JPWO2006016600A1 - Tartaric acid derivative and cross-linked polymer synthesized by the derivative - Google Patents
Tartaric acid derivative and cross-linked polymer synthesized by the derivative Download PDFInfo
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Abstract
これまで生体用接着剤や心臓弁などの医療用デバイス処理に用いられている架橋剤や縮合剤は、人工的に合成された非天然物であるため、生体内で代謝されずに毒性を示し、溶解するには有機溶媒が必要である。そのため、生体親和性の高い水溶性架橋剤の開発が望まれていた。
分子内に水酸基を持つ酒石酸及びその誘導体のカルボキシル基を電子吸引性基であるスクシンイミジル、スルホスクシンイミジル、マレイミジル、フタルイミジル、イミダゾールイル、ニトロフェニル、トレジル又はこれらの誘導体の１種又は２種以上の組み合わせによって修飾した水溶性酒石酸誘導体及び該酒石酸誘導体により得られる高分子架橋体。The cross-linking agents and condensing agents that have been used to treat biomedical adhesives and medical devices such as heart valves are non-natural products that have been artificially synthesized, and therefore are not metabolized in vivo and show toxicity. , An organic solvent is required for dissolution. Therefore, development of a water-soluble crosslinking agent having high biocompatibility has been desired.
One or more of succinimidyl, sulfosuccinimidyl, maleimidyl, phthalimidyl, imidazolylyl, nitrophenyl, and toresyl which are electron-withdrawing groups for carboxyl groups of tartaric acid and its derivatives having a hydroxyl group in the molecule, or two or more of these derivatives. A water-soluble tartaric acid derivative modified by a combination of the above and a crosslinked polymer obtained by the tartaric acid derivative.
Description
本発明は、酒石酸又はその誘導体のカルボキシル基を電子吸引性基により修飾した酒石酸誘導体と該酒石酸誘導体により合成された高分子架橋体および該酒石酸誘導体と生分解性高分子から構成される二成分系の生体内分解吸収性粘着性医用材料に関する。 The present invention relates to a tartaric acid derivative obtained by modifying the carboxyl group of tartaric acid or a derivative thereof with an electron-withdrawing group, a cross-linked polymer synthesized by the tartaric acid derivative, and a two-component system composed of the tartaric acid derivative and a biodegradable polymer. In vivo degradable and absorbable adhesive medical material.
これまで、生体用組織接着剤や生体由来の物質を用いた医療用デバイスには、人工的に化学合成されたグルタルアルデヒドなどのアルデヒドを有する架橋剤や1−エチル−3−（3−ジメチルアミノプロピル）カルボジイミドなどの縮合剤等が用いられていた（例えば、特許文献１、２）。本発明者らは、クエン酸回路内に存在するトリカルボン酸であるクエン酸のカルボキシル基を電子吸引性基によって少なくとも１つ以上修飾した生体低分子誘導体を開発した(特許文献３、非特許文献１〜３)。なお、アルカン二酸ジスクシンイミドによる分子間架橋タンパク質の生成方法が知られている（特許文献４）。 So far, medical devices using bio-tissue adhesives or substances of biological origin include cross-linking agents and 1-ethyl-3-(3-dimethylamino) having artificially chemically synthesized aldehydes such as glutaraldehyde. A condensing agent such as propyl)carbodiimide has been used (for example, Patent Documents 1 and 2). The present inventors have developed a low molecular weight biomolecule derivative in which at least one carboxyl group of citric acid, which is a tricarboxylic acid existing in the citric acid cycle, is modified with an electron-withdrawing group (Patent Document 3, Non-Patent Document 1 ~ 3). A method for producing an intermolecular crosslinked protein using alkanedioic acid disuccinimide is known (Patent Document 4).
これまで医療用デバイスの処置等に用いられている架橋剤や縮合剤は、主に人工的に合成された非天然物であり、生体内で代謝されず生体に対して毒性を示すことが指摘されている。そのため、医療現場で用いる際には用途や使用量が制限されている。さらに、これまで、強固な組織接着力を持ち、残留した架橋剤又は分解生成物の生体毒性が低い生体用組織接着剤は存在しなかった。また、血管の閉塞、止血、エアーリーク、動脈瘤の封止などに対し、従来の止血剤、血管塞栓剤、シーラントまたは動脈瘤の封止剤は、塗布部分から剥離し易く、十分な接着強度を持ったものが存在しなかった。 It has been pointed out that the cross-linking agents and condensing agents that have been used in the treatment of medical devices so far are mainly artificially synthesized non-natural products and are not metabolized in the body and are toxic to the body. Has been done. Therefore, the use and the amount used are limited when used in the medical field. Furthermore, until now, there has been no biological tissue adhesive having a strong tissue adhesive force and low biotoxicity of the residual crosslinking agent or decomposition product. In addition, conventional hemostasis agents, vascular embolic agents, sealants, or aneurysm sealants are easy to peel off from the applied part and have sufficient adhesive strength against vascular occlusion, hemostasis, air leak, aneurysm sealing, etc. There was no one with.
