JP3806241B2 - Peptide-containing adhesive for biological tissue - Google Patents

Description

で示されるペプチドが含有されていることを特徴とする。
【００１０】
本発明の請求項２記載のペプチド含有生体組織用接着剤は、更に、創傷治癒効果を有する栄養成分が含有されていることを特徴とする請求項１記載のペプチド含有生体組織用接着剤である。
【００１１】
本発明の請求項３記載のペプチド含有生体組織用接着剤は、式〔Ｉ〕で示されるペプチドの含有量が０．０００１〜１０重量％、並びに創傷治癒効果を有する栄養成分としてアスコルビン酸、薬理学的に許容されるアスコルビン酸のエステルおよび薬理学的に許容されるアスコルビン酸の塩からなる群より選ばれる少なくとも一種の含有量が０．００１〜９９重量％であることを特徴とする請求項２記載のペプチド含有生体組織用接着剤である。
【００１２】
本発明の請求項４記載のペプチド含有生体組織用接着剤は、式〔Ｉ〕で示されるペプチドの含有量が０．０００１〜１０重量％、並びに創傷治癒効果を有する栄養成分としてアルギニン、グルタミンおよびビタミンＡからなる群より選ばれる少なくとも一種の含有量が０．００１〜９９重量％であることを特徴とする請求項２記載のペプチド含有生体組織用接着剤である。
【００１３】
本発明の請求項５記載のペプチド含有生体組織用接着剤は、生体組織用接着剤がシアノアクリレート系接着剤、フィブリン接着剤またはゼラチン系接着剤であることを特徴とする請求項１〜４のいずれかに記載のペプチド含有生体組織用接着剤である。
【００１４】
本発明で用いられる上記ペプチドは、ストレプトマイセス属に属する該ペプチド生産菌株、例えば、放線菌ストレプトマイセス・ノビリス（Streptomyces nobilis、以下「Ｓ．ノビリス」と略記する）を培養し、得られた培養液または同液の乾固物もしくは培養菌体から有機溶剤によって抽出された抽出物を、各種カラムクロマトグラフィーに付し、目的物を含むカラムクロマトグラフィー画分を再結晶処理することにより得られる。
【００１５】
上記ペプチドを生産する放線菌Ｓ．ノビリスは、公的保存機関から入手可能であり、たとえば理化学研究所の保存菌（ＪＣＭ４２７４）（これは米国においてＡＴＣＣ１９２５２およびオランダにおいてＣＢＳ１９８．６５としても保存）などの菌が使用できる。
【００１６】
上記ペプチドを得る方法は、例えば、本発明者らが先に国際出願したＷＯ９６／１２７３２号公報に記載の方法によって得られる。
【００１７】
本発明で用いられる創傷治癒効果を有する栄養成分としては、例えば、アスコルビン酸、薬理学的に許容されるアスコルビン酸のエステル、薬理学的に許容されるアスコルビン酸の塩、アルギニン、グルタミンおよびビタミンＡなどが挙げられ、これらは医薬品として使用可能なものが挙げられる。
【００１８】
上記薬理学的に許容されるアスコルビン酸のエステルとしては、例えば、アスコルビン酸リン酸エステル、アスコルビン酸硫酸エステル、アスコルビン酸パルミチン酸エステル、アスコルビン酸ステアリン酸エステルなどを挙げることができるが、これらに限定されるわけではない。
【００１９】
上記薬理学的に許容されるアスコルビン酸の塩としては、例えば、アスコルビン酸と各種塩基との塩を挙げることができる。例えば、アルカリ金属塩（例えば、ナトリウム塩）、アルカリ土類金属塩（例えば、カルシウム塩、マグネシウム塩など）などを挙げることができるが、これらに限定されるわけではない。
【００２０】
本発明で用いられる生体組織用接着剤としては、従来から生体組織用接着剤として使用されてきたもののいずれも使用可能であり、特に限定されないが、従来技術の項で詳細に説明したシアノアクリレート系接着剤、フィブリン接着剤またはゼラチン系接着剤が好ましい。
【００２１】
本発明のペプチド含有生体組織用接着剤中に含有すべき上記ペプチドの量は、特に限定されず広範囲に適宜選択されるが、ペプチド含有生体組織用接着剤中に、好ましくは、１０^-7〜１０重量％の範囲である。
【００２２】
【作用】
本発明で用いられるペプチドは、肉芽形成作用があり、創傷治癒作用を有すると共に、強い抗菌作用も有することが知られている。このペプチドを生体組織用接着剤に含有させることにより、より高い創傷治癒促進作用を有すると共に、より高い組織接合効果をもつ生体組織用接着剤が得られた。
