JP7155544B2 - Polycarboxylic acid derivative - Google Patents

Description

The present invention provides a compound in which a propargyl amino acid is added to the carboxyl group of a carboxylic acid (polycarboxylic acid) having two or more carboxyl groups in the molecule, a base solution containing a biopolymer, and a cross-linking agent containing the compound. The present invention relates to a two-component adhesive consisting of a solution.

Medical adhesives are used to assist in rejoining tissue when joining sites where tissue has been destroyed by surgery or physical injury. Medical adhesives are used for joining soft tissues such as muscles, blood vessels and organs, skin, bones and teeth, and in soft tissues, they are used as sealants for closing wounds and blood vessels.

Three types of adhesives for soft tissue are mainly used: fibrin-based adhesives, gelatin-based adhesives, and cyanoacrylate-based adhesives. Although these adhesives have a long track record of use as medical products, safety problems have been pointed out. That is, since fibrin-based adhesives use blood products, there is concern about the risk of viral infections such as HIV and hepatitis C. In addition, the gelatin-based adhesive uses aldehydes as a raw material, and the problem of toxicity has been pointed out. It has been pointed out that cyanoacrylate-based adhesives exhibit toxicity due to formaldehyde produced by hydrolysis after polymerization.

Gelatin-based adhesives, also called GRF formulations, have been used since the 1966s as gelatin/resorcinol/formaldehyde mixed formulations. Gelatin is heat-denatured collagen into gelatin capable of sol-gel phase transition, and is a protein rich in amine and carboxylic acid groups. Since gelatin has excellent biodegradability, biocompatibility, tissue adhesion, and moderate elasticity, it is used as a main ingredient of adhesives for living tissue. GRF formulations utilize the hydromethylation reaction of resorcinol and formaldehyde, and use the principle of polymerization and adhesion as a crosslinkable polymer with gelatin.

GRF preparations are excellent adhesives with both hemostatic and adhesive properties. However, as described above, formaldehyde in GRF formulations is suspected of being carcinogenic. Therefore, Patent Document 1 discloses a technique using glutaraldehyde instead of highly toxic formaldehyde. The use of glutaraldehyde is believed to increase the stability of the adhesive due to the increased crosslink density. However, compounds having a formyl group such as glutaraldehyde are highly reactive with proteins and may exhibit skin irritation.

Therefore, as a new cross-linking agent that solves the problems of glutaraldehyde and formaldehyde, there is a method of utilizing amidation with an N-hydroxysuccinimidyl ester group (NHS). Patent Document 2 discloses a low-molecular-weight derivative based on citric acid or the like as an NHS-type cross-linking agent. Moreover, Patent Document 3 discloses an adhesive hydrogel using a polymeric NHS cross-linking agent and gelatin. This adhesive hydrogel is obtained by introducing N-hydroxysuccinimide into poly-α-(L-glutamic acid) with water-soluble carbodiimide and then cross-linking it with gelatin, and has a good hemostatic effect.

However, as described in Patent Document 3, polymeric NHS cross-linking agents such as polyglutamic acid must be prepared as alkaline solutions. Also, in general, carboxylic acids activated with NHS groups require alkaline conditions to accelerate the reaction.

For example, Non-Patent Document 1 describes that the cross-linking reaction between the NHS form of poly-γ-glutamic acid (γ-PGA) and an amine compound is carried out in an alkaline solution. The non-patent document describes that the NHS form of γ-PGA undergoes alkaline hydrolysis in the presence of alkali. Therefore, the NHS form of γ-PGA must be used by immediately mixing it with an amine compound after preparation of the alkaline solution. When it is assumed to be used as an adhesive, the hydrolysis of the NHS body proceeds at the same time, so there is a possibility that the adhesive strength will decrease after adhesion. Therefore, high-molecular-weight NHS forms such as γ-PGA have problems in clinical handling.

