Two-component curing adhesives, curing agents for two-component curing adhesives, and compounds

Abstract

To provide a rapidly curable two-part curable adhesive using a genipin derivative.SOLUTION: A two-part curable adhesive includes: a first agent containing a compound having, in each molecule, one or more primary amino groups and one or more groups represented by formula 1 in which * is a bonding position, and a basic compound; and a second agent containing an acidic compound.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a two-component curing adhesive, a curing agent for a two-component curing adhesive, and a compound. [Background technology]

[0002] Genipin has a lower cytotoxicity than glutaraldehyde and the like, and is used as a hardener for medical gels including xerogels. Patent Document 1 describes "a sponge for hemostatic material, comprising genipin and gelatin." [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2018-68723 A Summary of the Invention [Problem to be solved by the invention]

[0004] Genipin is difficult to use as a curing agent for two-component curing adhesives because the crosslinking reaction proceeds slowly. Therefore, the present invention aims to provide a two-component curing adhesive that contains a genipin derivative and can be cured quickly. Another object of the present invention is to provide a curing agent and a compound for a two-component curing adhesive. [Means for solving the problem]

[0005] As a result of extensive investigations aimed at achieving the above object, the present inventors have found that the above object can be achieved by the following configuration.

[0006] [1] A two-component curing adhesive comprising a first agent containing compound A having at least one primary amino group and at least one group represented by formula 1 in its molecule, and a basic compound, and a second agent containing an acidic compound. [2] The two-component curing adhesive described in [1], wherein at least one selected from the group consisting of the first component and the second component contains a solvent, and when the first component and the second component are mixed, the pH of the mixture is 8.0 or less. [3] The two-component curing adhesive according to [2], wherein the pH of the mixture is less than 7.0. [4] The two-component curing adhesive according to [2] or [3], wherein the pH of the mixture is 5.0 or higher. [5] The two-component curing adhesive according to any one of [1] to [4], wherein the second component contains a solvent and has a pH of less than 7.0. [6] The two-component curing adhesive according to [5], wherein the second component has a pH of 2.0 to 5.0. [7] The two-component curing adhesive according to any one of [1] to [6], wherein the first component contains a solvent and has a pH of greater than 7.0. [8] The two-component curing adhesive according to any one of [1] to [7], wherein compound A has a skeletal structure, the primary amino group bonded to the skeletal structure, and a group represented by formula 1 described below, and the skeletal structure is a skeletal structure derived from at least one compound selected from the group consisting of polysaccharides, peptides, proteins, gelatin, ethylenediamine, polyethyleneimine, polyalkylene glycols, polylactic acids, polyglycolic acids, polycaprolactones, and polyacrylic acids. [9] The two-component curing adhesive according to any one of [1] to [8], wherein compound A is a compound represented by formula 2 described below.

[10] L above 1 , the above L 2 , and the above L 3 contains an alkyleneoxy group having 1 to 10 carbon atoms.

[11] The two-component curing adhesive according to any one of [1] to

[10] , wherein compound A is a compound represented by the following formula 5:

[12] The two-component curing adhesive according to any one of [1] to

[11] , wherein the second component contains an extracellular matrix.

[13] The two-component curing adhesive according to any one of [2] to [7], wherein the first and second components have a pH of 5.0 to 8.0 when mixed together.

[14] The two-component curing adhesive according to any one of [1] to

[13] , wherein the ratio of the molar content of the group represented by the above formula 1 to the molar content of primary amino groups in the compound is 0.05 to 1.2.

[15] The two-component curing adhesive according to any one of [1] to

[14] , wherein compound A has 2 to 6 primary amino groups and 2 to 6 groups represented by formula 1.

[16] A curing agent for a two-component curing adhesive, comprising compound A having at least one primary amino group and at least one group represented by formula 1 in the molecule.

[17] The curing agent according to

[16] , further comprising a basic compound.

[18] The curing agent according to

[16] or

[17] , wherein compound A has a skeletal structure, the primary amino group bound to the skeletal structure, and a group represented by formula 1, and the skeletal structure is derived from at least one compound selected from the group consisting of polysaccharides, peptides, proteins, gelatin, ethylenediamine, polyethyleneimine, polyalkylene glycols, polylactic acids, polyglycolic acids, polycaprolactones, and polyacrylic acids.

[19] A compound represented by formula 2 below. Effect of the Invention

[0007] According to the present invention, it is possible to provide a two-component curing adhesive that contains a genipin derivative and is capable of curing rapidly. The present invention also provides a curing agent and a compound for a two-component curing adhesive. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 shows the results of calculating the amount of genipin introduced (vertical axis: pieces) against the reaction time (horizontal axis: minutes). [Diagram 2] FIG. 1 is a graph showing the results of calculating the average amount of genipin introduced per polyamine molecule versus the pH of the polyamine solution. [Diagram 3] This is a graph showing the change in shear modulus (Pa) over time, with the time when the first and second parts of the adhesive are mixed being set at 0 minutes. The solid circles represent the storage modulus, and the hollow circles represent the loss modulus. [Figure 4] FIG. 1 is a graph showing the change in shear modulus (Pa) over time for the second agent alone. [Diagram 5] FIG. 1 is a graph showing the change in shear modulus (Pa) over time, with the time when the second agent and genipin are mixed being set at 0 minutes. [Figure 6] 6 is a graph showing the change in shear modulus (Pa) over time, with the time when the second agent, genipin, and polyamine were mixed being set at 0 minutes. The solid circles represent the storage modulus, and the hollow circles represent the loss modulus. [Figure 7] This shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive used compound A, which was synthesized by adding 50 mol% genipin to 100 mol% of the primary amino groups of the polyamine, are mixed at 0 minutes. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. [Figure 8] This shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive used compound A, which was synthesized by adding 60 mol% genipin to 100 mol% of the primary amino groups of the polyamine, are mixed at 0 minutes. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. [Figure 9] This shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive used compound A, which was synthesized by adding 70 mol% genipin to 100 mol% of the primary amino groups of the polyamine, are mixed at 0 minutes. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. [Figure 10] This shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive used compound A, which was synthesized by adding 80 mol % genipin to 100 mol % of the primary amino groups of the polyamine, are mixed at 0 minutes. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. [Figure 11]This shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive using compound A obtained when the reaction time between genipin and polyamine was set to 3 minutes was mixed at 0 minutes. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. [Figure 12] This shows the change in shear modulus (Pa) over time, with the first and second parts of the adhesive using compound A obtained when the reaction time between genipin and polyamine was 10 minutes being taken as 0 minutes. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. [Figure 13] This shows the change in shear modulus (Pa) over time, with the first and second parts of the adhesive using compound A obtained when the reaction time between genipin and polyamine was 30 minutes being taken as 0 minutes. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. [Figure 14] This shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive using compound A obtained when the reaction time between genipin and polyamine was 60 minutes was set to 0 minutes. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. [Figure 15] This shows the change in shear modulus (Pa) over time, with the first and second parts of this adhesive mixed at 0 minutes when the pH of the UBM (Urinary bladder matrix) solution was 2.0. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. [Figure 16] This shows the change in shear modulus (Pa) over time, with the first and second parts of this adhesive mixed at 0 minutes when the pH of the UBM solution was 2.5. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. [Figure 17] This shows the change in shear modulus (Pa) over time, with the first and second parts of this adhesive mixed at 0 minutes when the pH of the UBM solution was 3.0. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. [Figure 18] This shows the change in shear modulus (Pa) over time, with the first and second parts of this adhesive mixed at 0 minutes when the pH of the UBM solution was 5.0. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. [Figure 19] This shows the change in shear modulus (Pa) over time, with the first and second parts of this adhesive mixed at 0 minutes when the pH of the UBM solution was 7.0. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. [Figure 20] This is the result of measuring the swelling degree of Hydrokel. [Figure 21] This is the stress-strain relationship of the hydrogel. [Figure 22] FIG. 1 shows cell viability (%). [Diagram 23] FIG. 13 is a graph showing adhesiveness to a collagen casing. [Figure 24] 1 shows the results of adhesiveness evaluation using porcine aorta. [Diagram 25] 1 shows the results of histological evaluation of the porcine aorta after pressure testing. [Figure 26] This shows the change in shear modulus (storage modulus: Pa) over time, with the time when the first and second parts of the adhesive, which includes a second part containing UBM, being 0 minutes. [Figure 27] This shows the change over time in shear modulus (storage modulus: Pa) when the first and second parts of this adhesive, which contains a second part that contains ECM (extracellular matrix) derived from the heart, are mixed at 0 minutes. [Figure 28] This shows the change in shear modulus (storage modulus: Pa) over time when the first and second parts of this adhesive, which includes a second part containing liver-derived ECM, are mixed at 0 minutes. [Figure 29] This shows the change in shear modulus (storage modulus: Pa) over time when the first and second parts of this adhesive, which includes a second part containing pancreatic-derived ECM, are mixed at 0 minutes. [Diagram 30] This shows the change in shear modulus (storage modulus: Pa) over time when the first and second agents of this adhesive, which includes a second agent containing ECM derived from the small intestine, are mixed at 0 minutes. [Diagram 31] This shows the change over time in the shear modulus (storage modulus: Pa) of this adhesive, with the second agent consisting only of PBS (phosphate-buffered saline) when the first and second agents are mixed together, with the time set at 0 minutes. [Diagram 32] It is a diagram showing the adhesiveness of a hydrogel prepared using a second agent containing various ECMs to a collagen casing. [Diagram 33] It is a diagram showing the introduction rate of genipin (introduction rate of the group represented by Formula 1) with respect to the charged amount (equivalent) of genipin. [Diagram 34] It is the change over time of the shear elastic modulus (storage elastic modulus: Pa) when the time at the mixing of the first agent and the second agent of this adhesive containing the first agent with a genipin introduction rate of 9% is set to 0 minutes. [Diagram 35] It is the change over time of the shear elastic modulus (storage elastic modulus: Pa) when the time at the mixing of the first agent and the second agent of this adhesive containing the first agent with a genipin introduction rate of 18% is set to 0 minutes. [Diagram 36] It is the change over time of the shear elastic modulus (storage elastic modulus: Pa) when the time at the mixing of the first agent and the second agent of this adhesive containing the first agent with a genipin introduction rate of 31% is set to 0 minutes. [Figure 37] It is the change over time of the shear elastic modulus (storage elastic modulus: Pa) when the time at the mixing of the first agent and the second agent of this adhesive containing the first agent with a genipin introduction rate of 33% is set to 0 minutes.