【００２３】
また、上記ペプチドに既に報告されているアスコルビン酸、アルギニン、グルタミンおよびビタミンＡなどの創傷治癒効果を有する栄養成分（医学のあゆみ、６月号別冊、３９〜４４頁、１９９６年）を組み合わせることにより、より一層高い創傷治癒促進作用を有すると共に、より一層高い組織接合効果をもつ生体組織用接着剤が得られた。
【００２４】
【発明の実施の形態】
参考例１
理化学研究所から入手した放線菌Ｓ．ノビリス（ＪＣＭ４２７４）を、酵母エキス０．２％（ｗ／ｖ）添加澱粉・アンモニウム培地５０ｍｌを含む５００ｍｌ容坂口スラスコ５本で、２６℃、１５０ｒｐｍ、１２０時間振盪培養（前々培養）した。続いて同培地１２リットルを含む２０リットル容ジャーファメンターに前々培養菌液２４０ｍｌを接種し、２６℃、４１０ｒｐｍ、通気量４リットル／分で２４時間培養（前培養）した。さらに澱粉・アンモニウム培地（蒸留水１００ｍｌ中に可溶性澱粉を１ｇ、リン酸水素二カリウムを０．０５ｇ、塩化アンモニウムを０．０５ｇ含む）１４０リットルを含む２００リットル容ジャーファメンターに、前培養菌液１２リットルを接種し、２６℃、２４時間種培養した。
【００２５】
さらに澱粉・アンモニウム培地１４００リットルを含む２０００リットル容タンクに、種培養菌液１４０リットルを接種し、２６℃、１４０ｒｐｍ、通気量７００リットル／分、ｐＨ７．５で７日間培養した。
【００２６】
培養終了後、濾過により菌体を濾別した。このようにして得られた菌体６．３４ｋｇ（湿重量）のうち、菌体１ｋｇ（湿重量）にジクロロメタン３リットルを加え、室温で１５時間攪拌後、菌体を濾別し、菌体抽出液を得た。菌体については、同操作を３回繰り返した。得られた菌体抽出液を濃縮後、シリカゲル担体１２０ｇに吸着させた。本吸着シリカゲル担体をシリカゲルカラムにより精製した。
【００２７】
シリカゲル担体８００ｇを充填した径８．０ｃｍのカラムに上記抽出物吸着担体約１２０ｇをチャージし、シリカゲルカラムを作成した。このシリカゲルカラムを下記の条件を用いて精製を行なった。溶出溶剤として、ａ）ヘキサン：酢酸エチル＝４：６を４リットル、ｂ）酢酸エチルを３．５リットル、ｃ）メタノールを２リットルを、この順に流速５００ｍｌ／時間で流した。分画は、溶剤組成を変更する毎に行い、特に酢酸エチルの溶出画分は５００ｍｌずつ分画した（従って、酢酸エチルについては、溶出画分数は合計７画分となる）。
【００２８】
上記の各溶出画分について、それぞれ、ＯＤＳ−８０ＴＭ、内径４．６ｍｍ×長さ２５．０ｃｍの東ソー社製のカラムを用いたＨＰＬＣ（日立社製、ポンプＬ−６０００、Ｌ−６２００、検出器Ｌ−３０００、カラムオーブン６５５Ａ−５２）によって、検出波長２１０ｎｍ、カラム温度４０℃、流速１ｍｌ／分の条件で、溶離液として水：アセトニトリル＝３：７を用いて、純度を確認した。
【００２９】
上記のＨＰＬＣによる純度確認において、リテンションタイムが１２〜１５分で溶出されるピーク面積が、全溶出ピーク面積の８０％以上を占めることが確認されたシリカゲルカラム溶出画分を合わせ、同一画分とした。本画分を濃縮乾固後、メタノール−ジクロロメタン系を用いて繰り返し再結晶を行い、柱状結晶３．５ｇを得た。
【００３０】
この物質の構造は、種々の機器分析データよりＷＯ９６／１２７３２号公報に記載された物質と同一であり、式〔Ｉ〕であると決定した。
【００３１】
構造分析データ
上記で得られた物質の機器分析データを以下に示す。
【００３２】

【００３３】

【００３４】
３．アミノ酸分析
加水分解物としてＤ−セリン、Ｌ−アラニンおよびＤ−Ｎ−メチル−フェニルアラニンが認められた。
【００３５】
（実施例１）
フィブリノーゲン末および血液凝固第XIII因子をアプロチニン溶液に溶解したＡ液と、トロンビン末を塩化カルシウム溶液に溶解したＢ液から構成されるフィブリン糊（ヘキスト社製、商品名 ベリプラストＰ）のＢ液に、参考例１で得られたペプチドをＡ液とＢ液との混合後の最終濃度が０．０１重量％となるように混合した。このようにして得られたＢ液とＡ液を混合し、本発明のペプチド含有生体組織用接着剤とした。後述の性能評価においては、このペプチド含有生体組織用接着剤を調製直後に切創に０．１ｇ塗布する方法により切創に投与した。
