Gel-forming material and gel composition

Abstract

One embodiment of the present invention relates to a gel-forming material comprising: at least one component (A) selected from the group consisting of modified hyaluronic acid having a thiol group and salts thereof; and at least one component (B) selected from the group consisting of a modified hyaluronic acid having a maleimide group and a salt thereof.

Description

Technical Field

[0001] The present invention relates to a gel-forming material and a gel composition. Background Art

[0002] Materials that gel in situ can be used as medical materials that can be injected into the body, and therefore, they can achieve minimally invasive treatment and are attracting attention in the fields of drug carriers and tissue engineering. For materials that gel in situ, in addition to gelling in a relatively short time in response to changes in temperature, pH, and chemical substances, biocompatibility and safety are also important issues. Among various gel-forming materials, hyaluronic acid has excellent biocompatibility and safety to the human body. As a composition for obtaining a hydrogel using hyaluronic acid, for example, Patent Document 1 discloses a composition comprising one or more hydrogel-forming polymers having an intrinsic viscosity [η] of at least 3 dL / g measured by an Ubbelohde viscometer in the composition, wherein the concentration C of the hydrogel-forming polymer in the composition is T The maximum is about 5 mg / ml. However, the hydrogel obtained using the composition disclosed in Patent Document 1 requires more than 10 hours for gelation, and is therefore not suitable as a gel-forming material for in-situ gelation. [Prior art literature] [Patent Document]

[0003] Patent Document 1: Japanese Patent Publication No. 2020-534427 Summary of the invention [Technical problem to be solved by the invention]

[0004] The present invention provides a gel-forming material and a gel composition using hyaluronic acid, which can gel in a short time. The gel-forming material and the gel composition are suitable for administering medical materials that need to gel in situ into the body by injection.

[0005] An object of one embodiment of the present invention is to provide a gel-forming material capable of forming a gel in a short time and a gel composition obtained by using the gel-forming material. An object of one embodiment of the present invention is to provide a novel in situ gel-forming material. [Technical solutions for solving technical problems]

[0006] The inventors of the present invention have found a gel-forming material comprising: at least one component A selected from the group consisting of modified hyaluronic acid having a thiol group and its salt; and at least one component B selected from the group consisting of modified hyaluronic acid having a maleimide group and its salt, and the gel-forming material can solve the above-mentioned technical problems, and a gel composition containing hyaluronic acid can be obtained in a short time of within several tens of minutes (for example, within 30 minutes). The present invention was completed based on this discovery.

[0007] That is, the present invention relates to, for example, the following technical aspects. [1] A gel-forming material comprises: at least one component A selected from the group consisting of modified hyaluronic acid having a thiol group and salts thereof; and at least one component B selected from the group consisting of modified hyaluronic acid having a maleimide group and salts thereof. [2] The gel-forming material according to [1], wherein the thiol group modification rate of the component A is 5% or more and 50% or less. [3] The gel-forming material according to [1] or [2], wherein the average molecular weight of the component A is 10,000 or more and 600,000 or less. [4] The gel-forming material according to any one of [1] to [3], wherein the maleimide group modification rate of the component B is 1% or more and 30% or less. [5] A material for in situ gel formation comprises at least one component selected from the group consisting of modified hyaluronic acid having a thiol group and salts thereof. [6] A material for in situ gel formation comprises at least one component selected from the group consisting of modified hyaluronic acid having a maleimide group and salts thereof. [7] A gel composition comprises: at least one component A selected from the group consisting of modified hyaluronic acid having a thiol group and salts thereof; and at least one component B selected from the group consisting of modified hyaluronic acid having a maleimide group and salts thereof, wherein the thiol group is bonded to the maleimide group. [Effects of the invention]

[0008] According to the present invention, a gel-forming material capable of forming a gel in a short time and a gel composition obtained by using the gel-forming material can be provided. According to the present invention, a novel in-situ gel-forming material can also be provided. DETAILED DESCRIPTION

[0009] Hereinafter, the mode for implementing the present invention will be described in detail. However, the present invention is not limited to the following embodiment.

[0010] <Features of the Invention> (Gel-forming materials) The present invention is characterized in that it provides a gel-forming material comprising: at least one component A selected from the group consisting of modified hyaluronic acid having a thiol group and salts thereof; and at least one component B selected from the group consisting of modified hyaluronic acid having a maleimide group and salts thereof. The gel-forming material of the present invention can gel in a short time of less than 1 hour and can be applied to medical materials that require in situ gelation.