Mode for Carrying Out the Invention

[0009] Hereinafter, the present invention will be described in detail. The description of the constituent elements described below may be made based on typical embodiments of the present invention, but the present invention is not limited to such embodiments. In this specification, a numerical range represented by "~" means a range including the numerical values described before and after "~" as the lower limit value and the upper limit value.

[0010] In the description of groups (atomic groups) in this specification, the description that does not indicate whether substituted or unsubstituted includes both unsubstituted and substituted groups, as long as it does not impair the effects of the present invention. For example, "alkyl group" includes not only alkyl groups that do not have a substituent (unsubstituted alkyl groups), but also alkyl groups that have a substituent (substituted alkyl groups). This also applies to each compound.

[0011] [Two-component curing adhesive] A two-component curing adhesive according to an embodiment of the present invention (hereinafter simply referred to as "the adhesive") is a two-component curing adhesive comprising compound A having, within the molecule, at least one primary amino group and at least one group represented by formula 1 when * is the bonding position, a first component containing a basic compound, and a second component containing an acidic compound. [ka]

[0012] The mechanism by which the problem of the present invention is solved by the above two-component curing adhesive is not entirely clear, but the inventor speculates as follows: Note that the technical scope of the present invention is not limited by the following speculation, and even if the problem is solved without relying on the following mechanism, the two-component curing adhesive etc. specified by the matters recited in each claim are considered to be included in the scope of the present invention.

[0013] In developing a two-component curing adhesive that cures quickly, the present inventors have intensively investigated the crosslinking (curing) mechanism of genipin and primary amines. In general, the crosslinking (curing) reaction mechanism of genipin is known to be (1) a nucleophilic attack on the C3 carbon atom of genipin by a primary amine, followed by ring-opening of the dihydropyran ring, followed by attack of the aldehyde group generated by the ring-opening by the secondary amine resulting from the nucleophilic attack to form an N-containing heterocycle (dihydropyridine ring) ("reaction scheme (1)" in the formula below), (2) an SN2 nucleophilic substitution reaction that replaces an ester group with a secondary amide bond ("reaction scheme (2)" in the formula below), and (3) a dimerization reaction that occurs in the presence of oxygen radicals (it is believed that there are multiple forms of the dimerization reaction).

[0014] The inventors conceived the idea that, among these reactions, reaction scheme (1) and reaction scheme (2), which are easier to control even in an atmospheric environment and are presumed to contribute to the formation of the main crosslinked structure in the cured product, could be applied to a more rapidly curing two-component curing adhesive by causing the reactions to occur in stages.

[0015] It has already been known that the reaction rates of reaction scheme (1) and reaction scheme (2) may vary depending on various factors. However, the present inventors have recently discovered that the reaction of reaction scheme (1) is more likely to proceed in a solution containing a basic compound, for example, in an aqueous solution having a pH of more than 7.0 (preferably pH 11.0 or less), while the reaction of reaction scheme (2) is more likely to proceed in a solution containing an acidic compound, specifically, in an aqueous solution having a pH of less than 7.0 (preferably pH 2.0 or more), and have focused on this finding.

[0016] Thus, the inventors have completed a two-component curing adhesive in which a genipin derivative having a primary amino group is synthesized, the pH is adjusted with a basic compound (preferably to a pH greater than 7.0) to form a first component, which is then mixed with a second component containing an acidic compound, the pH is lowered (preferably to a pH less than 7.0), and the adhesive is cured by causing a reaction according to reaction scheme (4) in the following formula.

[0017] Compound A contained in the first agent has a group represented by the above formula 1. The group represented by formula 1 is a group generated by the reaction of genipin with a primary amine according to reaction scheme (1), and the present inventors have surprisingly discovered that this group causes the reaction of reaction scheme (4) to proceed very quickly by lowering the pH of the reaction solution.

[0018] The following reaction formulas are schematic, and reactions derived from the dimerization mechanism of genipin may occur in parallel before, during, or after each reaction, but these are omitted. A , and R B Each represents a monovalent group.

[0019] [ka]

[0020] The present inventors have also investigated an embodiment in which the reaction of reaction scheme (2) is carried out in a low pH range, then a pH adjuster is added to raise the pH, and crosslinking is carried out in a high pH range through the reaction of reaction scheme (5). However, the desired effect was not obtained.

[0021] In other words, it is presumed that the adhesive achieves the desired effect by utilizing the relationship between the pH of the reaction solution and the crosslinking reaction rate of genipin, and by preparing in advance compound A having a group represented by formula 1 that facilitates the rapid reaction of reaction scheme (4), and using this as a crosslinking agent.

[0022] In the reaction scheme (4) above, the compound with a primary amino group, “H 2 NR B " is bonded to compound A to form a crosslinked structure. 2 NR B" may be compound A itself. Also, as described below, "H 2 NR B " may be another compound contained in the first or second agent (preferably the second agent). The components contained in the two-component curing adhesive according to the embodiment of the present invention will be described in detail below.

[0023] [First drug] The first agent contains compound A and a basic compound, and may further contain a solvent.

[0024] <Compound A> Compound A is a compound having one or more primary amino groups and one or more groups represented by formula 1 in the molecule. (Note that in the formula, * represents a bonding position.)

[0025] [ka]

[0026] The content of compound A in the first agent is not particularly limited, but is generally preferably 1 to 99 mass% based on the total mass of the solid content in the first agent, in order to obtain a two-agent curing adhesive having a more excellent effect of the present invention. The first agent may contain one type of compound A alone, or may contain two or more types. When the first agent contains two or more types of compound A, the total content is preferably within the above numerical range.

[0027] The number of primary amino groups contained in compound A is not particularly limited, but is preferably 2 or more, and more preferably 3 or more. The upper limit is not particularly limited, but is preferably 50 or less, preferably 10 or less, more preferably 8 or less, and even more preferably 6 or less.

[0028] The number of groups represented by formula 1 in one molecule of compound A is not particularly limited, but is preferably 2 or more, and the upper limit is not particularly limited, but is preferably 50 or less, preferably 10 or less, more preferably 8 or less, even more preferably 6 or less, and particularly preferably 4 or less.

[0029] The molar content ratio of the group represented by formula 1 to the molar content of the primary amino group in compound A (group represented by formula 1 / amino group) is not particularly limited, but is generally preferably 0.01 to 2.0. From the viewpoint of obtaining a two-component curing adhesive having a more excellent effect of the present invention, the content ratio is preferably 0.05 to 1.2. In particular, when compound A is a compound represented by formula 1 described later, from the viewpoint of obtaining a two-component curing adhesive having a more excellent effect of the present invention, the content ratio is more preferably 0.4 to 1.0, and even more preferably 0.5 to 0.9. On the other hand, when compound A is obtained by reacting gelatin with genipin as a polyamine described later, the above content ratio is preferably 0.05 to 0.6.

