JP6301896B2 - 抗生物質送達のための絹フィブロインシステム - Google Patents
抗生物質送達のための絹フィブロインシステム Download PDFInfo
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- JP6301896B2 JP6301896B2 JP2015222744A JP2015222744A JP6301896B2 JP 6301896 B2 JP6301896 B2 JP 6301896B2 JP 2015222744 A JP2015222744 A JP 2015222744A JP 2015222744 A JP2015222744 A JP 2015222744A JP 6301896 B2 JP6301896 B2 JP 6301896B2
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- Prior art keywords
- silk
- antibiotic
- scaffold
- silk fibroin
- gentamicin
- Prior art date
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Description
本出願は、その内容の全てが本明細書中に参照として組み入れられる、2009年3月4日に出願された米国仮出願第61/157,366号について合衆国法典第35章第119(e)節に基づく優先権の権利を主張する。
米国国立保険研究所より与えられたEB002520、及び、米国陸軍より与えられたW911NF-07-1-0618に基づく政府援助を受けて本発明は行われた。政府は本発明に一定の権利を有する。
本発明は、微生物汚染を予防または処置するための組成物、及び、そのような組成物を使用した微生物汚染を予防または処置する方法に関する。本発明の組成物は優れた安定性を示し、そして、医療移植片、組織工学、薬物送達系、または他の薬学若しくは医学用途に用いてもよい。
心血管及び筋骨格移植片を含め、組織工学により作られる軟骨、骨及び靭帯のための基質として、細胞増殖片及び薬物送達のための生育可能培地として、並びに、ヒト間葉幹細胞の特定組織への適切な分化を導くために、多様な応用目的で生物素材が開発されてきた。
[本発明1001]
三次元絹フィブロイン足場中に包埋された抗生物質装薬(loaded)絹フィブロインミクロスフェアを含む組成物。
[本発明1002]
足場がスポンジ、フィルム、スラブ、粉末、エレクトスピニングされたマット、多孔性マトリックス、ミクロスフェア、ゲル、光学装置、または電子機器である、本発明1001の組成物。
[本発明1003]
包帯を形成するための可撓性物質をさらに含む、本発明1001または1002の組成物。
[本発明1004]
足場がサージカルパック材料である、本発明1001または1002の組成物。
[本発明1005]
足場が、絹フィブロイン足場に包埋された抗生物質装薬絹フィブロインミクロスフェアを含む、本発明1001〜1004のいずれかの組成物。
[本発明1006]
抗生物質が、アクチノマイシン、アミノグリコシド、β-ラクタマーゼ阻害剤、グリコペプチド、アンサマイシン、バシトラシン、カルバセフェム、カルバペネム、セファロスポリン、イソニアジド、リネゾリド、マクロライド、ムピロシン、ペニシリン、オキソリン酸、ポリペプチド、キノロン、スルホンアミド、テトラサイクリン、モノバクタム、クロラムフェニコール、リンコマイシン、クリンダマイシン、エタンブトール、ムピロシン、メトロニダゾール、ペフロキサシン、ピラジンアミド、チアンフェニコール、リファンピシン、チアムフェニクル(thiamphenicl)、ダプソン、クロファジミン、キヌプリスチン、メトロニダゾール、リネゾリド、イソニアジド、ピラシル(piracil)、ノボビオシン、トリメトプリム、ホスホマイシン、及びフシジン酸からなる群より少なくとも選択される抗生物質である、本発明1001〜1005のいずれかの組成物。
[本発明1007]
抗生物質がセファゾリン、ゲンタマイシン、ペニシリン、アンピシリン、またはそれらの組み合わせである、本発明1006の組成物。
[本発明1008]
デフェンシン、マガイニン、ナイシン、溶菌バクテリオファージ、細胞、タンパク質、ペプチド、アミノ酸、核酸アナログ、ヌクレオチド、オリゴヌクレオチド、ペプチド核酸、アプタマー、抗体またはその断片若しくは部分、抗原またはエピトープ、ホルモン、ホルモンアンタゴニスト、成長因子または組換成長因子並びにその断片及びバリアント、細胞付着性メディエーター、サイトカイン、抗炎症剤、抗真菌剤、抗ウイルス剤、毒素、プロドラッグ、化学療法剤、小分子、薬物、染料、ビタミン、酵素、抗酸化剤、並びに、他の抗菌化合物からなる群より選択される少なくとも一つの剤をさらに含む、本発明1001〜1007のいずれかの組成物。