[0011] (Gel composition) The present invention is characterized in providing a gel composition comprising: at least one component A selected from the group consisting of modified hyaluronic acid having a thiol group and salts thereof; and at least one component B selected from the group consisting of modified hyaluronic acid having a maleimide group and salts thereof, wherein the thiol group is bonded to the maleimide group.

[0012] <Gel-forming materials> The "gel-forming material" in this specification is a material containing one or more components that can form a gel composition by reacting with other components, and in the present invention, it refers to a material that forms a gel composition within 1 hour from the start of the reaction with other components. The gel-forming material of the present invention is a material containing at least one component A selected from the group consisting of modified hyaluronic acid having a thiol group and salts thereof, and at least one component B selected from the group consisting of modified hyaluronic acid having a maleimide group and salts thereof, as described later.

[0013] <In situ gel formation> The gel-forming material of the present invention can form a gel in a short time of less than 1 hour, and therefore is suitable as an in-situ gel-forming material. The "in-situ gel-forming material" is a gel-forming material that can form a gel at a target location. The gel-forming material of the present invention can be sterilized in a state before gel formation (e.g., solution state). Therefore, sterilization is not necessarily required in a gelled state, and the sterilized gel-forming material can be administered to a target site in the body to form a gel.

[0014] <Modified hyaluronic acid having a thiol group and its salt> Component A is a compound or a salt thereof in which a thiol group (—SH) is introduced into at least a part of hyaluronic acid. The gel-forming material may contain one or more components A.

[0015] (Structure 1 of ingredient A) Component A may be a compound or a salt thereof in which at least a part of the functional groups contained in a part of the disaccharide units in the repeating unit constituting the hyaluronic acid is substituted with a functional group containing a thiol group. Component A may be a compound or a salt thereof in which at least one group selected from the group consisting of the hydroxyl group at the C4 position and the hydroxyl group at the C6 position of N-acetylglucosamine constituting the hyaluronic acid, and the hydroxyl group at the C2 position, the hydroxyl group at the C3 position, and the -OH group at the C6 position of the glucuronic acid constituting the hyaluronic acid is substituted with a functional group containing a thiol group.

[0016] (Structure 2 of Component A) Component A may be, for example, a compound represented by the following formula (1) or a salt thereof. [Chemical formula 1] In formula (1), R 1 represents -OH or a functional group containing a thiol group, R 2 ~R 5 Each of them independently represents a hydrogen atom or a functional group including a thiol group. n represents a number of 1 to 7500.

[0017] (Number and Substitution Position of Functional Groups Containing a Thiol Group in Formula (1)) In formula (1), R 1 ~R 5 At least one of the functional groups is a thiol group. In formula (1), R 1 ~R 5 1 to 4, 1 to 3 or 1 to 2 in the above formula may be a functional group containing a thiol group, and R 1 ~R 5 Any one of may be a functional group containing a thiol group. In formula (1), R 1 is a functional group containing a thiol group and R 2 ~R 5 They may all be hydrogen atoms.

[0018] (Structure 3 of ingredient A) Component A may be, for example, a compound represented by the following formula (1a) or a salt thereof. [Chemical formula 2] In formula (1a), R 1 and n are respectively the same as R in formula (1) 1 Has the same meaning as n.

[0019] (Structure of a functional group including a thiol group) The functional group containing a thiol group can be represented by the formula (I): -X 1 -Z1 A group represented by -SH.

[0020] (Z in formula (I) 1 Structure) Z 1 represents a divalent group, and X 1 A group connected to SH. 1 For example, it may be a divalent hydrocarbon group. The divalent hydrocarbon group may be linear or branched. The number of carbon atoms in the divalent hydrocarbon group may be, for example, 1 or more, 2 or more, or 10 or less, 8 or less, 6 or less, 4 or less, or 3 or less. The divalent hydrocarbon group may be, for example, an alkylene group. Z 1 It may be an alkylene group having 2 or more carbon atoms. 1 When Z is an alkylene group having 2 or more carbon atoms, the elasticity of the obtained gel composition is further improved. 1 In the case of an alkylene group having 3 or less carbon atoms, the solubility in water or PBS is further improved.

