JP4733283B2 - Liquid fibrinogen preparation - Google Patents

Description

【００１１】
【課題を解決するための手段】
そこで、本願発明者等は上述の問題に鑑み鋭意検討した結果、フィブリノゲン溶液に微量のカルシウムイオン源を共存させると、室温の高温域（１５℃以上）での保存において、カルシウムイオン存在によるゲル化を生じることなくフィブリノゲンの分解を抑制させうる知見を得、塗布用装置中にあらかじめ無菌的にフィブリノゲン溶液を分注し保存することにより、常温以上の室温高温域（１５〜３０℃）保存において溶液状態で安定に、しかも簡便性を伴って保存されうるフィブリノゲンを調製することを可能にした。さらに、フィブリノゲン溶液は低温環境下ではゲル化が進行しやすいことから、上記のカルシウムイオン源添加フィブリノゲン溶液に、ゲル化抑制作用をもつグアニジノ基を有する物質を共存させた結果、カルシウムイオン源のフィブリノゲン安定化効果を有意に減じることなくゲル化を抑制させうる知見を得、常温未満の室温低温域（１〜１５℃）においても、フィブリノゲン溶液を安定に保存させうることを発見した。以上のことから、フィブリノゲン溶液に至適濃度のカルシウムイオン源を添加することにより常温で安定に保存されうる液状フィブリノゲン製剤を、さらに好ましくはゲル化抑制作用をもつ物質を至適濃度で共存させることにより低温条件をも含む室温という広範囲な温度域の環境下で安定に保存されうる液状フィブリノゲン製剤を提供する本発明を完成した。以下、本発明をさらに詳細に説明する。
【００１２】
【発明の構成】
本発明の溶液状態で安定に保存されるフィブリノゲン製剤は、好適な緩衝液、例えばクエン酸緩衝液（具体例として、０．３Ｍ塩化ナトリウムを含むｐＨ７．０の０．０４Ｍクエン酸緩衝液）中の５％（ｗ／ｖ）以上の濃度のフィブリノゲン溶液にカルシウムイオン源を含有することを大きな特徴とする。カルシウムイオン源としては、塩化カルシウム、水酸化カルシウム、リン酸二水素カルシウム、硫酸カルシウム等が例として挙げられ、とりわけ塩化カルシウムは好適である。カルシウムイオン源の添加量は０．０５〜２ｍＭの濃度幅で選択され、特に０．２〜１ｍＭでの添加が好ましい。カルシウムイオン源によるフィブリノゲン分解抑制の作用機序は、カルシウムイオンがわずかなγ鎖の架橋形成もしくは構造変化を引き起こし、フィブリノゲン分子がプロテアーゼの攻撃に対して構造的に抵抗性を獲得するためと推定される。なお、フィブリノゲン溶液にカルシウムイオンが一定濃度以上存在するとゲル化することが既知であるが、上記の低濃度カルシウムイオン源を含有するフィブリノゲン溶液は、室温の高温域である常温以上（１５〜３０℃）での保存においてゲル化や粘性が上昇することなく、高温保存に伴う分解が抑制される。
【００１３】
また、上記フィブリノゲン溶液は低温環境下ではゲル化が進行するが、低温保存時のゲル化抑制剤としてグアニジノ基を有する物質の効果が公表されており（日本国公開特許公報 平成7-173073号）、これを室温の低温域（１〜１５℃）での保存に伴うゲル化を回避する目的で添加されうる。ここでいうグアニジノ基を有する物質とは特に限定されるものではないが、とりわけ、アルギニン、グアニジン等が好ましい態様であり、これらは単独あるいは組み合わせて添加することができる。添加量は０．１〜２．０Ｍの濃度が好ましく、０．１〜１．０Ｍの濃度での添加は最適な態様である。
【００１４】