【００３６】
（実施例２）
参考例１で得られたペプチドを、ゼラチン糊（Ｅ．Ｈ．Ｓ．社（フランス）製、商品名 ＧＲＦグルー）の中のゼラチン・レゾルシン混合液と混合し、上記ペプチドの最終濃度が０．０１重量％となるようにされた液を得た。後述の性能評価においては、この液を０．１ｇ切創に塗布し、その上にホルムアルデヒド添加液を数滴滴下する方法により切創に投与した。
【００３７】
（実施例３）
参考例１で得られたペプチドを、シアノアクリレート系生体組織用接着剤（三共社製、商品名 Ａｒｏｎ Ａｌｐｈａ Ａ）と混合し、上記ペプチドの最終濃度が０．０１重量％となるようにして、本発明のペプチド含有生体組織用接着剤とした。後述の性能評価においては、このペプチド含有生体組織用接着剤を調製直後に切創に０．１ｇ塗布する方法により切創に投与した。
【００３８】
（実施例４）
実施例１において、Ｂ液に参考例１で得られたペプチドを混合する代わりに、参考例１で得られたペプチドとアスコルビン酸（和光純薬社製）を混合し、混合後の最終濃度が共に０．０１重量％となるようにした他は、実施例１と同様に操作して、本発明のペプチド含有生体組織用接着剤とした。後述の性能評価においては、このペプチド含有生体組織用接着剤を調製直後に切創に０．１ｇ塗布する方法により切創に投与した。
【００３９】
（比較例１）
フィブリン糊（ヘキスト社製、商品名 ベリプラストＰ）のＡ液とＢ液を混合し、生体組織用接着剤とした。後述の性能評価においては、この生体組織用接着剤を調製直後に切創に０．１ｇ塗布する方法により切創に投与した。
【００４０】
（比較例２）
生体組織用接着剤としてゼラチン糊（Ｅ．Ｈ．Ｓ．社（フランス）製、商品名ＧＲＦグルー）を用いた。後述の性能評価においては、この生体組織用接着剤液を切創に０．１ｇ塗布し、その上にホルムアルデヒド添加液を数滴滴下する方法により切創に投与した。
【００４１】
（比較例３）
生体組織用接着剤としてシアノアクリレート系生体組織用接着剤（三共社製、商品名 Ａｒｏｎ Ａｌｐｈａ Ａ）を用いた。後述の性能評価においては、この生体組織用接着剤を切創に０．１ｇ塗布する方法により切創に投与した。
【００４２】
性能評価
実施例１〜４のペプチド含有生体組織用接着剤および比較例１〜３の生体組織用接着剤について、以下の方法により性能評価した。
【００４３】
１０週齢のウイスター系ラットの背部を毛刈りし、背部正中線に垂直な方向にメスで２ｃｍの切創を作成した。この切創部を等間隔で３ケ所で縫合した後、実施例１〜４および比較例１〜３の接着剤を前記のようにして投与処置した。処置の３日後にラットを犠牲死させ、抜糸後、切創中央部から左右に１ｃｍ幅の短冊状の皮膚切片を切り取った。レオメーターを用いて切創部の耐抗張力（ｇ／ｃｍ）を測定した。それぞれの実施例および比較例について５匹のラットを用いて行い、耐抗張力（ｇ／ｃｍ）はこれらラットについて得られた値の平均値を取った。結果を表１に示した。
【００４４】
【表１】

【００４５】
表１より、実施例１のペプチド含有生体組織用接着剤を投与した群では、比較例１の生体組織用接着剤を投与した群よりも、耐抗張力が大きく上昇したことが分かる。実施例２と比較例２、および実施例３と比較例３の関係においても同じことが言える。また、実施例４より、更にアスコルビン酸を含有するペプチド含有生体組織用接着剤においては、更に効果が上がることがわかる。
【００４６】
【発明の効果】
本発明の請求項１記載の発明の構成は、上記の通りであり、本発明によれば、より高い創傷治癒促進作用を有すると共に、より高い組織接合効果をもつ生体組織用接着剤が提供される。
【００４７】
本発明の請求項２記載の発明の構成は、上記の通りであり、本発明によれば、より一層高い創傷治癒促進作用を有すると共に、より一層高い組織接合効果をもつ生体組織用接着剤が提供される。
【００４８】
本発明の請求項３記載の発明の構成は、上記の通りであり、本発明によれば、より一層高い創傷治癒促進作用を有すると共に、より一層高い組織接合効果をもつ生体組織用接着剤が提供される。
【００４９】
本発明の請求項４記載の発明の構成は、上記の通りであり、本発明によれば、より一層高い創傷治癒促進作用を有すると共に、より一層高い組織接合効果をもつ生体組織用接着剤が提供される。
