derived tumors subcutaneously in nude mice. ] WiNA-21ならびにWiNA-201によるシグナル伝達タンパク質のERKの減少。 [WiNA-201、WiNA-21の投与によるERKの優位な減少が観察された。A:YKG1細胞株の培養液中でのERKの発現量の検討。B:HT1080細胞株の培養液中でのERKの発現量。C:ヌードマウス皮下のHT1080由来腫瘍でのERKの発現量。]Reduction of ERK of signaling protein by WiNA-21 and WiNA-201. [A significant decrease in ERK was observed with the administration of WiNA-201 and WiNA-21. A: Examination of the expression level of ERK in the culture solution of YKG1 cell line. B: ERK expression level in the culture medium of HT1080 cell line. C: ERK expression level in HT1080-derived tumors subcutaneously in nude mice. ]

1.本発明の「WiNA-21」又は「WiNA-201」の製造方法
アシュワガンダの葉をアルコール(水性アルコール)で抽出、濃縮して得られるアルコール抽出物(i-Extract)を得た後、特許文献1又は2の方法に従って、下記ウィザノン(式1)及びウィザフェリンA(式2)を精製する。粉末状のまま、両者を重量比で2:1又は20:1に配合して投与することもできるが、あらかじめDMSO溶媒にそれぞれ10mg/ml溶解した組成物として用いることが好ましい。本発明の実施例では、WiNA組成物として用いた。
なお、「2:1(重量比)」というとき、四捨五入して2:1となることを意味するから、「2.4〜1.5:1」、すなわち「24:10〜15:10」の場合を包含し、好ましくは22:10〜18:10である。同様に、「20:1(重量比)」という場合も「20.4〜19.5:1」すなわち「204:10〜195:10」の場合を包含し、好ましくは202:10〜198:10である。
<式1>ウィザノン
<式2>ウィザフェリンA
1. Method for Producing “WiNA-21” or “WiNA-201” of the Present Invention After obtaining an alcohol extract (i-Extract) obtained by extracting and concentrating Ashwagandha leaves with alcohol (aqueous alcohol), Patent Document 1 Alternatively, the following withanone (Formula 1) and witherferin A (Formula 2) are purified according to the method of 2. While both powders can be mixed and administered at a weight ratio of 2: 1 or 20: 1, it is preferable to use them as a composition in which 10 mg / ml is dissolved in DMSO solvent in advance. In the examples of the present invention, it was used as a WiNA composition.
When “2: 1 (weight ratio)” is used, it means rounding to 2: 1, so it includes “2.4 to 1.5: 1”, that is, “24:10 to 15:10”. And preferably 22:10 to 18:10. Similarly, “20: 1 (weight ratio)” includes “20.4 to 19.5: 1”, that is, “204: 10 to 195: 10”, preferably 202: 10 to 198: 10.
<Formula 1> Withanon
<Formula 2> Witherin A

2.in vitroでのWiNA-201の効果と、それを踏まえたin vivoにおけるウィザノライド化合物の最適配合割合(WiNA-21)
in vitroでのウィザノライド化合物の投与実験では、ウィザノン(上記式1)とウィザフェリンA(上記式2)とを重量比で20:1の割合で含んだ組成物の場合において、正常細胞への細胞毒性がなく、しかも、癌細胞に対する細胞増殖抑制能が強力であるという画期的な効果が奏せられる(WiNA-201)。
このように両者が特定の割合で組み合わされた場合には、正常細胞に対する毒性がなく、癌細胞に対しては細胞増殖抑制能が強力である数値範囲が存在することが明らかとなった。
そこで、実際の抗癌剤として、生体内に投与する場合についても最適配合割合を決定することとした。すなわち、in vivoにおける投与量は個体の重さに対する量となり、溶解性、半減期など生体内での挙動がin vitroとは必ずしも一致しないため、投与量を最適化する必要がある。
その際、従来はin vivoデータ採取の際に経口摂取、局所注射のみであったが、実際の抗癌剤投与形態が血流に投与(静脈注射)することが多いことを踏まえ、本実施例ではマウスの血流への注射を試みた。そのため、尾静脈への注射が可能なマウスの体重(kg)に対する最大投与量を検討したところ、マウスの体重(kg)に対し、ウィザノン-1mg、ウィザフェリンA-0.5mgが最大投与量であることがわかった。ウィザノンとウィザフェリンAとを重量比で2:1の割合で含む注射剤を製造し、マウス尾静脈へ注射したところ(「WiNA-21」)、投与し続けても体重の変化がなく、正常細胞、組織への毒性が極めて少ないことが判明した。
そして、この「WiNA-21」をヌードマウスに皮下移植した腫瘍細胞に適用したところ、強い腫瘍細胞増殖抑制効果のみならず、癌細胞の浸潤性抑制効果も確認し、癌細胞の転移抑制作用という優れた抗癌剤機能が実証できた。
2. Effect of WiNA-201 in vitro and optimal blending ratio of withanolide compound in vivo based on it (WiNA-21)
In vitro administration of withanolide compounds, in the case of a composition containing withanone (formula 1) and withferrin A (formula 2) in a weight ratio of 20: 1, cells to normal cells There is an epoch-making effect that it has no toxicity and has a strong ability to suppress cell growth against cancer cells (WiNA-201).
Thus, when both were combined in a specific ratio, it became clear that there was a numerical range in which there was no toxicity to normal cells and the ability to suppress cell growth was strong for cancer cells.
Therefore, as an actual anticancer agent, the optimum blending ratio was determined even when administered in vivo. In other words, the dose in vivo is the amount relative to the weight of the individual, and the behavior in vivo such as solubility and half-life does not always match that in vitro, so it is necessary to optimize the dose.
At that time, in the past, only ingestion and local injection were used at the time of collecting in vivo data. However, in this example, mice were used in this example because the actual anticancer drug administration form is often administered into the bloodstream (intravenous injection). Attempted injection into the bloodstream. Therefore, the maximum dose for body weight (kg) of mice that can be injected into the tail vein was examined. With respect to the body weight (kg) of mice, witherone-1 mg and witherferin A-0.5 mg are the maximum doses. I understood it. An injection containing 2: 1 by weight of Withanone and Witherin A was manufactured and injected into the tail vein of mice ("WiNA-21"). It was found that the toxicity to cells and tissues was extremely low.
And when this "WiNA-21" was applied to tumor cells transplanted subcutaneously in nude mice, it was confirmed not only a strong tumor cell growth inhibitory effect, but also a cancer cell invasion inhibitory effect, a cancer cell metastasis inhibitory effect Excellent anticancer drug function was demonstrated.