なお、本発明に係る製剤の主成分であるフィブリノゲンは公知の方法で調製される。そのようなものとして、例えば冷エタノール沈澱法にグリシンによるフィブリノゲンの溶解度低下効果を組み合わせた方法（Blomback,B. and Blomback,M., Arkiv Kemi,10,p.415〜443,(1956)）およびグリシンを単独で使用するグリシン沈澱法（Kazal,L.A., et al., Proc.Soc.Exp.Med.,113,p.989〜994,(1963)）等が報告されている。
【００１５】
本発明のフィブリノゲン製剤は、さらにフィブリンゲル−マトリックス内の架橋形成および線溶阻害の目的で、付加的に血液凝固第ＸIII因子および線溶阻害剤を含有することができる。線溶阻害剤は特に限定されるものではないが、アプロチニン、α₂プラスミンインヒビター、トラネキサム酸、εアミノカプロン酸等が好ましい。本発明に係る添加剤並びに付加的な添加剤の添加工程の順序については、最終製剤中に上記諸添加剤が所定の濃度で含有されるものであれば特別な制約はないが、例えば、夾雑ウイルス不活化のための凍結乾燥加熱処理を行った後、再溶解後の最終調製時に添加されるのが好ましい。
【００１６】
上記溶液組成において、フィブリノゲン溶液は室温保存条件下において溶液状態で安定に保存されうる。室温は１〜３０℃と定義され、季節や地域によりこの範囲内で変動するが、一般には常温（１５〜２５℃）付近である。
【００１７】
さらに、この液状フィブリノゲン製剤は注射器等の塗布用装置中にあらかじめ分注され使用することもできる。本発明のフィブリノゲン溶液を無菌濾過後、滅菌済みの注射器に分注し、先端をキャップしておくことにより臨床使用時に製剤を他の容器に移し換えることなく、直接使用することができる。この際の注射器はその利便性の観点からポリプロピレン等の合成樹脂性のものが好適に使用されうる。
【００１８】
また、本発明の上記液状フィブリノゲン製剤は、単独で使用されるのみならず、トロンビンを主成分とする成分と組み合わせることにより、ヒトおよび動物組織の生物学的接着剤を構成し、幅広い臨床の現場で応用される。
【００１９】
以下に、試験例並びに実施例を挙げて本発明を具体的に説明するが、本発明はこれらの例に何ら限定されるものではない。
【００２０】
【実施例】
試験例１
０．３Ｍ塩化ナトリウムを含むｐＨ７．０の０．０４Ｍクエン酸緩衝液に、８％（ｗ／ｖ）濃度で溶解されたフィブリノゲン溶液に対し、各種濃度の塩化カルシウムが添加され、３０℃の温度で経時的に安定性が試験された。安定性は、６カ月後までの３点の保存期間における目視による状態判定、濁度、凝固性蛋白質純度、および動粘度（２０℃での測定）を保存前の値と比較することによって評価された。凝固性蛋白質純度は、厚生省告示“生物学的製剤基準”収載の、乾燥人フィブリノゲン小分製品の試験である凝固性蛋白質含量及び純度試験に従って測定した。
【００２１】
その結果は表２（フィブリノゲン溶液を30℃保存した際の経時的性状変化）、表３および図２（フィブリノゲン溶液を30℃保存した際の凝固性蛋白質純度の経時的測定値）、表４および図３（フィブリノゲン溶液を30℃保存した際の動粘度の経時的測定値）のとおりである。３０℃での保存により、経時的に凝固性蛋白質純度が低下したが、塩化カルシウムの添加は濃度依存的にこの経時変化を抑制する効果を示し、フィブリノゲンの保存安定性に有効であることが判明した。目視での性状については、１ｍＭおよび２ｍＭ塩化カルシウム添加検体が経時的にゲル化が進行したが、それ以外の検体はいずれも経時的変化および塩化カルシウム添加濃度による差を認めなかった。しかし、動粘度測定値は０．５ｍＭ塩化カルシウム添加検体で経時的に上昇し、この検体では保存温度（３０℃）から動粘度測定条件（２０℃）に温度を低下させる際に粘性が上昇したことから、ゲル化に至らない塩化カルシウム添加上限濃度は保存温度に依存し、より低温保存ではより低濃度の塩化カルシウム添加検体もゲル化することが示唆された。
【００２２】
【表２】

