JP6058263B2 - 癌細胞においてアポトーシスを誘導する方法および使用 - Google Patents
癌細胞においてアポトーシスを誘導する方法および使用 Download PDFInfo
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Description
本出願は、2008年4月11日に出願された米国仮出願第61/044,085号に対する恩典および優先権を主張し、この全開示は、参照により本明細書に組み入れられる。
プログラム細胞死(アポトーシス)は、生命の維持に不可欠であり、何故ならば、一定の組織再生は、再生代謝についての生理学的な足場を提供するためである。アポトーシスは、全体的な組織健康を可能にする細胞再生の恒常性バランスを促進し、その結果、組織代謝の増殖、免疫調節、および血管新生成分の完全性が維持される。上述のプロセスのいずれか1つまたは上述のプロセスの組み合わせの調節障害は、アポトーシス制御の欠如を生じさせ得る。アポトーシス制御のこのような欠如は、任意で遺伝子突然変異と組み合わせて、好都合な腫瘍形成性環境を生じさせ得る。
本開示は、CoQ10またはその代謝産物を細胞中へ送達し、内因性CoQ10および膜脂質と複合体を形成させる方法を記載し、これは、Bcl-2サブファミリーメンバーの調節によって癌細胞中においてp53の活性化およびBcl-2媒介アポトーシスの開始を誘導する。
[本発明1001]
リン脂質を含むリポソームと補酵素Q10またはその代謝産物を含む生物活性剤とを含む組成物を細胞へ投与する工程;ならびに
補酵素Q10またはその代謝産物が、内因性補酵素Q10および膜脂質と複合体を形成することを可能にする工程、を含む方法であって、
該複合体の形成が、アポトーシスを受けるように該細胞を誘導する、前記方法。
[本発明1002]
膜脂質が、オレイン酸、メバロン酸およびキノンからなる群より選択される内因性脂質を含む、本発明1001の方法。
[本発明1003]
組成物が、薬学的に許容される担体をさらに含み、補酵素Q10が、組成物の約0.001%〜約60%(w/w)の量で存在する、本発明1001の方法。
[本発明1004]
組成物が、ゲル、軟膏剤、クリーム、軟膏、ローション、ムース、フォーム、スプレー、エアロゾル、液体、噴霧化散剤(nebulized powder)、および坐剤からなる群より選択される形態である、本発明1001の方法。
[本発明1005]
複合体の形成が、p53、Bcl-2、Bcl-2サブファミリーメンバー、およびBakからなる群より選択される細胞タンパク質の調節を誘導する、本発明1001の方法。
[本発明1006]
細胞タンパク質の調節が、p53タンパク質の再活性化を含む、本発明1005の方法。
[本発明1007]
細胞タンパク質の調節が、Bcl-2ファミリーの少なくとも1つのBH3結合ドメインを調節することを含む、本発明1005の方法。
[本発明1008]
Bcl-2ファミリーの少なくとも1つのBH3結合ドメインが、Bid、Bim、Bik、およびそれらの組み合わせからなる群より選択される、本発明1007の方法。
[本発明1009]
細胞が腫瘍形成性細胞である、本発明1001の方法。
[本発明1010]
リン脂質を含むリポソームと補酵素Q10またはその代謝産物を含む生物活性剤とを含む組成物を腫瘍形成性細胞へ投与する工程;ならびに
補酵素Q10またはその代謝産物が、内因性補酵素Q10および膜脂質と複合体を形成することを可能にする工程、を含む癌を治療するための方法であって、
該複合体の形成が、該腫瘍形成性細胞の血管新生因子を調節する、前記方法。
[本発明1011]
膜脂質が、オレイン酸、メバロン酸およびキノンからなる群より選択される内因性脂質を含む、本発明1010の方法。
[本発明1012]
組成物が、薬学的に許容される担体をさらに含み、補酵素Q10が、組成物の約0.001%〜約60%(w/w)の量で存在する、本発明1010の方法。
[本発明1013]
組成物が、ゲル、軟膏剤、クリーム、軟膏、ローション、ムース、フォーム、スプレー、エアロゾル、液体、噴霧化散剤、および坐剤からなる群より選択される形態である、本発明1010の方法。