また、本発明者らが開発した生体低分子誘導体及び特許文献４に開示されているアルカン二酸ジスクシンイミドは、水に対する溶解性が低いため、架橋体を合成するには有機溶媒であるジメチルスルホキシドなどを用いることが課題として挙げられる。そのため、生体親和性が高く、生体内で代謝・排泄される水溶性架橋剤の開発が求められている。 In addition, since the low molecular weight biomolecule derivatives developed by the present inventors and the alkanedioic acid disuccinimide disclosed in Patent Document 4 have low solubility in water, dimethyl sulfoxide, which is an organic solvent for synthesizing a crosslinked product, is used. The use of such as is an issue. Therefore, it is required to develop a water-soluble crosslinking agent that has high biocompatibility and is metabolized and excreted in vivo.
このような課題を解決するため、本発明では、水酸基を持つジカルボン酸である酒石酸を電子吸引性基により修飾した水溶性酒石酸誘導体および該酒石酸誘導体により生分解性高分子を架橋することにより合成された高分子架橋体、該酒石酸誘導体と生分解性高分子から構成される二成分系の生体内分解吸収性粘着性医用材料を開発した。 In order to solve such a problem, in the present invention, tartaric acid, which is a dicarboxylic acid having a hydroxyl group, is synthesized by crosslinking a biodegradable polymer with a water-soluble tartaric acid derivative modified with an electron-withdrawing group and the tartaric acid derivative. We have developed a two-component biodegradable and absorbable adhesive medical material composed of a crosslinked polymer, a tartaric acid derivative and a biodegradable polymer.
すなわち、本発明は、ジカルボン酸である酒石酸の２つのカルボキシル基を電子吸引性基により修飾したジカルボン酸誘導体、及び得られた酒石酸誘導体を用いて生分解性高分子を架橋することにより調製された高分子架橋体である。 That is, the present invention was prepared by cross-linking a biodegradable polymer with a dicarboxylic acid derivative obtained by modifying two carboxyl groups of tartaric acid, which is a dicarboxylic acid, with an electron-withdrawing group, and the obtained tartaric acid derivative. It is a cross-linked polymer.
また、本発明は、生分解性高分子の有機溶媒溶液若しくは水溶液、緩衝液、又は水−有機溶媒混合溶液を接着成分とし、ジカルボン酸である酒石酸の２つのカルボキシル基を電子吸引性基により修飾した酒石酸誘導体を硬化成分とすることを特徴とする二成分系の生体内分解吸収性粘着性医用材料である。 Further, the present invention uses an organic solvent solution or aqueous solution of a biodegradable polymer, a buffer solution, or a water-organic solvent mixed solution as an adhesive component, and modifies two carboxyl groups of tartaric acid, which is a dicarboxylic acid, with electron withdrawing groups. A two-component biodegradable and absorbable adhesive medical material, characterized by using the tartaric acid derivative as a hardening component.
また、用いる電子吸引性基には、スクシンイミジル、スルホスクシンイミジル、マレイミジル、フタルイミジル、イミダゾールイル、ニトロフェニル、トレジル又はこれらの誘導体の１種又は２種以上の組み合わせが挙げられる。 Examples of the electron-withdrawing group to be used include succinimidyl, sulfosuccinimidyl, maleimidyl, phthalimidyl, imidazolylyl, nitrophenyl, toresyl or one or a combination of two or more of these derivatives.
また、高分子架橋体又は生体内分解吸収性粘着性医用材料に用いる生分解性高分子は、タンパク質、グリコサミノグリカン、キトサン、ポリアミノ酸、ポリアルコール、又はこれらの２つ又はそれ以上の組み合わせが挙げられる。 The biodegradable polymer used in the crosslinked polymer or biodegradable and absorbable adhesive medical material is protein, glycosaminoglycan, chitosan, polyamino acid, polyalcohol, or a combination of two or more thereof. Is mentioned.