JP-A-06-070972 JP-A-2004-99562 JP-A-09-103479

Network Polymer, Vol. 36, No. 6 (2015), p. 282-287.

The present invention is a polycarboxylic acid derivative ( compound).

As a result of intensive studies, the inventors have found that the above-described problems can be solved by means shown below, and have completed the present invention.

（式中のＲは、水素または炭素数１から６の直鎖型または分鎖型のアルキル基又はアリール基を示し、それぞれ同じものであっても異なったものであってもよい。また、式中のｎは、１から３の整数を示す）
２． 前記ポリカルボン酸がポリペプチドである、１に記載の架橋剤。
３． 前記ポリカルボン酸がポリグルタミン酸またはポリアスパラギン酸である、１または２に記載の架橋剤。
４． 前記プロパルギルアミノ酸がプロパルギルグリシンである、１～３のいずれかに記載の架橋剤。
５． 前記ポリカルボン酸誘導体においてプロパルギルアミノ酸の置換率が１０～５５％である、１～４のいずれかに記載の架橋剤。
６． 主剤溶液と１～５のいずれかに記載の架橋剤を含む溶液とからなる、２液型接着剤。
７． 前記主剤が生体高分子である、６に記載の２液型接着剤。
８． 前記主剤がゼラチンである、６または７に記載の２液型接着剤。
９． 前記主剤と前記架橋剤とを１：０．０１～１：１の重量比で混合して使用する、６～８のいずれかに記載の２液型接着剤。
１０． 医療用の接着剤として用いられる、６～９のいずれかに記載の２液型接着剤。
That is, the present invention consists of the following configurations.
1. A cross-linking agent comprising a water-soluble polycarboxylic acid derivative in which a propargyl amino acid represented by the following general formula (1) is added to the carboxyl group of polycarboxylic acid.

(R in the formula represents hydrogen or a linear or branched alkyl group or aryl group having 1 to 6 carbon atoms, which may be the same or different. n in indicates an integer from 1 to 3)
2. 2. The cross- linking agent according to 1, wherein the polycarboxylic acid is a polypeptide.
3. 3. The cross- linking agent according to 1 or 2, wherein the polycarboxylic acid is polyglutamic acid or polyaspartic acid.
4. 4. The cross- linking agent according to any one of 1 to 3, wherein the propargyl amino acid is propargyl glycine.
5. 5. The cross- linking agent according to any one of 1 to 4, wherein the polycarboxylic acid derivative has a propargyl amino acid substitution rate of 10 to 55%.
6. A two-component adhesive comprising a main agent solution and a solution containing the cross-linking agent according to any one of 1 to 5 .
7. 7. The two-component adhesive according to 6 , wherein the main agent is a biopolymer.
8. 8. The two-component adhesive according to 6 or 7 , wherein the main agent is gelatin.
9. 9. The two-component adhesive according to any one of 6 to 8 , wherein the main agent and the cross-linking agent are mixed in a weight ratio of 1:0.01 to 1:1.
10. 10. The two-component adhesive according to any one of 6 to 9, which is used as a medical adhesive.

The polycarboxylic acid derivative of the present invention has a wide range of applications because it dissolves in water without the need for acids, alkalis, etc., and uses materials that are highly safe for living organisms, and can exhibit high adhesive strength. Excellent as a cross-linking agent for medical adhesives.

It is an example showing the results of ¹ H-NMR measurement of the polycarboxylic acid derivative of the present invention. It is a figure which shows an example of the test piece used for the measurement of tensile shear bond strength. FIG. 4 is a diagram showing a state after adhesion of a test piece used for measuring tensile shear bond strength. 1 is a graph showing the results of an adhesion test using the polycarboxylic acid derivative of the present invention as a cross-linking agent.

In the present invention, a polycarboxylic acid derivative is a derivative obtained by modifying a carboxylic acid having two or more carboxyl groups in the molecule with at least one or more propargyl amino acids.