[本発明1014]
複合体の形成が、VEGF、FGF、Hif-1α、およびアンジオスタチンからなる群より選択される血管新生因子を調節する、本発明1010の方法。
[本発明1015]
リン脂質を含むリポソームと補酵素Q10またはその代謝産物を含む生物活性剤とを含む組成物を腫瘍形成性細胞へ投与する工程;ならびに
補酵素Q10またはその代謝産物が、内因性補酵素Q10および膜脂質と複合体を形成することを可能にする工程、を含む癌を治療するための方法であって、
該複合体の形成が、該腫瘍形成性細胞の細胞周期因子を調節する、前記方法。
[本発明1016]
膜脂質が、オレイン酸、メバロン酸およびキノンからなる群より選択される内因性脂質を含む、本発明1015の方法。
[本発明1017]
組成物が、薬学的に許容される担体をさらに含み、補酵素Q10が、組成物の約0.001%〜約60%(w/w)の量で存在する、本発明1015の方法。
[本発明1018]
組成物が、ゲル、軟膏剤、クリーム、軟膏、ローション、ムース、フォーム、スプレー、エアロゾル、液体、噴霧化散剤、および坐剤からなる群より選択される形態である、本発明1015の方法。
[本発明1019]
複合体の形成が、smadタンパク質、TGF-β、サイクリン依存性キナーゼ、およびPI3K/aktからなる群より選択される細胞周期因子を調節する、本発明1015の方法。
本開示は、補酵素Q10(CoQ10)を含む薬学的組成物、および内因性脂質分子へ連結し、腫瘍形成性状態に関連する分子機構を調節する方法を提供する。本開示の範囲は、p53経路およびBcl-2遺伝子ファミリーの遺伝子調節に特有の分子医学および腫瘍学の分野に関する。
本開示に従って、本明細書において使用される場合、以下の用語は、特に記載されない限り、以下の意味で定義される。
多くの異なる種由来の被験体が、本開示の組成物で治療され得る。このような動物の非網羅的な例示的なリストは、哺乳動物、例えば、マウス、ラット、ウサギ、ヤギ、ヒツジ、ブタ、ウマ、ウシ、イヌ、ネコ、ならびに霊長類、例えば、サル、類人猿、およびヒトを含む。筋肉疲労、疼痛、創傷などに苦しむことが既知の動物被験体は、本開示における使用に好適であり得る。特に、損傷、手術、関節炎、筋肉疲労などに苦しむヒト患者は、本開示における使用についての好適な動物被験体である。医学または獣医学科学において公知の他の方法に本明細書において教示される方法を適合させること(例えば、被験体動物の体重に従って投与される物質の用量を調節すること)によって、本開示において利用される組成物は、他の動物における使用について容易に最適化され得る。
態様において、本開示は、癌の治療および予防用のCoQ10組成物を提供する。2,3-ジメトキシ-5-メチル-6-デカプレニル-1,4-ベンゾキノン(補酵素Q-10)の経皮、経口 静脈内、および他の非経口調製物は、特に、助剤、有効量の肺表面活性物質を、および/またはリポソームと組み合わせて、含み得る。
上述の組成物は、有効量で被験体へ投与され得る。有効量は、処置される動物または処理される細胞において所望の結果を生じさせることができる量である。医学および獣医学技術分野において周知であるように、いずれか1つの動物についての投薬量は、特定の動物のサイズ、体表面積、年齢、投与される特定の組成物、投与の時間および経路、全体的な健康、ならびに同時投与されている他の薬物を含む、多くの因子に依存する。本開示の組成物の局所投与について好適な投薬量は、約0.1〜約2.5 mg CoQ10/kg体重(例えば、約110〜約300 lbsの範囲の被験体について約10〜約500 mg)であると予想される。培養中の細胞での使用についての有効量はまた変化し得るが、経験的に(例えば、細胞へ異なる濃度を添加し、所望の結果を最もよく生じさせる濃度を選択することによって)容易に決定され得る。好適な濃度は約1〜約250 μMであると予想される。