また、グリコサミノグリカンには、コンドロイチン硫酸、デルマタン硫酸、ヒアルロン酸、ヘパラン硫酸、ヘパリン、ケラタン硫酸、又はこれらの誘導体の１種又は２種以上の組み合わせが挙げられる。これらのグリコサミノグリカンは、分子量及び由来する生物によらない。 Examples of glycosaminoglycan include chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparan sulfate, heparin, keratan sulfate, and one or a combination of two or more of these derivatives. These glycosaminoglycans are independent of molecular weight and the organism from which they are derived.
また、タンパク質は、コラーゲン（数１０種類のタイプによらない）、アテロコラーゲン（数１０種類のタイプによらない）、アルカリ処理コラーゲン（数１０種類のタイプによらない）、ゼラチン、ケラチン、ヘモグロビン、カゼイン、グロブリン、フィブリノーゲン、ヒト血液由来アルブミン、遺伝子組み換えアルブミン、アルブミンフラグメント、及び化学的に改変されたアルブミン等アミノ基を有する高分子が含まれる群より選択されるタンパク質の１種又は２種以上の組み合わせが挙げられる。 Proteins include collagen (not depending on tens of types), atelocollagen (not depending on tens of types), alkali-treated collagen (not depending on tens of types), gelatin, keratin, hemoglobin, and casein. , Globulin, fibrinogen, human blood-derived albumin, recombinant albumin, albumin fragments, and chemically modified albumin, or a combination of two or more proteins selected from the group consisting of macromolecules having an amino group. Is mentioned.
また、高分子架橋体、水溶性酒石酸誘導体と生分解性高分子から構成される二成分系の生体内分解吸収性粘着性医用材料の調製に用いるその他の生分解性高分子としてキトサン（脱アセチル化度、分子量によらない）、ポリアミノ酸（アミノ酸の種類、分子量によらない）、ポリアルコール（種類、分子量によらない）が挙げられる。 In addition, chitosan (deacetylated as another biodegradable polymer used for the preparation of a two-component biodegradable and absorbable adhesive medical material composed of a crosslinked polymer, a water-soluble tartaric acid derivative and a biodegradable polymer. Examples include polyamino acid (regardless of type and molecular weight), polyamino acid (regardless of amino acid type or molecular weight), and polyalcohol (regardless of type or molecular weight).
また、高分子架橋体、水溶性酒石酸誘導体と生分解性高分子から構成される二成分系の生体内分解吸収性粘着性医用材料を調製する際に、生分解性高分子を溶解するための溶媒には、塩酸塩、硫酸塩、硝酸塩、リン酸塩、炭酸塩、ホウ酸塩の１種又は２種以上の組み合わせにより調製された緩衝液が挙げられる。 Further, in preparing a two-component system biodegradable and absorbable adhesive medical material composed of a cross-linked polymer, a water-soluble tartaric acid derivative and a biodegradable polymer, a solution for dissolving the biodegradable polymer Examples of the solvent include buffer solutions prepared from one or a combination of two or more of hydrochloride, sulfate, nitrate, phosphate, carbonate, borate.
酒石酸又はその誘導体のカルボキシル基を電子吸引性基により修飾した酒石酸誘導体は、本発明者らが先に開発した生体低分子誘導体及びアルカン二酸ジスクシンイミドと比較して水溶性であり、高分子架橋体および該酒石酸誘導体と生分解性高分子から構成される二成分系の生体内分解吸収性粘着性医用材料を得る際に、緩衝液を用いる水溶液条件下にて生分解性高分子水溶液と均一に反応が進み、反応時間が短くなる。 The tartaric acid derivative obtained by modifying the carboxyl group of tartaric acid or its derivative with an electron-withdrawing group is more water-soluble than the low molecular weight biological derivative and the alkanedioic acid disuccinimide, which were previously developed by the present inventors, and has a high molecular weight. When obtaining a two-component biodegradable and absorbable adhesive medical material composed of the body and the tartaric acid derivative and a biodegradable polymer, the biodegradable polymer aqueous solution is homogeneous under the aqueous solution condition using a buffer solution. The reaction proceeds and the reaction time becomes shorter.
