JP7395113B2 - 止血組成物を調製する方法 - Google Patents
止血組成物を調製する方法 Download PDFInfo
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- JP7395113B2 JP7395113B2 JP2020558619A JP2020558619A JP7395113B2 JP 7395113 B2 JP7395113 B2 JP 7395113B2 JP 2020558619 A JP2020558619 A JP 2020558619A JP 2020558619 A JP2020558619 A JP 2020558619A JP 7395113 B2 JP7395113 B2 JP 7395113B2
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Description
本開示はトロンビンを含む止血ペースト組成物を調製する単純化された方法に関し、本方法は乾燥したトロンビンを、生体親和性ポリマーを含むペースト等のペーストの中で直接もどすステップを含む。トロンビンを含む止血組成物は、乾燥したトロンビン組成物及びペーストから単一ステップの操作で調製され、創傷の処置に用いられ得る。
コラーゲン及びゼラチン等のタンパク質系の止血材料は手術手順における使用のための固体スポンジ及び緩い又は詰め込まれていない粉末の形態で市販されている。この緩い又は詰め込まれていない粉末を食塩液又はトロンビン溶液等の流体と混合することによって、混合条件及び材料の相対的な割合に応じて、特に不均一な表面又は到達しにくい区域からの拡散した出血の場合における使用のための止血組成物として有用なペースト又はスラリーを形成することができる。
本開示は、止血ペーストの中へのトロンビンの組込みに関する上記の課題に対処し、乾燥したトロンビン組成物を、生体親和性ポリマーを含むペーストの中で直接もどし、トロンビンを含む止血ペーストを単一ステップの操作で産生する方法を提供する。止血組成物を調製するためのそのような単純で速い方法は、潜在的な出血を速くかつ効率的に制御しなければならない手術室において極めて価値がある。
a)第1の容器中に乾燥したトロンビン組成物を用意するステップ、
b)第2の容器中に生体親和性ポリマーを含むペーストを用意するステップ、
c)好適な連結手段を用いて第1の容器と第2の容器を連結するステップ、及び
d)容器の内容物を混合するステップ
を含む。
a)第1の容器中に乾燥したトロンビン組成物を用意するステップ、
b)第2の容器中に生体親和性ポリマーを含むペーストを用意するステップ、
c)好適な連結手段を用いて第1の容器と第2の容器を連結するステップ、及び
d)容器の内容物を混合するステップ
を含む。
「生物活性剤」は、in vivo又はin vitroで実証され得る、しばしば有益であるいくつかの薬理学的効果をもたらす任意の薬剤、薬物、化合物、物質の組成物又は混合物である。したがって、薬剤はそれがヒト又は動物の体内の細胞組織との相互作用又はこれへの影響を有するならば、生物活性と考えられる。本明細書で用いる場合、この用語には、個体に局所的又は全身的な影響を生じる任意の生理学的又は薬理学的に活性な物質がさらに含まれる。生物活性剤は酵素等のタンパク質であってもよい。生物活性剤のさらなる例には、それだけに限らないが、オリゴ糖、多糖、任意選択でグリコシル化されたぺプチド、任意選択でグリコシル化されたポリペプチド、オリゴヌクレオチド、ポリヌクレオチド、脂質、脂肪酸、脂肪酸エステル、及び二次代謝物が含まれるか、又はそれらからなる薬剤を含む。これは、ヒト又はその他の任意の動物等の個体の処置と併せて、予防的又は治療的に用いられ得る。本明細書で用いる用語「生物活性剤」は、真核細胞又は原核細胞等の細胞を包含しない。
本開示はトロンビンを含む止血ペースト組成物を調製する単純化された方法に関する。本方法は乾燥したトロンビン組成物を、生体親和性ポリマーを含むペースト等のペーストの中で直接もどす(reconstitute)ステップを含む。
a)第1の容器中に乾燥したトロンビン組成物を用意するステップ、
b)第2の容器中に生体親和性ポリマーを含むペーストを用意するステップ、
c)好適な連結手段を用いて第1の容器と第2の容器を連結するステップ、及び
d)容器の内容物を混合するステップ
を含む。
本開示は乾燥したトロンビン組成物をペーストの中で直接もどす方法に関する。
本開示の乾燥したトロンビン組成物は、生体親和性ポリマーを含むペースト等のペーストの中で直接もどされる。