[本発明1009]
以下を含む工程により調製された、抗生物質装薬三次元絹フィブロイン足場:
少なくとも一種の抗生物質剤を含む絹フィブロインミクロスフェアを調製する工程、
該抗生物質装薬絹フィブロインミクロスフェアを絹フィブロイン水性塩溶液と、三次元の形を限定する容器中で混合する工程、並びに
抗生物質装薬絹ミクロスフェアが包埋された三次元絹フィブロイン足場を形成するために、該溶液から塩及び水を除去する工程。
[本発明1010]
以下を含む工程により調製された、抗生物質装薬三次元絹フィブロイン足場:
少なくとも一種の抗生物質剤を含む絹フィブロインミクロスフェアを調製する工程、
該抗生物質装薬絹フィブロインミクロスフェアを絹フィブロイン溶液と混合する工程、並びに
抗生物質装薬絹ミクロスフェアを包埋したゲル足場を形成するよう、該溶液を超音波処理する工程。
[本発明1011]
少なくとも一つの活性剤をさらに含む、本発明1009または1010の抗生物質装薬三次元絹フィブロイン足場。
[本発明1012]
必要とする対象の一領域における微生物汚染を予防及び/または処置する方法であって、対象の該領域を本発明1001の組成物と接触させる段階を含む方法。
[本発明1013]
抗生物質を選択する段階、
該抗生物質を絹フィブロイン溶液中に組み入れる段階、及び
該溶液より足場を形成する段階
を含む、抗生物質長期貯蔵組成物を調製する方法。
[本発明1014]
前記溶液が水性溶液または水和型脂質溶液である、本発明1013の方法。
[本発明1015]
前記足場が、前記溶液を表面上に流し込んでフィルムまたはスラブ得ることにより形成される、本発明1013の方法。
[本発明1016]
前記足場が、前記溶液を鋳型または容器中に流し入れることにより形成され、次いで乾燥させて三次元足場が形成される、本発明1013の方法。
[本発明1017]
ゲル足場を形成するように前記溶液が超音波処理される、本発明1013の方法。
[本発明1018]
マット足場を形成するように前記溶液がエレクトロスピニングされる、本発明1013の方法。
[本発明1018]
抗生物質含有ミクロスフェアを作出するように前記溶液を処理する段階をさらに含む、本発明1013の方法。
[本発明1019]
抗生物質装薬ミクロスフェアを絹フィブロイン溶液に添加する段階、及び
該抗生物質装薬ミクロスフェアを含む足場を形成する段階
をさらに含む、本発明1018の方法。
[本発明1020]
前記足場を抗生物質含有絹フィブロイン溶液と少なくとも1回接触させて、前記足場上に抗生物質含有層を形成させる段階をさらに含む、本発明1013の方法。
[本発明1021]
前記溶液に追加の剤を添加する段階をさらに含む、本発明1013の方法。
[本発明1022]
前記抗生物質長期貯蔵組成物中の抗生物質が、少なくとも10日間、約4℃において、少なくとも50%の残存活性を維持する、本発明1013の方法。
[本発明1023]
前記抗生物質長期貯蔵組成物中の抗生物質が、少なくとも10日間、約25℃において、少なくとも50%の残存活性を維持する、本発明1013の方法。
[本発明1024]
前記抗生物質長期貯蔵組成物中の抗生物質が、少なくとも10日間、約37℃において、少なくとも50%の残存活性を維持する、本発明1013の方法。
本発明が、本明細書中に記載される特定の方法論、プロトコル及び試薬等々に限定されず、そして異なっていてもよいことが理解されるべきである。本明細書中で使用される用語は、特定の態様の記述のみを目的とし、そして、特許請求の範囲のみによって定義される本発明の範囲を限定することを意図していない。
絹フィブロイン水性原液を以前記載されたように調製した。Hofmannら、2006。簡単に述べると、カイコガの繭を20分間、0.02M Na2CO3の水性溶液中で煮、そしてその後、セリシンタンパク質を抽出するために蒸留水でよく洗い流した。抽出された絹フィブロインをその後9.3m LiBr溶液中に60℃で4時間溶解し、20%(重量/容量)溶液を得た。この溶液を蒸留水に対して、Slide-A-Lyzer透析カセット(MWCO3500g/mol, Pierce, Woburn, MA)を用いて室温で48時間、塩を除くため透析した。不純物及び透析の間に形成された凝集物を除くため、透析液を2回、各回4℃で20分間遠心した。絹フィブロイン水性溶液の最終濃度はおよそ8%(wt/v)であった。公知の容量の溶液の残存固体量を60℃における24時間の乾燥後に測定することによりフィブロイン濃度を決定した。