【００２３】
【表３】

【００２４】
【表４】

【００２５】
試験例２
０．３Ｍ塩化ナトリウムを含むｐＨ７．０の０．０４Ｍクエン酸緩衝液に、８％（ｗ／ｖ）濃度で溶解されたフィブリノゲン溶液に対し、各種濃度の塩化カルシウムが添加され、１０℃の温度で経時的に安定性が試験された。安定性は、保存した検体の目視による状態判定によって評価された。
【００２６】
その結果は表５（フィブリノゲン溶液を１０℃保存した際の経時的性状変化）のとおりである。フィブリノゲン溶液は１０℃での保存によりゲル化し、塩化カルシウム添加は濃度依存的にゲル化の進行を促進した。また、ゲル化した検体を３７℃前後の温度で加温して溶液状態に復帰させるのに要する時間が、塩化カルシウム添加濃度依存的に延長したことから、塩化カルシウムがゲル化を増強することを確認した。
【００２７】
【表５】

【００２８】
試験例３
試験例１および２において、塩化カルシウム添加によりゲル化が増強されることが示唆された結果を受け、ゲル化抑制作用をもつ物質の一態様であるアルギニンの添加効果について検討するため、以下の試験を実施した。０．３Ｍ塩化ナトリウムを含むｐＨ７．０の０．０４Ｍクエン酸緩衝液に、８％（ｗ／ｖ）濃度で溶解されたフィブリノゲン溶液に対し、各種濃度の塩化カルシウムおよびアルギニンが添加され、１０℃および３０℃の温度で経時的に安定性が試験された。安定性は、１０℃保存検体については４週間後までの２点、３０℃保存検体については６カ月後までの３点の保存期間における目視による状態判定、凝固性蛋白質純度、および動粘度（２０℃での測定）を保存前の値と比較することによって評価された。
【００２９】
その結果は表６（フィブリノゲン溶液を１０℃保存した際の経時的性状変化）、表７（フィブリノゲン溶液を１０℃保存した際の凝固性蛋白質純度の経時的測定値）、表８（フィブリノゲン溶液を１０℃保存した際の動粘度の経時的測定値）、並びに、表９（フィブリノゲン溶液を３０℃保存した際の経時的性状変化）、表１０（フィブリノゲン溶液を３０℃保存した際の凝固性蛋白質純度の経時的測定値）、表１１（フィブリノゲン溶液を３０℃保存した際の動粘度の経時的測定値）のとおりである。
【００３０】
１０℃での保存では、いずれの塩化カルシウムおよびアルギニン濃度の検体も凝固性蛋白質純度の低下はなく、安定に保存できることが確認された。保存に伴うゲル化や動粘度の上昇は、１ｍＭ以下の塩化カルシウム存在下では、０．３Ｍ以上のアルギニンを添加することにより抑制できることが判明した。また、３０℃保存では、０．５ｍＭ以上の塩化カルシウムを添加することにより保存による凝固性蛋白質純度の低下を有効に阻止でき、これら検体中に０．３Ｍ以下の濃度のアルギニンが共存しても、この安定化効果に影響を及ぼさないことが確認された。各検体の動粘度の上昇は塩化カルシウム添加濃度依存性を示したが、１ｍＭ以下の塩化カルシウム添加に対しては０．３Ｍ程度のアルギニンの添加で回避できることが示唆された。
【００３１】
【表６】

【００３２】
【表７】

【００３３】
【表８】

【００３４】
【表９】

【００３５】
【表１０】

【００３６】
【表１１】

【００３７】
試験例４
試験例３において、塩化カルシウムおよびアルギニンの適当な組み合わせが広い温度範囲の条件下での液状フィブリノゲンの安定化に有効に作用することが示唆された結果を受け、塩化カルシウムとアルギニンの組み合わせに関し、室温最下限である１℃での安定性について検討した。０．３Ｍ塩化ナトリウムを含むｐＨ７．０の０．０４Ｍクエン酸緩衝液に、８％（ｗ／ｖ）濃度で溶解されたフィブリノゲン溶液に対し、０．５ｍＭの塩化カルシウムおよび０．３Ｍのアルギニンが添加され、１℃の温度で経時的に安定性が試験された。安定性は１カ月まで保存した検体の目視による状態判定、凝固性蛋白質純度、および動粘度（２０℃での測定）を保存前の値と比較することによって評価された。
【００３８】
その結果は表１２（フィブリノゲン溶液を１℃保存した際の経時的変化）のとおりである。性状、凝固性蛋白質純度ならびに動粘度のいずれについても保存前後での有意な変動はなく、安定に保存できることが確認された。
【００３９】
【表１２】