3.本発明の「WiNA-201」「WiNA-21」の特性
本発明の組成物「WiNA-201」「WiNA-21」には、以下の特性がある。
(1)WiNA-201、WiNA-21はがん細胞特異的に毒性がある。正常細胞に対する毒性はない。(図1,図6)
(2)WiNA-21をマウスに静脈注射しても無害である。
WiNA-21の生体への無毒性は、10日間のマウス尾静脈注射により確認した。(図6)
(3)WiNA-201はin vitroで抗腫瘍効果を示す。(図1)
(4)WiNA-21はin vivoで抗腫瘍効果を示す。
WiNA-21の強力な抗癌抑制活性はヌードマウス腫瘍形成アッセイにより確認した。(図7,8)
(5)WiNA-201はがん細胞の浸潤能を弱める。
WiNA-201の癌細胞浸潤能抑制効果は、癌細胞の遊走能抑制効果の観察(図2)、Boydenチャンバーを用いた浸潤アッセイ(図3)、QCMTM細胞浸潤アッセイキットによる定量化(図4)により確認した。
(6)WiNA-201、WiNA-21はがん細胞の抗転移能を有する。
WiNA-21の抗転移能は、腫瘍転移アッセイで確認された。(図9)
また、WiNA-201、WiNA-21が、細胞接着タンパク質のNCAM-140の増加作用を示したことはWiNAが抗転移効果誘導に強く関与することを示すものであり、PSA-NCAMの減少効果も、細胞接着の増加と転移の減少を示す。(図10,11)
(7)WiNA-201はがん細胞の細胞外マトリクスへの付着を弱める。
WiNA-201の、癌細胞の細胞外マトリクス(フィブロネクチン)への接着能抑制効果を細胞接着アッセイにより確認した。(図5)
(8)WiNA-201はがん細胞の細胞接着タンパク質の発現を増加させる。
WiNA-201、WiNA-21は、細胞接着タンパク質のNCAM-140の増加作用を示す。このことはWiNAが抗転移効果誘導に強く関与することを示すものである。
細胞接着タンパク質NCAMと腫瘍退縮の関連性はすでに知られているが、本実施例8においては、WiNA-201、WiNA-21がNACM-140を増加させていることからみて、NACM-140の増加が腫瘍の縮小を引き起こしたことに強く関連していることが示された。(図10A,B、図11)
また、WiNA-201、WiNA-21には、NCAM-140発現増進剤、阻害剤としての効果が期待できる。
(9)WiNA-201、WiNA-21はがん細胞のメタロプロテアーゼMMP2の発現を減少させる。WiNA-201、WiNA-21は、マトリクスメタロプロテアーゼ(MMP)は、腫瘍に酸素や栄養を供給するための血管新生に関わっているが、とりわけ癌の増殖に最も関わりの深いとされており、阻害剤のターゲットとなっているMMP-2の発現を抑えることが実証された。すなわち、WiNA-201、WiNA-21には、MMP-2発現阻害剤としての効果が期待できる。(図12)
(10)WiNA-201、WiNA-21はがん細胞のシグナル伝達タンパク質ERKを減少させる。すなわち、WiNA-201、WiNA-21には、ERK発現阻害剤としての効果が期待できる。(図13)
3. Characteristics of “WiNA-201” and “WiNA-21” of the present invention The compositions “WiNA-201” and “WiNA-21” of the present invention have the following characteristics.
(1) WiNA-201 and WiNA-21 are toxic specifically for cancer cells. There is no toxicity to normal cells. (Fig. 1, Fig. 6)
(2) It is harmless even if WiNA-21 is intravenously injected into mice.
The non-toxicity of WiNA-21 in vivo was confirmed by 10 days of mouse tail vein injection. (Fig. 6)
(3) WiNA-201 exhibits an antitumor effect in vitro. (Figure 1)
(4) WiNA-21 exhibits an antitumor effect in vivo.
The strong anticancer suppressive activity of WiNA-21 was confirmed by nude mouse tumorigenesis assay. (Figs. 7 and 8)
(5) WiNA-201 weakens the invasive ability of cancer cells.
The inhibitory effect of WiNA-201 on the ability to infiltrate cancer cells was observed by observing the effect of inhibiting the migration ability of cancer cells (Fig. 2), invasion assay using Boyden chamber (Fig. 3), and quantification using QCM TM cell invasion assay kit (Fig. 4). ).
(6) WiNA-201 and WiNA-21 have anti-metastatic ability of cancer cells.
The anti-metastatic potential of WiNA-21 was confirmed by a tumor metastasis assay. (Fig. 9)
In addition, the fact that WiNA-201 and WiNA-21 increased NCAM-140, a cell adhesion protein, indicates that WiNA is strongly involved in the induction of anti-metastatic effects, and that PSA-NCAM also has a decreasing effect. Show increased cell adhesion and decreased metastasis. (Figs. 10 and 11)
(7) WiNA-201 weakens the adhesion of cancer cells to the extracellular matrix.
The inhibitory effect of WiNA-201 on the ability to adhere to the extracellular matrix (fibronectin) of cancer cells was confirmed by a cell adhesion assay. (Fig. 5)
(8) WiNA-201 increases the expression of cell adhesion proteins in cancer cells.
WiNA-201 and WiNA-21 show an increasing action of the cell adhesion protein NCAM-140. This indicates that WiNA is strongly involved in the induction of antimetastatic effects.
Although the relationship between the cell adhesion protein NCAM and tumor regression is already known, in this Example 8, the increase in NACM-140 is seen from the fact that WiNA-201 and WiNA-21 increase NACM-140. Was strongly associated with causing tumor shrinkage. (FIG. 10A, B, FIG. 11)
In addition, WiNA-201 and WiNA-21 are expected to be effective as NCAM-140 expression enhancers and inhibitors.
(9) WiNA-201 and WiNA-21 decrease the expression of metalloprotease MMP2 in cancer cells. WiNA-201 and WiNA-21 are matrix metalloproteases (MMPs) that are involved in angiogenesis to supply oxygen and nutrients to tumors. It was demonstrated to suppress the expression of MMP-2, which is the target of the drug. That is, WiNA-201 and WiNA-21 can be expected to have an effect as an MMP-2 expression inhibitor. (Fig. 12)
(10) WiNA-201 and WiNA-21 decrease cancer cell signaling protein ERK. That is, WiNA-201 and WiNA-21 can be expected to have an effect as an ERK expression inhibitor. (Fig. 13)

