JP2018528166A - ナノ粒子−硝子体基盤タンパク質複合体を有効成分として含む血管新生抑制用組成物およびその用途 - Google Patents
ナノ粒子−硝子体基盤タンパク質複合体を有効成分として含む血管新生抑制用組成物およびその用途 Download PDFInfo
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Abstract
【選択図】図9
Description
(1)in vitroで硝子体にナノ粒子を注入する段階;
(2)前記ナノ粒子と前記硝子体内のタンパク質との複合体を分離する段階;
(3)前記複合体と血管内皮細胞成長因子(VEGF)を結合させる段階;および
(4)前記血管内皮細胞成長因子と結合する複合体を選別する段階。
(1)in vitroで硝子体にナノ粒子を注入する段階;
(2)前記ナノ粒子と前記硝子体内のタンパク質との複合体を分離する段階;
(3)前記複合体と血管内皮細胞成長因子(VEGF)を結合させる段階;および
(4)前記血管内皮細胞成長因子と結合する複合体を選別する段階。
1−1.ナノ粒子の準備
硝子体タンパク質との結合のためのナノ粒子は、それぞれ20および100nmの直径を有する金およびシリカナノ粒子を準備した。前記ナノ粒子の具体的な情報およびTEMイメージをそれぞれ表1および図1に示した。
前記実施例1−1にて準備した直径20nmの金(Au20)とシリカ(Si20)ナノ粒子および直径100nmの金(Au100)とシリカ(Si100)ナノ粒子(1×1011個)それぞれを170μgのタンパク質を保有した硝子体(vitreous)と微小遠心管(microcentrifuge tube)中で4℃の条件で20rpmの回転を加えて6時間インキュベートした。その後、15,000rpmの条件で20分間遠心分離して沈殿物を獲得し、その後、蒸留水で2回洗浄して、非特異的に結合したタンパク質を除去した。この際、沈殿物は、ガラスナノ粒子およびタンパク質が結合したナノ粒子を含む。これを30μLのLaemmliバッファーに浮遊させ、100℃で3分間加熱して、ナノ粒子とタンパク質の分離を誘導した。その後、15,000rpmの条件で1分間遠心分離して上清液を使用してタンパク質を分析して、各ナノ粒子とコロナを形成した上位20個の硝子体基盤タンパク質を確認し、その結果を図2〜図5に示した。さらに、前記結果を全部総合して、上位20個の硝子体基盤タンパク質を確認し、その結果を図6に示した。
前記実施例1−2により確認された上位5個の硝子体基盤タンパク質がそれぞれ50ng、25ng、25ng、25ngおよび25ngからなる合計150ngのタンパク質(SALVAR complex)を準備し、1×109個のナノ粒子と4℃で20rpmの回転を加えて1時間インキュベートして、ナノ粒子−硝子体基盤タンパク質複合体を形成した。
金およびシリカナノ粒子の場合、水または細胞培養液で血管内皮細胞成長因子と結合する現象が知られているところ、このようなナノ粒子と比較する時、本発明によるナノ粒子−硝子体基盤タンパク質複合体が水または細胞培養液で血管内皮細胞成長因子と効果的に結合するか否かを確認するために、下記のように実験を行った。
水でナノ粒子に血管内皮細胞成長因子を結合させる場合と、ナノ粒子−硝子体基盤タンパク質複合体に血管内皮細胞成長因子を結合させる場合とを比較し、当該実験に対する概略的な過程を図7に示した。
硝子体内でナノ粒子に血管内皮細胞成長因子を結合させる場合と、ナノ粒子−硝子体基盤タンパク質複合体に血管内皮細胞成長因子を結合させる場合とを比較し、当該実験に対する概略的な過程を図8に示した。
20ng/mLの血管内皮細胞成長因子を血管内皮細胞に処理する時、血管新生過程を代表する血管内皮細胞の増殖や管形成を促進することが知られている。ナノ粒子−硝子体基盤タンパク質複合体の投与が血管内皮細胞成長因子によるin vitro血管新生過程を抑制することを確認するために、血管内皮細胞の増殖(proliferation)および管形成(tube formation)の試験を行った。血管内皮細胞の増殖の試験は、0.3%ゼラチンがコートされたプレートの各ウェルに2,000個の血管内皮細胞を1日間培養し、その後、血管内皮細胞成長因子、ナノ粒子、ナノ粒子−硝子体基盤タンパク質複合体、および ベバシズマブ(bevacizumab)を条件によって処理した後、48時間血管内皮細胞の増殖程度を比較する方式で進めた。血管内皮細胞の増殖程度は、トリパンブルー染色後、直接細胞の数を測定する方式と、 水溶性テトラゾリウム塩WST−1の処理後、450nmの吸光度を測定する方式で推定した。管形成の試験は、マトリゲルがコートされたプレートの各ウェルに100,000個の血管内皮細胞と血管内皮細胞成長因子、ナノ粒子、ナノ粒子−硝子体基盤タンパク質複合体、およびベバシズマブを条件によって処理した後、12時間後、血管内皮細胞の管形成の程度を比較する方式で進めた。50倍拡大した画面で形成された管(tube)の数字を確認して、定量的な比較を行った。