本発明の水溶性酒石酸誘導体の出発物質として使用する低分子は、酒石酸又はその誘導体、例えば、酒石酸中の水酸基（-OH）を硫酸基（-OSO3Na）あるいはカルボキシメチル基（-OCH2COONa）などに化学修飾したものである。また、本発明の水溶性酒石酸誘導体は、酒石酸又はその誘導体の２個のカルボキシル基を電子吸引性基、例えば、スクシンイミジル、スルホスクシンイミジル、マレイミジル、フタルイミジル、イミダゾールイル、ニトロフェニル、トレジル又はこれらの誘導体の１種又は２種以上の組み合わせと合成反応させ、活性エステルを導入したものである。The low molecule used as a starting material of the water-soluble tartaric acid derivative of the present invention is tartaric acid or its derivative, for example, a hydroxyl group (-OH) in tartaric acid is converted into a sulfate group (-OSO 3 Na) or a carboxymethyl group (-OCH 2 COONa). ) Etc. are chemically modified. Further, the water-soluble tartaric acid derivative of the present invention has two carboxyl groups of tartaric acid or a derivative thereof as electron withdrawing groups, for example, succinimidyl, sulfosuccinimidyl, maleimidyl, phthalimidyl, imidazolyl, nitrophenyl, toresyl or these. The active ester is introduced by a synthetic reaction with one or a combination of two or more of the above derivatives.
本発明の水溶性酒石酸誘導体は、下記の合成反応により得ることができる。有機溶媒１００重量％に対して、酒石酸又はその誘導体を０．００１〜１０重量％程度、より好ましくは、１〜３重量％程度加え、縮合剤、例えば、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）、又は、ジシクロヘキシルカルボジイミド（DCC）を該溶媒１００重量％に対して０．００１〜２０重量％程度、より好ましくは、５〜１５重量％程度を加え、その存在下で電子吸引性基となる分子、例えば、Ｎ−ヒドロキシスクシンイミドを該溶媒１００重量％に対して０．００１〜１０重量％程度、より好ましくは、３〜８重量％程度を加え、反応温度０〜１００℃、より好ましくは、１０〜３０℃、反応時間１〜４８時間、より好ましくは、１〜３時間の適宜の条件を選択し、反応させることにより酒石酸誘導体と反応副生成物であるＤＣＣウレアが沈殿生成する。 The water-soluble tartaric acid derivative of the present invention can be obtained by the following synthetic reaction. Tartaric acid or a derivative thereof is added in an amount of about 0.001 to 10% by weight, more preferably about 1 to 3% by weight, based on 100% by weight of the organic solvent, and a condensing agent such as 1-ethyl-3-(3-dimethyl) is added. Aminopropyl)carbodiimide (EDC) or dicyclohexylcarbodiimide (DCC) is added in an amount of 0.001 to 20% by weight, more preferably 5 to 15% by weight, relative to 100% by weight of the solvent. A molecule serving as an electron-withdrawing group, for example, N-hydroxysuccinimide is added in an amount of about 0.001 to 10% by weight, more preferably about 3 to 8% by weight, based on 100% by weight of the solvent, and a reaction temperature of 0 to 100 is added. C., more preferably 10 to 30.degree. C., a reaction time of 1 to 48 hours, more preferably 1 to 3 hours are selected and reacted to produce a tartaric acid derivative and a reaction by-product DCC urea. A precipitate is generated.
その後、反応副生成物であるＤＣＣウレアをガラスろ過フィルターにて濾過することによって酒石酸誘導体を単離する。ろ液を減圧留去した後、得られた残渣をn-ヘキサン等の有機溶媒で洗浄、ろ過することによって、精製を行う。上記の反応条件を外れると、酒石酸中のカルボキシル基を電子吸引性基であるN-ヒドロキシスクシンイミドなどで修飾することができなくなるので不適当である。この反応によって下記の構造式で示される水溶性の酒石酸誘導体が得られる。この水溶性酒石酸誘導体は、架橋剤、医療用接着剤の硬化剤、ブタ弁（Carpentier-Edowards valve：CE弁）などの固定剤などに有用である。 Then, the tartaric acid derivative is isolated by filtering the reaction by-product DCC urea with a glass filter. The filtrate is evaporated under reduced pressure, and the obtained residue is washed with an organic solvent such as n-hexane and filtered to purify it. If the above reaction conditions are not satisfied, the carboxyl group in tartaric acid cannot be modified with an electron-withdrawing group such as N-hydroxysuccinimide, which is not suitable. By this reaction, a water-soluble tartaric acid derivative represented by the following structural formula is obtained. This water-soluble tartaric acid derivative is useful as a cross-linking agent, a curing agent for medical adhesives, a fixing agent for pig valves (Carpentier-Edowards valve: CE valve), and the like.