本開示では生体親和性ポリマーを含むペーストを調製するために水性媒体が用いられ得る。
一実施形態では、止血組成物は1つ又は複数の親水性化合物を含む。親水性化合物は通常、極性又は電荷を有する機能性基を含み、それにより水溶性になっている。本開示の止血組成物に1つ又は複数の親水性化合物が含まれていることがトロンビンの安定性に有利な効果を有していると考えられ、それにより、乾燥したトロンビン組成物のもどし効率が改善され得る。親水性化合物はまた、止血組成物の稠度を改善し得る。
本発明の一実施形態では、止血組成物は、止血、創傷の治癒、骨の治癒、組織の治癒、及び/又は腱の治癒を刺激することができる1つ又は複数のさらなる生物活性剤を含む。1つ又は複数の生物活性剤は、乾燥したトロンビン組成物、生体親和性ポリマーを含むペーストの成分であってよく、及び/又は乾燥したトロンビン組成物をもどした後の別のステップにおいて止血組成物中に組み込んでもよい。好ましくは、そのような生物活性剤は、さらなる混合ステップを避けるために、乾燥したトロンビン組成物の中又は生体親和性ポリマーを含むペーストの中に含まれる。生物活性剤が保存及びもどしの間にその生物活性を保持すること、即ち、その薬剤が最終の止血組成物中でその生物学的機能を保持していることが重要である。多くの生物活性剤、特に水が存在すると分解するか又はその二次構造を失うおそれがある酵素及びその他のタンパク質は、溶液中で不安定である。
本発明の止血組成物は、DMSO(ジメチルスルホキシド)及び/又は2-メチル-2,4-ペンタンジオール(MPD)のうちの1つ又は複数をさらに含んでよい。
本開示は、乾燥したトロンビン組成物をペーストの中で直接もどして創傷の処置における特に止血目的のための使用に適した止血組成物を産生する方法に関する。
a)第1の容器中に乾燥したトロンビン組成物を用意するステップ、
b)第2の容器中に生体親和性ポリマーを含むペーストを用意するステップ、
c)好適な連結手段を用いて第1の容器と第2の容器を連結するステップ、及び
d)容器の内容物を混合するステップ
を含む。
a)第1の容器中に乾燥したトロンビン組成物を用意するステップ、
b)第2の容器中に生体親和性ポリマーを含むペーストを用意するステップ、
c)好適な連結手段を用いて第1の容器と第2の容器を連結するステップ、及び
d)容器の内容物を混合するステップ
を含む。
本開示の止血組成物は、乾燥したトロンビン組成物を、生体親和性ポリマーを含むペーストの中で直接もどすことによって調製される。ペーストのもどしは本明細書に記載した方法によって実施し得る。止血組成物は、止血組成物中のトロンビンの実質的に均一な分布を有する。
止血組成物を調製するために、当業者には既知の任意の好適な容器、例えばバイアル、ジャー、チューブ、トレイ、カートリッジ、又はシリンジが用いられ得る。
一実施形態では、例えば本明細書で開示したシリンジ等のシリンジ、又はその他の封入ユニットの中に含まれる乾燥したトロンビン組成物及び/又は生体親和性ポリマーを含むペーストは外部包装の中にさらに含まれ、それにより製品は使用まで無菌に保たれる。これにより使用者は外部包装を取り除き、止血組成物の成分を無菌野に移すことができる。
本開示の乾燥したトロンビン組成物、生体親和性ポリマーを含むペースト、及び/又は止血組成物は、好ましくは無菌である。当技術で既知の任意の好適な滅菌手法を利用することができる。滅菌は、伝染性の物質(例えば真菌、細菌、ウイルス、プリオン、及び胞子の形態等)を効果的に殺滅し又は排除する任意のプロセスを意味する。滅菌は例えば熱、化学物質、及び/又は照射の適用によって達成することができる。
本開示は、本開示の方法によって得られる止血組成物の、止血及び/又は創傷の治癒を促進するための使用にさらに関する。
本開示はさらに、本開示の乾燥したトロンビン組成物及び乾燥したトロンビン組成物の量に適合した生体親和性ポリマーを含むペーストを含み、混合に際して止血における使用に適したトロンビン含量を有する止血組成物が形成される止血用キットに関する。
a)乾燥したトロンビン組成物を含む第1のシリンジ、
b)生体親和性ポリマーを含むペーストを含む第2のシリンジ、及び
c)任意選択で外部包装
を含む止血用キットであって、
2つのシリンジが相互連結可能である、止血用キットに関する。
a)外部包装中の乾燥したトロンビン組成物を含む第1のシリンジ、
b)外部包装中の生体親和性ポリマーを含むペーストを含む第2のシリンジ、及び
c)任意選択でa)及びb)の成分を含む外部包装
を含み、2つのシリンジは相互連結可能である。
[実施例1:ペースト中のトロンビンの分布]
