JP2007527881A - 薬学的に活性な物質を細胞特異的に濃縮するためのタンパク質ベースのナノ粒子形態の支持システム - Google Patents
薬学的に活性な物質を細胞特異的に濃縮するためのタンパク質ベースのナノ粒子形態の支持システム Download PDFInfo
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Abstract
Description
EP1 392 255は、ヒト血清アルブミンベースのナノ粒子であって、これにアポリポタンパク質Eが共有結合的に、もしくはアビジン/ビオチン系を介して結合しており、血液脳関門の通過を可能にしたものを開示している。
− タンパク質水溶液を脱溶媒和する工程、
− 脱溶媒和によって形成されるナノ粒子を架橋することにより安定化させる工程、
− 安定化したナノ粒子の表面の官能基を部分的に反応性のチオール基に変換する工程、
− 官能性タンパク質、好ましくはアビジンを、二官能性スペーサー分子により共有結合的に付着させる工程、
− 必要に応じて、抗体をビオチン化させる工程、
− アビジン修飾したナノ粒子に、ビオチン化した抗体を負荷する工程、
− アビジン修飾したナノ粒子に、ビオチン化した、薬学的にもしくは生物学的に活性な物質を負荷する工程、
を含む方法を包含する。
タンパク質ナノ粒子を調製するため、500mgのゼラチンAを10.0mlの精製水中に加温しながら溶解し、10.0mlのアセトンを加えることにより沈殿させて沈澱物とした。沈殿したゼラチンを分離し、加温しながら10.0mlの水に再溶解し、溶液のpH値を2.5に調節した。30mlのアセトンを滴加すること(脱溶媒和処理)により、この溶液中からナノ粒子を得た。
1.表面抗原であるCD3を有するリンパ球性の標的細胞(Jurkat T細胞)
ナノ粒子にはビオチン化した抗CD3抗体を負荷した。
2.HER2表面抗原を発現したヒト乳がん細胞系(SK−Br−3細胞、MCF−7細胞、BT474細胞)
ナノ粒子には、あらかじめビオチン化した、認可された抗体トラスツズマブ(Herceptin(登録商標))を負荷した。
リンパ球性標的細胞(Jurkat T細胞)
FACSとCLSMの両方により、細胞特異的抗CD3抗体で修飾された形で用いたナノ粒子が細胞によって取り込まれたことが示された。粒子を添加する前に細胞を遊離特異抗体で処理した場合、細胞による取り込みを回避することができた。しかしながら、遊離非特異IgG抗体での前処置は、粒子の取り込みにいかなる影響も示さなかった。特異的抗CD3抗体のかわりに非特異IgG抗体でナノ粒子を修飾しても、同様に、標的細胞における取り込みは起こらなかった。CD3表面抗原を持たない乳がん細胞(MCF−7細胞)を用いてさらに対照実験を行った。これらの対照実験において、ナノ粒子製剤の取り込みは選択されたいかなる条件下においても見られなかった。
用いた細胞は、程度は異なるが、抗体修飾したナノ粒子の細胞による取り込みのための攻撃点として使用されるHER2表面抗原の発現を示した。細胞における発現は、ナノ粒子とのインキュベーションに先だってウエスタンブロット解析により決定した(表1)。
Claims (14)
- 少なくとも1種の薬学的に活性な物質の細胞特異的な細胞内濃縮のための担体システムであって、該担体システムが、タンパク質をベースとする、好ましくはゼラチンおよび/または血清アルブミンをベースとする、特に好ましくはヒト血清アルブミンをベースとするナノ粒子の形態で存在し、かつ、反応性基により結合した構造を有し、該構造が、ナノ粒子の細胞特異的付着と細胞による取り込みとを可能にすることを特徴とする、前記担体システム。
- 反応性基がアミノ、チオール、カルボキシル基、またはアビジン誘導体であることを特徴とする、請求項1に記載の担体システム。
- 結合した構造が抗体であることを特徴とする、請求項1または2に記載の担体システム。
- 抗体がモノクローナル抗体であることを特徴とする、請求項3に記載の担体システム。
- 吸着、組み込み、または共有結合もしくは複合体形成結合により反応性基を用いて担体システムに結合した薬学的に活性な物質を追加的に含むことを特徴とする、請求項1〜4のいずれかに記載の担体システム。
- 薬学的に活性な物質の、特定細胞への、または特定細胞における濃縮のための医薬の製造のための、請求項1〜5のいずれかに記載の担体システムの使用。
- 少なくとも1種の薬学的に活性な物質の細胞特異的濃縮のための、タンパク質ベースのナノ粒子の形態の担体システムの製造方法であって、以下の工程:
− タンパク質水溶液を脱溶媒和する工程、
− 脱溶媒和によって形成されたナノ粒子を架橋により安定化する工程、
− 安定化されたナノ粒子の表面の官能基の一部を、反応性チオール基に変換する工程、
− 官能性タンパク質、好ましくはアビジンを、二官能性スペーサー分子を用いて共有結合的に付着させる工程、
− 必要に応じて、抗体をビオチン化する工程、
− アビジン修飾ナノ粒子に、ビオチン化した抗体を負荷する工程、
− アビジン修飾ナノ粒子に、ビオチン化した、薬学的もしくは生物学的に活性な物質を負荷する工程、
を含む、前記方法。 - タンパク質のベースが、ゼラチンおよび/または血清アルブミン、好ましくはヒト血清アルブミンであることを特徴とする、請求項7に記載の方法。
- 脱溶媒和が、攪拌とタンパク質に対する水混和性の非溶媒の添加とにより、または塩析により行われることを特徴とする、請求項7または8に記載の方法。
- タンパク質に対する水混和性の非溶媒が、エタノール、メタノール、イソプロパノールおよびアセトンを含む群から選択されることを特徴とする、請求項9に記載の方法。
- 熱処理、または、二官能性アルデヒドもしくはホルムアルデヒドが、ナノ粒子の安定化に用いられることを特徴とする、請求項7〜10のいずれかに記載の方法。
- グルタルアルデヒドが二官能性アルデヒドとして用いられることを特徴とする、請求項11に記載の方法。
- チオール基修飾剤として、2−イミノチオラン、1−エチル−3−(3−ジメチルアミノプロピル)カルボジイミドとシステインとの組み合わせ、または1−エチル−3−(3−ジメチルアミノプロピル)カルボジイミドとジトレイトールのみならずシスタミニウム二塩化物との組み合わせを含む群から選択される物質が用いられることを特徴とする、請求項7〜12のいずれかに記載の方法。
