JP2011256204A - オリゴヌクレオチド含有マイクロスフェア、1型糖尿病を処置する医薬の製造のための、その使用 - Google Patents
オリゴヌクレオチド含有マイクロスフェア、1型糖尿病を処置する医薬の製造のための、その使用 Download PDFInfo
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Abstract
【解決手段】AS−オリゴヌクレオチドが、特に、非肥満性糖尿病(NOD)マウスモデルにおいて、樹状細胞寛容を誘導するためにマイクロスフェア形態で送達される。このマイクロスフェアは、アンチセンス(AS)オリゴヌクレオチドをとりこむ。プロセスは、インビボおよびインサイチュでのNODマウスにおける自己免疫性糖尿病状態を予防するためにアンチセンスアプローチを使用する工程を包含する。このオリゴヌクレオチドは、主要転写物CD40、CD80、CD86およびそれらの組み合わせへ結合するように標的化される。
【選択図】なし
Description
2004年5月12日に出願された、仮特許出願シリアル番号60/570,273、および2004年11月5日に出願された、仮特許出願シリアル番号60/625,483。
(発明の分野)
本発明は、一般的に、特に、非肥満性糖尿病(NOD)マウスモデルにおいて、樹状細胞寛容を誘導するためのAS−オリゴヌクレオチドのマイクロスフェア送達に関する。より詳しくは、本発明は、完全に水性の条件を使用して製造されるマイクロスフェア(マイクロスフェアは、アンチセンス(AS)オリゴヌクレオチドをとりこむ)による薬物送達技術に関する。これらのマイクロスフェアは、インビボおよびインサイチュでのNODマウスにおける自己免疫性糖尿病状態を予防する、アンチセンスアプローチのために使用される。
微粒子、マイクロスフェアおよびマイクロカプセルは、1mm未満の直径、より好ましくは100ミクロン未満の直径を有する、固体または半固体の粒子であり、これらは、種々の材料(合成ポリマー、タンパク質および多糖類が挙げられる)から形成され得る。マイクロスフェアは、多くの異なる用途(主に、分離、診断および薬物送達)において使用されてきた。
本発明にしたがって、樹状細胞に送達されるべきDNAは、マイクロスフェアとして送達される。このような送達アプローチは、マイクロスフェア中の核酸へのヌクレアーゼの接近を防止すると考えられる。AS−オリゴヌクレオチドのマイクロスフェア送達は、特に、NODマウスモデルにおいて、樹状細胞の寛容を誘導するために実行される。マイクロスフェアは、マイクロスフェアがアンチセンス(AS)オリゴヌクレオチドを取り込む、水性条件を使用して製造される。これらのマイクロスフェアは、遺伝子発現を阻害し、そしてインビボおよびインサイチュでのNODマウスにおける自己免疫性糖尿病状態を予防するために使用される。
例えば、本願発明は以下の項目を提供する。
(項目1)
1型糖尿病の処置のためのオリゴヌクレオチドを含むマイクロスフェアであって、該オリゴヌクレオチドは、該マイクロスフェアの全重量に基づいて、該マイクロスフェアの約30重量%と約100重量%との間を構成し、該マイクロスフェアは、約50ミクロンを超えない平均粒子サイズを有する、マイクロスフェア。
(項目2)
前記オリゴヌクレオチドが、CD40、CD80およびCD86主要転写物ならびにその組み合わせからなる群から選択される主要転写物に結合するように標的化される、項目1に記載のマイクロスフェア。
(項目3)
前記オリゴヌクレオチドは、配列番号1、配列番号2または配列番号3およびその組み合わせからなる群から選択される、項目2に記載のマイクロスフェア。
(項目4)
1型糖尿病を有する個体へ、マイクロスフェアの形態で核酸を送達するためのプロセスであって、該マイクロスフェアの送達のための投与経路は、静脈内、筋内、皮下、局所、皮内、腹腔内、経口、肺、眼、経鼻または直腸からなる群から選択される、プロセス。
(項目5)
項目1に記載のマイクロスフェアを皮下注射する工程を包含する、自己免疫性の破壊から非肥満性糖尿病マウスの膵臓β細胞を防御するためのプロセス。
(項目6)
項目2に記載のマイクロスフェアを皮下注射する工程を包含する、自己免疫性の破壊から非肥満性糖尿病マウスの膵臓β細胞を防御するためのプロセス。
(項目7)
項目1に記載のマイクロスフェアを皮下注射する工程を包含する、自己免疫性の破壊から個体の膵臓β細胞を防御するためのプロセス。
(項目8)
項目2に記載のマイクロスフェアを皮下注射する工程を包含する、自己免疫性の破壊から個体の膵臓β細胞を防御するためのプロセス。
(項目9)
項目1に記載のマイクロスフェアを皮下注射する工程を包含する、自己免疫性の破壊からヒトの膵臓β細胞を防御するためのプロセス。
(項目10)
項目2に記載のマイクロスフェアを皮下注射する工程を包含する、自己免疫性の破壊からヒトの膵臓β細胞を防御するためのプロセス。
(項目11)
項目1に記載のマイクロスフェアを皮下注射する工程を包含する、自己免疫性の破壊および1型糖尿病の発症から個体の膵臓β細胞を防御するためのプロセス。
(項目12)
項目2に記載のマイクロスフェアを皮下注射する工程を包含する、自己免疫性の破壊および1型糖尿病の発症から個体の膵臓β細胞を防御するためのプロセス。
必要とされるように、本発明の詳細な実施形態が、本明細書で開示される;しかしながら開示される実施形態は、本発明の例示であるにすぎず、本発明は種々の形態に具体化され得ることが理解されるべきである。したがって、本明細書で開示される特定の詳細は、限定として解釈されるべきではなく、特許請求のための単なる根拠として、そして実質的に任意の適切な様式で本発明を種々に使用するように当業者へ教示するための代表的な根拠として解釈されるべきである。
CD40、CD80およびCD86主要転写物に標的化される、3つのAS−オリゴヌクレオチドを、University of Pittsburgh(Pittsburgh、PA)にあるDNA合成設備により合成した。AS−オリゴヌクレオチドの配列は、以下である:
Prussia、PAによるポリ−L−リジン・HBr 50,000まで)。ポリ−L−リジン・HBrを、1:1の体積比で、このオリゴヌクレオチド溶液に添加した。この混合物を、穏やかにボルテックスした。pH=5.5でのlM 酢酸ナトリウム(Spectrum、Gardena、CA)中に、12.5%のPVP(ポリビニルピロリドン、40,000ダルトン、Spectrum Chemicals、Gardena、CA)と12.5%のPEG(ポリエチレングリコール、3,350ダルトン、Spectrum Chemicals、Gardena、CA)とを含む25%ポリマー溶液を作製した。このポリマー溶液を以下:750μ1のAS−オリゴヌクレオチド、0.75mlのポリ−L−リジン・HBr、3.0mlのPEG/PVPのように2:1の体積比で添加し、そして4.50mlの全容積にした。
CD40、CD80およびCD86主要転写物に標的化される、AS−オリゴヌクレオチドは、実施例1のAS−オリゴヌクレオチド配列であった。このオリゴヌクレオチド混合物の水溶液を、3つのオリゴヌクレオチド溶液(各々は、1つの型のオリゴヌクレオチドを含んだ)のアリコートを組み合わせることにより調製し、3つの型のオリゴヌクレオチドの10[mg/ml]溶液を生成した。オリゴヌクレオチド混合物の溶液を調製した。diH2O中で5[mg/ml]のポリ−L−オルニチン・HBrを調製した(Sigmaによるポリ−L−オルニチン・HBr 11,900(vis))。ポリ−L−オルニチン・HBrを、このオリゴヌクレオチド溶液に添加した。この混合物を、穏やかにボルテックスした。pH=5.5での0.1M 酢酸ナトリウム(Spectrum Chemicals、Gardena、CA)中に、12.5%のPVP(40,000ダルトン、Spectrum Chemicals、Gardena、CA)と12.5%のPEG(3,350ダルトン、Spectrum Chemicals、Gardena、CA)とを含む25%ポリマー溶液を作製した。このポリマー溶液を添加した。実施例1に記載されるように、インキュベーションとリンスが続いた。1.5mlのAS−オリゴヌクレオチド、1.5mlのポリ−L−オルニチン・HBr、3.0mlのPEG/PVP、そして全容積、6.0mlを調製した。
インビボ研究を、1型真性糖尿病のNODマウスモデルを使用して実行した。1型糖尿病は、図1に示されるように、膵臓のインシュリン産生β細胞の自己免疫性の破壊により発現する。AS−オリゴヌクレオチドを、β細胞の自己免疫性の破壊を妨害する試みにおいて、3つの適用により使用した。この目標は、T細胞の活性化に必要とされる樹状細胞(dendritric cell)表面タンパク質をコードするCD40、CD80およびCD86主要転写物を標的化することにより、樹状細胞の機能を妨害することである。低レベルのCD40、CD80およびCD86を有する樹状細胞は、インビボにおいて、抑制性の免疫細胞ネットワークを促進することが公知である。これらのカスケードは、インビボにおいて、β細胞に対するT細胞の反応低下をもたらし得る。
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- 本願明細書に記載された発明。
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1999053903A1 (en) * | 1998-04-23 | 1999-10-28 | The Regents Of The University Of Michigan | Microspheres containing condensed polyanionic bioactive agents and methods for their production |
WO2000041679A1 (en) * | 1999-01-13 | 2000-07-20 | Johns Hopkins University School Of Medicine | Genetic immunization with co-delivery of nucleic acid and cytokines |
JP2002538174A (ja) * | 1999-03-02 | 2002-11-12 | ウエスト・ファーマシューティカル・サービセズ・ドラッグ・デリバリー・アンド・クリニカル・リサーチ・センター・リミテッド | ポリヌクレオチドデリバリーのためのポリマー組成物 |
WO2003083089A2 (en) * | 2002-03-28 | 2003-10-09 | Revivicor Inc. | Tolerogenic antigen-presenting cells |
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AU2005244851A1 (en) | 2005-12-01 |
US7884085B2 (en) | 2011-02-08 |
ES2442115T3 (es) | 2014-02-10 |
US20100260855A1 (en) | 2010-10-14 |
EP1758558B1 (en) | 2013-10-16 |
CA2566199A1 (en) | 2005-12-01 |
EP1758558A2 (en) | 2007-03-07 |
CN103432079A (zh) | 2013-12-11 |
CA2566199C (en) | 2013-10-22 |
WO2005112885A3 (en) | 2006-02-09 |
PT1758558E (pt) | 2013-12-05 |
DK1758558T3 (da) | 2014-01-20 |
AU2005244851B2 (en) | 2010-08-26 |
WO2005112885A2 (en) | 2005-12-01 |
JP2007537288A (ja) | 2007-12-20 |
US9115357B2 (en) | 2015-08-25 |
MXPA06012989A (es) | 2007-06-12 |
US20060024240A1 (en) | 2006-02-02 |
JP5457414B2 (ja) | 2014-04-02 |
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