【００４０】
実施例１
クエン酸塩血漿から得られた寒冷沈殿物を可溶化し、１．５Ｍグリシン溶液を用いて沈殿させたフィブリノゲン沈殿を０．１５Ｍ塩化ナトリウムを含むｐＨ８．０の０．０２５Ｍクエン酸緩衝液に３７℃で溶解させた。このフィブリノゲン溶液を−２℃に冷却した後、８％（ｖ／ｖ）エタノールを添加し沈殿を回収した。得られた沈殿を０．３Ｍ塩化ナトリウム、０．５ｍＭ塩化カルシウム、０．３Ｍアルギニンおよび１０００ＫＩＥ／ｍｌアプロチニンを含むｐＨ７．０の０．０４Ｍクエン酸緩衝液に３７℃で溶解させた。溶解しなかった部分は遠心分離して除去した。フィブリノゲン濃度を約８％（ｗ／ｖ）に調整した後、無菌濾過し、容器に分注した。
【００４１】
実施例２
クエン酸塩血漿から得られた寒冷沈殿物を可溶化し、１．５Ｍグリシン溶液を用いて沈殿させたフィブリノゲン沈殿を０．１５Ｍ塩化ナトリウムを含むｐＨ８．０の０．０２５Ｍクエン酸緩衝液に３７℃で溶解させた。このフィブリノゲン溶液を−２℃に冷却した後、８％（ｖ／ｖ）エタノールを添加し沈殿を回収した。得られた沈殿を０．０７５Ｍ塩化ナトリウムを含むｐＨ７．０の１０ｍＭクエン酸緩衝液に３７℃で溶解させた。フィブリノゲン濃度を約２％（ｗ／ｖ）に調整した後、フィブリノゲン溶液１ｍｌあたり２５単位の第ＸIII因子が添加された。この溶液を無菌濾過し、凍結乾燥およびそれに続く乾燥加熱処理を行った後、得られた凍結乾燥粉末を０．５ｍＭ塩化カルシウム、０．３Ｍアルギニンおよび１０００ＫＩＥ／ｍｌアプロチニンを含むｐＨ７．０の溶液でフィブリノゲン濃度約８％（ｗ／ｖ）となるように可溶化した。この溶液を無菌濾過し、滅菌済みのポリプロピレン製５ｍｌ容注射器に３ｍｌ分注し、先端にキャップをつけ、注射器入りの液状フィブリノゲン製剤を得た。
【００４２】
【発明の効果】
本発明の利点は、フィブリノゲンの溶解操作や注射器等への移し換えを要さない液状フィブリノゲン製剤の室温保存を可能とし、緊急時の使用における利便性を向上させることに加え、製剤を保存するための特別な設備を不要とすることである。
【図面の簡単な説明】
【図１】 フィブリノゲン溶液の各温度下（４℃、１５℃、２５℃、３０℃、３７℃）における保存に伴う凝固性蛋白質純度の経時的変化を示す図。
【図２】 各濃度の塩化カルシウム添加フィブリノゲン溶液を３０℃保存した際の凝固性蛋白質純度の経時的変化を示す図。
【図３】 各濃度の塩化カルシウム添加フィブリノゲン溶液を３０℃保存した際の動粘度（測定温度：２０℃）の経時的変化を示す図。[0001]
[Industrial application fields]
The present invention relates to a fibrinogen-containing preparation that is a constituent of a tissue adhesive existing as a two-component preparation. More specifically, the present invention relates to a liquid fibrinogen preparation that can be stored at room temperature or room temperature in a solution state.
[0002]
[Prior art and problems to be solved by the invention]
Fibrinogen (fibrinogen) is a very important coagulation factor that exists in the final stage of the so-called coagulation cascade. Fibrinogen is converted by thrombin from its soluble form to insoluble fibrin, which makes an important contribution to hemostasis and wound healing, for example in the activation of the clotting system after injury.
[0003]
Fibrinogen is important for hemostasis and wound healing and is used clinically as an intravenous formulation in replacement therapy in congenital and acquired fibrinogen deficiencies, for example, by increasing the fibrinogen concentration in the blood Stop bleeding. Furthermore, in recent years fibrinogen has been used as a surgical substitute for sutures of soft organs such as the liver or spleen, or as a suturing aid by mixing with thrombin, and at the same time applied in a wide range of clinical settings. Has been.
[0004]
As disclosed in the claims of Japanese Patent Publication No. 63-40546 and the detailed description of the invention, a tissue adhesive containing fibrinogen and blood coagulation factor XIII (factor XIII) Manufacturing methods are known. It has been known that freeze-dried lyophilized fibrinogen or a plasma protein mixture containing a high proportion of fibrinogen is gradually re-dissolved only at an elevated temperature when reconstituted with a lysate.
[0005]
In order to sufficiently obtain an adhesive action as a fibrin adhesive, it is necessary to dissolve fibrinogen at a high concentration, and the higher the concentration of fibrinogen that can be coagulated, the more advantageous. However, it takes time to prepare such a high-concentration fibrinogen solution from a lyophilized fibrinogen product and cannot be used for emergency use. Furthermore, if the time required for the dissolution operation of fibrinogen lyophilizate becomes longer, there is a concern that the patient may be adversely affected.