In the present invention, propargyl amino acid is an amino acid derivative represented by general formula (1). R in the formula represents hydrogen or a linear or branched alkyl group or aryl group having 1 to 6 carbon atoms, which may be the same or different. Also, n in the formula represents an integer of 1 to 3.

Any of the amino acids constituting the propargyl amino acid of general formula (1) may be used without any limitation, and commercially available ones may be used. It is preferred to use amino acids that constitute Amino acids that constitute living body proteins include asparagine, aspartic acid, alanine, arginine, cysteine/cystine, glutamine, glutamic acid, glycine, proline, serine, tyrosine, isoleucine, leucine, valine, histidine, lysine, methionine, Examples include phenylalanine, threonine (threonine), and tryptophan, and it is preferable to use the L-form of each of them.

The propargyl amino acid of general formula (1) can be obtained by condensing the amino acid with propargyl alcohol, propargyl halide, or the like, as shown in the following reaction formula (2). X in the formula represents a hydroxyl group or a halogen group.

In the present invention, the carboxylic acid constituting the polycarboxylic acid derivative is a carboxylic acid having two or more carboxyl groups in the molecule. Specifically, low-molecular-weight carboxylic acids such as malonic acid, succinic acid, glutaric acid, adipic acid, citric acid, and tartaric acid, high-molecular-weight synthetic polymers such as polyacrylic acid and polymethacrylic acid, pectin, alginic acid, and hyaluronic acid. Acids, natural polysaccharides such as carboxymethylcellulose, and polypeptides such as poly-γ-glutamic acid, poly-α-glutamic acid and polyaspartic acid. Polypeptides such as poly-γ-glutamic acid and polyasparagine are preferred among these. Any poly-γ-glutamic acid can be used without limitation, and any commercially available one may be used.

In the present invention, the polycarboxylic acid derivative can be obtained by condensing the carboxyl group of the polycarboxylic acid with the propargyl amino acid of general formula (1), as shown in the following reaction formula (3). As a method for condensing the polycarboxylic acid and the propargylamino acid, the polycarboxylic acid and the propargylamino acid can be reacted at room temperature in the presence of a dehydration condensing agent.

In the present invention, the dehydration condensation agent is N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC), O-(benzotriazol-1-yl). -N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and the like.

In the present invention, the reaction solvent for the condensation reaction is not particularly limited as long as it dissolves the polycarboxylic acid, and examples thereof include water, dimethylformamide (DMF), dimethylsulfoxide (DMSO), tetrahydrofuran (THF), and the like. .

In the present invention, a base, a catalyst, or an active ester may be used to promote the condensation reaction. Examples of the base include amines such as triethylamine and N,N-diisopropylethylamine, and carbonates such as sodium carbonate and sodium hydrogen carbonate. The catalyst includes N,N-dimethyl-4-aminopyridine and the like. Active esters include 1-hydroxybenzotriazole and N-hydroxysuccinimide as reaction intermediate esters.

In the above condensation reaction, the rate of modification of polycarboxylic acid with propargyl amino acid can be changed by changing the charged amount of each raw material such as propargyl amino acid and dehydration condensation agent. When polycarboxylic acid is used, the propargyl amino acid modification rate is preferably 1 to 55%, more preferably 15 to 50%, per carboxylic acid unit. If it exceeds 55%, the solubility in solvents becomes poor.

In the present invention, the polycarboxylic acid derivative can be used as an adhesive because it forms a crosslinked product when mixed with a main agent such as a biopolymer having an amino group.

In the present invention, biopolymers having amino groups include proteins such as plasma albumin, ovalbumin, casein, collagen, and gelatin, and any of them may be used.

