JP2004267791A - 過酢酸架橋非抗原性icl移植片 - Google Patents
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- JP2004267791A JP2004267791A JP2004134191A JP2004134191A JP2004267791A JP 2004267791 A JP2004267791 A JP 2004267791A JP 2004134191 A JP2004134191 A JP 2004134191A JP 2004134191 A JP2004134191 A JP 2004134191A JP 2004267791 A JP2004267791 A JP 2004267791A
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Abstract
【解決手段】哺乳動物患者に移植されたときに、身体部分又は組織構造体の機能的代用品として役立ち、同時に患者の生活細胞による生物学的再構築を伴って生じる制御された生分解を受けるプロテーゼを、有意な体液性免疫応答を誘発しないように非抗原性になるように処理する。本プロテーゼはその種々な実施態様において二重の性質を有する。第一に、これは置換身体部分として機能し、第二には、宿主細胞の内部増殖のための生物学的再構築鋳型として機能する。
【選択図】なし
Description
本発明は移植可能な生物学的プロテーゼの分野に属する。本発明は、多様な形に加工されて、哺乳動物の組織及び器官の修復、増大又は置換に用いられることができる非抗原性で弾性の生体適合性組織プロテーゼである。プロテーゼは宿主細胞によって徐々に分解され、改造され(remodeled)、宿主細胞は移植されたプロテーゼに代わって、構造と機能を復活するので、プロテーゼは器官修復と再構成のために用いられる。
医療技術がますます精緻になるにも拘わらず、損傷した器官の修復と置換は依然として健康ケアにおいて頻発する、費用のかかる重大な問題である。移植可能なプロテーゼは現在、多くの合成材料及び処理された天然材料から製造される。理想的なプロテーゼ材料は化学的に不活性で、非発癌性であり、機械的応力に耐えることができ、必要な形状で製造されることができ、滅菌可能であるべきであり、しかも組織液(tissue fluid)によって物理的に変化されるべきではなく、炎症性反応及び異物反応を惹起すべきではなく、アレルギー反応若しくは過敏症を誘導すべきではなく、場合によっては内蔵癒着を促進すべきではない(Jenkins S.D.等,Surgery94(2):392〜398,1983)。
本発明は現在入手可能な材料の問題点を克服して、損傷した組織及び器官の修復、増大又は置換に用いるためのプロテーゼデバイスを提供する。本発明は、哺乳動物宿主に移植されたときに、制御された生分解を受け、それに伴って充分な生細胞置換又は新組織形成が生じて、最初に移植されたプロテーゼが最終的に改造され、宿主由来組織及び細胞によって置換されるようなプロテーゼに関する。本発明のプロテーゼ(組織修復のための材料)は、哺乳動物組織に由来する非抗原性コラーゲン材料を含む。このコラーゲン材料は積層して、相互に結合して、多層状シート、管、又は複雑な形状のプロテーゼを形成することもできる。本発明の結合したコラーゲン層は構造的に安定で、柔軟であり、半透性かつ縫合可能である。
他の目的は、移植部位での組織内部及び/又は組織再生を可能にし、促進するプロテーゼ材料であって、哺乳動物コラーゲン組織に由来する、無菌の、非発熱性かつ非抗原性材料を提供することである。この材料から製造されたプロテーゼは、レシピエント宿主又は患者に移植されたときに、有意な免疫応答を誘発しない。この材料から形成されたプロテーゼは、充分な生細胞置換を伴って生ずる制御された生物学的改造(bioremodeling)を同時に示すので、最初の移植されたプロテーゼは患者の生細胞によって改造されて、再生された器官又は組織を形成する。
本発明のさらに他の目的は、自家移植、同種移植又は異種移植適応症における新規な多目的修復布帛の使用方法を提供することである。
発明の詳細な説明