4.本発明における「WiNA-21」を有効成分とする抗癌剤
本発明は「WiNA-21」を有効成分とする「抗癌剤」に係るものであるが、本発明において「抗癌剤」というとき、ヒト又は哺乳動物に対して経口若しくは非経口で投与した場合に抗癌活性又は抗腫瘍活性を有する医薬組成物であって、癌の予防又は治療若しくは処置のために用いることができる医薬組成物を指す。
本発明の医薬用組成物の投与方法は特に限定されるものではなく、経口、経鼻、非経口、経肺、経皮、経粘膜などが可能であり、種々の剤形とすることができるが、注射剤による投与が特に適している。例えば、経口投与のためには、錠剤、カプセル剤などとすることができ、薬剤的に許容できる種々の担体、例えば、賦形剤、結合剤、崩壊剤、滑沢剤、着香剤、着色剤、甘味剤、矯味剤、溶解補助剤、懸濁化剤、乳化剤、コーティング剤、ビタミンC、抗酸化剤を含むことができるが、これらに限定されない。
本発明の医薬用組成物の投与量は、投与するヒトの年齢、体重、症状によって異なるが、一般的には成人1日用量として1mg〜1000mg、好ましくは100mg〜500mgを使用し、1日あたりの量を数回に分けて投与することもできる。また、他の抗腫瘍剤や治療法と組み合わせて投与することもできる。
本発明の組成物は、食品又は栄養補助食品の形態とすることも、医薬部外品または化粧品の形態とすることもできる。
また、「WiNA-201」の場合は、患者の血液、リンパ球、樹状細胞などを生体外に取り出し、生体外で他の免疫活性剤、サイトカイン類などと共に投与する場合に有効である。
4). The present invention relates to an “anticancer agent” comprising “WiNA-21” as an active ingredient. The term “anticancer agent” as used herein refers to a human or mammal. It refers to a pharmaceutical composition having anticancer activity or antitumor activity when administered orally or parenterally, which can be used for the prevention, treatment or treatment of cancer.
The administration method of the pharmaceutical composition of the present invention is not particularly limited, and can be oral, nasal, parenteral, pulmonary, transdermal, transmucosal, etc., and can be in various dosage forms. However, administration by injection is particularly suitable. For example, for oral administration, it can be tablets, capsules, etc., and various pharmaceutically acceptable carriers, such as excipients, binders, disintegrants, lubricants, flavoring agents, coloring agents. May include, but is not limited to, agents, sweeteners, flavoring agents, solubilizers, suspending agents, emulsifiers, coating agents, vitamin C, antioxidants.
The dosage of the pharmaceutical composition of the present invention varies depending on the age, weight and symptoms of the human being administered, but generally 1 mg to 1000 mg, preferably 100 mg to 500 mg is used as an adult daily dose. Can be divided into several doses. It can also be administered in combination with other anti-tumor agents and treatments.
The composition of the present invention can be in the form of a food or dietary supplement, or in the form of a quasi-drug or cosmetic.
In the case of “WiNA-201”, the blood, lymphocytes, dendritic cells, etc. of the patient are taken out of the living body and administered together with other immunoactive agents, cytokines and the like in vitro.

本発明を以下の実施例でさらに詳しく説明するが、本発明はこれに限定されない。
なお、本願明細書において引用した文献中の記載事項は、本願明細書中の記載として組み入れることとする。また、特に断りがない場合は、公知の方法、例えばMolecular Cloning 3rd edition Sambrook Jら、Cold Spring Harbor Laboratory Press,2001年、細胞工学別冊「バイオ実験イラストレイテッド」(秀潤社、2001年)などの記載に従って実施可能であり、市販の試薬やキット等の指示書に従って実施可能である。
The present invention will be described in more detail in the following examples, but the present invention is not limited thereto.
In addition, the description items in the documents cited in the present specification are incorporated as descriptions in the present specification. Unless otherwise specified, known methods such as Molecular Cloning 3rd edition Sambrook J et al., Cold Spring Harbor Laboratory Press, 2001, Cell Engineering Supplement "Bio-Experiment Illustrated" (Shyujunsha, 2001), etc. And can be carried out according to the instructions of commercially available reagents and kits.

(実施例1)2種類のウィザノライドの配合割合の検討
2種類のウィザノライドとしてウィザノンとウィザフェリンAを選択し、in vitroにおける両者の最適な組み合わせを検討した。
本発明者らは、以前にウィザノンとウィザフェリンAとの1:1の比率での組み合わせ(WiNA-11)を検討していたが、正常細胞に対する毒性があり、抗癌剤の有効成分としては適さないことをすでに確認していた(図示せず)。
そこで、ヒト骨肉腫細胞(U20S株)及び正常細胞(TIG-1株)の培養液(3×105個/0.2ml)に対し、ウィザノン5.0μg/ml、ウィザフェリンA0.250μg/ml(WiNA-201[20:1])、ウィザノン2.0μg/ml、ウィザフェリンA0.4μg/ml(WiNA-102 [10:2])、ウィザノン5.0μg/ml、ウィザフェリンA0.250μg/ml(WiNA-201[20:1])を添加し、さらに30時間培養後の写真を示す(図1)。コントロールとしては、2μlのDMSOを添加した。
ウィザノンとウィザフェリンAの20:1の組み合わせ(WiNA-201)は、がん細胞を殺傷するが、正常細胞には無害である。一方、ウィザノンとウィザフェリンAの10:2の組み合わせ(WiNA-102)およびウィザノンとウィザフェリンAの10:3の組み合わせ(WiNA-103)は正常細胞に対し、毒性をもつ。
以上の結果から、in vitroにおけるウィザノンとウィザフェリンAの最適な配合割合が20:1であると確定した。当該「WiNA-201」は正常細胞への毒性のない高い抗癌活性を有する優れた抗癌剤としての効果が期待されるので、以下の実施例で、「WiNA-201」の抗癌剤としての効果について詳細に検討する。
(Example 1) Examination of blending ratio of two kinds of withanolides Withanone and Witherin A were selected as two kinds of withanolides, and the optimal combination of both in vitro was examined.
The inventors of the present invention have previously studied a combination of Wizanone and Witherin A in a 1: 1 ratio (WiNA-11), which is toxic to normal cells and is not suitable as an active ingredient of an anticancer agent. I have already confirmed that (not shown).
Therefore, with respect to the culture solution (3 × 10 5 cells / 0.2 ml) of human osteosarcoma cells (U20S strain) and normal cells (TIG-1 strain), Withanone 5.0 μg / ml, Witherin A0.250 μg / ml (WiNA -201 [20: 1]), Withanone 2.0 μg / ml, Witherin A 0.4 μg / ml (WiNA-102 [10: 2]), Withanone 5.0 μg / ml, Witherin A 0.250 μg / ml (WiNA-201 [20: 1]) is added, and a photograph after further culturing for 30 hours is shown (FIG. 1). As a control, 2 μl of DMSO was added.
The 20: 1 combination of Withanone and Witherin A (WiNA-201) kills cancer cells but is harmless to normal cells. On the other hand, the 10: 2 combination of Wizanon and Witherin A (WiNA-102) and the 10: 3 combination of Withanone and Witherin A (WiNA-103) are toxic to normal cells.
From the above results, it was determined that the optimal blending ratio of Withanone and Witherin A in vitro was 20: 1. Since “WiNA-201” is expected to be an excellent anticancer agent having high anticancer activity without toxicity to normal cells, details of the effect of “WiNA-201” as an anticancer agent will be described in the following examples. To consider.