- 二官能性スペーサー分子として、m−マレイミドベンゾイル−N−ヒドロキシスルフォスクシンイミドエステル、スルフォスクシンイミジル−4−[N−マレイミド−メチル]シクロヘキサン−1−カルボキシレート、スルフォスクシンイミジル−2−[m−アジド−o−ニトロベンザミド]−エチル−1,3’ジチオプロピオネート、ジメチル−3,3’−ジチオビスプロピオンイミデート−ジヒドロクロライド、3,3’−ジチオビス[スルフォスクシンイミジルプロピオネート]を含む群から選択される物質が用いられることを特徴とする、請求項7〜13のいずれかに記載の方法。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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DE102004011776A DE102004011776A1 (de) | 2004-03-09 | 2004-03-09 | Trägersystem in Form von Nanopartikeln auf Proteinbasis zur zellspezifischen Anreicherung von pharmazeutisch aktiven Wirkstoffen |
PCT/EP2005/002185 WO2005089797A2 (de) | 2004-03-09 | 2005-03-02 | Trägersystem in form von nanopartikeln auf proteinbasis zur zellspezifischen anreicherung von pharmazeutisch aktiven wirtstoffen |
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US (1) | US20080095857A1 (ja) |
EP (1) | EP1722816A2 (ja) |
JP (1) | JP2007527881A (ja) |
KR (1) | KR20070006828A (ja) |
CN (1) | CN1993145A (ja) |
AU (1) | AU2005223986B2 (ja) |
BR (1) | BRPI0508134A (ja) |
CA (1) | CA2558730A1 (ja) |
DE (1) | DE102004011776A1 (ja) |
IL (1) | IL177879A0 (ja) |
NZ (1) | NZ549355A (ja) |
RU (1) | RU2388463C2 (ja) |
WO (1) | WO2005089797A2 (ja) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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JP2013513609A (ja) * | 2009-12-11 | 2013-04-22 | バイオリテック ファーマ マーケティング リミテッド | 光力学的療法のためのヒト血清アルブミンをベースにしたナノ粒子キャリアシステム |
JP2019533665A (ja) * | 2016-10-10 | 2019-11-21 | アブラクシス バイオサイエンス, エルエルシー | ナノ粒子製剤ならびにその作製方法および使用方法 |
WO2020241562A1 (ja) * | 2019-05-24 | 2020-12-03 | ユーハ味覚糖株式会社 | ナノ粒子及びその製造方法 |
Families Citing this family (12)
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DE102005062440B4 (de) * | 2005-12-27 | 2011-02-24 | Lts Lohmann Therapie-Systeme Ag | Proteinbasiertes Trägersystem zur Resistenzüberwindung von Tumorzellen |
DE102006011507A1 (de) | 2006-03-14 | 2007-09-20 | Lts Lohmann Therapie-Systeme Ag | Wirkstoffbeladene Nanopartikel auf Basis hydrophiler Proteine |
JP2008162981A (ja) * | 2006-12-28 | 2008-07-17 | Japan Science & Technology Agency | ビオチン化ないしホーミングペプチド提示型バイオナノカプセル |
GB0724360D0 (en) * | 2007-12-14 | 2008-01-23 | Glaxosmithkline Biolog Sa | Method for preparing protein conjugates |
US9125949B2 (en) * | 2008-12-30 | 2015-09-08 | University Of North Texas | Direct utilization of plasma proteins for the in vivo assembly of protein-drug/imaging agent conjugates, nanocarriers and coatings for biomaterials |
RU2542417C2 (ru) * | 2013-05-17 | 2015-02-20 | Александр Александрович Кролевец | Способ биоинкапсуляции лекарственных препаратов группы цефалоспоринов |
RU2576239C2 (ru) * | 2014-03-26 | 2016-02-27 | Александр Александрович Кролевец | Способ получения нанокапсул антисептика-стимулятора дорогова (асд) 2 фракция |
US20170112777A1 (en) * | 2014-05-16 | 2017-04-27 | Dana-Farber Cancer Institute, Inc. | Protein-based particles for drug delivery |
WO2015018380A2 (en) * | 2014-07-03 | 2015-02-12 | Cspc Zhongqi Pharmaceutical Technology(Shijiazhuang)Co., Ltd. | Therapeutic nanoparticles and the preparation methods thereof |