[0030] Compound A has at least one primary amino group and at least one group represented by formula 1 in the molecule. As already described, the group represented by formula 1 is typically a group formed by bonding genipin with a primary amine, and compound A is a genipin derivative.

[0031] As shown in the Examples (Figure 22), the inventors' studies have revealed that free genipin (shown as "UBM+Genipinn" in Figure 22), which was previously thought to have low cytotoxicity, does not have sufficiently low cytotoxicity. On the other hand, in the two-component curing adhesive of the present embodiment, the component (compound A) that contributes to crosslinking is a genipin "derivative." In other words, genipin, which has cytotoxicity, is fixed to the skeletal structure described below, and has the excellent feature that the toxicity of genipin itself (free genipin) is unlikely to manifest regardless of the progress of the crosslinking reaction.

[0032] Compound A is typically a compound having a skeleton structure, a primary amino group bonded to the skeleton structure, and a group represented by formula 1. This skeleton structure is not particularly limited, and structures derived from various organic compounds can be used. For example, the skeletal structure may be derived from polysaccharides, peptides, proteins, gelatin, ethylenediamine, polyethyleneimine, polyalkylene glycol, polylactic acid, polyglycolic acid, polycaprolactone, polyacrylic acid, or the like.

[0033] In particular, from the viewpoint of obtaining a two-component curing adhesive having superior effects of the present invention, the skeletal structure is preferably a structure derived from at least one selected from the group consisting of ethylenediamine, polyethyleneimine, polyalkylene glycol, polylactic acid, polyglycolic acid, polycaprolactone, and polyacrylic acid, more preferably a structure derived from at least one selected from the group consisting of polyalkylene glycol, polylactic acid, polyglycolic acid, polycaprolactone, and polyacrylic acid, and even more preferably a structure derived from polyalkylene glycol.

[0034] As compound A, the compound represented by formula 2 is preferred from the viewpoint of obtaining a two-component curing adhesive having better effects of the present invention.

[0035] [ka]

[0036] In formula 2, s1 is an integer of 1 or more, and although there is no particular upper limit, it is preferably 8 or less, more preferably 6 or less, even more preferably 4 or less, and particularly preferably 2 or less. In formula 2, t1 is an integer of 1 or more, preferably 2 or more, and although there is no upper limit, it is preferably 10 or less, more preferably 8 or less, even more preferably 6 or less, and particularly preferably 4 or less. In formula 2, u1 is an integer of 0 or more, preferably 2 or less, and more preferably 1 or less.

[0037] M 1 represents a group with a valence of s1+t1+u1. M 1 When is a divalent group, M 1 is, for example, -C(O)-, -C(O)O-, -OC(O)-, -O-, -S-, -NR20 -(R 20 represents a hydrogen atom or a monovalent organic group), an alkylene group (preferably having 1 to 10 carbon atoms), a cycloalkylene group (preferably having 3 to 10 carbon atoms), an alkenylene group (preferably having 2 to 10 carbon atoms), and combinations thereof. Of these, from the viewpoint of obtaining a two-component curing adhesive having superior effects of the present invention, -C(O)-, -NH-, -O-, alkylene groups having 1 to 5 carbon atoms, and combinations of these are preferred.

[0038] Also, M 1 When is a trivalent or higher group, it is not particularly limited, and examples thereof include groups represented by (3a) to (3d). [ka]

[0039] In formula 3a, Q 3 represents a trivalent group. 3 represents a single bond or a divalent group, and three T 3 may be the same or different from each other. Q 3 Examples of Q include a tertiary amino group, a trivalent hydrocarbon group (preferably having 1 to 10 carbon atoms. The hydrocarbon group may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group), or a trivalent heterocyclic group (preferably a 5- to 7-membered heterocyclic group), and the hydrocarbon group may contain a heteroatom (e.g., -O-). 3 Specific examples of the alkyl group include a glycerin residue, a trimethylolpropane residue, a phloroglucinol residue, and a cyclohexanetriol residue. In addition, T 3 The divalent group of is not particularly limited, but may be any of the above-described M 1 Among them, an alkylene group having 1 to 5 carbon atoms which may have a substituent is preferred.

[0040] In formula 3b, Q 4 represents a tetravalent group. 4represents a single bond or a divalent group, and four T 4 may be the same or different from each other. In addition, Q 4 Examples of Q include a tetravalent hydrocarbon group (preferably having 1 to 10 carbon atoms. The hydrocarbon group may be either an aromatic hydrocarbon group or an aliphatic hydrocarbon group), and a tetravalent heterocyclic group (preferably a 5- to 7-membered heterocyclic group), and the hydrocarbon group may contain a heteroatom (for example, -O-). 4 Specific examples of the residue include a pentaerythritol residue and a ditrimethylolpropane residue. In addition, T 4 The divalent group of is not particularly limited, but may be any of the above-described M 1 Among them, -O-, an alkylene group having 1 to 5 carbon atoms, or a combination thereof is preferable.

[0041] In formula 3c, Q 5 represents a pentavalent group. 5 represents a single bond or a divalent group, and five T 5 may be the same or different from each other. In addition, Q 5 Examples of Q include a pentavalent hydrocarbon group (preferably having 2 to 10 carbon atoms. The hydrocarbon group may be an aromatic hydrocarbon group or an aliphatic hydrocarbon group) or a pentavalent heterocyclic group (preferably a 5- to 7-membered heterocyclic group), and the hydrocarbon group may contain a heteroatom (for example, -O-). 5 Specific examples of the alkyl group include an arabinitol residue, a phloroglucidol residue, and a cyclohexanepentanol residue. In addition, T 5 The divalent group of is not particularly limited, but may be any of the above-described M 1 The divalent groups include the same groups as those of the above divalent groups, and the preferred forms are also the same.

[0042] In formula 3d, Q 6 represents a hexavalent group. 6 represents a single bond or a divalent group, and six T 6 may be the same or different from each other. In addition, Q 6 Examples of Q include a hexavalent hydrocarbon group (preferably having 2 to 10 carbon atoms. The hydrocarbon group may be either an aromatic hydrocarbon group or an aliphatic hydrocarbon group) or a hexavalent heterocyclic group (preferably a 6- to 7-membered heterocyclic group), and the hydrocarbon group may contain a heteroatom (for example, -O-). 6 Specific examples of the hydroxyalkyl group include mannitol residue, sorbitol residue, dipentaerythritol residue, hexahydroxybenzene residue, and hexahydroxycyclohexane residue. In addition, T 6 The divalent group of is not particularly limited, but may be any of the above-described M 1 The divalent groups include the same groups as those of the above divalent groups, and the preferred forms are also the same.

[0043] In addition, M 1 When is a group with a valence of 7 or more, a group formed by combining the groups represented by formulae 3a to 3d can be used.

[0044] In formula 2, L 1 , L 2 , and L 3 are each independently a single bond or a divalent group. The divalent group is not particularly limited, but 1 Examples of the divalent group include the same groups as those already explained. Also, L 1 , L 2 , and L 3 It is preferable that the alkyleneoxy group contains an alkyleneoxy group (oxyalkylene group) having a carbon number of 1 to 10. The group containing an alkyleneoxy group also includes a polyoxyalkylene group. L 1 , L 2 , and L 3 When the adhesive contains an alkyleneoxy group (oxyalkylene group) having 1 to 10 carbon atoms, the cured product of the adhesive has superior hydrophilicity, and as a result, has superior tissue adhesion and / or biocompatibility.

[0045] Among these, from the viewpoint of obtaining a two-component curing adhesive having a more excellent effect of the present invention, L1 , L 2 , and L 3 The divalent group is preferably a group represented by the following formula (4).

[0046] [ka]

[0047] In formula 4, R 1 are preferably an alkylene group having 1 to 10 carbon atoms (more preferably 2 to 6 carbon atoms) each of which may independently have a substituent, and -CH 2 CH 2 - is more preferable. Also, L 4 is a single bond or a divalent group, L 4 As the divalent group, M 1 Examples of the divalent group include the same groups as those explained above. Furthermore, p1 represents a number of 2 or more, and is not particularly limited, but is more preferably 50 or more, even more preferably 100 or more, and is preferably 10,000 or less, more preferably 5,000 or less, even more preferably 2,000 or less, and particularly preferably 1,000 or less. Also, * stands for M 1 represents the bonding position to the other one, and ** represents the bonding position to the other one. In addition, L 1 , L 2 , and L 3 may be the same or different, but it is preferable that they are all the same.