本発明の水溶性酒石酸誘導体と生分解性高分子との架橋反応は、溶媒中の生分解性高分子の濃度０．１〜５０重量％程度に対し、該酒石酸誘導体の濃度０．０１％〜５０重量％程度とし、好ましくは、１０〜５０℃程度で反応させる。上記反応条件を外れると反応速度が遅くなり、得られる架橋体の架橋密度が低くなり、架橋体が得られない場合もあるので不適当である。なお、両者の配合に際しては、均一に反応させるため、該酒石酸誘導体も上記溶媒に溶解し、双方を上記濃度範囲となるように適宜濃度の溶液として混合するのが好ましい。 The cross-linking reaction between the water-soluble tartaric acid derivative and the biodegradable polymer of the present invention is carried out at a concentration of the tartaric acid derivative of 0.01% to about 50% by weight with respect to the concentration of the biodegradable polymer in the solvent. The reaction is performed at about 50% by weight, preferably at about 10 to 50°C. If the above reaction conditions are not satisfied, the reaction rate becomes slow, the crosslink density of the obtained crosslinked product becomes low, and the crosslinked product may not be obtained in some cases, which is not suitable. In addition, in the case of blending both, it is preferable that the tartaric acid derivative is also dissolved in the above-mentioned solvent in order to cause a uniform reaction, and both are mixed as a solution having an appropriate concentration so as to be in the above-mentioned concentration range.
溶媒としては、ジメチルスルホキシド溶液若しくは塩酸塩、硫酸塩、硝酸塩、リン酸塩、炭酸塩、ホウ酸塩の１種又は２種以上の組み合わせからなる緩衝溶液又は該緩衝溶液−ジメチルスルホキシド混合溶液等を用いることができる。 As the solvent, a dimethylsulfoxide solution or a buffer solution consisting of one or a combination of two or more kinds of dimethylsulfoxide solution, hydrochloride, sulfate, nitrate, phosphate, carbonate, borate or a mixed solution of the buffer solution and dimethylsulfoxide is used. Can be used.
この反応によって、図１に示すような構造の架橋体が得られる。この架橋体は、含水率が約80〜99重量％のハイドロゲルである。含水率が低くなると強度が強く、架橋密度が高くなるため、接着剤等への有用性が向上する。この架橋体は、医療用接着剤、止血剤、血管塞栓剤、動脈瘤封止剤などに使用できる。 By this reaction, a crosslinked product having a structure as shown in FIG. 1 is obtained. This crosslinked product is a hydrogel having a water content of about 80 to 99% by weight. When the water content is low, the strength is high and the crosslink density is high, so that the usefulness as an adhesive or the like is improved. This crosslinked product can be used as a medical adhesive, a hemostatic agent, a vascular embolus agent, an aneurysm sealant, and the like.
以上のようにして生成する高分子架橋体は、生体用接着剤、止血剤、血管塞栓剤、動脈瘤の封止剤のいずれかに適用する場合は、架橋反応を直接患部で行う。また、一旦架橋反応させた後用いることにより、癒着防止剤、組織再生用足場材料、薬物担体として好適に用いられる。 When the crosslinked polymer produced as described above is applied to any one of a bioadhesive agent, a hemostatic agent, a vascular embolus agent, and a sealant for an aneurysm, the crosslink reaction is directly performed on the affected area. Further, once used after cross-linking reaction, it is suitably used as an adhesion preventive agent, a tissue regeneration scaffold material, and a drug carrier.
生体内分解吸収性粘着性医用材料に用いる本発明の水溶性酒石酸誘導体と生分解性高分子との架橋反応は、溶媒中の生分解性高分子の濃度０．１〜５０重量％程度に対し、該酒石酸誘導体の濃度０．０１％〜５０重量％程度とし、好ましくは、１０〜５０℃程度で反応させる。上記反応条件を外れると接着時間が長くなり、さらに接着強度も弱くなる。なお、両者の配合に際しては、均一に反応させるため、該酒石酸誘導体も上記溶媒に溶解し、双方を上記濃度範囲となるように適宜濃度の溶液として混合するのが好ましい。 The cross-linking reaction between the water-soluble tartaric acid derivative of the present invention and the biodegradable polymer used in the biodegradable and absorbable adhesive medical material is performed at a concentration of 0.1 to 50% by weight of the biodegradable polymer in the solvent. The concentration of the tartaric acid derivative is about 0.01% to 50% by weight, and preferably the reaction is performed at about 10 to 50°C. If the reaction conditions are not satisfied, the bonding time will be longer and the bonding strength will be weaker. In addition, in the case of blending both, it is preferable that the tartaric acid derivative is also dissolved in the above-mentioned solvent in order to cause a uniform reaction, and both are mixed as a solution having an appropriate concentration so as to be in the above-mentioned concentration range.