[0006]
Japanese Patent Publication Showa 63-40547 discloses a technique for freezing a fibrinogen solution having the above-described composition to improve stability. Furthermore, in order to provide a preparation containing a high concentration of fibrinogen, focusing on the solvent that increases the solubility of fibrinogen, a 6-10% (w / v) fibrinogen solution containing various fibrinogen dissolution promoters was prepared. In addition, a technique has been developed that is stored in a frozen state and melted at the time of use (Japanese Laid-Open Patent Publication No. 57-149229). Although the above-mentioned frozen preparation has improved convenience in use compared to the freeze-dried preparation, there are still problems such as the point that it takes time for thawing at the time of use and the equipment for storing in the frozen state is required. Was included.
[0007]
Therefore, Japanese Published Patent Publication No. Hei 7-73073 discloses a low temperature that avoids freezing so that it can be used for emergency use as a constituent of tissue adhesive, and also as a fibrinogen concentrate intended for intravenous administration. A technique for preparing a preparation based on fibrinogen that can be stably stored in a solution state under storage conditions is disclosed. However, although liquid storage preparations have improved convenience in use, there is still room for improvement, such as requiring time to return to room temperature when used, and requiring equipment for storage at low temperatures. It was.
[0008]
Under such circumstances, an object of the present invention is to prepare a preparation based on fibrinogen that can be stably stored in a solution state at room temperature or room temperature storage conditions. The room temperature and room temperature are defined as 1-30 ° C. at room temperature and 15-25 ° C. at room temperature in the Japanese Pharmacopoeia.
[0009]
A problem in the above problem is that fibrinogen decomposes with time by storage in a solution state, and the degree of decomposition depends on the storage temperature. Table 1 and FIG. 1 show the results of evaluating the degree of fibrinogen degradation under each storage temperature condition based on changes in the purity of coagulable protein, which is an index of fibrinogen degradation. As a result, the purity of the clotting protein, which indicates the degree of fibrinogen degradation, hardly changes when stored at a low temperature of 15 ° C or lower, but the purity of the clotting protein decreases with time when stored at 25 ° C or higher. The higher the temperature, the more prominent. For this reason, it is important to suppress fibrinogen decomposition when the fibrinogen solution is stored for a long period of time at a room temperature range (1 to 30 ° C.), particularly in a high temperature range (15 ° C. or higher). It becomes.
[0010]
[Table 1]