[0048] In addition, in formula 2, Y 1 is a hydrogen atom or a monovalent group that does not have a primary amino group. The monovalent group is not particularly limited, and examples thereof include a hydrocarbon group that may have a hetero atom, a halogen atom, a carboxy group, a hydroxyl group, and an alkoxy group. 1 may be a group represented by the following formula: In the formula, * represents the bonding position.

[0049] [ka]

[0050] In addition, in formula 2, X 1 is a group represented by formula 1.

[0051] From the viewpoint of obtaining a two-component curing adhesive having a more excellent effect of the present invention, compound A is preferably a compound represented by the following formula 5. [ka]

[0052] In formula 5, X 1 , L 1 , and L 2 Each of the symbols has the same meaning as in formula 2, and the preferred embodiments are also the same. 5 is a tetravalent group, and specific examples and preferred embodiments are 1 In addition, s5 is an integer of 1 to 3.

[0053] The molecular weight of compound A is not particularly limited, but is typically preferably from 100 to 300,000, more preferably from 1000 to 200,000, and even more preferably from 2000 to 100,000.

[0054] (Method of synthesizing compound A) Compound A can typically be obtained by reacting genipin with a polyamine (which in this specification means a compound containing two or more primary amino groups) in a basic solution.

[0055] As the polyamine, any polyamine having a plurality of primary amino groups, such as chitosan, albumin, gelatin, ethylenediamine, polyethyleneimine, etc., can be used. In addition, polyalkylene glycol, polylactic acid, polyglycolic acid, polycaprolactone, polyacrylic acid, etc., into which a primary amino group has been introduced can also be used.

[0056] The polyamine is preferably a compound represented by the following formula 6, in that the resulting two-component curing adhesive containing compound A has a more excellent effect of the present invention.

[0057] [ka]

[0058] In formula 6, s6 represents an integer of 2 or more, and although there is no particular upper limit, it is preferably 9 or less, more preferably 7 or less, even more preferably 5 or less, and particularly preferably 3 or less. In formula 2, u6 represents an integer of 0 or more, preferably 2 or less, and more preferably 1 or less.

[0059] In formula 6, M 1 , L 1 , L 3 , and Y 1 have the same meanings as the respective symbols in formula 2, and the preferred embodiments are also the same. Among them, Y 1 is preferably a hydrogen atom, a hydroxyl group, or the following groups: In the following formulae, * represents a bonding position.

[0060] [ka]

[0061] Specific examples of polyamines include polysaccharides having amino sugars as constituent units, such as chitosan, and proteins, such as elastin, albumin, and collagen. In addition, gelatin having a primary amino group derived from lysine can also be used as a polyamine.

[0062] Other examples of polyamines include ethylenediamine and polyethyleneimine. In this case, the molecular weight of the polyethyleneimine is not particularly limited, but is preferably 300 to 100,000 in order to obtain better effects of the present invention.

[0063] As another example of a polyamine, a derivative in which a plurality of primary amino groups are introduced into at least one polymer selected from the group consisting of polyalkylene glycol, polylactic acid, polyglycolic acid, polycaprolactone, and polyacrylic acid can also be used. Since it has superior hydrophilicity and superior biocompatibility compared to the above polymers, when the above polyamines are used, the cured product of the two-component curing adhesive has superior biocompatibility.

[0064] The molecular weight of the polyamine is not particularly limited, but is preferably 300 or more in order to provide a cured product with better mechanical properties, and is preferably 200,000 or less in order to provide the first agent with better fluidity.

[0065] <Basic compounds> The first agent contains a basic compound. In this specification, a basic compound means a compound that, when 0.05 mol of the compound is dissolved in 1 kg of ultrapure water at 25° C. in an inert gas atmosphere not containing carbon dioxide, causes the pH of the aqueous solution to exceed 7.0 (rounded up to one decimal place).

[0066] The basic compound is not particularly limited, but examples thereof include sodium hydroxide, potassium hydroxide, sodium hydrogen carbonate, sodium carbonate, disodium hydrogen phosphate, and sodium borate. The form in which the first agent contains a basic compound is not particularly limited, and for example, a pH adjuster (such as sodium hydroxide) or a buffer (such as disodium hydrogen phosphate) used in the synthesis process of compound A may ultimately remain in the first agent.

[0067] More specifically, compound A is preferably obtained by reacting genipin with polyamine in an alkaline aqueous solution, and in this case, the solution contains a basic compound as a pH adjuster or buffer. The first agent may be in a form containing a basic compound due to the basic compound used in the synthesis process of compound A remaining.

[0068] The content of the basic compound in the first agent is not particularly limited, but is generally preferably 0.0001 to 99 mass% based on the total mass of the solid content of the first agent, in order to obtain a two-component curing adhesive having a better effect of the present invention. The first agent may contain one kind of basic compound alone, or may contain two or more kinds. When the first agent contains two or more kinds of basic compounds, the total content thereof is preferably within the above numerical range.

[0069] <Solvent> The first agent may contain a solvent. The solvent is not particularly limited, but is preferably water, a water-soluble organic solvent such as alcohol, or a mixture thereof, and more preferably water. The content of the solvent in the first agent is not particularly limited, but from the viewpoints of handleability and curability, the solid content of the first agent is preferably adjusted to 0.01 to 99 mass%, more preferably 0.1 to 40 mass%, and even more preferably 1 to 30 mass%. When the first agent contains a solvent, the pH of the first agent is not particularly limited, but it is preferably greater than pH 7.0 and less than pH 11.

[0070] The first agent may contain other ingredients within the scope of the effects of the present invention. The other ingredients are the same as the "other ingredients" that may be contained in the second agent, and will be described later.

[0071] [Second drug] The second agent contains an acidic compound. In this specification, an acidic compound means a compound that, when 0.05 mol of the compound is dissolved in 1 kg of ultrapure water at 25° C. in an inert gas atmosphere not containing carbon dioxide, gives an aqueous solution with a pH of less than 7.0 (rounded up to one decimal place).

[0072] The content of the acidic compound in the second agent is not particularly limited, but in relation to the first agent, it is preferable that the pH of the mixed solution be adjusted to 8.0 or less when the two agents (including the solvent) are mixed at the time of use. The pH of the mixed solution is affected by the type and amount of the basic compound contained in the first agent, and also by the type and amount of the solvent if the first agent contains a solvent. It is also affected by factors such as the temperature of the mixed solution during use. Therefore, the content of the acidic compound may be appropriately selected according to the above conditions. Such pH adjustment is easy for those skilled in the art.

[0073] In terms of the fact that the curing of the two-component curing adhesive is more likely to be faster, it is more preferable that the pH of the mixed solution of the two components is adjusted to 7.0 or less, and it is preferably adjusted to less than 7.0. On the other hand, from the viewpoint of ensuring a more sufficient time (more sufficient pot life) from the preparation of the mixed solution to curing, it is preferable that the pH of the mixed solution is adjusted to be 2.0 or more, more preferably adjusted to exceed 2.0, and even more preferably adjusted to be 5.0 or more.

[0074] The pH of the second agent is not particularly limited, but from the viewpoint of easy adjustment of the pH of the mixed solution, it is preferably less than 7.0, preferably less than 5.0, more preferably less than 5.0, even more preferably 3.0 or less, preferably 2.0 or more, preferably exceeding 2.0, and even more preferably 2.5 or more.

[0075] The acidic compound is not particularly limited, and examples thereof include hydrochloric acid, acetic acid, boric acid, citric acid, citrate, etc. The content of the acidic compound in the second agent is not particularly limited, but in terms of obtaining a two-component curing adhesive having more excellent effects of the present invention, generally, 0.001 to 99% by mass is preferable based on the total mass of the solid content of the second agent. Note that the first agent may contain one type of acidic compound alone or may contain two or more types. When the first agent contains two or more types of acidic compounds, it is preferable that the total content is within the above numerical range.

[0076] <Solvent> The second agent may contain a solvent. The solvent is not particularly limited, but is preferably water, a water-soluble organic solvent such as alcohol, or a mixture thereof, and more preferably water. The content of the solvent in the second agent is not particularly limited, but from the viewpoints of handleability and curability, the solid content of the second agent is preferably adjusted to 0.01 to 99 mass%, and more preferably adjusted to 0.01 to 10 mass%.

[0077] Examples of the water-soluble organic solvent include alcohols having 5 or less carbon atoms, such as methanol, ethanol, and isopropyl alcohol, and aprotic polar solvents, such as dimethyl sulfoxide.