溶媒としては、ジメチルスルホキシド溶液若しくは塩酸塩、硫酸塩、硝酸塩、リン酸塩、炭酸塩、ホウ酸塩の１種又は２種以上の組み合わせからなる緩衝溶液又は該緩衝溶液−ジメチルスルホキシド混合溶液等を用いることができる。上記反応によって得られる生体組織と接着剤の界面の構造は、図２に示すように、該酒石酸誘導体が生体組織内の生体高分子および接着剤中の生分解性高分子と架橋反応をすることにより、接着する。 As the solvent, a dimethylsulfoxide solution or a buffer solution consisting of one or a combination of two or more kinds of dimethylsulfoxide solution, hydrochloride, sulfate, nitrate, phosphate, carbonate, borate or a mixed solution of the buffer solution and dimethylsulfoxide is used. Can be used. As shown in FIG. 2, the structure of the interface between the biological tissue and the adhesive obtained by the above reaction is such that the tartaric acid derivative undergoes a crosslinking reaction with the biopolymer in the biological tissue and the biodegradable polymer in the adhesive. To adhere.
以下、本発明について実施例を挙げて詳細に説明をする。
＜酒石酸誘導体の合成＞
酒石酸をテトラヒドロフラン（THF）溶媒１００重量％に対して、５重量％加えた溶液に対し、ジシクロヘキシルカルボジイミド（DCC）を溶媒１００重量％に対して１４重量％加え、その存在下でＮ−ヒドロキシスクシンイミドを溶媒１００重量％に対して８重量％を加え、１時間攪拌し、その後、室温にて２時間攪拌を行った。これによりＤＣＣウレアと酒石酸誘導体の混合溶液が得られた。Hereinafter, the present invention will be described in detail with reference to examples.
<Synthesis of tartaric acid derivative>
To a solution of 5% by weight of tartaric acid in 100% by weight of tetrahydrofuran (THF) solvent, 14% by weight of dicyclohexylcarbodiimide (DCC) in 100% by weight of solvent was added, and N-hydroxysuccinimide was added in the presence thereof. 8% by weight was added to 100% by weight of the solvent, and the mixture was stirred for 1 hour and then at room temperature for 2 hours. As a result, a mixed solution of DCC urea and a tartaric acid derivative was obtained.
続いて、析出したＤＣＣウレアをガラスろ過フィルターにて取り除き、反応系の溶媒であるＴＨＦを減圧留去した。得られた残渣をｎ−へキサン、２−プロパノールにより洗浄、濾過することによって精製を行い、酒石酸の２つのカルボキシル基が、Ｎ−ヒドロキシスクシンイミドに修飾された酒石酸誘導体（TAD）を合成した。酒石酸の２つのカルボキシル基が完全にN-ヒドロキシスクシンイミドによって置換されたことを１Ｈ−ＮＭＲにより確認した。合成したＴＡＤは、水に対し、０．１〜５０重量％の濃度で溶解することが可能であった。 Subsequently, the deposited DCC urea was removed by a glass filtration filter, and the solvent of the reaction system, THF, was distilled off under reduced pressure. The obtained residue was purified by washing with n-hexane and 2-propanol and filtering to synthesize a tartaric acid derivative (TAD) in which two carboxyl groups of tartaric acid were modified to N-hydroxysuccinimide. It was confirmed by 1H-NMR that the two carboxyl groups of tartaric acid were completely replaced by N-hydroxysuccinimide. The synthesized TAD could be dissolved in water at a concentration of 0.1 to 50% by weight.