[0021] (X in formula (I) 1 Structure) X 1 At the bonding site is R 1 In the case of, it may be -NH- or -O-. 1 At the bonding site is R 2 ~R 5 In the case of , it may be -(C=O)- or a single bond.

[0022] (Specific examples of functional groups including thiol groups) The functional group containing a thiol group is R 1 In the case of, it may be -NH-CH2-CH2-SH or -O-CH2-CH2-SH. The functional group containing a thiol group is R 2 ~R 5 In the case of , it may be -(C=O)-CH2-SH or -CH2-CH2-SH.

[0023] (Salt of modified hyaluronic acid having a thiol group) The salt of the modified hyaluronic acid having a thiol group may be a pharmaceutically acceptable salt. Examples of the salt of the modified hyaluronic acid having a thiol group include sodium salts, potassium salts, and ammonium salts.

[0024] (Preferred upper limit of average molecular weight of component A) The mean molecular weight of component A can be below 1,500,000, below 1,400,000, below 1,300,000, below 1,200,000, below 1,100,000 or below 1,000,000 from the viewpoint of solubility relative to water or PBS isosolvents. When the mean molecular weight of component A is below 1,000,000, the solubility relative to water or PBS isosolvents is further excellent. The mean molecular weight of component A is preferably below 800,000, below 700,000, below 600,000, below 500,000, below 400,000, below 300,000 or below 200,000 from the viewpoint of solubility relative to water or PBS isosolvents.

[0025] (Preferred lower limit of average molecular weight of component A) The average molecular weight of component A may be 10,000 or more, 20,000 or more, 30,000 or more, 40,000 or more, 50,000 or more, 60,000 or more, 70,000 or more, or 80,000 or more from the viewpoint of obtaining a gel composition having elasticity. When the average molecular weight of component A is 80,000 or more, the elasticity of the obtained gel composition is further improved. The average molecular weight of component A is preferably 100,000 or more from the viewpoint of obtaining a gel composition having elasticity.

[0026] (Preferred numerical range of average molecular weight of component A) The average molecular weight of component A can be 10,000 to 1,500,000, 80,000 to 1,000,000, or 100,000 to 800,000 from the viewpoint of further improving solubility in solvents such as water or PBS. The average molecular weight of component A is preferably 10,000 to 600,000, more preferably 100,000 to 300,000 from the viewpoint of further improving solubility in solvents such as water or PBS.

[0027] (Method for determining average molecular weight) The average molecular weight can be measured by the following method. First, using gel filtration column, a plurality of (purification) hyaluronic acids (reference substances) with known molecular weight are subjected to liquid chromatography analysis, thus a standard curve is made according to their retention time. Similarly, the modified hyaluronic acid as the object of determination is subjected to liquid chromatography analysis, and the standard curve made is used to obtain the molecular weight, thus the molecular weight of the modified hyaluronic acid can be obtained.

[0028] (Measurement apparatus and measurement conditions of average molecular weight) The 2690 separation module manufactured by Waters Corporation of Japan was used as a liquid chromatography analysis device. The 996 photodiode array manufactured by Waters Corporation of Japan was used as a photodiode array. A column formed by connecting 1 TSK guard column PWXL (manufactured by Tosoh Corporation) and 2 TSK gel GMPW (manufactured by Tosoh Corporation) in series in the order of recording was used as a column. In the determination of the average molecular weight, the conditions of column temperature 40°C, measurement wavelength 210nm, flow rate 0.8mL / min, sample injection volume 20μL, analysis time 40 minutes, and mobile phase 0.003mol / L phosphate buffer-0.15mol / LNaCl (pH7.0) were used. The details of other experimental conditions can be as described in the embodiments described later.

[0029] (Definition of thiol group modification rate) The thiol group modification rate of component A refers to the number of thiol groups contained in each of the two sugar units constituting hyaluronic acid as one unit, specifically, the ratio (%) of the number of thiol groups contained in each of the two sugar units constituting hyaluronic acid to the one unit when the one unit is set to 100%. In this specification, "the two sugar units constituting hyaluronic acid" refers to one unit consisting of two sugars (glucuronic acid and N-acetylglucosamine) that are adjacently bonded to constitute hyaluronic acid.

[0030] (Method for determining the thiol group modification rate) The thiol group modification rate of component A can be calculated using 1 The measurement was performed by H-NMR. Specific examples of the method for preparing the sample for measurement and the measurement method are as described in the examples described later.