本発明は、哺乳動物宿主に移植されたときに、機能的な修復、増大又は置換身体部分又は組織構造体として作用することができ、宿主細胞による改造と同時に生ずる制御された生分解を受ける組織工学プロテーゼに関する。本発明のプロテーゼは、本発明の種々な実施態様において、二重の性質を有する:第一に、このプロテーゼは代用身体部分として機能し、第二には、なおも代用身体部分として機能しながら、宿主細胞の内部成長のための改造鋳型として機能する。これをするために、非抗原性にされ、相互に結合されることができる哺乳動物由来コラーゲン組織を含む、本発明のプロテーゼ材料(組織修復布帛)が開発された。このプロテーゼを種々なデバイス及び構築体の構築によって説明するが、本発明はこれによって限定されない。デバイスの形状及び厚さにおけるデバイスの設計が構築体の最終的な適応に依存して選択されるべきであることは理解されるであろう。
代用身体部分又はサポートとして機能することの他に、プロテーゼの第2機能は、生物学的改造のための鋳型又は足場としての機能である。本明細書で用いる“生物学的改造”なる用語は、移植されたプロテーゼの、宿主細胞及び酵素による生物学的分解速度にほぼ等しい機能的速度での宿主細胞の内部増殖による構造コラーゲンの産生、血管形成及び上皮形成を意味する。組織修復布帛は、宿主によって全ての又は実質的に全ての宿主組織に改造されながら、最初に移植されたプロテーゼの特性を保有し、それゆえに、それが修復若しくは置換する組織の類似体として機能する。
ブタの小腸を回収し、機械的作用と熱水を用いた洗浄との組合せを用いて粘膜下組織から脂肪、筋肉及び粘膜層を強制的に除くBitterling腸洗浄装置(Nottingham,UK)によって、機械的にストリップした。機械的作用は、ローラーの間を完全な腸が通されるときに粘膜下組織から連続層を圧縮し、ストリップする、一連のローラーとして説明されることができる。小腸の粘膜下組織は周囲組織よりも硬く、剛性であるので、ローラーは粘膜下組織から軟組織を圧搾する。装置洗浄の結果、腸の粘膜下組織層のみが残存した。最終的に、粘膜下組織を4℃において18時間0.3%過酢酸で汚染除去し、次にリン酸緩衝化生理的食塩水において洗浄した。残存した生成物は腸コラーゲン層(ICL)であった。
溶着強度に対する、溶着温度(その後急冷)、溶着時間、溶着後の、1−エチル−3−(3−ジメチルアミノ)プロピル)カルボジイミド(EDC)濃度、アセトン濃度及び架橋時間の効果を、ICL二層状管適用に関して調べた。ICLは、実施例1に述べたようにブタから得た。強度特性は、縫合保持試験及び極限引張り強さ(UTS)試験を用いて測定した。
溶着強度に対する溶着温度と溶着後急冷との影響をICL二層状管適用に関して試験した。
ブタ粘膜下組織腸層の新鮮なサンプルを、実施例1に述べたような浄化工程後に、得た。次に、サンプルを未処理のままで水中に保存し、生理的濃度のリン酸塩緩衝化食塩水中に浸漬し、0.1%過酢酸で処理し、又は0.1%過酢酸で処理してから、1−エチル−3−(3−ジメチルアミノプロピル)カルボジイミド塩酸塩(EDC)によって架橋させた。次に、サンプルを0.5M NaCl/0.1M 酒石酸の溶液によって約18時間抽出した。
実施例5:腹壁パッチとしての六層状組織修復布帛
ブタ腸コラーゲンの6層をガラスプレート上で相互にスーパーインポーズさせた。第2ガラスプレートを腸コラーゲン層の頂部に載せ、第1プレートにきつくクランプした。この装置を慣用型のオーブンに62℃において1時間入れた。加熱の直後に、装置を4℃の水浴に10分間入れた。装置を分解し、腸コラーゲン層を取り出し、50%アセトン中の100mM 1−エチル−3−(3−ジメチルアミノプロピル)カルボジイミド塩酸塩(EDC)によって25℃において4時間処理した。この材料を袋に入れ、γ線照射(2.5Mrad)によって滅菌した。
ブタ腸コラーゲンの2層をガラスプレート上で相互にスーパーインポーズさせた。第2ガラスプレートを腸コラーゲン層の頂部に載せ、第1プレートにきつくクランプした。この装置を慣用型のオーブンに62℃において1時間入れた。加熱の直後に、装置を4℃の水浴に10分間入れた。装置を分解し、腸コラーゲン層を取り出し、50%アセトン中の10mM 1−エチル−3−(3−ジメチルアミノプロピル)カルボジイミド塩酸塩(EDC)によって25℃において4時間処理した。この材料を袋に入れ、γ線照射(2.5Mrad)によって滅菌した。