【００５０】
本発明の請求項５記載の発明の構成は、上記の通りであり、本発明によれば、より一層高い創傷治癒促進作用を有すると共に、より一層高い組織接合効果をもつ生体組織用接着剤が提供される。
【００５１】
また、請求項１〜５記載の発明は、その高い創傷治癒促進効果からみて、従来の生体組織用接着剤に見られた毒性も緩和しているものと推定される。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an adhesive for living tissue.
[0002]
[Prior art]
In surgery and the like, closing and joining wounds such as skin, organs, and blood vessels is one of the most basic techniques, and now sutures are generally used. However, it is conceivable that the wound is closed and bonded quickly without using sutures, and with a biocompatible adhesive that can withstand moderate tension. It is used clinically. Hitherto, biological tissue adhesives that have been clinically used or developed include cyanoacrylate adhesives, polyurethane adhesives, gelatin adhesives, fibrin adhesives, and the like.
[0003]
The cyanoacrylate-based adhesive is widely used industrially as an instantaneous adhesive. As an application method of the adhesive, a direct method of directly applying and bonding between tissues to be joined, There is an indirect method in which a cloth piece is covered and adhered from above. However, cyanoacrylate adhesives are superior in terms of high adhesive strength, but the cured product is too hard and the hemostasis of the wound surface tends to be incomplete, and formaldehyde, a hydrolysis product of the cured polymer, is toxic. It is said that there are defects such as inhibiting the repair of tissue (Kimura Satoshi: Journal of Adhesion Society of Japan, 22 (2), page 77 (1986)). For this reason, it cannot be used for a part directly touching the central nerve or blood vessel, and there is a limit to an adaptation part. Moreover, since decomposition | disassembly rate is slow, there also exists a problem that it becomes a foreign material and tends to prevent wound healing.