本開示に付随のデータを生じさせるための実験について利用した材料は、以下を含んだ:Skmel-28(HTB-72)、PC-3(CRL-1435)、およびSkBr3(HBT-30)をATCCから購入した。細胞株を、DMEM/F12培地(ダルベッコ改変イーグル培地:栄養混合物F-12、Invitrogen Corporationから市販)中において増殖させ、5%仔ウシ血清を補った。Bcl-2(カタログ番号:2872)、Bax(カタログ番号:2774)、Bid(カタログ番号:2002)、p53(カタログ番号:9282)、Bcl-xl(カタログ番号:2762)、カスパーゼ-3(カタログ番号:9662)、Mcl-1(カタログ番号:4572)、Bax(カタログ番号:2772)、抗ウサギIgG(カタログ番号:7074)、および抗マウスIgG(カタログ番号:7076)抗体を、Cell Signaling Technology (Boston, MA)から購入した。試薬および化学物質をSigma Aldrich (St Louis, MO)から購入した。ウエスタンブロットゲルおよび緩衝液をBio-Rad (Hercules, CA)から購入した。
タンパク質発現プロトコル(図:3、4、5、6、7、10a〜10d、13、14、16、18、19a、および25において見られるデータを生じた)
Skmel-28、PC-3、およびSkBr3細胞を、80%コンフルエンシーへ増殖させ、ペトリ皿中において継代培養した。24時間後、細胞はプレートへ付着し、培地を抜き取った。処理培地を各プレートへ添加した。意図したインキュベーション時間後、培地を除去し、細胞を冷リン酸緩衝生理食塩水(PBS)で洗浄した。細胞を冷PBS中においてこすり落とし、遠心分離チューブ中に回収した。次いで、細胞をペレット化し、冷PBSで洗浄した(3回)。PBSを除去し、その後、溶解緩衝液を添加し、超音波処理し、タンパク質構造体を分散させた。サンプル緩衝液を各チューブへ添加し、溶液を5分間沸騰させた。BCA(ビシンコニン酸)タンパク質分析キットを使用して、タンパク質の濃度を各サンプルについて定量した。これらの値は、各サンプルについてのローディング体積を決定した。
タンパク質発現についての手順を使用し、タンパク質発現の写真画像を得た。これらの画像(imaged)をコンピュータ分析のための画像ファイルへスキャンした。米国国立衛生研究所(NIH)によって開発されたImageJソフトウェアを使用して、タンパク質発現のレベルを定量した。次いで、サンプルのローディングコントロールであったアクチンの発現レベルに基づいて、発現を計算した。数値を、統計的有意性について統計分析した。
Skmel-28細胞を、5%血清補充DMEM/F12培地中において、80%コンフルエンシーまで増殖させた。細胞をトリプシン処理し、遠心分離機を使用してペレット化した。次いで、ペレットを冷PBS中に再懸濁した。この研究についての被験体は、無胸腺ヌードマウスであった。各被験体に、マウスの背部において、細胞懸濁液の2回の注射を受容させた。腫瘍ができたことを視覚的に評価した後、局所適用での処置を開始した。処置の30日後に、腫瘍をマウスから切除し、ホルマリン中に置いた。各腫瘍サンプルを、パラフィン中に包埋し、ミクロトームを使用してスライスした。スライドをH & EまたはS-100染色した。次いで、これらのサンプルを病理学者が分析し、腫瘍の血管完全性を評価した。
上述したように、本開示の組成物は、癌の治療のために利用され得る。このような組成物は、薬学的に許容される担体中にCoQ10またはその代謝産物を含み得る。このような組成物は、これらに限定されないがメバロン酸およびオレイン酸に加えて内因性補酵素Q10またはその代謝産物の細胞接触を達成し、細胞内複合体を形成させ得る。態様において、このような組成物は、補酵素Q10を約0.001%〜約60%(w/w)含み得る。このような組成物は、局所組成物であり得、これは、今度は、ゲル、軟膏剤、液体、クリーム、軟膏、ローション、スプレー、エアロゾル、ムース、フォーム、それらの組み合わせなどであり得る。
1)減少した酸素(低酸素)
2)増加したフリーラジカル形成
3)調節されないアポトーシス(細胞死)
4)グルコース代謝の依存
5)増加した血管形成
6)変化した免疫認識(自己制御状態の開始)