[0078] <Other ingredients> The second agent may contain ingredients other than those mentioned above. An example of the component is a polyamine, which has been described as a compound that can be used in the synthesis of compound A. When the second part contains a polyamine, a two-part curing adhesive can be obtained that cures faster and / or can form a cured product with better adhesive properties.

[0079] The other component is preferably an extracellular matrix. As used herein, the term "extracellular matrix" (ECM) is synonymous with "extracellular matrix" and refers to a substance that exists between somatic cells, regardless of whether they are epithelial cells or non-epithelial cells. Extracellular matrix is ​​a biological material generally produced by cells. It is involved in the formation of the internal environment necessary for the survival of somatic cells as well as the support of tissues. Extracellular matrix is ​​generally produced by connective tissue cells, but some is also secreted by cells themselves that possess a basement membrane, such as epithelial cells and endothelial cells.

[0080] The extracellular matrix is ​​broadly divided into fibrous components and the matrix that fills the gap between them, and the fibrous components include collagen fibers and elastic fibers. The basic component of the matrix is ​​glycosaminoglycan (acidic mucopolysaccharide), most of which binds with non-collagenous proteins to form a polymer of proteoglycan (acidic mucopolysaccharide-protein complex). In addition, the matrix also contains glycoproteins such as laminin in the basement membrane, microfibrils around elastic fibers, fibers, and fibronectin on the cell surface. Representative examples of the extracellular matrix include collagen I, collagen III, collagen V, elastin, vitronectin, fibronectin, laminin, thrombospondin, and proteoglycans (e.g., decorin, biglycan, fibromodulin, lumican, hyaluronic acid, aggrecan, etc.).

[0081] When the second part contains an extracellular matrix, the cured product of the two-part curing adhesive has better tissue regeneration ability, better tissue adhesive strength, and cures faster.

[0082] The extracellular matrix may be purchased or may be prepared by a known method. The method for preparing the extracellular matrix is ​​not particularly limited, but for example, a method can be used in which an organ derived from a living body is washed, then a process for removing nucleic acids as necessary, and then digested with acid proteases such as trypsin and pepsin in an acidic solution, and the solvent is removed from the resulting digestive solution by a method such as freeze-drying. Also, the matrix secreted by cultured cells can be used. When the second agent contains an extracellular matrix, the acidic compound contained in the second agent may be one that is contained (transferred) in the second agent as a result of the preparation process of the extracellular matrix, due to its preparation method.

[0083] The type of organ derived from a living body used for preparing the extracellular matrix is ​​not particularly limited, but examples thereof include the urinary bladder, heart, liver, pancreas, and small intestine of a mammal, and from the viewpoint of providing the resulting two-component curing adhesive with superior adhesive properties (compression resistance: FIG. 32), at least one selected from the group consisting of the urinary bladder, heart, liver, and small intestine is preferred, and at least one selected from the group consisting of the urinary bladder and small intestine is more preferred. In addition, examples of cultured cells include fibroblasts derived from human fetuses.

[0084] When the second agent contains an extracellular matrix, the content of the extracellular matrix in the second agent is not particularly limited, but generally, when the total mass of the solids in the second agent is taken as 100% by mass, it is preferably, but not particularly limited to, 0.1 to 99.99% by mass, and may be appropriately adjusted depending on the content of other components, particularly acidic compounds, contained in the second agent.

[0085] The second agent may contain various other drugs in addition to those mentioned above. When the second part contains these drugs, the cured adhesive can be used as a local delivery vehicle or a sustained release delivery vehicle for the drugs. Drugs include, but are not limited to, anticancer drugs, anti-inflammatory drugs, antithrombotic drugs, antibiotics, and biological agents, as well as growth factors such as fibroblast growth factor, vascular endothelial growth factor, and hepatocyte growth factor. Furthermore, when a virus and / or a cancer antigen protein is contained as a vaccine, the cured adhesive can be used as a vaccine carrier.

[0086] [Structure of two-component curing adhesive] This adhesive has a first agent and a second agent, and by mixing these agents, the curing reaction proceeds and the adhesive cures quickly. Moreover, the pot life can be freely controlled by adjusting the relative relationship between the content of the basic compound in the first agent and the content of the acidic compound in the second agent, that is, by adjusting the degree to which the pH of the mixture is lowered (details are shown in the examples). Therefore, when the purpose is to repair a damaged area in a living body, the adhesive can be directly injected into the damaged area with a syringe or the like after mixing the two agents, and the adhesive can be used to cure quickly at the damaged area by adjusting the degree to which the pH of the mixture is lowered, taking into consideration the working time in advance.

[0087] The content mass ratio (percentage) of the first agent relative to the total solid content of the two-agent curing adhesive is not particularly limited, but is preferably 0.01 mass% or more, more preferably 70 mass% or more, even more preferably 80 mass% or more, particularly preferably 90 mass% or more, most preferably 95 mass% or more, and preferably 99.99 mass% or less.

[0088] On the other hand, the content of the second agent relative to the total solid content of the two-agent curing adhesive is not particularly limited, but in general, it is preferably 0.01 mass% or more, preferably 30 mass% or less, more preferably 20 mass% or less, even more preferably 10 mass% or less, and particularly preferably 5 mass% or less.

[0089] Since the adhesive has a first agent and a second agent, these may be filled in a syringe in advance to make a prefilled syringe. In that case, as shown in the examples below, the mechanical properties, crosslinking time, pot life, etc. can be freely controlled, so that the adhesive can be applied to healing muscle damage, preventing pancreatic juice leakage, preventing thinning after myocardial infarction, preventing adhesions, treating spinal cord injuries, treating bedsores, and wound healing in the digestive tract mucosa.

[0090] The method for curing the adhesive is not particularly limited, but for example, it is preferable to leave it at a temperature of 20 to 40° C. for about 10 minutes to 2 hours, and more preferably for 30 to 60 minutes.

[0091] [Cured product] The cured product of the adhesive has a three-dimensional network structure due to a crosslinked structure formed by compound A and, if necessary, an extracellular matrix, etc. From the viewpoint of biocompatibility and tissue regeneration ability, the cured product is preferably a hydrogel containing water. The water may be derived from the first agent and / or the second agent, or may be present around the adherend.

[0092] A hydrogel is a gel that contains water and has no fluidity. The water content in the hydrogel is not particularly limited, but is preferably 80 to 99% by weight when the total mass of the hydrogel is taken as 100% by weight.

[0093] [Uses of two-component curing adhesives] This adhesive can be rapidly cured using the genipin derivative. In addition, when the second agent contains ECM, it can form a cured product (hydrogel) with higher biocompatibility, higher tissue adhesiveness, and higher tissue regeneration ability, and can be used as a medical tissue adhesive.

[0094] In addition, when the first or second agent contains a drug, the cured material can be fixed to the lesion and used to release the drug. In addition, when the second agent contains ECM, the biocompatibility is particularly high, so it can be used as a scaffold for cells during cell transplantation in the field of regenerative medicine. EXAMPLES

[0095] The present invention will be described below with reference to examples, but the present invention is not limited to these.

[0096] <Preparation of the first agent containing compound A> A 4-branched polyethylene glycol having an amino group at the end (NOF AMERICA CORPORATIO, product name "SUNBRIGHT PTE-200PA", molecular weight 20,000) was dispersed in a phosphate buffered saline (PBS, pH = 7.4). Hereinafter, this aqueous dispersion is referred to as the "polyamine solution."

[0097] The above-mentioned "SUNBRIGHT PTE-200PA" corresponds to the polyamine represented by formula 6, where M 1 is the pentaerythritol residue, L 1 is a group represented by formula 4, and R 1 Ga-CH 2 CH 2 -, L 4 Ga-CH 2 CH 2 CH 2 -, u6 is 0, and s6 is 4. The main component is a compound (molecular weight 20,000).

[0098] Genipin was added to the polyamine liquid in an amount of 50-125 mol % when the amount of amino groups (n) in the 4-branched polyethylene glycol was taken as 100 mol %, and the mixture was stirred. Compound A was synthesized in the liquid by stirring at 20-30°C for 3-60 minutes, and a compound corresponding to formula 5 was obtained under all conditions. After the reaction was completed, the dispersion was flash frozen and the sample was stored. This corresponds to the first agent (a form containing a solvent) of this adhesive. The first agent contains disodium hydrogen phosphate (a buffer agent) and sodium hydroxide (a pH adjuster).