US20220387338A1 (en) * | 2019-10-04 | 2022-12-08 | Association For The Advancement Of Tissue Engineering And Cell Based Technologies & Therapies A4Tec | Hydrogel-like particles, methods and uses thereof |
CN112451679A (zh) * | 2020-11-25 | 2021-03-09 | 天津医科大学第二医院 | 结合了纳米药物载体的卡介苗复合体及其制备方法 |
CN113588523B (zh) * | 2021-07-26 | 2022-03-29 | 浙江大学 | 一种用于质谱流式细胞技术的基于框架结构的纳米颗粒及其制备方法 |
Citations (1)
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WO2002089776A1 (de) * | 2001-05-05 | 2002-11-14 | Lts Lohmann Therapie-Systeme Ag | Nanopartikel aus protein mit gekoppeltem apolipoprotein e zur überwindung der blut-hirn-schranke und verfahren zu ihrer herstellung |
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US6207195B1 (en) * | 1997-06-13 | 2001-03-27 | The Johns Hopkins University | Therapeutic nanospheres |
JP2003535063A (ja) * | 2000-06-01 | 2003-11-25 | ザ・ボード・オブ・リージェンツ・フォー・オクラホマ・ステート・ユニバーシティー | 放射線医薬としてのナノ粒子のバイオコンジュゲート |
JP2004198915A (ja) * | 2002-12-20 | 2004-07-15 | Shin Etsu Chem Co Ltd | ポジ型レジスト組成物及びパターン形成方法 |
EP1598419A1 (en) * | 2003-02-28 | 2005-11-23 | Mitsubishi Pharma Corporation | Monoclonal antibody, gene encoding the same, hybridoma, medicinal composition and diagnostic reagent |
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- 2005-03-02 RU RU2006130260/15A patent/RU2388463C2/ru not_active IP Right Cessation
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- 2005-03-02 CN CNA2005800073779A patent/CN1993145A/zh active Pending
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- 2005-03-02 AU AU2005223986A patent/AU2005223986B2/en not_active Ceased
- 2005-03-02 US US10/590,601 patent/US20080095857A1/en not_active Abandoned
- 2005-03-02 KR KR1020067020921A patent/KR20070006828A/ko not_active Application Discontinuation
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WO2002089776A1 (de) * | 2001-05-05 | 2002-11-14 | Lts Lohmann Therapie-Systeme Ag | Nanopartikel aus protein mit gekoppeltem apolipoprotein e zur überwindung der blut-hirn-schranke und verfahren zu ihrer herstellung |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2013513609A (ja) * | 2009-12-11 | 2013-04-22 | バイオリテック ファーマ マーケティング リミテッド | 光力学的療法のためのヒト血清アルブミンをベースにしたナノ粒子キャリアシステム |
JP2019533665A (ja) * | 2016-10-10 | 2019-11-21 | アブラクシス バイオサイエンス, エルエルシー | ナノ粒子製剤ならびにその作製方法および使用方法 |
WO2020241562A1 (ja) * | 2019-05-24 | 2020-12-03 | ユーハ味覚糖株式会社 | ナノ粒子及びその製造方法 |
Also Published As
Publication number | Publication date |
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AU2005223986A1 (en) | 2005-09-29 |
US20080095857A1 (en) | 2008-04-24 |
IL177879A0 (en) | 2006-12-31 |
EP1722816A2 (de) | 2006-11-22 |
RU2388463C2 (ru) | 2010-05-10 |
RU2006130260A (ru) | 2008-02-27 |
AU2005223986B2 (en) | 2010-12-23 |
BRPI0508134A (pt) | 2007-07-17 |
WO2005089797A2 (de) | 2005-09-29 |
CN1993145A (zh) | 2007-07-04 |
WO2005089797A3 (de) | 2006-11-23 |
CA2558730A1 (en) | 2005-09-29 |
KR20070006828A (ko) | 2007-01-11 |
DE102004011776A1 (de) | 2005-11-03 |
NZ549355A (en) | 2009-09-25 |
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