[0011]
[Means for Solving the Problems]
Therefore, as a result of intensive investigations by the inventors of the present invention in view of the above-mentioned problems, when a small amount of calcium ion source is allowed to coexist in the fibrinogen solution, gelation due to the presence of calcium ions in storage at room temperature (15 ° C. or higher). In order to prevent the degradation of fibrinogen without causing a problem, the fibrinogen solution is aseptically dispensed and stored in advance in a coating apparatus, so that the solution can be stored in a room temperature high temperature range (15 to 30 ° C.). It has made it possible to prepare fibrinogen that can be stored stably in a state and with simplicity. Furthermore, since the fibrinogen solution is prone to gelation in a low-temperature environment, the result of coexisting a substance having a guanidino group having a gelation-inhibiting action in the above-mentioned fibrinogen solution to which calcium ion source is added is a fibrinogen of the calcium ion source. It has been found that gelling can be suppressed without significantly reducing the stabilizing effect, and that the fibrinogen solution can be stably stored even at room temperature and low temperature (1 to 15 ° C.) below room temperature. Based on the above, a liquid fibrinogen preparation that can be stored stably at room temperature by adding an optimal concentration of calcium ion source to the fibrinogen solution, and more preferably a substance having a gelation-inhibiting action coexist at an optimal concentration. Thus, the present invention has been completed which provides a liquid fibrinogen preparation that can be stably stored in a wide temperature range of room temperature including room temperature conditions. Hereinafter, the present invention will be described in more detail.
[0012]
[Structure of the invention]
The fibrinogen preparation stably stored in the solution state of the present invention is in a suitable buffer solution, for example, citrate buffer solution (specifically, 0.04M citrate buffer solution having pH 7.0 containing 0.3M sodium chloride). The main feature is that a calcium ion source is contained in a fibrinogen solution having a concentration of 5% (w / v) or more. Examples of the calcium ion source include calcium chloride, calcium hydroxide, calcium dihydrogen phosphate, calcium sulfate and the like, and calcium chloride is particularly preferable. The addition amount of the calcium ion source is selected in a concentration range of 0.05 to 2 mM, and addition of 0.2 to 1 mM is particularly preferable. The mechanism of action to suppress fibrinogen degradation by the calcium ion source is presumed to be that calcium ions cause slight γ-chain crosslink formation or structural changes, and the fibrinogen molecule gains structural resistance to protease attack. The It is known that the fibrinogen solution is gelled when calcium ions are present at a certain concentration or more. However, the fibrinogen solution containing the low-concentration calcium ion source is a room temperature or higher (15-30 ° C.) which is a high temperature range of room temperature. ), The degradation associated with high temperature storage is suppressed without increasing gelation or viscosity.
[0013]
In addition, the fibrinogen solution progresses in a low-temperature environment, but the effect of a substance having a guanidino group as a gelation inhibitor during low-temperature storage has been published (Japanese Published Patent Publication No. 7-173073). This can be added for the purpose of avoiding gelation associated with storage in a low temperature range (1 to 15 ° C.) at room temperature. The substance having a guanidino group here is not particularly limited, but arginine, guanidine and the like are particularly preferable, and these can be added alone or in combination. The addition amount is preferably a concentration of 0.1 to 2.0M, and the addition at a concentration of 0.1 to 1.0M is an optimum mode.
[0014]
Fibrinogen, which is the main component of the preparation according to the present invention, is prepared by a known method. As such, for example, a method combining the ethanol ethanol precipitation method with the effect of reducing the solubility of fibrinogen by glycine (Blomback, B. and Blomback, M., Arkiv Kemi, 10 , p.415-443, (1956)) and A glycine precipitation method (Kazal, LA, et al., Proc. Soc. Exp. Med., 113 , p. 989 to 994, (1963)) using glycine alone has been reported.
[0015]
The fibrinogen preparation of the present invention may further contain blood coagulation factor XIII and a fibrinolysis inhibitor for the purpose of cross-linking in the fibrin gel-matrix and fibrinolysis inhibition. The fibrinolysis inhibitor is not particularly limited, but aprotinin, α ₂ plasmin inhibitor, tranexamic acid, ε aminocaproic acid and the like are preferable. There are no particular restrictions on the order of the additive step according to the present invention and the additive additive step, as long as the additives are contained in the final formulation at a predetermined concentration. It is preferably added at the time of final preparation after re-dissolution after performing freeze-drying heat treatment for virus inactivation.
[0016]
In the above solution composition, the fibrinogen solution can be stably stored in a solution state at room temperature storage conditions. The room temperature is defined as 1 to 30 ° C. and varies within this range depending on the season and region, but is generally around normal temperature (15 to 25 ° C.).
[0017]
Furthermore, this liquid fibrinogen preparation can be dispensed and used in advance in a coating apparatus such as a syringe. The fibrinogen solution of the present invention can be used directly without being transferred to another container during clinical use by aseptic filtration and dispensing into a sterilized syringe and capping the tip. In this case, from the viewpoint of convenience, a synthetic resin material such as polypropylene can be preferably used.
[0018]
In addition, the liquid fibrinogen preparation of the present invention is not only used alone, but also forms a biological adhesive for human and animal tissues by combining with a component mainly composed of thrombin, and has a wide clinical field. Applied in.
[0019]
Hereinafter, the present invention will be specifically described with reference to test examples and examples, but the present invention is not limited to these examples.
[0020]
【Example】
Test example 1
Various concentrations of calcium chloride are added to a fibrinogen solution dissolved at a concentration of 8% (w / v) in a 0.04 M citrate buffer solution containing 0.3 M sodium chloride at pH 7.0, and a temperature of 30 ° C. The stability was tested over time. Stability is assessed by comparing visual status determination, turbidity, coagulable protein purity, and kinematic viscosity (measured at 20 ° C.) over three storage periods up to 6 months after storage. It was. The clotting protein purity was measured according to the clotting protein content and purity test, which is a test of dried human fibrinogen fraction product, published in the Ministry of Health and Welfare “Biological Formulation Standards”.
[0021]
The results are shown in Table 2 (Change in property with time when fibrinogen solution is stored at 30 ° C.), Table 3 and FIG. 2 (Measured value of coagulation protein purity with time when fibrinogen solution is stored at 30 ° C.), Table 4 and FIG. 3 (measured value of kinematic viscosity over time when fibrinogen solution is stored at 30 ° C.). Although the coagulation protein purity decreased with time by storage at 30 ° C, the addition of calcium chloride showed an effect of suppressing this change over time in a concentration-dependent manner and proved effective for the storage stability of fibrinogen. did. As for visual properties, gelation progressed with time in 1 mM and 2 mM calcium chloride-added specimens, but no change due to changes with time and calcium chloride addition concentration was observed in any other specimens. However, the measured value of kinematic viscosity increased with time in the sample added with 0.5 mM calcium chloride. In this sample, the viscosity increased when the temperature was lowered from the storage temperature (30 ° C.) to the kinematic viscosity measurement condition (20 ° C.). This suggests that the upper limit concentration of calcium chloride addition that does not lead to gelation depends on the storage temperature, and that samples with a lower concentration of calcium chloride are gelled at lower temperature storage.
[0022]
[Table 2]