In the present invention, when a polycarboxylic acid derivative is used as a cross-linking agent for a (medical) adhesive, a cross-linking agent solution containing the polycarboxylic acid derivative and a solution containing the main agent are mixed and applied to a living tissue or the like. . Then, a crosslinked body of polycarboxylic acid and biopolymer is produced through the reaction shown in Reaction Formula (4). Here, the concentration of the polycarboxylic acid derivative in the cross-linking agent solution is preferably 0.1 to 20% by weight, more preferably 0.5 to 20% by weight. The concentration of the main agent in the main agent solution is preferably in the range of 0.1 to 30% by weight, more preferably in the range of 0.5 to 20% by weight. From the viewpoint of safety, the solvent used for the solution is preferably water, physiological saline, low-concentration ethanol, or the like. The weight ratio of the main agent and the polycarboxylic acid derivative is preferably 1:0.01 to 1:1, more preferably 1:0.05 to 1:1.

In the present invention, the polycarboxylic acid derivative is a highly safe material for living organisms and is easily soluble in water. It can be applied to medical applications such as adhesives used for bonding soft tissue and skin, sealants for closing wounds and blood vessels, and the like.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.

(Measurement of propargyl amino acid modification rate)
The propargyl amino acid modification rate for the carboxyl group constituting the polycarboxylic acid derivative was determined by measuring the ¹ H-NMR spectrum (BRUKER, MR400) in DMSO-d ₆ . The modification rate was calculated by measuring the integrated intensity ratio of the α-hydrogen (FIG. 1) of the carboxyl group modified with propargyl amino acid and the carboxyl group not modified with the propargyl amino acid, and calculated by the following formula.
Modification rate (%) = [α hydrogen of modified carboxyl group] / [(α hydrogen of unmodified carboxyl group) + (α hydrogen of modified carboxyl group)] × 100

(Measurement of adhesive strength)
Tensilon universal material testing machine (A&A, RTG-1310) was used to measure the tensile shear bond strength (JIS K6850) of the obtained test piece. The temperature during the measurement was 23° C., the humidity was 50% Rh, and the tensile speed was 10 mm/min. The obtained strain-stress curve was used to determine the tensile shear bond strength. The tensile shear bond strength was the average value of four measurements.

(Production of glycine, 2-propyn-1-yl, ester (GPE))
A mixture of glycine (2.1 g) and 2-propyn-1-ol (30 mL) was prepared and thionyl chloride (2.4 mL) was added at room temperature. The reaction solution was stirred at room temperature for 2 hours and then at 50° C. for 2 hours. The reaction solution was cooled to 5° C. and ethyl acetate (90 mL) was added to obtain a precipitate. The precipitate was separated by filtration, washed with ethyl acetate and dried to give glycine, 2-propyn-1-yl, ester (GPE) in 84% yield.

(Production Example 1) Production of GPE-modified γ-PGA (10) Toyobo poly-γ-glutamic acid (γ-PGA) (0.5 g) and DMSO (8 mL) were added and dissolved by stirring at 60°C for 1 hour. . The solution was cooled to room temperature, and 0.15 equivalents of glycine, 2-propyn-1-yl, ester (GPE) and O-(benzotriazole-1- yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) was added. Furthermore, 2 equivalents of triethylamine were added and the mixture was stirred at room temperature for 24 hours to react. After the reaction, acetone (35 mL) was added to precipitate the polymer. The resulting polymer was washed with acetone and dried. After drying, the crude polymer was dissolved in water (5.5 mL), precipitated again by adding acetone (60 mL) and separated by filtration. Vacuum drying was performed at 60° C. for 12 hours to obtain the desired GPE-γ-PGA. From ¹ H-NMR (DMSO-D ₆ ), it was confirmed that the GPE modification rate for the carboxylic acid unit of γ-PGA was 10%. The polycarboxylic acid derivative obtained in Production Example 1 was designated as GPE-γ-PGA (10).