(実施例2)ヒト神経膠芽腫細胞の浸潤抑制
WiNA-201のヒト神経膠芽腫細胞株の浸潤抑制能を確認するために、「創傷治癒アッセイ」で細胞遊走能を観察した。ヒト神経膠芽腫細胞(YKG1)細胞を6穴の培養皿で培養し、細胞が100%コンフルエントに近付いた時点で細胞層に均一な傷を付けた(20ゲージ注射針)。
i-Extract、ウィザフェリンA、ウィザノン及びWiNA-201をそれぞれ培養液中に2μg/ml、0.25μg/ml、5μg/ml、5μg/ml+0.25μg/mlずつ添加し、8時間後の細胞の動きを位相差顕微鏡で観察した。コントロールとウィザノライド類添加細胞の動きを継時的に写真に収めた。なお、コントロール(比較対照群)としては2μlのDMSOを添加したものを用いている。コントロールでは8時間ほどで周囲の細胞が傷部分に遊走したが培養液にi-Extract、ウィザノンもしくはWiNA-201を処理した細胞では、傷への遊走(=細胞層の修復)が遅れた。このうち、WiNA-201が最も効果的に遊走を遅らせた(図2)。
(Example 2) Infiltration suppression of human glioblastoma cells
In order to confirm the ability of WiNA-201 to suppress invasion of human glioblastoma cell lines, the cell migration ability was observed by “wound healing assay”. Human glioblastoma cells (YKG1) cells were cultured in a 6-well culture dish, and when the cells approached 100% confluence, the cell layer was uniformly damaged (20 gauge needle).
i-Extract, Witherin A, Withanone and WiNA-201 were added to the culture solution at 2 µg / ml, 0.25 µg / ml, 5 µg / ml, 5 µg / ml + 0.25 µg / ml, respectively, and cell movement after 8 hours. Was observed with a phase-contrast microscope. The movements of the control and withanolide added cells were photographed over time. A control (comparison control group) to which 2 μl of DMSO was added was used. In the control, the surrounding cells migrated to the wound in about 8 hours, but migration to the wound (= repair of the cell layer) was delayed in cells treated with i-Extract, withanone or WiNA-201 in the culture medium. Of these, WiNA-201 most effectively delayed migration (Figure 2).

(実施例3)ヒト線維肉腫細胞の浸潤抑制
(3−1)Boydenチャンバーを用いた浸潤アッセイ
WiNA-201によるヒト線維肉腫細胞株HT1080の浸潤能の抑制効果を、Boydenチャンバーを用いた浸潤アッセイ(Corning Inc.,Corning,NY)により検討した。
Boydenチャンバーの仕組みは以下の通りである。培養皿を上層と下層のポリカーボネートのフィルター膜で隔てる。この膜には8μmの穴が開いており、マトリゲル(50μl of 5mg/ml)を加えて37℃で2時間放置、ゲル化させる。1×106個の細胞を100μlの無血清培養液に浮遊させて、ゲル化したマトリゲルの上層に注ぐ。培養皿の下層には、細胞接着基質としてフィブロネクチン(5μg/ml)を含む培養液(600μl)で満たされている。この状態でチャンバーを37℃で24時間放置する。浸潤能を有する細胞はマトリゲルを分解してフィルター膜を通過、上層から下層に移動する。培養終了後、フィルター膜の上層に残っている細胞は綿棒で取り除く。下層に浸潤した細胞は、培養液をメタノール・アセトン(1:1)と交換して固定、クリスタルバイオレットで染色する。染色した細胞は乾燥させて、培養皿から取り外し、顕微鏡下で写真に収める。
(Example 3) Infiltration suppression of human fibrosarcoma cells (3-1) Invasion assay using Boyden chamber
The inhibitory effect of WiNA-201 on the invasion ability of human fibrosarcoma cell line HT1080 was examined by invasion assay using Boyden chamber (Corning Inc., Corning, NY).
The mechanism of the Boyden chamber is as follows. The culture dish is separated by an upper and lower polycarbonate filter membrane. This membrane has an 8 μm hole, and Matrigel (50 μl of 5 mg / ml) is added and left at 37 ° C. for 2 hours to gel. 1 × 10 6 cells are suspended in 100 μl of serum-free medium and poured onto the upper layer of the gelled Matrigel. The lower layer of the culture dish is filled with a culture solution (600 μl) containing fibronectin (5 μg / ml) as a cell adhesion substrate. In this state, the chamber is left at 37 ° C. for 24 hours. Cells having invasive ability decompose Matrigel, pass through the filter membrane, and move from the upper layer to the lower layer. After completion of the culture, the cells remaining on the upper layer of the filter membrane are removed with a cotton swab. Cells infiltrating the lower layer are fixed by exchanging the culture medium with methanol / acetone (1: 1) and stained with crystal violet. Stained cells are dried, removed from the culture dish and photographed under a microscope.