[0031] (Preferred lower limit of thiol group modification rate) The thiol group modification rate of component A may be 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, or 10% or more from the viewpoint of gel forming ability. When the thiol group modification rate of component A is 5% or more, the gel forming ability is further improved even when the concentration of the modified hyaluronic acid in the gel forming material is a low concentration of less than 1%.

[0032] (Preferred upper limit of thiol group modification rate) The thiol group modification rate of component A may be 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, or 50% or less from the viewpoint of solubility in solvents such as water or PBS. When the thiol group modification rate of component A is 50% or less, the solubility in solvents such as water or PBS is further improved.

[0033] (Preferred numerical range of thiol group modification rate) The thiol group modification ratio of Component A is preferably 1% to 80%, more preferably 5% to 50% or 6% to 45%, from the viewpoint of further improving the gel-forming ability in a short period of time.

[0034] (Form of Component A) In the gel-forming material, component A may be in liquid or solid form (e.g., powder form). The gel-forming material may include a solution containing component A and a solvent or a powder containing component A. As the solvent, water, a buffer solution, etc. may be listed. As the buffer solution, a phosphate buffer solution, etc. may be listed. From the viewpoint of easily obtaining a gel in a shorter time, the solvent may be a phosphate buffer solution.

[0035] (Solution containing component A) In the solution containing component A, the content of component A can be 0.1 mg or more, 0.5 mg or more, 1 mg or more, or 1.5 mg or more, and can be 25 mg or less, 15 mg or less, 10 mg or less, 7.5 mg or less, or 5 mg or less relative to 1 mL of the solvent, from the viewpoint of gel-forming ability.

[0036] (Method for producing component A) Component A can be obtained, for example, by a method including a reaction step A in which hyaluronic acid is reacted with raw material compound A in a reaction solution containing hyaluronic acid and raw material compound A containing a group capable of reacting with a functional group in hyaluronic acid and a thiol group. The method for producing component A may further include a post-treatment step A in which the reactant obtained in the reaction step A is post-treated.

[0037] (Raw material compound A) The raw material compound A can be a compound of formula (Ia): X 2 -Z 1 -SH or a salt thereof. Z in formula (Ia) 1 It can be the same as Z in formula (I) 1 In formula (Ia), X 2 Represents a group that can react with a functional group in hyaluronic acid. As a group that can react with a functional group in hyaluronic acid, for example, an amino group (-NH2) can be cited. The amount of raw material compound A used can be appropriately adjusted according to the target thiol group modification rate, etc.

[0038] (Specific example of raw material compound A) Examples of the raw material compound A include cysteamine (NH2-CH2-CH2-SH), 2-hydroxyethanethiol (HO-CH2-CH2-SH), thioglycolic acid (HOOC-CH2-SH), 2-chloroethanethiol (Cl-CH2-CH2-SH) or disulfide compounds thereof.

[0039] (Solvent in Reaction Step A) The reaction solution may contain a solvent. Examples of the solvent include water or a mixed solution of water and a water-soluble organic solvent such as ethanol.

[0040] (Reactants in Reaction Step A) The reaction solution may contain a condensing agent. As the condensing agent, for example, a carbodiimide condensing agent can be cited. As the carbodiimide condensing agent, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) can be cited. In addition to the condensing agent, the reaction solution may further contain a condensation aid. As the condensation aid, for example, 1-hydroxybenzotriazole (HOBT) can be cited.

[0041] (Post-processing step A) As the post-treatment step A, for example, a step of precipitating component A or its precursor, a step of washing component A or its precursor, a step of reacting the precursor of component A with a reducing agent, etc. can be performed. As a method for precipitating component A or its precursor, for example, a method of mixing an organic solvent such as ethanol in the reaction solution obtained after the reaction step A can be cited. As a method for washing component A or its precursor, a method of washing the precipitate containing component A or its precursor with an ethanol aqueous solution can be cited. As a reducing agent when reacting the precursor of component A with a reducing agent, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol, and mercaptoethanol can be cited.