マウスでレーザー誘導脈絡膜新生血管モデルを作製し、ナノ粒子またはナノ粒子−硝子体基盤タンパク質複合体を硝子体腔内注射して効果を確認した。マウスの網膜に400 mWの強さで50ms持続時間のダイオードレーザーを照射すると、網膜層と脈絡膜層との間のブルッフ膜の破壊が起こるが、レーザーの照射後、ナノ粒子(109個/mL、1μL)、ナノ粒子−硝子体基盤タンパク質複合体(109個/mL、1μL)、抗血管内皮細胞成長因子抗体(1μg)を硝子体腔内に注射し、レーザーの照射後に7日目に、脈絡膜新生血管の形成程度を免疫蛍光染色を利用して確認した。
Claims (8)
- ナノ粒子と前記ナノ粒子の表面を取り囲む硝子体基盤タンパク質からなる複合体を有効成分として含む血管新生抑制用薬学的組成物。
- 前記ナノ粒子は、金またはシリカであることを特徴とする、請求項1に記載の組成物。
- 前記ナノ粒子は、20〜100nmの直径を有することを特徴とする、請求項1に記載の組成物。
- 前記血管新生は、未熟児網膜症、糖尿病黄斑浮腫(DME、Diabetic Macular Edema)、糖尿病網膜症(diabetic retinopathy)、中心性漿液性脈絡網膜症(Central serous(chorio)retinopathy)、加齢黄斑変性(Age−related macular degeneration)または増殖網膜症に伴うことを特徴とする、請求項1に記載の薬学的組成物。
- 前記硝子体基盤タンパク質は、ビトリン(Vitrin)、分泌されたフリッツルド関連タンパク質2(Secreted Frizzled Related Protein 2)、血清アルブミン、レチノール結合タンパク質3(retinol−binding protein 3)、およびα−クリスタリンA鎖(alpha−crystallin A chain)よりなる群から一つ以上選択されることを特徴とする、請求項1に記載の組成物。
- 請求項1〜5のいずれかに記載の組成物を有効成分として含む網膜疾患の予防または治療用薬学的組成物。
- 前記網膜疾患は、未熟児網膜症、糖尿病黄斑浮腫(DME、Diabetic Macular Edema)、糖尿病網膜症(diabetic retinopathy)、中心性漿液性脈絡網膜症(Central serous(chorio)retinopathy)、加齢黄斑変性(Age−related macular degeneration)、および増殖網膜症よりなる群から選択されることを特徴とする、請求項6に記載の組成物。
- 下記段階を含む網膜疾患の治療に適したタンパク質スクリーニング方法:
(1)in vitroで硝子体にナノ粒子を注入する段階;
(2)前記ナノ粒子と前記硝子体内のタンパク質との複合体を分離する段階;
(3)前記複合体と血管内皮細胞成長因子(VEGF)を結合させる段階;および
(4)前記血管内皮細胞成長因子と結合する複合体を選別する段階。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020150099308A KR101748120B1 (ko) | 2015-07-13 | 2015-07-13 | 나노입자-유리체 기반 단백질 복합체를 유효성분으로 포함하는 혈관신생억제용 조성물 및 이의 용도 |
KR10-2015-0099308 | 2015-07-13 | ||
PCT/KR2016/007570 WO2017010790A1 (ko) | 2015-07-13 | 2016-07-12 | 나노입자-유리체 기반 단백질 복합체를 유효성분으로 포함하는 혈관신생억제용 조성물 및 이의 용도 |
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EP3323411A1 (en) | 2018-05-23 |
US10406110B2 (en) | 2019-09-10 |
KR101748120B1 (ko) | 2017-06-16 |
EP3323411B1 (en) | 2020-09-09 |
CN108348544A (zh) | 2018-07-31 |
KR20170008058A (ko) | 2017-01-23 |
US20190015348A1 (en) | 2019-01-17 |
EP3323411A4 (en) | 2018-08-15 |
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CN108348544B (zh) | 2021-01-22 |
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