上記のように調製したヒト線維肉腫細胞株(HT1080)上層のゲル化マトリゲル上に、コントロールのDMSO、ウィザフェリンA、ウィザノンおよびWiNA-201をそれぞれ2μl、0.25μg/ml、5μg/ml、5μg/ml+0.25μg/mlずつ添加し、37℃で24時間培養後、メンブレンより下に浸潤した細胞をメタノール・アセトン(1:1)で固定した後、クリスタルバイオレットで染色し、乾燥して写真撮影した。ウィザフェリンA、ウィザノンおよびWiNA-201の全てに於いて浸潤能の抑制が観察されたが、WiNA-201が最も強力な作用を示した。(図3)   On the gelled matrigel of the human fibrosarcoma cell line (HT1080) prepared as described above, 2 μl, 0.25 μg / ml, 5 μg / ml, and 5 μg / ml of control DMSO, Witherin A, Withanone and WiNA-201, respectively. ml + 0.25μg / ml was added, cultured at 37 ° C for 24 hours, cells infiltrated below the membrane were fixed with methanol / acetone (1: 1), stained with crystal violet, dried and photographed . Inhibition of invasive ability was observed in all of Witherin A, Withanon and WiNA-201, but WiNA-201 showed the strongest effect. (Figure 3)

(3−2)QCMTM細胞浸潤アッセイキットによる定量化
WiNA-201によるHT1080細胞株の浸潤能抑制効果は更に、QCMTM細胞浸潤アッセイキット(Milipore社のQCMTM キット)を用い、蛍光測定により定量化した。なお、細胞は6穴培養皿で48時間培養後、実験に使用した。
Boydenチャンバー同様、このキットも培養皿の上層と下層を細胞外マトリクス層で隔てられており、上層に注いだ細胞が浸潤能を持てば、細胞外マトリクスを分解して下層に移動する。細胞外マトリクス層を温めた無血清培養液300μlで15分から30分濡らしておく。その後膜を破ることなく培養液を250μlほど取り除き、細胞(250μl,1×106cells/ml)を加える。下層には10%胎児ウシ血清を含む培養液で満たされている。培養液中に、コントロールのDMSO、ウィザフェリンA、ウィザノン及びWiNA-201をそれぞれ2μl、0.25μg/ml、5μg/ml、5μg/ml+0.25μg/mlずつ添加し、この状態で37℃、24時間培養した後、上層の培養液を取り除く。浸潤チャンバーインサートを取り外し、温めた細胞剥離液(225μl)に漬けて37℃で30分間放置する。遊離させた細胞はその後NP40細胞溶解液で溶解し、核酸染色液(CyQuant GRRダイ,Molecular Probes)で染色した(室温で15分)。染色した細胞の細胞数はプレートリーダー(480/520nmフィルター)で計測した。(図4)
その結果、ウィザフェリンA、ウィザノンおよびWiNA-201の全てに於いて浸潤能の抑制が観察されたが、WiNA-201が最も強力な抑制作用を示した。
(3-2) Quantification with QCM TM cell invasion assay kit
The inhibitory effect of WiNA-201 on the invasion ability of the HT1080 cell line was further quantified by fluorescence measurement using a QCM cell invasion assay kit (QCM kit from Millipore). The cells were cultured in a 6-well culture dish for 48 hours and then used for experiments.
Like the Boyden chamber, this kit also separates the upper and lower layers of the culture dish with an extracellular matrix layer. If the cells poured into the upper layer have invasive ability, the extracellular matrix is decomposed and moved to the lower layer. Wet the extracellular matrix layer with 300 μl of warm serum-free medium for 15 to 30 minutes. Then, remove about 250 μl of the culture without breaking the membrane, and add cells (250 μl, 1 × 10 6 cells / ml). The lower layer is filled with a culture solution containing 10% fetal bovine serum. Add 2 μl, 0.25 μg / ml, 5 μg / ml, 5 μg / ml + 0.25 μg / ml of control DMSO, witherferin A, withanone and WiNA-201 to the culture solution, respectively, in this state at 37 ° C. for 24 hours. After culturing, the upper culture solution is removed. Remove the invasion chamber insert, soak in warm cell detachment solution (225 μl) and leave at 37 ° C. for 30 minutes. The released cells were then lysed with an NP40 cell lysate and stained with a nucleic acid stain (CyQuant GR R dye, Molecular Probes) (15 minutes at room temperature). The number of stained cells was counted with a plate reader (480/520 nm filter). (Fig. 4)
As a result, suppression of invasive ability was observed in all of Witherin A, Withanone and WiNA-201, but WiNA-201 showed the strongest inhibitory action.

(実施例4)細胞接着アッセイ
この実験では、ヒト線維肉腫細胞(HT1080)の細胞外マトリクス構成因子(フィブロネクチン)への接着能へのWiNA-201の抑制効果を検討する。
HT1080株は6穴培養皿で48時間培養後、実験に使用した。96穴培養皿をフィブロネクチンで室温2時間コーティングして、0.5% BSA/PBSで30分間ブロッキングしたものを使用した。細胞数を5×105cells/mlになるように、無血清培養液に浮遊させてから、100μlの細胞を上記セッティングに乗せて、ウィザフェリンA、ウィザノン及びWiNA-201をそれぞれ0.25μg/ml、5μg/ml、5μg/ml+0.25μg/mlずつ37℃で45分間培養した。その後培養液を注意深く取り除き、培養皿をPBSで3回洗浄、底に接着した細胞数を計測した。細胞数の計測には、MTT比色分析アッセイを行った。培養液に、3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT)(0.5mg/ml)を添加して4時間放置、その後100・lのDMSOで置換すると結晶化したフォルムアザンが溶解して赤紫色になる。550nmの吸光度をプレートリーダー(Wallac)で計測下結果を図5に示す。
ウィザフェリンA、ウィザノンおよびWiNA-201の全てに於いてフィブロネクチンとの細胞接着が抑制されたが、WiNA-201が最も強力な作用を示した。
Example 4 Cell Adhesion Assay In this experiment, the inhibitory effect of WiNA-201 on the ability of human fibrosarcoma cells (HT1080) to adhere to the extracellular matrix constituent factor (fibronectin) is examined.
The HT1080 strain was cultured in a 6-well culture dish for 48 hours and then used for the experiment. A 96-well culture dish was coated with fibronectin at room temperature for 2 hours and then blocked with 0.5% BSA / PBS for 30 minutes. After suspending in a serum-free culture solution so that the number of cells becomes 5 × 10 5 cells / ml, 100 μl of cells are placed on the above setting, and witherferin A, withanone and WiNA-201 are each 0.25 μg / ml. The cells were cultured at 37 ° C. for 45 minutes at 5 μg / ml, 5 μg / ml + 0.25 μg / ml. Thereafter, the culture solution was carefully removed, the culture dish was washed three times with PBS, and the number of cells adhered to the bottom was counted. To measure the number of cells, an MTT colorimetric assay was performed. Add 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazoliumbromide (MTT) (0.5 mg / ml) to the culture and leave it for 4 hours. Formazane is dissolved and turns reddish purple. FIG. 5 shows the results of measuring the absorbance at 550 nm with a plate reader (Wallac).
Witherferin A, withanone and WiNA-201 all inhibited cell adhesion to fibronectin, but WiNA-201 showed the most potent action.