[0099] Furthermore, the above dispersion was freeze-dried to obtain a dry powder containing compound A (and a basic compound). This corresponds to the first agent of the adhesive in a form that does not contain a solvent. In each of the following tests, this was redissolved in ultrapure water as necessary and used as the liquid first agent.

[0100] <Measurement using an ultraviolet-visible spectrophotometer> When compound A was synthesized according to the above method, the number of genipin introduced per molecule of polyamine was measured using an ultraviolet-visible spectrophotometer. Genipin (70 mol%) and polyamine solution (polyamine content 20 mass%, pH = 10.5) were mixed, and samples were taken at each time (up to 60 minutes), and the amount of genipin introduced (the amount of the group represented by formula 1 introduced) was calculated from the absorption spectrum. For the calibration curve, genipin was reacted with ethanolamine and the absorption at 290 nm was measured.

[0101] As a result, the absorbance at 290 nm increased significantly one minute after mixing the polyamine solution with genipin, and the absorbance increased with increasing reaction time. FIG. 1 is a graph showing the results of calculation of the average amount of genipin (group represented by formula 1) introduced into one polyamine molecule (unit: piece) against the reaction time (minutes) on the horizontal axis. FIG. 1 shows that, in a reaction time of 30 minutes, an average of 1.7 genipin molecules (groups represented by formula 1) were introduced per one molecule of polyamine (4-branched polyethylene glycol).

[0102] <Effect of pH during synthesis of compound A on genipin incorporation rate> The pH of a polyamine solution (20% by mass) was changed from 2 to 10, and genipin (100% by mole) was dissolved therein and reacted. The number of genipin molecules introduced was measured using an ultraviolet-visible spectrophotometer. A calibration curve was prepared by reacting genipin with ethanolamine and measuring the absorbance at 290 nm.

[0103] FIG. 2 is a graph showing the results of calculating the average amount of genipin (group represented by formula 1) introduced into one polyamine molecule versus the pH of the polyamine solution. According to FIG. 2, it was found that the amount of genipin introduced was small at low pH, i.e., the reaction rate was significantly decreased, and that genipin was more easily introduced at pH levels above 7.0, and even more easily introduced at pH levels of 9.0 or higher.

[0104] <Curing speed test> The second part was prepared according to the following procedure, and reacted with the first part to examine the hardening speed (gelation speed). First, a decellularized matrix derived from porcine urinary bladder (Urinary Bladder Matrix, UBM) was prepared as a component of the second agent.

[0105] UBM was prepared as follows. First, epithelial tissue from a pig bladder was excised with a scalpel and washed with physiological saline. It was then washed with a treatment solution (0.1% by weight peracetic acid, 4% ethanol) for 2 hours, and then washed with physiological saline and ultrapure water. It was then treated with DNase solution (0.1 mg / mL) for 24 hours to remove DNA. It was further washed with physiological saline and ultrapure water, and 10 mg of the obtained powder was treated with 1 mg of pepsin and 1 mL of 0.01 M hydrochloric acid for 48 hours. PBS was added to adjust the pH to 7 to inactivate the pepsin, and the UBM powder was obtained by lyophilization.

[0106] The UBM powder was redissolved by adding ultrapure water, and the pH was adjusted by adding 1M hydrochloric acid to obtain a UBM solution. This UBM solution (1% by mass of UBM powder, pH=2.5, containing hydrochloric acid, corresponding to the second agent) and compound A solution (20% by mass of compound A obtained above, pH=10.5, corresponding to the first agent) were mixed in equal amounts and stirred at 37°C, and the time until hardening was measured. The hardening (hydrogelation) time was defined as the time when the stirrer stopped rotating. As a result, it was revealed that the solution hardened 30 seconds after mixing. This was significantly faster than conventional genipin-containing adhesives, which took several hours to a day to harden.

[0107] <Viscosity measurement> The viscoelasticity of the cured product (hydrogel) was measured using a viscoelasticity measuring device (rheometer "Rheoplus", Anton Paar). 100 μL of the pre-gel solution (a mixture of the first and second agents) was placed on the stage of the rheometer and clamped with a jig having a diameter of 10 mm. The temperature of the stage was kept at 37°C during the measurement.

[0108] The results are shown in Figures 3 to 6. Figure 3 shows the shear modulus (Pa) that changes over time, with the mixing of the first and second parts of this adhesive taken as 0 minutes, where the solid circle (filled circle) represents the storage modulus and the hollow circle (open circle) represents the loss modulus. As mentioned above, Figures 4 to 6 show the results for only the second agent (UBM solution), Figure 5 shows the results for a mixture of the second agent and genipin, and Figure 6 shows the results for a mixture of the second agent, genipin, and polyamine.

[0109] Although UBM (Fig. 4) gels by collagen self-assembly even when used alone, it has a small storage modulus (G'). On the other hand, in the pre-gel solution (Fig. 3) consisting of UBM solution (1% by mass, pH = 2.5) and compound A solution (20% by mass, pH = 10.5), G' rose immediately after mixing the first and second agents, and a hardened product (hydrogel) with high strength was formed.

[0110] On the other hand, the combinations of UBM / genipin (Figure 5) and UBM / genipin / polyamine (Figure 6) caused gelation, but the reaction time was slow. These results demonstrated that the adhesive hardened quickly after mixing the two components and could easily form a decellularized matrix hydrogel.

[0111] Next, compound A was synthesized by varying the amount of genipin reacted with polyamine, and compound A solution (20% by mass, pH 10.5) was prepared from each compound A solution, which was then mixed with UBM solution (1% by mass, pH 2.5) to prepare a pregel solution. Viscoelasticity measurements were performed on this pregel solution in the same manner as above.

[0112] The results are shown in Figures 7 to 10. Figure 7 shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive used compound A, which was synthesized by adding 50 mol% genipin to 100 mol% of the primary amino groups of the polyamine, were mixed at 0 minutes. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. As in the above, FIGS. 8 to 10 show the results when the genipin concentrations were 60 mol %, 70 mol %, and 80 mol %, respectively.

[0113] 7 to 10, when the amount of genipin added was 60 mol % or more, the curing (gelation) became faster. On the other hand, when the amount of genipin added was 70 mol % or less, the time from mixing the two parts to curing (hereinafter referred to as "pot life") became longer. At any equivalent ratio, the curing time was sufficiently fast.

[0114] Next, multiple compound A solutions were synthesized by varying the reaction time between polyamine and genipin, and compound A solutions (20% by mass, pH 10.5) were prepared, which were then mixed with UBM solution (1% by mass, pH 2.5) to prepare pregel solutions. Viscoelasticity measurements were performed on these pregel solutions in the same manner as above.

[0115] The results are shown in Figures 11 to 14. Figure 11 shows the change in shear modulus (Pa) over time when the first and second parts of the adhesive using compound A obtained when the reaction time between genipin and polyamine was 3 minutes was set to 0 minutes. The solid circle indicates the storage modulus, and the hollow circle indicates the loss modulus. As in the above, FIGS. 12 to 14 show the results when the reaction times were 10 minutes, 30 minutes, and 60 minutes, respectively.

[0116] 11 to 14, it was found that the curing rate increased when the reaction time between genipin and polyamine was 10 minutes or more.

[0117] Next, the content of the acidic compound in the UBM solution (second agent) was adjusted to change the pH, and the solution was mixed with Compound A solution (20% by mass, pH 10.5) to prepare a pre-gel solution. Viscoelasticity measurements were performed on this pre-gel solution in the same manner as above.

[0118] The results are shown in Figures 15 to 19. Figure 15 shows the change in shear modulus (Pa) over time when the first and second parts of this adhesive were mixed at 0 minutes when the pH of the UBM solution was 2.0. The solid circles represent the storage modulus and the hollow circles represent the loss modulus. As in the above, FIGS. 16 to 19 show the results when the pH of the UBM solution was 2.5, 3.0, 5.0, and 7.0, respectively.

[0119] 15 to 19 show that when the pH of the UBM solution is less than 7.0, the hardening becomes faster, when the pH is 5.0 or less, the hardening becomes even faster, when the pH is less than 5.0, the hardening becomes even faster, and when the pH is 3.0 or less, the hardening becomes the largest. It is also found that when the pH of the UBM solution exceeds 2.0, a more sufficient pot life is obtained, and when the pH is 2.5 or more, an even more sufficient pot life is obtained.