[0042] <Modified hyaluronic acid having a maleimide group and its salt> Component B is hyaluronic acid or a salt thereof in which a maleimide group represented by the following formula is introduced into at least a part of the hyaluronic acid. The gel-forming material may contain one or more components B. [Chemical formula 3]

[0043] (Structure 1 of ingredient B) Component B may be a compound or a salt thereof in which at least a part of the functional groups contained in a part of the disaccharide units in the repeating unit constituting the hyaluronic acid is substituted with a functional group containing a maleimide group. Component B may be a compound or a salt thereof in which at least one group selected from the group consisting of -OH in the hydroxyl group at the C4 position and the hydroxyl group at the C6 position of N-acetylglucosamine constituting the hyaluronic acid, and the hydroxyl group at the C2 position, the hydroxyl group at the C3 position, and the carboxyl group at the C6 position of the glucuronic acid constituting the hyaluronic acid is substituted with a functional group containing a maleimide group.

[0044] (Structure 2 of ingredient B) Component B may be, for example, a compound represented by the following formula (2) or a salt thereof. [Chemical formula 4] In formula (2), R 6 represents -OH or a functional group containing a maleimide group, R 7 ~R 10 Each of them independently represents a hydrogen atom or a functional group including a maleimide group. m represents a number of 1 to 7500.

[0045] (Number and substitution position of functional groups including maleimide group) In formula (2), R 6 ~R 10 At least one of the functional groups is a maleimide group. 6 ~R 10 1 to 4, 1 to 3 or 1 to 2 in the above formula may be a functional group containing a maleimide group, and R 6 ~R 10 Any one of them may be a functional group containing a maleimide group. In formula (2), R 6 is a functional group containing a maleimide group and R 7 ~R 10 They may all be hydrogen atoms.

[0046] (Structure 3 of ingredient B) Component B may be, for example, a compound represented by the following formula (2a) or a salt thereof. [Chemical formula 5] In formula (2a), R 6 and m are respectively the same as R in formula (2) 6 Has the same meaning as m.

[0047] (Structure containing a maleimide functional group) The functional group containing a maleimide group can be represented by formula (II): -X 2 -Z2 -A group represented by Y. Y represents a maleimide group.

[0048] (Z in formula (II) 2 Structure) Z 2 represents a divalent group, and X 2 The group connected to Y. Z 2 For example, it may be a divalent hydrocarbon group. The divalent hydrocarbon group may be linear or branched. The number of carbon atoms in the divalent hydrocarbon group may be, for example, 1 or more, 2 or more, or 10 or less, 8 or less, 6 or less, 4 or less, or 3 or less. The divalent hydrocarbon group may be, for example, an alkylene group. Z 2 It may be an alkylene group having 2 or more carbon atoms. 2 When Z is an alkylene group having 2 or more carbon atoms, the elasticity of the obtained gel composition is further improved. 2 In the case of an alkylene group having 3 or less carbon atoms, the solubility in a solvent such as water or PBS is further improved.

[0049] (X in formula (II) 2 Structure) X 2 At the bonding site is R 6 In the case of, it may be -NH- or -O-. 2 At the bonding site is R 7 ~R 10 In the case of , it may be -(C=O)- or a single bond.

[0050] (Specific examples of functional groups including maleimide groups) The functional group containing maleimide group is R 6 In the case of, it may be -NH-CH2-CH2-Y, -NH-CH2-Y, -NH-CH2-CH2-CH2-Y or -O-CH2-CH2-Y. The maleimide-containing functional group is R 7 ~R 10 In the case of , it may be -(C=O)-CH2-Y or -CH2-CH2-Y.

[0051] (Salt of modified hyaluronic acid having a maleimide group) The salt of the modified hyaluronic acid having a maleimide group may be a pharmaceutically acceptable salt. Examples of the salt of the modified hyaluronic acid having a maleimide group include sodium salts, potassium salts, and ammonium salts.

[0052] (Preferred upper limit of average molecular weight of component B) The molecular weight average of component B can be below 100000, below 90000, below 80000, below 70000, below 60000 or below 50000 from the viewpoint of solubility with respect to water or PBS isosolvents. When the molecular weight average of component B is below 50000, the solubility with respect to water or PBS isosolvents is further excellent. The molecular weight average of component B is preferably below 10000 from the viewpoint of solubility with respect to water or PBS isosolvents.

[0053] (Preferred lower limit of average molecular weight of component B) From the viewpoint of gel forming ability, the average molecular weight of component B may be 2000 or more, 3000 or more, 5000 or more, 6000 or more, 7000 or more, or 8000 or more. When the average molecular weight of component B is 8000 or more, the gel forming ability is further improved even when the concentration of the modified hyaluronic acid in the gel forming material is a low concentration of less than 1%.