[0004]
As the polyurethane adhesive, there is a reactive urethane prepolymer adhesive developed as an elastic adhesive capable of maintaining the flexibility of the joint after curing. The reactive urethane prepolymer adhesive has a problem that the aromatic diisocyanate which is a synthetic raw material has biotoxicity. For this reason, attempts have been made to use non-toxic fluorinated aliphatic diisocyanates in spite of some delay in curing speed (Takehisa Matsuda: Biomaterials, 7 (1), p. 32 (1989)).
[0005]
Gelatin used for the gelatin-based adhesive is a protein obtained by hydrolyzing collagen and has an adhesive action alone. However, when formaldehyde or glutaraldehyde is added as a polymerization agent, it quickly gels and shrinks. Further, when resorcinol is added to gelatin, the adhesive layer becomes hydrophobic and the adhesive strength is increased. Since this gelatin-based adhesive uses formaldehyde, which is harmful to the tissue, as a polymerization agent, there is a problem that when the adhesive is applied directly to the surgical field, the tissue touches the tissue and exhibits biotoxicity.
[0006]
The fibrin adhesive is based on high-concentration human fibrinogen, thrombin, an enzyme that converts fibrinogen into fibrin, blood coagulation factor XIII, a factor that stabilizes fibrin, calcium ions necessary for these reactions, and It contains aprotinin, a fibrinolytic activity inhibitor that protects the generated fibrin clot from degradation by fibrinolytic enzymes such as plasmin and trypsin and maintains adhesion. A fibrin adhesive mixes these substances at the time of use, and generates a fibrin clot at the site of use. This fibrin adhesive is less likely to become a foreign body, has no toxicity, does not inhibit the wound healing process of the wound surface, and has many advantages such as promoting it. However, it is flexible and has low fixing and adhesive strength. There's a problem. In addition, as a side effect, fibrin glue can become a bacterial culture medium, and there is a risk of infection. Moreover, there is also a problem that the operation time becomes longer due to the longer preparation time.
[0007]
Thus, biological tissue adhesives such as cyanoacrylate adhesives, polyurethane adhesives, and gelatin adhesives have a problem that they are biotoxic to biological tissues and prevent wound healing of the tissues. In addition, fibrin adhesive does not inhibit the wound healing process of the wound surface, but rather works in an accelerated manner, but has a problem of weak fixation and adhesion. Under the present circumstances, there is no adhesive for living tissue having an action of more actively promoting wound healing, and an adhesive for living tissue that promotes wound healing and has higher adhesive force is desired.
[0008]
[Problems to be solved by the invention]
The present invention solves the above problems, and an object of the present invention is to provide a biological tissue adhesive having a higher wound healing promoting effect and a higher tissue bonding effect.
[0009]
[Means for Solving the Problems]
The peptide-containing adhesive for biological tissue according to claim 1 of the present invention is applied to the biological tissue adhesive with the following formula [I].
[Chemical 2]

The peptide shown by is contained, It is characterized by the above-mentioned.
[0010]
The peptide-containing adhesive for biological tissue according to claim 2 of the present invention further comprises a nutritional component having a wound healing effect. .
[0011]
The peptide-containing adhesive for biological tissue according to claim 3 of the present invention comprises 0.0001 to 10% by weight of the peptide represented by the formula [I], and ascorbic acid, a medicine as a nutritional component having a wound healing effect The content of at least one selected from the group consisting of a physically acceptable ester of ascorbic acid and a pharmacologically acceptable salt of ascorbic acid is 0.001 to 99% by weight. 2. The peptide-containing adhesive for biological tissue according to 2.
[0012]
The peptide-containing adhesive for biological tissue according to claim 4 of the present invention contains 0.0001 to 10% by weight of the peptide represented by the formula [I], and arginine, glutamine and nutritive ingredients having a wound healing effect. The peptide-containing adhesive for biological tissue according to claim 2, wherein the content of at least one selected from the group consisting of vitamin A is 0.001 to 99% by weight.
[0013]
The peptide-containing biological tissue adhesive according to claim 5 of the present invention is characterized in that the biological tissue adhesive is a cyanoacrylate adhesive, fibrin adhesive or gelatin adhesive. The peptide-containing adhesive for biological tissue according to any one of the above.