ヘルニア修復の基本型デバイスを中空内部領域を有するようにICLを用いて開発した。このデバイスは、完成したときに、周囲に結合した丸い形態と、生理的濃度のリン酸塩緩衝化食塩水の封入(inclusion)によって膨潤した膨潤内部領域とを有した。内部領域は任意に、剛性を加えるためにワイヤメッシュを、又は構造的サポート若しくは物質の投与のための他の物質を収容することができる。
ICL、濃厚なフィブリルコラーゲン及びヒアルロン酸を椎間円板の解剖学的構造と組成とに密接に近似するように設計した。
ブタの近位空腸を回収し、腸浄化装置(Bitterling,Nottingham,英国)によって処理し、実施例1に述べたように、過酢酸溶液によって汚染除去した。過酢酸処理したICL(PA−ICL)を縦に切断して開いて、リンパ管タグ部分を除去して、ICLシートを形成した。このICLシートを6.0mm直径ステンレス鋼マンドレルに巻き付けて、二層構築体を形成した。これらの構築体(マンドレル上)を62℃に設定された平衡化THERMOCENTERTMオーブン室に約1時間入れた。構築体を室から取り出し、4℃の水浴に約2〜5分間入れた。構築体を50/50水/アセトン溶液中の100mM 1−エチル−3−(3−ジメチルアミノプロピル)カルボジイミド(EDC)(50ml)中で18時間架橋させて、過酢酸処理済みEDC架橋(PA/EDC−ICL)血管移植片構築体を形成した。構築体をマンドレルから取り外し、残留EDC溶液を除去するために水ですすぎ洗いした。
25匹の雑種イヌ(体重15〜25kg)を一晩絶食させ、静脈内チオペンタル(30mg/kg)によって麻酔し、エンツベートし(entubated)、ハロタンと亜酸化窒素とによって維持した。Cefazolin(1000mg)を手術前と手術後に静脈内投与した。各イヌは大動脈バイパス移植片か又は大腿挿入移植片(femoral interposition graft)を受容した。大動脈バイパス移植片のためには、正中線腹部切開をおこない、大動脈を腎動脈から大動脈分岐まで露出した後に、静脈内ヘパリン(100U/kg)を投与した。遠位腎臓下大動脈(末端−側部吻合)と分岐近接大動脈(末端−側部吻合)との間に移植片(6mmx8cm)を挿入した。大動脈を近位吻合の遠位に結紮した。切開部を閉じて、イヌを手術後30日間アスピリンで維持した。大腿挿入移植片では、動物を両側から開いて、大腿動脈を露出させ、5cm長さを切除した。移植片(4mmx5cm)を末端−末端式に大腿動脈に吻合した。反対側では、対照移植片を挿入した。切開部を閉じ、動物を手術後30日間アスピリンで維持した。手術後フォローアップは30日間〜360日間の範囲であった。移植前、移植後4週間及び8週間の血液を採取した。動物を種々な時点(30日間、60日間、90日間、180日間及び360日間)で殺した。
ブタ粘膜下組織腸層の新鮮なサンプルを実施例1に述べた浄化工程後に得たが、これらは過酢酸処理しなかった。次に、サンプルを未処理のまま残し(NC−ICL)、0.1%過酢酸で処理し(PA−ICL)、又は0.1%過酢酸で処理してから、1−エチル−3−(3−ジメチルアミノプロピル)カルボジイミド塩酸塩(EDC)によって架橋させた(PA/EDC−ICL)。
NC−ICL、PA−ICL、又はPA/EDC−ICLから酒石酸を用いて(Bellon,G.等(1988)Anal.Biochem.175:263〜273)又はTRITON X−100(Rohm and Haas)を用いて、タンパク質を抽出した。粉末化NC−ICL、PA−ICL、又はPA/EDC−ICL(10%w/v)を酒石酸(0.1M酒石酸、0.5M NaCl)又はTRITON X−100(Rohm and Haas)抽出用緩衝液(TEB:20mM Tris HCl(pH7.2)中の1%TRITON X−100、2mM EGTA、2mM EDTA、1mM フェニルメチルスルホニルフルオリド、及びアプロチニン、ロイペプチン及びペプスタチン(Sigma,St.Louis,MO)の各25mg/ml)のいずれかと混合した。混合物を4℃において一晩インキュベートした。抽出物をガーゼで濾過して残屑を除去し、PBSに対して透析し、Centriprep−30(Amicon,Danvers,MA)を用いて濃縮した。抽出物は用いるまで−80℃において保存した。