(実施例5)WiNA-21注射による毒性試験
マウスの尾静脈への注射を行うため、ウィザノン及びウィザフェリンの尾静脈注射が可能なマウスの体重(kg)に対する最大投与量を検討した結果、マウスの体重(kg)に対し、ウィザノン-1mg、ウィザフェリンA-0.5mgが最大投与量であることがわかった。
WiNA-21(ウィザノン-1mg/kg体重+ウィザフェリンA-0.5mg/kg体重)をヌードマウスに尾静脈に注射した。毎回200μlを、1日間隔で10日間投与した。比較のために、コントロール(PBS)、ウィザフェリンA(0.5mg/kg体重)及びウィザノン(1mg/kg体重)も200μlずつ同様に投与した。マウスの体重は3日おきに計測した。餌の摂食と行動は毎日記録した(図6)。
その結果、ウィザフェリンAを投与したマウスは食欲も減退し体重が減少するという明らかな毒性を示したが、WiNA-21はウィザノンと同様に順調な体重増加が見られ、マウスに毒性を示さなかった。
(Example 5) Toxicity test by WiNA-21 injection In order to inject into the tail vein of mice, the maximum dose with respect to body weight (kg) of mice capable of tail vein injection of withanone and withferrin was examined. It was found that with the body weight (kg), witherone-1 mg and witherferin A-0.5 mg were the maximum doses.
WiNA-21 (withanone-1 mg / kg body weight + witherferin A-0.5 mg / kg body weight) was injected into nude mice into the tail vein. Each time 200 μl was administered at daily intervals for 10 days. For comparison, 200 μl of control (PBS), witherin A (0.5 mg / kg body weight) and withanone (1 mg / kg body weight) were similarly administered. Mice were weighed every 3 days. Feeding and behavior were recorded daily (Figure 6).
As a result, mice administered Witherin A showed clear toxicity that appetite decreased and body weight decreased, but WiNA-21 showed a steady weight gain similar to Withanone and showed no toxicity to mice. It was.

(実施例6)ヌードマウス腫瘍形成アッセイ
この実験では、WiNA-21によるヌードマウス皮下移植下でのヒト線維肉腫細胞株(HT1080)の増殖抑制効果を、WiNA-21注射群とコントロール群とで比較検討した。
Balb/cヌードマウス(4週齢雌)を業者から購入(日本クレア)し、動物施設で1週間慣れさせた。ヒト線維肉腫細胞株(HT1080)3×106個を0.2ml培養液に浮遊させ、下腹部の皮下に注射した(一匹当たり二か所)。小さい腫瘍が現れた後(8日後)、腫瘍マウスをランダムに4匹ずつグループに分けた。コントロール群にはリン酸緩衝生理食塩水(PBS)を投与する。試験群にはWiNA-21(ウィザノン-1mg/kg体重+ウィザフェリンA-0.5mg/kg体重)を尾静脈注射で1日おきに5回投与した。腫瘍の増殖と動物の健康は毎日モニターした。
その結果、コントロール群では、湿潤性の巨大な腫瘍を形成するものが多かったが、WiNA-21注射群では腫瘍がほとんど消失したマウスもあり、他のマウスの腫瘍も増大する速度は遅いことが観察された(図7)。なお、図7では、図に示した日に写真を撮影した。
同時に、WiNA-21注射群と比較対照群におけるヌードマウス移植腫瘍をノギスで測定し、皮下の腫瘍の体積をV=(長さ×幅×厚さ)/2として計算した。4匹のマウスの平均値をプロットした(図8)。
以上の結果は、WiNA-21の強力ながん抑制作用を示すものである。
(Example 6) Nude mouse tumor formation assay In this experiment, the growth inhibitory effect of human fibrosarcoma cell line (HT1080) under subcutaneous transplantation of nude mice by WiNA-21 was compared between the WiNA-21 injection group and the control group. investigated.
Balb / c nude mice (4 weeks old female) were purchased from a vendor (Claire Japan) and habituated to the animal facility for 1 week. 3 × 10 6 human fibrosarcoma cell lines (HT1080) were suspended in 0.2 ml culture medium and injected subcutaneously into the lower abdomen (two sites per animal). After the appearance of a small tumor (8 days later), the tumor mice were randomly divided into groups of 4 mice. The control group receives phosphate buffered saline (PBS). In the test group, WiNA-21 (withanone-1 mg / kg body weight + witherferin A-0.5 mg / kg body weight) was administered five times every other day by tail vein injection. Tumor growth and animal health were monitored daily.
As a result, in the control group, many of the tumors formed a huge wet tumor, but in the WiNA-21 injection group, there were some mice in which the tumor almost disappeared, and the tumor growth rate of other mice may be slow. Observed (FIG. 7). In FIG. 7, a photograph was taken on the day shown in the figure.
At the same time, nude mouse transplanted tumors in the WiNA-21 injection group and the comparative control group were measured with calipers, and the volume of the subcutaneous tumor was calculated as V = (length × width × thickness) / 2. Average values of 4 mice were plotted (FIG. 8).
The above results show the strong cancer inhibitory action of WiNA-21.

(実施例7)腫瘍転移アッセイ
この実験では、WiNA-21によるin vivoでのヒト線維肉腫細胞株(HT1080)の転移抑制効果を、転移アッセイにより検討する。
転移アッセイでは、ヒト線維肉腫細胞株(HT1080)を3×105個、0.2mlの培養液に浮遊させて、尾静脈注射した。8日後、8匹のマウスをランダムに2グループに分ける。上記実施例6と同様に、コントロール群にはリン酸緩衝生理食塩水を投与、試験群にはWiNA-21を1日おきに5回投与した(尾静脈注射)。マウスは5週間後に安楽死させて、肺組織への癌細胞の転移を観察した。(図9)
WiNA-21注射群では、腫瘍の転移は殆ど観察されないか、あるとしてもごく小さなものだった。
(Example 7) Tumor metastasis assay In this experiment, the metastasis inhibitory effect of WiNA-21 on human fibrosarcoma cell line (HT1080) in vivo is examined.
In the metastasis assay, 3 × 10 5 human fibrosarcoma cell lines (HT1080) were suspended in 0.2 ml of culture medium and injected into the tail vein. After 8 days, 8 mice are randomly divided into 2 groups. As in Example 6 above, phosphate buffered saline was administered to the control group, and WiNA-21 was administered to the test group 5 times every other day (tail vein injection). Mice were euthanized after 5 weeks and observed for cancer cell metastasis to lung tissue. (Fig. 9)
In the WiNA-21 injection group, little or no tumor metastasis was observed.