[0014]
The peptide used in the present invention was obtained by culturing the peptide-producing strain belonging to the genus Streptomyces, for example, Streptomyces nobilis (hereinafter abbreviated as “S. nobilis”). Obtained by subjecting the culture solution or a dried product of the same solution or an extract extracted from the cultured cells with an organic solvent to various column chromatography and recrystallizing the column chromatography fraction containing the target product. .
[0015]
Actinomycetes that produce the peptide Nobilis can be obtained from public preservation institutions, for example, bacteria such as a preserved bacterium from RIKEN (JCM4274) (which is also preserved as ATCC 19252 in the United States and CBS 198.65 in the Netherlands).
[0016]
The method for obtaining the peptide can be obtained, for example, by the method described in WO96 / 12732, which was previously filed by the present inventors.
[0017]
Examples of nutritional components having wound healing effect used in the present invention include ascorbic acid, pharmacologically acceptable esters of ascorbic acid, pharmacologically acceptable salts of ascorbic acid, arginine, glutamine and vitamin A. These include those that can be used as pharmaceuticals.
[0018]
Examples of the pharmacologically acceptable esters of ascorbic acid include, but are not limited to, ascorbic acid phosphate, ascorbic acid sulfate, ascorbyl palmitate, ascorbic acid stearate, and the like. It is not done.
[0019]
Examples of the pharmacologically acceptable salt of ascorbic acid include salts of ascorbic acid and various bases. Examples thereof include, but are not limited to, alkali metal salts (for example, sodium salts), alkaline earth metal salts (for example, calcium salts and magnesium salts).
[0020]
As the biological tissue adhesive used in the present invention, any of those conventionally used as biological tissue adhesives can be used, and is not particularly limited. However, the cyanoacrylate system described in detail in the section of the prior art is used. Adhesives, fibrin adhesives or gelatin based adhesives are preferred.
[0021]
The amount of the peptide to be contained in the peptide-containing biological tissue adhesive of the present invention is not particularly limited and is appropriately selected within a wide range, but in the peptide-containing biological tissue adhesive, preferably 10 ⁻⁷ to It is in the range of 10% by weight.
[0022]
[Action]
It is known that the peptide used in the present invention has a granulation action, has a wound healing action, and also has a strong antibacterial action. By including this peptide in an adhesive for living tissue, an adhesive for living tissue having a higher wound healing promoting effect and a higher tissue bonding effect was obtained.
[0023]
In addition, by combining the above-mentioned peptides with nutritional ingredients having wound healing effects such as ascorbic acid, arginine, glutamine and vitamin A (Ayumi Medicine, June issue, 39-44, 1996) Thus, an adhesive for living tissue having an even higher wound healing promoting effect and an even higher tissue bonding effect was obtained.
[0024]
DETAILED DESCRIPTION OF THE INVENTION
Reference example 1
Actinomycetes obtained from RIKEN Nobilis (JCM4274) was subjected to shaking culture (pre-culture) at 26 ° C. and 150 rpm for 120 hours with five 500 ml Sakaguchi Slasco containing 50 ml of starch / ammonium medium supplemented with 0.2% (w / v) yeast extract. Then, a 20 liter jar fermenter containing 12 liters of the same medium was inoculated with 240 ml of the previously cultured bacterial solution and cultured (precultured) at 26 ° C., 410 rpm, with an aeration rate of 4 liters / minute for 24 hours. Furthermore, in a 200 liter jar fermenter containing 140 liters of starch / ammonium medium (1 g of soluble starch, 0.05 g of dipotassium hydrogen phosphate and 0.05 g of ammonium chloride in 100 ml of distilled water) 12 liters were inoculated and seeded at 26 ° C. for 24 hours.
[0025]
Further, a 2000 liter tank containing 1400 liters of starch / ammonium medium was inoculated with 140 liters of the seed culture solution and cultured at 26 ° C., 140 rpm, aeration rate of 700 liters / minute, and pH 7.5 for 7 days.