(Production Example 2) Production of GPE-γ-PGA (15) The method described in Production Example 2 was followed except that 0.25 equivalents of GPE and HBTU were used as the amounts of raw materials charged. From ¹ H-NMR (DMSO-D ₆ ), it was confirmed that the GPE modification rate for the carboxylic acid unit of γ-PGA was 15%. The polycarboxylic acid derivative obtained in Production Example 2 was used as GPE-γ-PGA (15).

(Production Example 3) Production of GPE-γ-PGA (25) The procedure described in Production Example 2 was followed, except that 0.5 equivalents of GPE and HBTU were used as the amounts of raw materials charged. From ¹ H-NMR (DMSO-D ₆ ), it was confirmed that the GPE modification rate for the carboxylic acid unit of γ-PGA was 25%. The polycarboxylic acid derivative obtained in Production Example 3 was used as GPE-γ-PGA (25).

(Production Example 4) Production of GPE-γ-PGA (45) The method described in Production Example 2 was followed, except that 0.75 equivalents of GPE and HBTU were used as the amounts of raw materials charged. From ¹ H-NMR (DMSO-D ₆ ), it was confirmed that the GPE modification rate for the carboxylic acid unit of γ-PGA was 45%. The polycarboxylic acid derivative obtained in Production Example 4 was used as GPE-γ-PGA (45).

(Production Example 5) Production of GPE-γ-PGA (50) The method described in Production Example 2 was followed, except that 0.9 equivalents of GPE and HBTU were used as the amounts of raw materials charged. From ¹ H-NMR (DMSO-D ₆ ), it was confirmed that the GPE modification rate for the carboxylic acid unit of γ-PGA was 50%. The polycarboxylic acid derivative obtained in Production Example 5 was designated as GPE-γ-PGA (50).

(Production Example 6) Production of GPE-γ-PGA (55) The method described in Production Example 2 was followed, except that 1.0 equivalent of GPE and HBTU were used as the amounts of raw materials charged. However, because of its poor solubility in water, purification using water was abandoned. From ¹ H-NMR (DMSO-D ₆ ), it was confirmed that the GPE modification rate for the carboxylic acid unit of γ-PGA was 55%. The polycarboxylic acid derivative obtained in Production Example 6 was designated as GPE-γ-PGA (57).

(Production Example 7) Production of GPE-modified PAA (25) Production example except that polyaspartic acid (PAA) was used instead of γ-PGA, and 0.15 equivalents of GPE and HBTU were used as the amount of raw materials charged. 2 described method. From ¹ H-NMR (DMSO-D ₆ ), it was confirmed that the GPE modification rate with respect to the carboxylic acid unit of PAA was 25%. The polycarboxylic acid derivative obtained in Production Example 7 was designated as GPE-PAA (25).

(Production Example 8) Production of NHS-modified γ-PGA (50) NHS (N-hydroxysuccinimidyl)-modified γ-PGA is manufactured according to Network Polymer, Vol. 36, No. 6 (2015), p. 282-287. Manufactured by the method described in. From ¹ H-NMR (DMSO-D ₆ ), it was confirmed that the NHS modification rate of the carboxylic acid unit of γ-PGA was 50%. The polycarboxylic acid derivative obtained in Production Example 8 was designated as NHS-γ-PGA (50).

(Production Example 9) NHS-modified γ-PGA (25)
In the same manner as in Production Example 8, NHS-modified γ-PGA (25) having a NHS modification rate of 25% with respect to the carboxylic acid units of γ-PGA was produced.

(Production Example 10)
In the same manner as in Production Example 8, NHS-modified γ-PGA (7) having a NHS modification rate of 7% with respect to the carboxylic acid units of γ-PGA was produced.

(Solubility test)
Table 1 shows the results of examining the solubility in water of each polycarboxylic acid derivative obtained in Production Examples 1-10. In the solubility test in water, each polycarboxylic acid derivative was added to pure water so as to be 20 wt %, stirred at 350 rpm at 25° C. for 1 hour, and then the degree of dissolution was visually confirmed. The state in which the polycarboxylic acid derivative in the liquid was completely dissolved was defined as "soluble", and the state in which there was an undissolved residue was defined as "insoluble".