(実施例8)WiNA-201及びWiNA-21による神経細胞接着因子(NCAM)の誘導効果
(8−1)Westernブロッティングの操作条件
本実施例、及び以下の各実施例で用いる免疫ブロッティング(Westernブロッティング)は以下の条件下で行った。
細胞溶解にはRIPAバッファーを用いた。タンパク質量をBCA法で計測して、20mgのタンパク質をSDS-PAGEで分離し、イモビロン-Pメンブレン(Millipore)にセミドライトランスファー機器で転写した。免疫ブロッティングに用いた抗体は以下の通り。NCAM抗体(AbCys SA)、MMP2抗体(Santa Cruz)、リン酸化ERK抗体、リン酸化p38抗体(Cell Signalling)、およびactin抗体(Chemicon International)。2次抗体として西洋わさびペルオキシダーゼ結合ウサギIgG抗体を用い、発色にはECLキットを用いた(GE Healthcare)。
(Example 8) Induction effect of neural cell adhesion factor (NCAM) by WiNA-201 and WiNA-21 (8-1) Western blotting operating conditions Immunoblotting used in this example and the following examples (Western blotting) ) Was performed under the following conditions.
RIPA buffer was used for cell lysis. The amount of protein was measured by the BCA method, 20 mg of protein was separated by SDS-PAGE, and transferred to Immobilon-P membrane (Millipore) with a semi-dry transfer instrument. The antibodies used for immunoblotting are as follows. NCAM antibody (AbCys SA), MMP2 antibody (Santa Cruz), phosphorylated ERK antibody, phosphorylated p38 antibody (Cell Signaling), and actin antibody (Chemicon International). Horseradish peroxidase-conjugated rabbit IgG antibody was used as the secondary antibody, and ECL kit was used for color development (GE Healthcare).

(8−2)神経細胞接着因子(NCAN)のin vitro誘導
ヒト神経芽腫細胞株(IMR32株)を、ウィザフェリンA、ウィザノン及びWiNA-201をそれぞれ0.25μg/ml、5μg/ml、5μg/ml+0.25μg/mlの存在下37℃で48時間培養した。ウィザノンとWiNA-201は細胞接着タンパク質のNCAM-140を強力に誘導した。ウィザノンがNCAM-180を誘導したのに対して、 WiNA-201は専らNCAM-140を誘導した。NCAM-120の合成には影響がなかった。(図10A)。
細胞接着タンパク質のNCAM-140の増加はWiNAが抗転移効果誘導に強く関与することを示している。
(8-2) In vitro induction of neuronal cell adhesion factor (NCAN) Human neuroblastoma cell line (IMR32 line), Withaferin A, Withanon and WiNA-201, 0.25 μg / ml, 5 μg / ml and 5 μg / The cells were cultured at 37 ° C. for 48 hours in the presence of ml + 0.25 μg / ml. Withanon and WiNA-201 strongly induced NCAM-140, a cell adhesion protein. WiNA-201 induced NCAM-180, whereas WiNA-201 exclusively induced NCAM-140. There was no effect on the synthesis of NCAM-120. (FIG. 10A).
An increase in the cell adhesion protein NCAM-140 indicates that WiNA is strongly involved in inducing antimetastatic effects.

(8−3)HT1080細胞株により形成された腫瘍(in vivo)でのNCAM誘導実験
前記実施例6と同様の操作により、ヌードマウス皮下でHT1080細胞の腫瘍を作らせた。ウィザフェリンA、ウィザノン及びWiNA-21を注射した腫瘍内での18日後のNCAMの発現を上記(8−1)の免疫ブロッティング法により観察した。WiNA-21を注射した腫瘍は、NCAM-140のタンパク質レベルがコントロール群よりも高かった。同様の結果はウィザフェリンAでも得られた。その一方でNCAM-120は、ウィザノンおよびWiNA-21を注射した腫瘍で減少していた。結論として、WiNA-21はin vivoで腫瘍の縮小を引き起こし、細胞接着タンパク質のNCAM-140を増加させる。(図10B)。
細胞接着タンパク質NCAMによる細胞接着と腫瘍退縮の関連性はすでに知られているが、本実施例8においては、WiNA-201、WiNA-21が、NACM-140を増加させたことで腫瘍の縮小を引き起こしたことが示唆される。
(8-3) NCAM induction experiment in tumor (in vivo) formed by HT1080 cell line By the same operation as in Example 6, a tumor of HT1080 cells was made subcutaneously in nude mice. NCAM expression after 18 days in the tumor injected with Witherin A, Withanone and WiNA-21 was observed by the immunoblotting method described in (8-1) above. Tumors injected with WiNA-21 had higher NCAM-140 protein levels than the control group. Similar results were obtained with Witherin A. On the other hand, NCAM-120 was decreased in tumors injected with Withanone and WiNA-21. In conclusion, WiNA-21 causes tumor shrinkage in vivo and increases the cell adhesion protein NCAM-140. (FIG. 10B).
Although the relationship between cell adhesion by the cell adhesion protein NCAM and tumor regression is already known, in this Example 8, WiNA-201 and WiNA-21 reduced the tumor size by increasing NACM-140. It is suggested that it was caused.

(8−4)HT1080細胞培養液中でのNCAM誘導実験
HT1080細胞培養液を用いて、前記(8−2)と同様の培養を行った。HT1080細胞培養液においてはWiNA-201によるNCAM-140の増加が最も強く観察された。一方で発達中の神経細胞によく見られる長鎖のポリシアル酸(PSA)をもったPSA-NCAMは減少していた。(図10C)。
ここで、PSA-NCAMの減少は、細胞接着の増加と転移の減少を示すものである。
(8-4) NCAM induction experiment in HT1080 cell culture medium
Using the HT1080 cell culture solution, the same culture as in (8-2) was performed. In HT1080 cell culture medium, the increase of NCAM-140 by WiNA-201 was observed most strongly. On the other hand, PSA-NCAM with long-chain polysialic acid (PSA) often found in developing neurons was decreased. (FIG. 10C).
Here, a decrease in PSA-NCAM indicates an increase in cell adhesion and a decrease in metastasis.