[0026]
After completion of the culture, the cells were separated by filtration. From 6.34 kg (wet weight) of the cells thus obtained, 3 liters of dichloromethane was added to 1 kg (wet weight) of the cells, and after stirring at room temperature for 15 hours, the cells were filtered and extracted. A liquid was obtained. For the cells, the same operation was repeated three times. The obtained bacterial cell extract was concentrated and then adsorbed onto 120 g of a silica gel carrier. The adsorbed silica gel carrier was purified by a silica gel column.
[0027]
About 120 g of the above-mentioned extract adsorption carrier was charged in a column having a diameter of 8.0 cm packed with 800 g of silica gel carrier to prepare a silica gel column. This silica gel column was purified using the following conditions. As elution solvents, a) hexane: ethyl acetate = 4: 6 4 liters, b) ethyl acetate 3.5 liters, and c) methanol 2 liters were flowed in this order at a flow rate of 500 ml / hour. Fractionation was carried out every time the solvent composition was changed, and in particular, the ethyl acetate elution fraction was fractionated by 500 ml (thus, for ethyl acetate, the total number of elution fractions was 7 fractions).
[0028]
About each said elution fraction, HPLC using the column made by Tosoh Corporation of ODS-80TM, internal diameter 4.6mm x length 25.0cm (Hitachi, pumps L-6000, L-6200, detector) L-3000, column oven 655A-52), purity was confirmed using water: acetonitrile = 3: 7 as an eluent under the conditions of a detection wavelength of 210 nm, a column temperature of 40 ° C., and a flow rate of 1 ml / min.
[0029]
In the above purity confirmation by HPLC, the peak areas eluted at a retention time of 12 to 15 minutes were combined with silica gel column elution fractions that were confirmed to occupy 80% or more of the total elution peak area, did. This fraction was concentrated to dryness and then recrystallized repeatedly using a methanol-dichloromethane system to obtain 3.5 g of columnar crystals.
[0030]
The structure of this substance was the same as the substance described in WO96 / 12732 from various instrumental analysis data, and was determined to be of the formula [I].
[0031]
Structural analysis data The instrumental analysis data of the substance obtained above is shown below.
[0032]

[0033]

[0034]
3. D-serine, L-alanine and DN-methyl-phenylalanine were recognized as amino acid analysis hydrolysates.
[0035]
Example 1
In the B liquid of fibrin glue (Hexist, trade name Veriplast P) composed of a liquid A in which fibrinogen powder and blood coagulation factor XIII are dissolved in an aprotinin solution and a liquid B in which thrombin powder is dissolved in a calcium chloride solution. The peptide obtained in Reference Example 1 was mixed so that the final concentration after mixing the liquid A and the liquid B was 0.01% by weight. The thus obtained B solution and A solution were mixed to obtain the peptide-containing adhesive for biological tissue of the present invention. In the performance evaluation described later, this peptide-containing biological tissue adhesive was administered to the cut by a method of applying 0.1 g to the cut immediately after preparation.
[0036]
(Example 2)
The peptide obtained in Reference Example 1 was mixed with a gelatin / resorcin mixture in gelatin paste (trade name GRF Glue, manufactured by EHS Corporation (France), and the final concentration of the peptide was 0.00. A liquid adjusted to be 01% by weight was obtained. In the performance evaluation described later, 0.1 g of this solution was applied to a cut and administered to the cut by a method in which a few drops of a formaldehyde-added solution was dropped thereon.
[0037]
Example 3
The peptide obtained in Reference Example 1 was mixed with a cyanoacrylate biological tissue adhesive (trade name Aron Alpha A, manufactured by Sankyo Co., Ltd.) so that the final concentration of the peptide was 0.01% by weight, The peptide-containing adhesive for biological tissue of the present invention was used. In the performance evaluation described later, this peptide-containing biological tissue adhesive was administered to the cut by a method of applying 0.1 g to the cut immediately after preparation.
[0038]
Example 4
In Example 1, instead of mixing the peptide obtained in Reference Example 1 with Liquid B, the peptide obtained in Reference Example 1 and ascorbic acid (manufactured by Wako Pure Chemical Industries, Ltd.) were mixed, and the final concentration after mixing was The peptide-containing adhesive for biological tissue of the present invention was prepared in the same manner as in Example 1 except that both were 0.01% by weight. In the performance evaluation described later, this peptide-containing biological tissue adhesive was administered to the cut by a method of applying 0.1 g to the cut immediately after preparation.