絹フィブロイン足場の調製のため、ディスク型容器中の2mlの6%絹フィブロイン水性溶液への4gの顆粒状NaCl2(粒径600μm〜710μm)の添加により水性由来絹フィブロイン足場を調製した。Kimら、2005。容器を覆いそして24時間室温に置いた。容器を蒸留水に漬け、そして48時間NaCl2を抽出した。足場を該容器から取り、そして所望の寸法に切った。
抗生物質を含有する足場、及び、抗生物質を含有しない対照の足場を6mmの直径の円筒に切った。3mlの蒸留水中に足場を浸漬し、そして、振盪することなく室温でインキュベートした。24時間の間隔で、168時間に亘って各100μlの溶液を取り出し、そして水を補充した。放出された抗生物質量は分光光度的に分析した(SPECTRAMAX(登録商標)分光光度計, Molecular Devices, Sunnyvale, CA)。
抗生物質装薬足場に対する大腸菌ATCC25922及び黄色ブドウ球菌ATCC25923(どちらもAmerican Type Culture Collection, Manassas, VAから)の感受性は、臨床研究所規格委員会(NCCLS)の「好気的に生育する細菌についての希釈抗菌感受性試験方法、承認標準M7-A5(Methods for dilution antimicrobial susceptibility testing for bacteria that grow aerobically Approved standard M7-A5)」(Nat. Committee Clin. Lab. Standards, Wayne, PA, 2000)に従った、寒天上の改変Kirby-Bauerディスク拡散方法により決定した。1ミリリットル(1ml)の各細菌バイアルを5mlの#18 Tryptic Soy Broth(Beckton Dickenson, Sparks, MD)に懸濁し、そして懸濁液の密度は、3.0×108CFU/mlの濁度標準(McFarland Standard, BioMerieux, Marcy l’Etoile, France)に適合すると見積もられた。ミューラー・ヒントン寒天100mmプレートに1mlの細菌懸濁液を接種した。寒天プレートの表面上に懸濁液が確実に完全に覆うように、滅菌1mlシリンジ及び棒を用いて広げた。
多くの材料形式試験についてより長い期間の放出プロフィールが試験されたものの、移植片が表面細菌コロニー形成に特に感受性である移植後6時間の「決定的期間」について報告する文献(Zilberman & Elsner, 130 Contr. Release 202-15(2008))のため、絹フィルムからのペニシリン及びアンピシリン放出の最初の24時間のみを特徴付けた。絹フィルムからの最初の24時間の放出はそのため、細菌接着の予防及び長期に亘る移植片の成功において最も重大であると考えられた。
高装薬=8%(w/v)絹溶液中に5mg/mLアンピシリン(理論的装薬量=フィルム当たり0.4mg)
低装薬=8%(w/v)絹溶液中に2.5mg/mLアンピシリン(理論的装薬量=フィルム当たり0.2mg)
高装薬=8%(w/v)絹溶液中に10mg/mLペニシリン(理論的装薬量=フィルム当たり0.8mg)
低装薬=8%(w/v)絹溶液中に5mg/mLペニシリン(理論的装薬量=フィルム当たり0.4mg)
抗生物質放出絹生物素材の注射可能な送達システムも研究した。バルク装薬したゲル及び薬物放出絹ミクロスフェアを装薬したゲルの両方からの薬物放出を特徴付けた。Branson Digital Sonifier 450を15%振幅で60秒から90秒間用いて絹溶液を超音波処理し、その後抗生物質溶液に混合し、その後ゲル化が起こるのを待って、それにより薬物を封入することによって、バルク装薬したゲルを調製した。ミクロスフェアは本明細書中に記載する脂質鋳型プロトコルに従って調製した。バルク装薬の場合と同様に、ゲル化直前に絹を超音波処理し、ミクロスフェア懸濁液と混合した。
絹フィルムへの組み入れが、酵素について観察されるのと同様の安定化効果を有するかどうかを決定するため、8%(w/v)の絹フィルム、または、4℃(冷蔵)、25℃(室温)、及び37℃(体温)の溶液中で貯蔵されたペニシリンの残存活性を比較する長期安定化試験を行った。最初の5ヶ月の安定化データの結果を図13に示す。