NC−ICL、PA−ICL、又はPA/EDC−ICLによって免疫化されたウサギからの血清(実施例11に述べたように調製された血清)又は酸抽出されたI型コラーゲン(Organogenesis,Canton,MA)をELISAによってI型コラーゲン特異性抗原に関して試験した。ELISAプレート(Immulon II,NUNC,Bridgeport,NJ)を0.05M炭酸塩緩衝液(pH9.6)中の1mg/mlの酸抽出I型コラーゲン(200ml/孔)によって4℃において一晩被覆した。プレートをPBS/Tween20(0.1%)によって2回洗浄した。動物又はウサギ抗I型コラーゲン抗体(Southern Biotechnology,Birmingham,AL)からの血清サンプルを孔に加え(100ml/孔)、室温において1時間インキュベートした。プレートをPBS/Tweenによって3回洗浄した。二次抗体:ALPH標識ヤギ抗ウサギIg又はALPH標識ヤギ抗イヌIg(Southern Biotechnology)を適当な孔に加え、室温において1時間インキュベートした。プレートをPBS/Tweenによって3回洗浄した。p−ニトロフェニルホスフェート(PNPP)基質(1mg/ml)を各孔に加えた(100ml/孔)。SpectraMaxマイクロプレート読み取り機(Molecular Devices,Sunnydale,CA)上で吸光度を405nmにおいて読み取った。
ICLの抗原性に対するPAとEDCとの効果を抗NC−ICL抗血清を用いて調べて、PA又はPA/EDC処理ICLの酒石酸又はTEB抽出物中に存在する免疫反応性タンパク質を検出した。
イヌをICL移植片成分に対する体液性免疫応答に関して調べて、移植片中への細胞内部増殖を刺激するためにICLはその抗原性を有するべきであるかどうかを判定した。PA/EDC−ICL血管移植片を受容した15匹のイヌから、移植前、移植後4週間後及び8週間後に血液サンプルを採取した。各血液サンプルからの血清をNC−ICLの酒石酸抽出物及びTEB抽出物の両方中のタンパク質に対する抗体に関して検査した。血清の1:40の希釈度においても、検査したいずれのイヌもICLタンパク質と反応する抗体を有さなかった。これらの同じ血清サンプルを抗I型コラーゲン抗体の存在に関してELISAによって調べた。全ての血清サンプルは1:40の血清希釈度においてもI型コラーゲンに対する抗体に関して陰性であった。これらのイヌからの外植パラフィン切片のMassonトリクロム染色は宿主細胞の浸潤を示した。これらの結果は、宿主が活性に材料を再構築(改造)するときにPA/EDC−ICLが抗体反応を誘発しないことを実証する。
Claims (36)
- 2個以上の重ね合わせて結合したコラーゲン組織層を含むプロテーゼであって、哺乳動物患者に移植されたときに、本来の移植されたプロテーゼが患者の生細胞によって再構築されるような、充分な生細胞置換を伴って生ずる制御された生分解を受けるプロテーゼ。
- その形状がフラット、管状、又は複雑な形状である、請求項1記載のプロテーゼ。
- 前記コラーゲン組織が哺乳動物ソースから得られ、腸材料、大腿筋膜、硬膜及び心膜である、請求項1記載のプロテーゼ。
- 前記コラーゲン組織が小腸の粘膜下組織である、請求項3記載のプロテーゼ。
- コラーゲン組織層を結合させるために充分な時間及び条件での加熱溶着によって、前記コラーゲン層が一緒に結合される、請求項1記載のプロテーゼ。
- 架橋剤によって架橋される、請求項1記載のプロテーゼ。
- 架橋剤の1−エチル−3−(3−ジメチルアミノプロピル)カルボジイミド塩酸塩によって、架橋される、請求項6記載のプロテーゼ。
- 架橋剤にスルホ−N−ヒドロキシスクシンイミドが加えられる、請求項6記載のプロテーゼ。
- 架橋剤にアセトンが加えられる、請求項6記載のプロテーゼ。
- 過酢酸によって滅菌される、請求項1記載のプロテーゼ。
- 非抗原性である、請求項10記載のプロテーゼ。
- その1つ以上の表面が平滑な流動面として作用するコラーゲン分散系又はゲルによって被覆されている、請求項1記載のプロテーゼ。
- さらに孔を含有する、請求項1記載のプロテーゼ。