＜コラーゲン架橋体の合成＞
実施例１にて合成したＴＡＤを用いて、アルカリ処理コラーゲン（AlCol）の高分子架橋体を合成した。０．１Ｍリン酸緩衝溶液（pH7.0）にＴＡＤ100μlを溶解し、AlCol濃度２．５ｗ／ｖ％のリン酸緩衝溶液（0.1M,pH7.0）４００μl中に添加し、攪拌した。ＴＡＤの最終濃度は、２０，５０，１００，１５０ｍＭになるように加えた。その後、３７℃で３０分間静置し、コラーゲン架橋体生成の有無を確認した。結果を表１に示す。表１の結果から、０．１Ｍリン酸緩衝溶液、すなわち水溶液条件下におけるコラーゲン架橋体の生成が確認された。含水率が低くなると強度が強く、架橋密度が高くなるため、接着剤等への有用性が向上する。<Synthesis of cross-linked collagen>
The TAD synthesized in Example 1 was used to synthesize a crosslinked polymer of alkali-treated collagen (AlCol). 100 μl of TAD was dissolved in a 0.1 M phosphate buffer solution (pH 7.0), added to 400 μl of a phosphate buffer solution (0.1 M, pH 7.0) having an AlCol concentration of 2.5 w/v%, and stirred. The final concentration of TAD was added to be 20, 50, 100, 150 mM. Then, the mixture was allowed to stand at 37° C. for 30 minutes, and it was confirmed whether or not a crosslinked collagen product was formed. The results are shown in Table 1. From the results in Table 1, it was confirmed that a 0.1M phosphate buffer solution, that is, a collagen crosslinked product was formed under an aqueous solution condition. When the water content is low, the strength is high and the crosslink density is high, so that the usefulness as an adhesive or the like is improved.
＜アルブミン架橋体の合成＞
実施例1にて合成したＴＡＤを用いて、ヒト血清アルブミン（HSA）の高分子架橋体を合成した。０．１Ｍリン酸緩衝溶液（pH7.0）にＴＡＤ１００μｌを溶解し、HＳＡ濃度４５ｗ／ｖ％のリン酸緩衝溶液（0.1M,pH7.0）４００μl中に添加し、攪拌した。ＴＡＤの最終濃度は、１００，１５０，２００ｍＭになるように加えた。その後、３７℃で３０分間静置し、ＨＳＡ架橋体生成の有無を確認した。結果を表２に示す。表２の結果から、０．１Ｍリン酸緩衝溶液、すなわち水溶液条件下における生体高分子のゲル化が確認された。<Synthesis of cross-linked albumin>
The TAD synthesized in Example 1 was used to synthesize a crosslinked polymer of human serum albumin (HSA). 100 μl of TAD was dissolved in a 0.1 M phosphate buffer solution (pH 7.0), added to 400 μl of a phosphate buffer solution (0.1 M, pH 7.0) having an HSA concentration of 45 w/v%, and stirred. The final concentration of TAD was added to 100, 150, 200 mM. Then, the mixture was allowed to stand at 37° C. for 30 minutes, and it was confirmed whether or not HSA crosslinked products were formed. The results are shown in Table 2. From the results in Table 2, gelation of the biopolymer under a 0.1 M phosphate buffer solution, that is, an aqueous solution condition was confirmed.
＜生体組織接着剤の調製＞
生体組織接着剤を下記のようにして調製した。ヒト血清由来のアルブミン（シグマアルドリッチジャパン（株）製A1653）を０．１Ｍリン酸ナトリウム緩衝液（pH7.0）に４５重量％となるように溶解した。このアルブミン溶液４００μlに対し、硬化成分としてＴＡＤの０．１Mリン酸緩衝溶液（pH7.0）を１００μl加え、２５℃で数秒攪拌した。最終体積(500ul)に対するＴＡＤ濃度は、１００ｍＭ，１５０ｍＭ，２００ｍＭ，２５０ｍＭとなるように調製した。<Preparation of biological tissue adhesive>
A biological tissue adhesive was prepared as follows. Albumin derived from human serum (A1653 manufactured by Sigma-Aldrich Japan Co., Ltd.) was dissolved in 0.1 M sodium phosphate buffer (pH 7.0) to a concentration of 45% by weight. To 400 μl of this albumin solution, 100 μl of 0.1 M phosphate buffer solution (pH 7.0) of TAD was added as a hardening component, and the mixture was stirred at 25° C. for several seconds. The TAD concentration with respect to the final volume (500 ul) was adjusted to be 100 mM, 150 mM, 200 mM, 250 mM.