[0039]
(Comparative Example 1)
A liquid B and B liquid of fibrin glue (manufactured by Hoechst, trade name Beriplast P) were mixed to obtain an adhesive for living tissue. In the performance evaluation described later, this biological tissue adhesive was administered to the cut by a method of applying 0.1 g to the cut immediately after preparation.
[0040]
(Comparative Example 2)
Gelatin paste (manufactured by EHS Corporation (France), trade name GRF Glue) was used as an adhesive for living tissue. In the performance evaluation described later, 0.1 g of this biological tissue adhesive solution was applied to the cut, and a few drops of a formaldehyde additive solution were added onto the cut, and then administered to the cut.
[0041]
(Comparative Example 3)
A cyanoacrylate biological tissue adhesive (trade name: Aron Alpha A, manufactured by Sankyo Co., Ltd.) was used as the biological tissue adhesive. In the performance evaluation described later, this biological tissue adhesive was administered to the cut by a method of applying 0.1 g to the cut.
[0042]
Performance Evaluation The performance of the peptide-containing biological tissue adhesives of Examples 1 to 4 and Comparative Examples 1 to 3 were evaluated by the following methods.
[0043]
The back of 10 week old Wistar rats was shaved and a 2 cm cut was made with a scalpel in a direction perpendicular to the midline of the back. After this cut portion was sutured at three positions at equal intervals, the adhesives of Examples 1 to 4 and Comparative Examples 1 to 3 were administered as described above. Three days after the treatment, the rats were sacrificed and after stripping, a strip-shaped skin section having a width of 1 cm was cut from the center of the cut. The tensile strength (g / cm) of the cut portion was measured using a rheometer. Each example and comparative example was carried out using 5 rats, and the tensile strength (g / cm) was the average of the values obtained for these rats. The results are shown in Table 1.
[0044]
[Table 1]

[0045]
From Table 1, it can be seen that the tensile strength of the group administered with the peptide-containing biological tissue adhesive of Example 1 was significantly increased as compared with the group administered with the biological tissue adhesive of Comparative Example 1. The same applies to the relationship between Example 2 and Comparative Example 2, and Example 3 and Comparative Example 3. Further, Example 4 shows that the effect is further improved in the peptide-containing adhesive for biological tissue further containing ascorbic acid.
[0046]
【The invention's effect】
The constitution of the invention described in claim 1 of the present invention is as described above, and according to the present invention, there is provided a biological tissue adhesive having a higher wound healing promoting action and a higher tissue joining effect. The
[0047]
The constitution of the invention according to claim 2 of the present invention is as described above, and according to the present invention, there is provided an adhesive for living tissue having a higher wound healing promoting action and a higher tissue bonding effect. Provided.
[0048]
The constitution of the invention according to claim 3 of the present invention is as described above, and according to the present invention, there is provided an adhesive for living tissue having a higher wound healing promoting action and a higher tissue bonding effect. Provided.
[0049]
The constitution of the invention according to claim 4 of the present invention is as described above, and according to the present invention, there is provided a biological tissue adhesive having a higher wound healing promoting action and a higher tissue bonding effect. Provided.
[0050]
The constitution of the invention according to claim 5 of the present invention is as described above, and according to the present invention, there is provided an adhesive for living tissue having a higher wound healing promoting action and a higher tissue bonding effect. Provided.
[0051]
In addition, in the inventions of claims 1 to 5, it is presumed that the toxicity seen in the conventional adhesive for living tissue is mitigated from the viewpoint of the high wound healing promoting effect.

Claims (2)

A biological tissue adhesive selected from a cyanoacrylate adhesive, a fibrin adhesive, or a gelatin adhesive has the following formula [I]

A peptide-containing adhesive for biological tissue, comprising the peptide represented by Furthermore, the ascorbic acid is contained, The peptide-containing adhesive for biological tissues of Claim 1 characterized by the above-mentioned.