- 細断されたコラーゲン繊維をさらに含む、請求項1記載のプロテーゼ。
- コラーゲン糸をさらに含む、請求項1記載のプロテーゼ。
- 前記コラーゲン糸がフェルト、束、織物又はブレードを形成するように配置される、請求項15記載のプロテーゼ。
- 前記コラーゲン繊維又は糸が部分的に又は完全に架橋される、請求項14〜16のいずれかに記載のプロテーゼ。
- さらに抗凝固剤、1種以上の抗生物質、又は1種以上の成長因子を含有する、請求項1記載のプロテーゼ。
- 2個以上の重ね合わせて結合したコラーゲン組織層を有するプロテーゼの製造方法であって、
(a)コラーゲン組織層を結合させて、プロテーゼを形成するために充分な時間及び条件でコラーゲン組織層を加熱することによる加熱溶着を用いて、2個以上のコラーゲン組織層を一緒に結合させる工程と;
(b)前記加熱されたプロテーゼを冷却する工程と;
(c)前記プロテーゼを架橋させる工程と
を含み、このように形成された前記プロテーゼが、哺乳動物患者に移植されたときに、本来の移植されたプロテーゼが患者の生細胞によって再構築されるような、充分な生細胞置換を伴って生ずる制御された生分解を受ける、上記方法。 - 前記コラーゲン組織層が、哺乳動物ソースから得られる、腸材料、大腿筋膜、硬膜及び心膜であるコラーゲン組織の2層以上から形成される、請求項19記載の方法。
- 前記コラーゲン組織が小腸の粘膜下組織である、請求項20記載の方法。
- 前記加熱溶着が約50℃〜約75℃、より好ましくは約60℃〜約65℃、最も好ましくは約62℃においておこなわれる、請求項19記載の方法。
- 前記冷却が急冷によって達成される、請求項19記載の方法。
- 前記加熱溶着が約7分間〜約24時間、好ましくは約1時間で達成される、請求項19記載の方法。
- 前記プロテーゼを架橋剤の1−エチル−3−(3−ジメチルアミノプロピル)カルボジイミド塩酸塩によって架橋する、請求項19記載の方法。
- 前記プロテーゼを過酢酸によって滅菌する、請求項19記載の方法。
- 前記プロテーゼが非抗原性である、請求項26記載の方法。
- 損傷した組織の修復又は置換方法であって、2個以上の重ねられて結合したコラーゲン組織層を含み、哺乳動物患者に移植されたときに、本来の移植されたプロテーゼが患者の生細胞によって再構築されるような、充分な生細胞置換を伴って生ずる制御された生分解を受けるプロテーゼを患者に移植することを含む方法。
- レシピエント患者に移植するための哺乳動物由来コラーゲン組織から形成される、無菌で、非発熱性かつ非抗原性プロテーゼであって、移植されたプロテーゼがコラーゲン組織中の成分に対して体液性免疫応答を誘発せず、かつ前記プロテーゼが付随的に、本来の移植されたプロテーゼが患者の生細胞によって再構築されるような、充分な生細胞置換を伴って生ずる生物学的再構築を受けるプロテーゼ。
- プロテーゼレシピエントから得た血液血清をコラーゲン組織の抽出物中のタンパク質に対する抗体に関して調べた場合に、前記コラーゲン組織由来の成分に対する前記体液性免疫応答が、基礎力価レベルから、コラーゲン組織タンパク質に対する抗体の抗体力価の有意な上昇を示さない、請求項29記載のプロテーゼ。
- 前記抗体力価レベルが、コラーゲン組織タンパク質に対して予め感作されていない患者又は宿主に対して1:40以下である、請求項30記載のプロテーゼ。
- 哺乳動物ソースに由来するコラーゲン組織から製造される非抗原性プロテーゼの製造方法であって、
(a)前記材料の生物学的再構築可能な特性を維持する作用剤によってコラーゲン組織を消毒する工程と;
(b)前記消毒済みコラーゲン組織を架橋剤によって架橋させる工程と
を含み、
このような形成されたプロテーゼが哺乳動物患者に移植されたときに、本来の移植されたプロテーゼが体液性免疫応答を誘発せずに患者の生細胞によって再構築されるような、充分な生細胞置換を伴って生ずる制御された生分解を受ける、上記方法。 - 前記哺乳動物ソースが腸材料、大腿筋膜、硬膜及び心膜から成る群から選択される、請求項32記載の方法。
- 前記コラーゲン組織が小腸の粘膜下組織である、請求項33記載の方法。
- 前記コラーゲン組織が2層以上の重ねられて結合したコラーゲン組織層から形成される、請求項32記載の方法。