材料:
10mLガラスシリンジ中、2000IUの乾燥したトロンビン(第1のシリンジ)
10mLシリンジ中、5mLのゼラチンペースト(第2のシリンジ)
ルアーロックを介して2つのシリンジを相互連結し、ゼラチンペーストの内容物を、乾燥したトロンビン組成物を含むシリンジに移送する(第1の移送)。次いで得られるトロンビンとゼラチンペーストの混合物を第1と第2のシリンジの間を5回往復させて移送し、第2のシリンジの中に含まれた止血組成物を得る(図1)。移送の全回数は6回である。得られる止血組成物はトロンビンを含む流動性ペースト製剤であり、本明細書では「6tomix」又は「6TM」ペーストと特定する。混合の結果として全体で約3mLの空気がペーストの中に入り、ペーストの最終体積は約8mLになる。
最終の止血組成物は体積約8mLのペーストである。
トロンビン含量の変動は、2つの部分の間の平均トロンビン活性の百分率での差異として計算する。
(36/(31+36))-0.50*100=3.7%
として計算される。
目的
本研究の目的は、実施例1の6TMペーストの止血効率を試験することであった。ペーストの止血効率を市販のSurgiflo(登録商標)ペーストの止血効率と比較した。Surgiflo(登録商標)は出血している表面に塗布することによる止血用途に承認された無菌の吸収性ブタゼラチンペースト製剤であり、確立された市販の製品である。
ブタ脾臓生検パンチモデルを用い、最初10秒の圧迫時間、続いて120秒の評価時間、その後10秒の圧迫時間で8mmの穿刺(深さ3mm)を脾臓に適用した。
ブタは大量の血液(70mL/kg)及び大血管の脾臓を有し、単一の動物で多くの止血の比較が可能であるので、このモデルのために選択される動物である。本研究の雌ブタは体重約40kg(±5kg)、月齢約3か月であった。体重をブタの組み入れ基準とした。体重の下限は試験すべき臓器が適切な大きさであることを保証することとし、上限はブタの大きさが標準化されていることを保証する指針であった。
ブタは手術前、少なくとも6時間絶食させた。以下の混合物、即ち6.25mLのNarcoxyl(キシラジン20mg/mL)、1.25mLのKetaminol(ケタミン100mg/mL)、2mLのTurbogesic(ブトルファノール10mg/mL)、及び2mLのMetadon(メタドン10mg/mL)をZoletil 50Vetバイアルに添加したもの(125mgのチレタミン及び125mgのゾラゼパムを含む)を筋肉内注射(1mL/10kg)することによって麻酔を誘導した。
試料の調製
実施例1に記載したように、即ち食塩液中でトロンビンを事前にもどすことなく、湿潤したゼラチンペーストを乾燥したトロンビンの調製物と混合することによって、本発明の6TMペーストを調製した。
正中腹部切開を作成して脾臓を露出した。脾臓に8mmのパンチ(深さ3mm)を作成した。出血強度は本明細書で下記するように0~5のスケールで評価した。出血強度3及び4のみを許容可能とみなした。パンチは今や対照試料又は試験試料のいずれかに準備できた。試験試料ごとに新たなパンチを作成した。
それぞれのパンチの出血強度は、外科医によって0~5のスケールで評価された(下表を参照)。
湿潤したガーゼをパンチの上に直接載せた。指圧を30秒間印加し、続いて120秒を止血評価時間とした。止血を評価した(血液が試験物品の下から30秒間漏出しないことと定義)。120秒以内に止血が達成されない場合には、さらに30秒間の指圧を印加し、120秒の止血再評価を実施した。出血が停止して止血が達成されるまで、又は試験時間が12分に達するまで、タンポンの適用及び観察時間を実施した。陰性対照では止血は12分の試験時間以内で達成されず、したがって研究を通してブタが出血する能力が示された。
アプリケーターチップを用いて、ほぼ1~2mLのペーストを直接パンチに塗布した。塗布の間、チップをパンチの中に進入させ、組織との接触を確実にした。塗布の後、0.9%の食塩液で湿潤したガーゼをパンチの上に載せた。指圧(タンポン)を10秒間印加した。加圧を停止し、ガーゼを取り除きし、続いて止血を評価した。試験物品の下からの血液の漏出が120秒間見られない場合には、止血が達成されたと結論し、実験を終了する。血液が試験物品の下から120秒の時間枠内で漏出した場合には、漏出に要する時間を記録し、再び指圧を10秒間印加し、その後、止血を検査した。止血が達成されるか、12分のいずれか早い方まで、この手順を継続した。
指圧10秒、止血の検査:39秒後に血液漏出、指圧さらに10秒、止血の検査120秒間:漏出なし-結論:止血は10+39+10秒=59秒後に達成された。即ち、最後の観察時間はTTHの計算に含めない。