[0023]
[Table 3]

[0024]
[Table 4]

[0025]
Test example 2
Various concentrations of calcium chloride are added to a fibrinogen solution dissolved at a concentration of 8% (w / v) in a 0.04 M citrate buffer solution containing 0.3 M sodium chloride at pH 7.0, and a temperature of 10 ° C. The stability was tested over time. Stability was evaluated by visual judgment of the stored specimen.
[0026]
The results are as shown in Table 5 (Change in properties over time when the fibrinogen solution was stored at 10 ° C.). The fibrinogen solution gelled by storage at 10 ° C., and the addition of calcium chloride promoted the gelation in a concentration-dependent manner. In addition, since the time required to warm the gelled specimen at a temperature of about 37 ° C. to return to the solution state is extended depending on the concentration of calcium chloride added, it indicates that calcium chloride enhances gelation. confirmed.
[0027]
[Table 5]

[0028]
Test example 3
In Test Examples 1 and 2, in order to examine the effect of addition of arginine, which is one embodiment of a substance having a gelation-inhibiting action, based on the results suggested that the gelation is enhanced by the addition of calcium chloride, the following test was conducted. Carried out. Various concentrations of calcium chloride and arginine were added to a fibrinogen solution dissolved at a concentration of 8% (w / v) in a 0.04 M citrate buffer solution containing 0.3 M sodium chloride at pH 7.0, 10 ° C. And stability was tested over time at a temperature of 30 ° C. Stability was determined by visual inspection, coagulant protein purity, and kinematic viscosity (20 points for samples stored at 10 ° C. up to 2 weeks after 4 weeks, and 3 points for samples stored at 30 ° C. up to 6 months after storage. (Measured in ° C.) was compared with the value before storage.
[0029]
The results are shown in Table 6 (Change in properties over time when fibrinogen solution is stored at 10 ° C.), Table 7 (Measured value of coagulant protein purity when stored at 10 ° C.), Table 8 (Fibrinogen solution) Measured value of kinematic viscosity over time when stored at 10 ° C.), Table 9 (Change in properties over time when fibrinogen solution is stored at 30 ° C.), Table 10 (Coagulable protein when fibrinogen solution is stored at 30 ° C.) Purity measurement value over time) and Table 11 (measurement value of kinematic viscosity over time when fibrinogen solution was stored at 30 ° C.).
[0030]
In storage at 10 ° C., it was confirmed that specimens having any calcium chloride and arginine concentrations could be stably stored without any decrease in the coagulable protein purity. It was found that gelation and kinematic viscosity increase during storage can be suppressed by adding 0.3 M or more arginine in the presence of 1 mM or less calcium chloride. In addition, when stored at 30 ° C., it is possible to effectively prevent a decrease in coagulation protein purity due to storage by adding calcium chloride of 0.5 mM or more. Even if arginine having a concentration of 0.3 M or less coexists in these samples. It was confirmed that this stabilizing effect was not affected. The increase in kinematic viscosity of each specimen showed calcium chloride addition concentration dependence, but it was suggested that the addition of about 0.3 M arginine can be avoided for the addition of 1 mM or less calcium chloride.
[0031]
[Table 6]

[0032]
[Table 7]

[0033]
[Table 8]

[0034]
[Table 9]

[0035]
[Table 10]

[0036]
[Table 11]

[0037]
Test example 4
In Test Example 3, in response to the results suggested that an appropriate combination of calcium chloride and arginine effectively acts to stabilize liquid fibrinogen under conditions of a wide temperature range, The stability at 1 ° C., which is the lowest limit, was examined. 0.5 mM calcium chloride and 0.3 M arginine were added to a fibrinogen solution dissolved at a concentration of 8% (w / v) in a 0.04 M citrate buffer solution containing 0.3 M sodium chloride at pH 7.0. Added and tested for stability over time at a temperature of 1 ° C. The stability was evaluated by comparing the visual judgment of the specimen stored up to 1 month, the coagulable protein purity, and the kinematic viscosity (measured at 20 ° C.) with the values before storage.
[0038]
The results are shown in Table 12 (change over time when fibrinogen solution was stored at 1 ° C.). It was confirmed that there was no significant change before and after storage in terms of properties, clotting protein purity, and kinematic viscosity, and that it could be stored stably.
[0039]
[Table 12]