(実施例9)免疫染色法を用いた腫瘍細胞内のNCAM発現誘導の観察
本実施例では、免疫染色法を用いて、WiNA-201の腫瘍細胞内でのNCAMの発現誘導を観察する。具体的には以下の条件下で行った。
HT1080細胞株を12穴の培養皿に沈めたカバースリップ上で培養して接着させる。その際の培養条件は、上記実施例1と同様であり、培養液中にはウィザフェリンA、ウィザノン及びWiNA-201をそれぞれ0.25μg/ml、5μg/ml、5μg/ml+0.25μg/mlずつ添加されている。
48時間培養後、リン酸緩衝生理食塩水(PBS)で洗浄、冷やしたメタノール・アセトン(1:1)で5分から10分間固定する。固定した細胞をPBSで洗浄後、0.2%のTriton X-100/PBSで10分間漬けて、細胞膜に穴を空ける。2%ウシ血清アルブミン/PBSでブロッキングして、NCAM抗体(Santa Cruz Biotechnology)あるいはPSA NCAM抗体(Cell signaling)で反応させる。二次抗体として、Alexa488結合ヤギ抗ウサギIgG抗体を用いる(Molecular probes)。0.2% Triton X-100/PBSで2、3回洗浄後、マウンティング液で封入する。観察にはCarl Zeissの蛍光顕微鏡を用いた。
WiNA-201を培養液に添加した細胞および比較対照群のNCAMおよびPSA-NCAMの発現を免疫染色で検討した。WiNA-201投与によってNCAMが上昇したのに対して、PSA-NCAMは減少した。(図11)
(Example 9) Observation of NCAM expression induction in tumor cells using immunostaining method In this example, the induction of NCAM expression in tumor cells of WiNA-201 is observed using the immunostaining method. Specifically, it was performed under the following conditions.
The HT1080 cell line is cultured and adhered on a coverslip submerged in a 12-well culture dish. The culture conditions at that time were the same as in Example 1 above, and Withaferin A, Withanone and WiNA-201 in the culture solution were 0.25 μg / ml, 5 μg / ml, 5 μg / ml + 0.25 μg / ml, respectively. It has been added.
After incubation for 48 hours, wash with phosphate buffered saline (PBS) and fix with cold methanol / acetone (1: 1) for 5 to 10 minutes. The fixed cells are washed with PBS and then immersed in 0.2% Triton X-100 / PBS for 10 minutes to make a hole in the cell membrane. Block with 2% bovine serum albumin / PBS, and react with NCAM antibody (Santa Cruz Biotechnology) or PSA NCAM antibody (Cell signaling). As a secondary antibody, Alexa488-conjugated goat anti-rabbit IgG antibody is used (Molecular probes). Wash 2 or 3 times with 0.2% Triton X-100 / PBS and seal with mounting solution. For observation, a fluorescence microscope of Carl Zeiss was used.
The expression of NCAM and PSA-NCAM in cells added with WiNA-201 and the control group was examined by immunostaining. NSA increased with WiNA-201 administration, while PSA-NCAM decreased. (Fig. 11)

(実施例10)マトリクスメタロプロテアーゼMMP2の発現抑制
本実施例では、ヒト神経芽腫細胞株(IMR32株)及びヒト線維肉腫細胞株(HT1080)を用いて、血管新生を促進する因子として知られているマトリクスメタロプロテアーゼMMPのうち、腫瘍の増殖に最も関わりの深いとされるMMP-2発現に対するWiNA-201の抑制効果を、前記実施例(8−1)に記載したウエスタンブロッティング法により検討した。
YKG1およびHT1080細胞株を培養し(培養条件は実施例1と同様。)、培養液中にウィザフェリンA、ウィザノンあるいはWiNA-201を添加して、48時間培養後のMMP2タンパク質の発現を検討した。WiNA-201を添加したYKG1およびHT1080細胞株のいずれにおいてもMMP2タンパク質の優位な減少が観察された。(図12A,B)
一方、実施例6と同様の操作でヌードマウス皮下で増殖させたHT1080細胞株においては、WiNA-21の投与により、in vitroにおけるWiNA-201を添加した場合と同様、MMP2の減少を顕著に引き起こした(図12C)。
(Example 10) Inhibition of expression of matrix metalloprotease MMP2 In this example, human neuroblastoma cell line (IMR32 line) and human fibrosarcoma cell line (HT1080) are known as factors that promote angiogenesis. Among the matrix metalloproteases MMPs, the inhibitory effect of WiNA-201 on MMP-2 expression, which is considered to be most closely related to tumor growth, was examined by the Western blotting method described in Example (8-1).
YKG1 and HT1080 cell lines were cultured (culture conditions are the same as in Example 1), witherferin A, withanone or WiNA-201 was added to the culture solution, and the expression of MMP2 protein after 48 hours of culture was examined. . A significant decrease in MMP2 protein was observed in both YKG1 and HT1080 cell lines supplemented with WiNA-201. (Fig. 12A, B)
On the other hand, in the HT1080 cell line grown subcutaneously in nude mice in the same manner as in Example 6, administration of WiNA-21 caused a significant decrease in MMP2 as in the case of adding WiNA-201 in vitro. (FIG. 12C).

(実施例11)細胞外シグナル制御キナーゼ(ERK)の発現抑制
ERKは、非常に多くのシグナル伝達経路に係わっているシグナル伝達分子であり、細胞の分化や増殖などを制御していると考えら、癌細胞のシグナル伝達にも関わっていることが知られている。本実施例では、YKG1株およびHT1080細胞株を用いて、ERK発現に対するWiNA-201の抑制効果を、実施例10と同様に行った。
その結果、YKG1およびHT1080細胞株の培養液中にWiNA-201を添加すると、ERKタンパク質の減少が観察された。しかしながら同様の実験をウィザフェリンAあるいはウィザノンで行ってもERKの発現には変化がなかった(図13A,B)。同様の結果は、WiNA-21を投与したヌードマウス皮下のHT1080由来腫瘍でも観察された(図13C)。
(Example 11) Inhibition of expression of extracellular signal-regulated kinase (ERK)
ERK is a signaling molecule that is involved in a large number of signal transduction pathways, and is thought to be involved in cancer cell signal transduction because it is thought to regulate cell differentiation and proliferation. Yes. In this example, the inhibitory effect of WiNA-201 on ERK expression was performed in the same manner as in Example 10 using the YKG1 strain and the HT1080 cell line.
As a result, a decrease in ERK protein was observed when WiNA-201 was added to the culture solution of YKG1 and HT1080 cell lines. However, even when the same experiment was performed with Witherin A or Withanone, the expression of ERK was not changed (FIGS. 13A and 13B). Similar results were observed in HT1080-derived tumors subcutaneously in nude mice administered with WiNA-21 (FIG. 13C).

Claims (2)

ウィザノン及びウィザフェリンAを有効成分とし、両成分を2:1(重量比)の割合で含む抗癌剤。   An anticancer agent comprising Withanone and Witherin A as active ingredients and containing both ingredients in a ratio of 2: 1 (weight ratio). 注射剤として投与することを特徴とする、請求項1に記載の抗癌剤。 The anticancer agent according to claim 1 , which is administered as an injection.
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