本研究の結果を下表に示し、さらに図3に示す。
Claims (20)
- 止血組成物を調製する方法であって、
a)第1のシリンジ中に乾燥したトロンビン組成物を用意するステップ、
b)第2のシリンジ中に生体親和性ポリマーを含むペーストを用意するステップ、
c)好適な連結手段を用いて前記第1のシリンジと前記第2のシリンジを連結するステップ、及び、
d)前記第1及び第2のシリンジの内容物を混合するステップ、
を含み、
前記生体親和性ポリマーが、ゼラチン、コラーゲン、キチン、キトサン、アルギネート、セルロース、酸化セルロース、ポリグリコール酸、及び、ポリ酢酸からなる群から選択される、方法。 - 前記混合するステップが、前記第1及び第2の容器の内容物を数回往復して移送することによって実施され、それによりトロンビンの実質的に均一な分布が得られる、請求項1に記載の方法。
- 前記移送の回数が10回未満である、請求項2に記載の方法。
- 前記止血組成物の全体にわたる前記トロンビンの均一な分布が10%未満のトロンビン含量の変動によって特徴付けられる、請求項1~3のいずれか一項に記載の方法。
- 前記止血組成物が、止血及び/又は創傷治癒における使用に適したペーストである、請求項1~4のいずれか一項に記載の方法。
- 前記止血組成物が流動性組成物である、請求項1~5のいずれか一項に記載の方法。
- 前記乾燥したトロンビン組成物が噴霧乾燥又は凍結乾燥によって調製されている、請求項1~6のいずれか一項に記載の方法。
- 前記生体親和性ポリマーが水性媒体に実質的に不溶な粉末粒子からなる、請求項1~7のいずれか一項に記載の方法。
- 前記生体親和性ポリマーが架橋されている、請求項1~8のいずれか一項に記載の方法。
- ステップb)における前記ペーストが生体親和性ポリマーを7%~20%の含量で含む、請求項1~9のいずれか一項に記載の方法。
- 前記止血組成物が1つ又は複数の親水性化合物を含む、請求項1~10のいずれか一項に記載の方法。
- 前記1つ又は複数の親水性化合物が1つ又は複数のポリオールである、請求項11に記載の方法。
- 前記止血組成物が、止血、創傷の治癒、骨の治癒、組織の治癒、及び/又は腱の治癒を刺激することができる1つ又は複数のさらなる生物活性剤を含む、請求項1~12のいずれか一項に記載の方法。
- 前記止血組成物が、押出し促進剤をさらに含み、前記押出し促進剤がアルブミンである、請求項1~13のいずれか一項に記載の方法。
- 請求項11に記載の親水性化合物、請求項13に記載の生物活性剤、及び/又は、請求項14に記載の押出し促進剤が、
ステップa)における前記乾燥したトロンビン組成物の成分である、
ステップb)における前記ペーストの成分である、及び/又は、
ステップd)の後の別のステップにおいて前記止血組成物中に組み込まれる、請求項1~25のいずれか一項に記載の方法。 - 前記第1のシリンジがガラスシリンジであるか、又は前記第1のシリンジが前記乾燥したトロンビン組成物を含むガラスインサートを含む、請求項1~15のいずれか一項に記載の方法。
- 前記第2のシリンジがプラスチックシリンジである、請求項1~16のいずれか一項に記載の方法。
- ステップa)における前記乾燥したトロンビン組成物、ステップb)における前記ペースト、及び/又は、前記止血組成物を滅菌するさらなるステップを含む、請求項1~17のいずれか一項に記載の方法。
- a)乾燥したトロンビン組成物を含む第1のシリンジ、
b)生体親和性ポリマーを含むペーストを含む第2のシリンジ、及び、
c)任意選択で外部包装、
を含むキットであって、前記2つのシリンジが相互連結可能であり、
前記生体親和性ポリマーが、ゼラチン、コラーゲン、キチン、キトサン、アルギネート、セルロース、酸化セルロース、ポリグリコール酸、及び、ポリ酢酸からなる群から選択さる、キット。 - 乾燥したトロンビン組成物をもどす方法であって、
a)第1のシリンジ中に乾燥したトロンビン組成物を用意するステップ、
b)第2のシリンジ中に生体親和性ポリマーを含むペーストを用意するステップ、
c)好適な連結手段を用いて前記第1のシリンジと前記第2のシリンジを連結するステップ、及び、
d)前記第1及び第2のシリンジの内容物を混合するステップ、
を含み、
前記生体親和性ポリマーが、ゼラチン、コラーゲン、キチン、キトサン、アルギネート、セルロース、酸化セルロース、ポリグリコール酸、及び、ポリ酢酸からなる群から選択される、方法。
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