[0040]
Example 1
The fibrinogen precipitate obtained by solubilizing the cryoprecipitate obtained from citrate plasma and precipitated with 1.5 M glycine solution was added to 0.025 M citrate buffer pH 8.0 containing 0.15 M sodium chloride. It was dissolved at ° C. The fibrinogen solution was cooled to −2 ° C., and 8% (v / v) ethanol was added to recover the precipitate. The resulting precipitate was dissolved at 37 ° C. in 0.04 M citrate buffer pH 7.0 containing 0.3 M sodium chloride, 0.5 mM calcium chloride, 0.3 M arginine and 1000 KIE / ml aprotinin. The undissolved part was removed by centrifugation. After adjusting the fibrinogen concentration to about 8% (w / v), it was aseptically filtered and dispensed into containers.
[0041]
Example 2
The fibrinogen precipitate obtained by solubilizing the cryoprecipitate obtained from citrate plasma and precipitated with 1.5 M glycine solution was added to 0.025 M citrate buffer pH 8.0 containing 0.15 M sodium chloride. It was dissolved at ° C. The fibrinogen solution was cooled to −2 ° C., and 8% (v / v) ethanol was added to recover the precipitate. The obtained precipitate was dissolved at 37 ° C. in 10 mM citrate buffer pH 7.0 containing 0.075 M sodium chloride. After adjusting the fibrinogen concentration to about 2% (w / v), 25 units of Factor XIII were added per ml of fibrinogen solution. This solution was subjected to sterile filtration, lyophilization followed by drying and heat treatment, and the resulting lyophilized powder was then added with a pH 7.0 solution containing 0.5 mM calcium chloride, 0.3 M arginine and 1000 KIE / ml aprotinin. Solubilization was performed so that the fibrinogen concentration was about 8% (w / v). This solution was sterile filtered, and 3 ml was dispensed into a sterilized polypropylene 5 ml syringe, and a cap was attached to the tip to obtain a liquid fibrinogen preparation with a syringe.
[0042]
【The invention's effect】
The advantage of the present invention is that it enables storage at room temperature of a liquid fibrinogen preparation that does not require dissolution operation of fibrinogen or transfer to a syringe, etc., in addition to improving convenience in emergency use, in addition to storing the preparation This eliminates the need for special equipment.
[Brief description of the drawings]
FIG. 1 is a graph showing changes over time in the purity of a coagulable protein during storage of a fibrinogen solution at various temperatures (4 ° C., 15 ° C., 25 ° C., 30 ° C. and 37 ° C.).
FIG. 2 is a graph showing changes with time in the purity of coagulable proteins when calcium chloride-added fibrinogen solutions at various concentrations are stored at 30 ° C.
FIG. 3 is a graph showing changes with time in kinematic viscosity (measurement temperature: 20 ° C.) when calcium chloride-added fibrinogen solutions at various concentrations are stored at 30 ° C.

Claims (11)

A liquid fibrinogen preparation that can be stored in a solution under normal temperature storage conditions, comprising fibrinogen as a main component and a calcium ion source at a concentration of 0.05 to 2 mM . Liquid fibrinogen formulation according to claim 1, further comprising the source of calcium ions in a concentration of 0.2 to 1 mm. A liquid fibrinogen preparation which can be stored in a solution state at room temperature and normal temperature storage conditions, characterized by comprising a fibrinogen as a main component, a calcium ion source at a concentration of 0.05 to 2 mM , and a substance having a guanidino group. The liquid fibrinogen preparation according to claim 3 , comprising the calcium ion source at a concentration of 0.2 to 1 mM. The liquid fibrinogen preparation according to claim 3, wherein the substance having a guanidino group is selected from arginine and guanidine. The liquid fibrinogen preparation according to claim 3 , comprising the substance having a guanidino group at a concentration of 0.1 to 2.0M. The liquid fibrinogen preparation according to claim 3, which contains the substance having a guanidino group at a concentration of 0.1 to 1.0M. The liquid fibrinogen preparation according to claim 1 or 3 , further comprising blood coagulation factor XIII. The liquid fibrinogen preparation according to claim 1 or 3 , further comprising a fibrinolysis inhibitor. By combining the components consisting mainly of thrombin, liquid fibrinogen preparation according to any one of claims 1 to 9, characterized in that configuring the biological adhesive of human and animal tissues. The liquid fibrinogen preparation according to any one of claims 1 to 10 , which is dispensed in advance into a coating apparatus.

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