幾層ものナノフィルム被覆を、水溶性抗生物質及び多孔性三次元基質についての潜在的な装薬方法として試験した。被覆前、ゲンタマイシン被覆足場の絹足場の平均質量は25.1mg±3.4mgであり、そして、セファゾリン被覆足場については25.0mg±4.8mgであった。
Claims (8)
- 三次元絹フィブロイン足場中に包埋された抗生物質装薬(loaded)絹フィブロインミクロスフェアを含む組成物であって、該足場がゲルであってかつ基質上の被覆である、組成物。
- 足場が光学装置もしくは電子機器中に組み入れられる、または光学装置もしくは電子機器を含む、請求項1記載の組成物。
- 基質が包帯または移植片である、請求項1または2記載の組成物。
- 足場がサージカルパック材料である、請求項1または2記載の組成物。
- 抗生物質が、アクチノマイシン、アミノグリコシド、β-ラクタマーゼ阻害剤、グリコペプチド、アンサマイシン、バシトラシン、カルバセフェム、カルバペネム、セファロスポリン、イソニアジド、リネゾリド、マクロライド、ムピロシン、ペニシリン、オキソリン酸、ポリペプチド、キノロン、スルホンアミド、テトラサイクリン、モノバクタム、クロラムフェニコール、リンコマイシン、クリンダマイシン、エタンブトール、ムピロシン、メトロニダゾール、ペフロキサシン、ピラジンアミド、チアンフェニコール、リファンピシン、チアムフェニクル(thiamphenicl)、ダプソン、クロファジミン、キヌプリスチン、メトロニダゾール、リネゾリド、イソニアジド、ピラシル(piracil)、ノボビオシン、トリメトプリム、ホスホマイシン、及びフシジン酸からなる群より少なくとも選択される抗生物質である、請求項1〜4のいずれか一項記載の組成物。
- 抗生物質がセファゾリン、ゲンタマイシン、ペニシリン、アンピシリン、またはそれらの組み合わせである、請求項5記載の組成物。
- デフェンシン、マガイニン、ナイシン、溶菌バクテリオファージ、細胞、タンパク質、ペプチド、アミノ酸、核酸アナログ、ヌクレオチド、オリゴヌクレオチド、ペプチド核酸、アプタマー、抗体またはその断片若しくは部分、抗原またはエピトープ、ホルモン、ホルモンアンタゴニスト、成長因子または組換成長因子並びにその断片及びバリアント、細胞付着性メディエーター、サイトカイン、抗炎症剤、抗真菌剤、抗ウイルス剤、毒素、プロドラッグ、化学療法剤、小分子、薬物、染料、ビタミン、酵素、抗酸化剤、並びに、他の抗菌化合物からなる群より選択される少なくとも一つの剤をさらに含む、請求項1〜6のいずれか一項記載の組成物。
- 必要とする対象の一領域における微生物汚染を予防及び/または処置するための組成物を調製するための、三次元絹フィブロイン足場中に包埋された抗生物質装薬(loaded)絹フィブロインミクロスフェアの使用であって、該足場がゲルであってかつ基質上の被覆である、使用。
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KR20190119501A (ko) * | 2018-04-12 | 2019-10-22 | 숙명여자대학교산학협력단 | 식품 신선도 유지를 위한 항균성 하이드로겔 |
KR102166485B1 (ko) | 2018-04-12 | 2020-10-15 | 숙명여자대학교산학협력단 | 식품 신선도 유지를 위한 항균성 하이드로겔 |
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JP2012519698A (ja) | 2012-08-30 |
CA2791580A1 (en) | 2010-12-09 |
US20190175785A1 (en) | 2019-06-13 |
WO2010141133A2 (en) | 2010-12-09 |
JP2020058834A (ja) | 2020-04-16 |
AU2010257120A1 (en) | 2011-10-27 |
WO2010141133A3 (en) | 2011-03-31 |
JP5909362B2 (ja) | 2016-04-26 |
US20140105995A1 (en) | 2014-04-17 |
EP2403551A4 (en) | 2014-02-26 |
EP2403551A2 (en) | 2012-01-11 |
JP2016104724A (ja) | 2016-06-09 |
JP2018027342A (ja) | 2018-02-22 |
CA2791580C (en) | 2017-12-05 |
US20120052124A1 (en) | 2012-03-01 |
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