本出願に引用された全ての刊行物および特許文献は、各個々の刊行物または特許文献がそのように個々に表示されているのと同程度まで、全ての目的について適切な箇所における参照により組み入れられる。この文献における種々の参考文献のそれらの引用によって、本出願人は、特定の参考文献が本出願人の開示に対する「先行技術」であることを認めるものではない。
Claims (8)
- リン脂質と、組成物の0.001%〜60%(w/w)の量の補酵素Q10と、薬学的に許容される担体とを含む薬学的組成物であって、
癌細胞中の腫瘍形成マーカーであるBcl-2、Bax及びカスパーゼ3の発現を標準化し、かつ、p53タンパク質の活性を標準化するための、前記薬学的組成物。 - Bid、Bcl-xlおよびMcl-1からなる群より選択される腫瘍形成マーカーの発現をさらに標準化するための、請求項1記載の薬学的組成物。
- 少なくとも1つの腫瘍形成マーカーの発現レベルが、標準化腫瘍形成マーカーレベルと50%未満異なる、請求項1記載の薬学的組成物。
- 少なくとも1つの腫瘍形成マーカーの発現レベルが、標準化腫瘍形成マーカーレベルと25%未満異なる、請求項1記載の薬学的組成物。
- 少なくとも1つの腫瘍形成マーカーの発現レベルが、標準化腫瘍形成マーカーレベルと10%未満異なる、請求項1記載の薬学的組成物。
- ゲル、軟膏剤、クリーム、軟膏、ローション、ムース、フォーム、スプレー、エアロゾル、液体、噴霧化散剤(nebulized powder)、および坐剤からなる群より選択される形態である、請求項1記載の薬学的組成物。
- 全身または局所投与される、請求項1記載の薬学的組成物。
- 経口、直腸、経皮、経膣、経粘膜、腸内、非経口、筋肉内、皮下、髄内、鞘内、脳室内、静脈内、腹腔内、鼻腔内、または眼内投与で投与される、請求項1記載の薬学的組成物。
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US20150010614A1 (en) | 2015-01-08 |
US20120309086A1 (en) | 2012-12-06 |
US20150231091A1 (en) | 2015-08-20 |
MX2010011032A (es) | 2011-03-01 |
US20210077424A1 (en) | 2021-03-18 |
KR20100136997A (ko) | 2010-12-29 |
CN103462896A (zh) | 2013-12-25 |
US10668028B2 (en) | 2020-06-02 |
SG189687A1 (en) | 2013-05-31 |
EP2271325A1 (en) | 2011-01-12 |
CA2721071A1 (en) | 2009-10-15 |
CN102083424B (zh) | 2013-08-28 |
JP2017036341A (ja) | 2017-02-16 |
CN102083424A (zh) | 2011-06-01 |
BRPI0911658A2 (pt) | 2015-10-13 |
AU2009233785A1 (en) | 2009-10-15 |
IL208579A0 (en) | 2010-12-30 |
US20110136231A1 (en) | 2011-06-09 |
EA023827B1 (ru) | 2016-07-29 |
EA201001624A1 (ru) | 2011-06-30 |
JP2011516568A (ja) | 2011-05-26 |
JP2015108009A (ja) | 2015-06-11 |
IL208579A (en) | 2017-05-29 |
CO6311073A2 (es) | 2011-08-22 |
CA2721071C (en) | 2017-10-17 |
EP2271325A4 (en) | 2011-11-09 |
EP3015104A1 (en) | 2016-05-04 |
WO2009126764A1 (en) | 2009-10-15 |
AU2009233785B2 (en) | 2015-08-20 |
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