[0120] <Swelling degree measurement> Equal amounts of UBM solution (1% by mass, pH=2.5) and Compound A solution (20% by mass, pH=10.5) were mixed and gelled at 37°C for 60 minutes. The resulting gel was cut into a disk shape with a diameter of 5 mm, immersed in PBS, and allowed to stand at 37°C to swell. The gel was collected at predetermined intervals, the swollen weight (Ws) was measured, and the dry weight (Wd) was calculated by freeze-drying. The swelling degree was calculated from the formula: (Ws-Wd) / Wd. As a result, it was found that the swelling degree of this hydrogel was about 17, and equilibrium swelling was reached in 1 hour (FIG. 20 shows the results).

[0121] <Mechanical strength measurement by tensile test> Equal amounts of UBM solution (1% by weight, pH=2.5) and compound A solutions (5, 10, and 20% by weight, pH=10.5) were mixed, poured into a silicon mold of ISO 37-2 size, and cured (gelled) at 37° C. for 60 minutes. Next, a tensile test of the cured product (gel) was carried out using a texture analyzer.

[0122] Fig. 21 shows the stress-strain relationship of the cured product. In the figure, 10%, 5%, and 2.5% respectively represent the contents of compound A in the first agent used to prepare the pre-gel solution. From Fig. 21, it was found that the mechanical strength of the cured product (hydrogel) can be controlled by the content of compound A. From the above results, it was found that from the viewpoint of increasing the breaking strength of the cured product, the content of compound A in the mixed liquid is preferably 75 mass% or more, and more preferably 85 mass% or more. In addition, it was found that from the viewpoint of increasing the maximum elongation of the cured product, the content of compound A is preferably 90 mass% or less, and more preferably 80 mass% or less.

[0123] <Cytotoxicity test of hardened material (hydrogel)> Cytotoxicity was evaluated by immersing the cured product (hydrogel) in a culture medium and adding the supernatant to the cells to be evaluated. Equal amounts of UBM solution (1% by mass, pH = 2.5) and compound A solutions (5, 10, 20% by mass, pH = 10.5) were mixed and cured (gelled) at 37°C for 60 minutes.

[0124] 10 mL of RPMI (Roswell Park Memorial Institute medium) 1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin) was added per 1 g of the cured material (hydrogel), and the mixture was left to stand at 37° C. for 24 hours, and the supernatant was collected. The toxicity of the cured material (hydrogel) was evaluated using mouse fibroblast cells (L929 cells) as the evaluation cells.

[0125] L929 cells were cultured in RPMI 1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin) at 37°C and 5% CO 2 The cells were cultured in an incubator at 1 × 10 4L929 cells were seeded in a 96-well plate and pre-cultured for 24 hours. The collected supernatant or untreated medium alone as a control was added to each well and cultured for another 24 hours. After the culture was completed, the cell number was quantified using a cell counting kit (WST-8, DOJINDO).

[0126] Fig. 22 is a graph showing cell viability (%). From the results in Fig. 22, the hydrogel which is a cured product of UBM-compound A (shown as "UBM+GeniPEG" in Fig. 22) showed high cell viability. In addition, a hydrogel that does not contain UBM (shown as "GeniPEG" in Figure 22, which is similar to "UBM + GeniPEG" except that the second agent does not contain UBM) also showed high cell viability.

[0127] On the other hand, in samples containing underivatized genipin and UBM (labeled "UBM+Genipin"), cell viability was significantly lower. This was thought to be due to the effect of genipin, as the "UBM" sample, which did not contain genipin, showed high cell viability.

[0128] Genipin has been considered to be a crosslinking agent with low cytotoxicity, but as mentioned above, according to the study by the present inventor, the cell viability rate is not sufficiently low. However, in the compound A, which is the crosslinking agent contained in the first agent of the two-agent curing adhesive of the present invention, genipin is derivatized, and genipin is difficult to dissolve in the cured material. Therefore, it is presumed that the crosslinking function of genipin is maintained while the cytotoxicity is significantly reduced. In other words, it was revealed that the hydrogel, which is the cured product of this adhesive, exhibits high cytocompatibility.

[0129] <Adhesion to collagen casing> In accordance with ASTM-F2392-04R, adhesiveness evaluation was performed using collagen casing as a model tissue for evaluating tissue adhesiveness. A pinhole with a diameter of 3 mm was made in a collagen casing with a diameter of 35 mm, and 200 μL of the pregel solution was added. The pre-gel solution was prepared by mixing 100 μL of the UBM solution (1% by mass, pH=2.5) and 100 μL of the compound A solution (20% by mass, pH=10.5).

[0130] This was left to stand at 37°C for 60 minutes and the compressive strength was measured. Figure 23 shows the results. As shown in Figure 23, the cured product of this adhesive (labeled "UBM+GeniPEG" in Figure 23) showed a high compressive strength of 50 mmHg. On the other hand, UBM alone was below the detection limit (0.75 mmHg or less), and "UBM+Genipin" showed a low value of 5.5 mmHg. These results make it clear that the cured product of this adhesive has high tissue adhesion.

[0131] <Adhesion to porcine aorta> Adhesion evaluation was performed using porcine aorta in accordance with ASTM-F2392-04R. A pinhole with a diameter of 3 mm was created in a porcine aorta cut to a diameter of 30 mm, and 200 μL of pregel solution was added to the outside of the aorta. This was left to stand at 37°C for 60 minutes, and the pressure resistance was measured. Figure 24 shows the results. The pre-gel solution was prepared by mixing 100 μL of the UBM solution (1% by mass, pH=2.5) and 100 μL of the compound A solution (20% by mass, pH=10.5).

[0132] As shown in Figure 24, the cured product of this adhesive (shown as "UBM+GeniPEG" in Figure 24) exhibited a high pressure resistance of 100mmHg. On the other hand, UBM alone was below the detection limit (0.75mmHg or less), and "UBM+Genipin" showed a low value of 3.3mmHg. These results demonstrate that the cured product of this adhesive has high tissue adhesion even to the aorta.

[0133] Moreover, Figure 25 shows the results of histological evaluation of the porcine aorta after the pressure test. Figure 25 reveals that "UBM+Genipin" did not remain on the tissue surface, whereas "UBM+GeniPEG" strongly adhered to the tissue surface and remained.

[0134] <Viscoelasticity measurements of hydrogels prepared using second agents containing various ECMs> A second agent containing a decellularized matrix (ECM) other than UBM was prepared, and the viscoelasticity of the cured product (hydrogel) was measured.

[0135] First, a portion of the pig heart, liver, pancreas, and small intestine was excised using a scalpel and washed with physiological saline. After that, it was washed with a treatment solution (0.1% by weight peracetic acid, 4% ethanol) for 2 hours, and then washed with physiological saline and ultrapure water. After that, it was washed with a 1% Triton / 0.1% SDS solution for 24 hours, and then washed with ultrapure water. After that, it was treated with a DNase solution (0.5 mg / mL) for 24 hours to remove DNA. It was further washed with physiological saline and ultrapure water, treated with pepsin (1 mg / mL) for 48 hours, and freeze-dried to obtain various matrix powders.

[0136] Using the above powder, 100 μL of pregel solution was prepared by mixing 100 μL of ECM powder solution (ECM 1% by mass, pH=2.5, second agent) prepared from UBM, heart, liver, pancreas, and small intestine, respectively, with 100 μL of compound A solution (20% by mass, pH=10.5, first agent).

[0137] 100 μL of the pregel solution was placed on the stage of the viscoelasticity measuring device and clamped with a jig with a diameter of 10 mm. The temperature of the stage was kept at 37°C during the measurement. The results are shown in Figures 26 to 31. Figure 26 shows the change in shear modulus (storage modulus: Pa) over time when the first and second agents of the adhesive containing the second agent containing urinary bladder-derived ECM (UBM) are mixed at 0 minutes. As above, Figures 27 to 30 show the results for the adhesive containing the second agent containing ECM derived from the heart, liver, pancreas, and small intestine, respectively. Also, Figure 31 shows the results for the adhesive containing a second agent consisting of PBS only (pH = 2.5) without ECM.

[0138] 26 to 31 show that all pre-gel solutions hardened (gelled) within 30 minutes. In particular, it was confirmed that the adhesive containing the second agent containing UBM or ECM derived from the small intestine hardened more quickly. 26 to 30 and FIG. 31, it was confirmed that when the second agent contains ECM, hardening occurs more quickly.

[0139] <Adhesion of hydrogels prepared using second agents containing various ECMs to collagen casings> In accordance with ASTM-F2392-04R, adhesiveness evaluation was performed using collagen casing as a model tissue for evaluating tissue adhesiveness. A 3 mm pinhole was created in a 35 mm collagen casing, and 200 μL of pregel solution was added. This was left to stand at 37° C. for 60 minutes, and the pressure resistance was measured. Figure 32 shows the results. The pregel solution was prepared by mixing 100 μL of ECM powder solution (ECM 1% by mass, pH=2.5, second agent) prepared from UBM, heart, liver, pancreas, and small intestine, respectively, with 100 μL of compound A solution (20% by mass, pH=10.5).