- 前記過酢酸が水中約0.01〜0.3%v/vの濃度である、請求項32記載の方法。
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1996
- 1996-03-12 WO PCT/US1996/003336 patent/WO1996031157A1/en active IP Right Grant
- 1996-03-12 EP EP05077508.9A patent/EP1623681B1/en not_active Expired - Lifetime
- 1996-03-12 ES ES96909660T patent/ES2252753T3/es not_active Expired - Lifetime
- 1996-03-12 CA CA2217581A patent/CA2217581C/en not_active Expired - Fee Related
- 1996-03-12 JP JP53029496A patent/JP3756187B2/ja not_active Expired - Fee Related
- 1996-03-12 AU AU53083/96A patent/AU711900B2/en not_active Ceased
- 1996-03-12 AT AT96909660T patent/ATE308926T1/de not_active IP Right Cessation
- 1996-03-12 ES ES05077508T patent/ES2426668T3/es not_active Expired - Lifetime
- 1996-03-12 DE DE69635414T patent/DE69635414T2/de not_active Revoked
- 1996-03-12 EP EP96909660A patent/EP0828453B1/en not_active Revoked
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2004
- 2004-04-28 JP JP2004134191A patent/JP2004267791A/ja not_active Withdrawn
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- 2011-12-27 JP JP2011286563A patent/JP2012101100A/ja active Pending
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MX9707655A (es) | 1997-11-29 |
JPH11503051A (ja) | 1999-03-23 |
EP1623681A1 (en) | 2006-02-08 |
DE69635414T2 (de) | 2006-08-17 |
CA2217581C (en) | 2012-05-08 |
JP2012101100A (ja) | 2012-05-31 |
US5733337A (en) | 1998-03-31 |
CA2217581A1 (en) | 1996-10-10 |
ES2252753T3 (es) | 2006-05-16 |
DE69635414D1 (de) | 2005-12-15 |
AU5308396A (en) | 1996-10-23 |
ES2426668T3 (es) | 2013-10-24 |
WO1996031157A1 (en) | 1996-10-10 |
EP0828453A4 (en) | 2001-01-10 |
AU711900B2 (en) | 1999-10-21 |
EP1623681B1 (en) | 2013-05-15 |
JP3756187B2 (ja) | 2006-03-15 |
ATE308926T1 (de) | 2005-11-15 |
EP0828453A1 (en) | 1998-03-18 |
EP0828453B1 (en) | 2005-11-09 |
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