<生体組織接着剤の接着強度測定>
生体組織に対する接着強度測定用の被接着物としてコラーゲンケーシング（新田ゼラチン（株）製、組成：コラーゲン44％、セルロース 18％、グリセリン15％、植物性油脂3％、カルボキシメチルセルロース2％）を用いて接着強度を測定した。コラーゲンケーシング（幅10mm×長さ25mm）の１０ｍｍ×１０ｍｍの領域に厚さが均一になるように硬化前の混合溶液を５０μl塗布し、同等の大きさのコラーゲンケーシングをその接着面上に重ね合わせた。さらに、その接着面上に５０ｇの錘をのせ、３７℃で１時間放置した。接着強度は、引っ張り試験機（英弘精機（株）製TA-XT2i）により測定した。測定条件は２５℃、測定スピード２ｍｍ/ｓで行った。結果を表３に示す。表３の結果から、ＴＡＤ濃度依存的に接着強度が強くなることが確認された。<Adhesion strength measurement of biological tissue adhesive>
A collagen casing (manufactured by Nitta Gelatin Co., Ltd., composition: 44% collagen, 18% cellulose, 15% glycerin, 3% vegetable oil and fat, 3% carboxymethylcellulose) is used as an adherend for measuring the adhesive strength to biological tissues. And the adhesive strength was measured. 50 μl of the mixed solution before curing is applied to the area of 10 mm × 10 mm of the collagen casing (width 10 mm × length 25 mm) so that the thickness is uniform, and the collagen casing of the same size is overlaid on the adhesive surface. It was Further, a weight of 50 g was placed on the adhesive surface and left at 37° C. for 1 hour. The adhesive strength was measured by a tensile tester (TA-XT2i manufactured by Eiko Instruments Co., Ltd.). The measurement conditions were 25° C. and the measurement speed was 2 mm/s. The results are shown in Table 3. From the results shown in Table 3, it was confirmed that the adhesive strength increases depending on the TAD concentration.
また、市販されている生体用組織接着剤であるフィブリン系接着剤（藤沢薬品工業社製、商品名 ボルヒール）とシアノアクリレート系接着剤（ETHICON社製、商品名 ダーマボンド）を用い、同様に接着強度を測定した。結果を表４に示す。表４の結果から、ＴＡＤ系接着剤は、シアノアクリレート系接着剤には及ばないものの、フィブリン系接着剤と比較して１０倍以上接着強度が高いことが確認された。 In addition, a fibrin-based adhesive (trade name: Volheel, manufactured by Fujisawa Pharmaceutical Co., Ltd.), which is a commercially available tissue adhesive for living organisms, and a cyanoacrylate-based adhesive (trade name: Dermabond, manufactured by ETHICON, Inc.), are used to similarly bond strength. Was measured. The results are shown in Table 4. From the results shown in Table 4, it was confirmed that the TAD-based adhesive was not as good as the cyanoacrylate-based adhesive, but the adhesive strength was 10 times or more higher than that of the fibrin-based adhesive.
本発明の水溶性酒石酸誘導体は、生体用接着剤、止血剤、血管塞栓剤、動脈瘤の封止剤などの医用材料の架橋剤として有用である。また、一旦、生分解性高分子と架橋反応させた後用いることにより、癒着防止剤、組織再生用足場材料、薬物担体としても用いられる。 INDUSTRIAL APPLICABILITY The water-soluble tartaric acid derivative of the present invention is useful as a cross-linking agent for medical materials such as biomedical adhesives, hemostatic agents, vascular embolic agents, and aneurysm sealants. Further, once used after being cross-linked with a biodegradable polymer, it can be used as an adhesion preventive agent, a tissue regeneration scaffold material, and a drug carrier.
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| US4101380A (en) * | 1975-06-12 | 1978-07-18 | Research Products Rehovot Ltd. | Process for the cross-linking of proteins |
| JP2004099562A (en) * | 2002-09-11 | 2004-04-02 | National Institute For Materials Science | Low-molecular derivative of biomolecule |
| JP2004261222A (en) * | 2003-02-13 | 2004-09-24 | National Institute For Materials Science | Intravital decomposing and absorbing adhesive material for medical use |
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| US4101380A (en) * | 1975-06-12 | 1978-07-18 | Research Products Rehovot Ltd. | Process for the cross-linking of proteins |
| JP2004099562A (en) * | 2002-09-11 | 2004-04-02 | National Institute For Materials Science | Low-molecular derivative of biomolecule |
| JP2004261222A (en) * | 2003-02-13 | 2004-09-24 | National Institute For Materials Science | Intravital decomposing and absorbing adhesive material for medical use |
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