[0140] From Figure 32, it was confirmed that each cured material (hydrogel) had a high compressive strength of 40 mmHg or more. In particular, the two-agent composition containing UBM and the second agent containing ECM derived from small intestine showed a high compressive strength. The cured product of the curing adhesive had higher tissue adhesive strength. In FIG. 32, "Heart" indicates ECM derived from the heart, "Liver" indicates ECM derived from the liver, and "Pancreas" indicates ECM derived from the pancreas.

[0141] <Measurement using an ultraviolet-visible spectrophotometer> Compound A was synthesized in the same manner as above, except that a polyamine solution containing gelatin was used instead of the 4-branched polyethylene glycol. At that time, in order to measure the number of genipin (groups represented by formula 1) introduced per molecule of polyamine (gelatin), measurements were performed using an ultraviolet-visible spectrophotometer. Genipin was added to a gelatin solution (content 20% by mass, pH=10.5, PBS) so that the amount of genipin was 10-40 mol% relative to the primary amino group of gelatin, and mixed at 50°C. After 2 minutes, a sample was taken and the amount of genipin introduced (the amount of the group represented by formula 1 introduced) was calculated from the absorption spectrum. For the calibration curve, genipin was reacted with ethanolamine and the absorption at 290 nm was measured.

[0142] As a result, when gelatin solution was mixed with genipin, the absorption at 290 nm increased significantly, and the absorption increased with an increase in the amount of genipin added. Figure 33 shows the results of calculating the amount of genipin (group represented by formula 1) introduced (unit: %) to the amino groups in gelatin, with the horizontal axis representing the reaction time (minutes) and the vertical axis representing the amount of genipin introduced (unit: %) to the amino groups in gelatin. It was found that the introduction rate increased as the amount of genipin added (equivalent to the amino group) increased, and was 9% to 33%.

[0143] <Rheology> Compound A was synthesized by varying the amount of genipin reacted with gelatin, and a compound A solution was prepared from each (20% by mass, pH 10.5, first agent), which was then mixed with a UBM solution (1% by mass, pH 2.5, second agent) to prepare a pregel solution. Viscoelasticity measurements were performed on this pregel solution in the same manner as above.

[0144] Figures 34 to 37 show the change over time in shear storage modulus (Pa) when the time when the first and second agents of the adhesive are mixed is set to 0 minutes, the time being 0 minutes, and the adhesive contains a first agent containing compound A in which 9 to 33% genipin has been introduced relative to 100 mol % of the primary amino groups of gelatin.

[0145] 34 to 37, when the introduction rate of genipin was 18 mol % or more, hardening (gelation) became faster. On the other hand, when the introduction rate of genipin was less than 18 mol %, the pot life became longer after mixing of the two parts. The curing time was sufficiently fast at all equivalent ratios. [Industrial Applicability]

[0146] The hydrogel that is the cured product of the two-component curing adhesive of the present invention is a hydrogel that has high biocompatibility, tissue adhesiveness, and tissue regeneration ability, and is extremely useful as a tissue adhesive for medical applications. In addition, by incorporating anticancer drugs into the hydrogel, it is expected that it can be used in cancer treatment. Furthermore, in the field of regenerative medicine, it can be used as a scaffold for cells during cell transplantation.

Claims

1. a first agent including a compound A having, in the molecule, at least one primary amino group and at least one group represented by formula 1 when * is the bonding position, and a basic compound; and a second agent containing an acidic compound. 【Chemistry 1】

2. At least one selected from the group consisting of the first agent and the second agent contains a solvent; 2. The two-component curing adhesive according to claim 1, wherein when the first and second components are mixed, the resulting mixture has a pH of 8.0 or less.

3. The two-component curing adhesive according to claim 2 , wherein the mixed solution has a pH of less than 7.

0.

4. The two-component curing adhesive according to claim 2 or 3, wherein the mixed liquid has a pH of 5.0 or higher.

5. the second part comprises a solvent; The two-component curing adhesive according to any one of claims 1 to 4, wherein the second part has a pH of less than 7.

0.

6. The two-component curing adhesive according to claim 5, wherein the second component has a pH of 2.0 to 5.

0.

7. The first agent includes a solvent, The two-component curing adhesive according to any one of claims 1 to 6, wherein the pH of the first component is greater than 7.

0.

8. the compound A has a skeleton structure, the primary amino group bonded to the skeleton structure, and a group represented by formula 1; The two-component curing adhesive according to any one of claims 1 to 7, wherein the skeletal structure is derived from at least one compound selected from the group consisting of polysaccharides, peptides, proteins, gelatin, ethylenediamine, polyethyleneimine, polyalkylene glycols, polylactic acids, polyglycolic acids, polycaprolactones, and polyacrylic acids.

9. The two-component curing adhesive according to any one of claims 1 to 8, wherein compound A is a compound represented by the following formula 2: 【Chemistry 2】 (In formula 2, s1 is an integer of 1 or more, t1 is an integer of 1 or more, u1 is an integer of 0 or more, and L １ , L ２ , and L ３ are each independently a single bond or a divalent group; M １ is a s1+t1+u1 valent group, and Y １ is a hydrogen atom or a monovalent group not having a primary amino group, １ is a group represented by formula 1.

10. Said L １ , said L ２ , and the L ３ The two-component curing adhesive according to claim 9, wherein the alkyleneoxy group has 1 to 10 carbon atoms.

11. The two-component curing adhesive according to any one of claims 1 to 10, wherein compound A is a compound represented by the following formula 5: 【Chemistry 3】 (In formula 5, S5 is an integer of 1 to 3, L １ , and L ２ are each independently a divalent group containing an alkyleneoxy group having 1 to 10 carbon atoms; １ is a group represented by formula 1, M ５ is a tetravalent group.)

12. The two-component curing adhesive according to any one of claims 1 to 11, wherein the second component comprises an extracellular matrix.

13. The two-component curing adhesive according to any one of claims 2 to 7, wherein the first and second components have a pH of 5.0 to 8.0 when mixed together.

14. The two-component curing adhesive according to any one of claims 1 to 13, wherein the content ratio of the molar content of the group represented by formula 1 to the molar content of the primary amino group in compound A is 0.05 to 1.

2.

15. The two-component curing adhesive according to any one of claims 1 to 14, wherein compound A has 2 to 6 primary amino groups and 2 to 6 groups represented by formula 1.

16. A curing agent for a two-component curing adhesive, comprising compound A having, in the molecule, at least one primary amino group and at least one group represented by formula 1 when * is the bonding position. 【Chemistry 4】

17. The curing agent according to claim 16, further comprising a basic compound.

18. the compound A has a skeleton structure, the primary amino group bonded to the skeleton structure, and a group represented by formula 1; The curing agent according to claim 16 or 17, wherein the skeletal structure is derived from at least one compound selected from the group consisting of polysaccharides, peptides, proteins, gelatin, ethylenediamine, polyethyleneimine, polyalkylene glycols, polylactic acids, polyglycolic acids, polycaprolactones, and polyacrylic acids.

19. A compound having a backbone structure, a primary amino group bonded to the backbone structure, and a group represented by formula 1: the skeletal structure is derived from at least one compound selected from the group consisting of polysaccharide, gelatin, ethylenediamine, polyethyleneimine, polyalkylene glycol, polylactic acid, polyglycolic acid, polycaprolactone, and polyacrylic acid, A compound represented by the following formula 2. 【Chemistry 5】 (In formula 2, s1 is an integer of 1 or more, t1 is an integer of 1 or more, u1 is an integer of 0 or more, and L １ , L ２ , and L ３ are each independently a single bond or a divalent group; M １ is a s1+t1+u1 valent group, and Y １ is a hydrogen atom or a monovalent group having no primary amino group, and X １ is a group represented by the following formula 1 when * is the bonding position. 【Chemistry 6】 20. The compound according to claim 19, wherein L 1 , L 2 and L 3 each comprise an alkyleneoxy group having 1 to 10 carbon atoms.

21. The compound described in claim 19, represented by the following formula 5. 【Chemistry 7】 (In formula 5, S5 is an integer of 1 to 3, L 1 and L 2 are each independently a divalent group containing an alkyleneoxy group having 1 to 10 carbon atoms, X 1 is a group represented by formula 1, and M 5 is a tetravalent group.)

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