(Tensile shear bond strength)
Wako Pure Chemical bovine bone-derived gelatin was dissolved in warm water (50° C.) to prepare a 20 wt % aqueous solution (hereinafter referred to as a base solution). Further, each polycarboxylic acid derivative obtained in Production Examples 1-10 was dissolved in water to prepare an aqueous solution having a concentration shown in Table 1 (hereinafter referred to as a cross-linking agent solution). Pigskin of 1 cm x 1 cm x 5 mm was attached to the end (1 cm x 1 cm) of a PET film with a width of 1 cm, a length of 5 cm, and a thickness of 188 µm with a cyanoacrylate adhesive (Aron Alpha (registered trademark), Toagosei Co., Ltd., model number 201). Two adhesively fixed test pieces (Fig. 2) were prepared. 25 μL of the main solution was added dropwise to the prepared pork skin portion, followed by 25 μL of the cross-linking agent solution, and mixed uniformly. After applying the mixed adhesive to the entire surface of the pigskin, another test piece was pasted so that the pigskins overlap each other, sandwiched with cross pins, and left at room temperature for 1 hour. The obtained test piece (Fig. 3) was used to measure the tensile shear bond strength. These results are shown in Table 1 and FIG.

As is clear from the results in Table 1 and FIG. 4, the polycarboxylic acid derivatives of Examples 1-18 had good solubility in pure water. Moreover, when mixed with the main agent solution, it had a tensile shear adhesive strength exceeding that of gelatin alone. On the other hand, the polycarboxylic acid derivative of Comparative Example 1 had a high modification rate of 57% and poor solubility (insolubility) in pure water. Therefore, an aqueous cross-linking agent solution could not be prepared. Also, the polycarboxylic acid derivatives of Comparative Examples 2-7 were not soluble in pure water and required the addition of alkali. In addition, Comparative Example 8 had a certain degree of tensile shear adhesive strength in a dry state because it was bonded only with the main agent without using a cross-linking agent, but the test piece peeled off quickly when it got wet with water.

The polycarboxylic acid derivative of the present invention has a wide range of applications because it dissolves in water without the need for acids or alkalis, etc., and uses materials that are highly safe for living organisms. Excellent as a cross-linking agent for adhesives.

1 PET film 2 Pigskin 3 Adhesive

Claims (10)

A cross-linking agent comprising a water-soluble polycarboxylic acid derivative in which a propargyl amino acid represented by the following general formula (1) is added to the carboxyl group of polycarboxylic acid.

(R in the formula represents hydrogen or a linear or branched alkyl group or aryl group having 1 to 6 carbon atoms, which may be the same or different. n in indicates an integer from 1 to 3) 2. The cross- linking agent of claim 1, wherein said polycarboxylic acid is a polypeptide. 3. The cross- linking agent according to claim 1 or 2, wherein said polycarboxylic acid is polyglutamic acid or polyaspartic acid. The cross- linking agent according to any one of claims 1 to 3, wherein said propargyl amino acid is propargyl glycine. The cross- linking agent according to any one of claims 1 to 4, wherein the polycarboxylic acid derivative has a propargyl amino acid substitution rate of 10 to 55%. A two-component adhesive comprising a base solution and a solution containing the cross-linking agent according to any one of claims 1 to 5 . 7. The two-component adhesive according to claim 6 , wherein the main agent is a biopolymer. 8. The two-component adhesive according to claim 6 or 7 , wherein said main ingredient is gelatin. The two-component adhesive according to any one of claims 6 to 8 , wherein the main agent and the cross-linking agent are mixed in a weight ratio of 1:0.01 to 1:1. The two-component adhesive according to any one of claims 6 to 9, which is used as a medical adhesive.

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