[0054] (Preferred numerical range 1 of average molecular weight of component B) The average molecular weight of Component B may be 2,000 to 100,000, 8,000 to 50,000, or 9,000 to 30,000, preferably 5,000 to 20,000, from the viewpoint of further improving solubility in solvents such as water and PBS.

[0055] (Method for measuring average molecular weight of component B) The average molecular weight of component B can be measured in the same manner as the average molecular weight of component A.

[0056] (Definition of maleimide group modification rate) The maleimide modification rate of component B refers to the number of maleimide groups contained in each of the two sugar units constituting hyaluronic acid as one unit, specifically, when the one unit is taken as 100%, the ratio (%) of the number of maleimide groups contained in each of the one unit to the one unit.

[0057] (Method for determining the maleimide group modification rate) The maleimide modification rate of component B can be 1 The measurement was performed by H-NMR. Specific examples of the method for preparing the sample for measurement and the measurement method are as described in the examples described later.

[0058] (Preferred lower limit of maleimide group modification ratio) The maleimide modification rate of component B may be 1% or more, 2% or more, 3% or more, 4% or more, 5% or more, 8% or more, 10% or more, or 12% or more from the viewpoint of gel forming ability. When the maleimide modification rate of component B is 5% or more, the gel forming ability is further improved even when the concentration of the modified hyaluronic acid in the gel forming material is a low concentration of less than 1%. The maleimide modification rate of component B is preferably 15% or more from the viewpoint of further improving the gel forming ability in a short time.

[0059] (Preferred upper limit of maleimide group modification ratio) The maleimide modification rate of component B can be 80% or less, 70% or less, 60% or less, or 50% or less from the viewpoint of solubility in solvents such as water or PBS. When the maleimide modification rate of component B is 50% or less, the solubility in solvents such as water or PBS is further improved. The maleimide modification rate of component B is preferably 30% or less, more preferably 20% or less from the viewpoint of solubility in solvents such as water or PBS.

[0060] (Preferred numerical range of maleimide group modification rate) The maleimide modification rate of component B can be 1% or more and 80% or less, 5% or more and 50% or less, or 10% or more and 45% or less from the viewpoint of making the solubility with respect to solvents such as water or PBS further excellent. The maleimide modification rate of component B is preferably 1% or more and 30% or less from the viewpoint of making the solubility with respect to solvents such as water or PBS further excellent, and is more preferably 15% or more and 25% or less from the viewpoint of making the solubility with respect to solvents such as water or PBS further excellent.

[0061] (Form of Component B) In the gel-forming material, component B may be in liquid or solid form (e.g., powder form). The gel-forming material may include a solution containing component B and a solvent or a powder containing component B. As the solvent, water, a buffer solution, etc. may be listed. As the buffer solution, a phosphate buffer solution, etc. may be listed. From the viewpoint of easily obtaining a gel in a shorter time, the solvent may be a phosphate buffer solution.

[0062] (Solution containing ingredient B) In the solution containing component B, from the viewpoint of gel forming ability, the content of component B can be 0.1 mg or more, 1 mg or more, or 1.5 mg or more, and can be 25 mg or less, 15 mg or less, 10 mg or less, 7.5 mg or less, or 5 mg or less relative to 1 mL of solvent.

[0063] (Method for producing component B) Component B can be obtained, for example, by a method including a reaction step B, in which hyaluronic acid is reacted with raw material compound B in a reaction solution containing hyaluronic acid and raw material compound B containing a group capable of reacting with a functional group in hyaluronic acid and a maleimide group. The method for producing component B may further include a post-treatment step B, in which the reactant obtained in the reaction step B is post-treated.

[0064] (Raw material compound B) The raw material compound B can be a compound of formula (IIa): X 2 -Z 2 -Y or a salt thereof. Z in formula (IIa) 2 It can be the same as Z in formula (II) 2 In formula (IIa), X 2 Represents a group that can react with the functional group in hyaluronic acid. As a group that can react with the functional group in hyaluronic acid, for example, amino (-NH2) can be listed. The usage amount of raw material compound B can be appropriately adjusted according to the maleimide modification rate as the target, etc.

[0065] (Specific example of raw material compound B) Examples of the raw material compound B include N-(2-aminoethyl)maleimide or a salt thereof (eg, hydrochloride), N-(2-hydroxyethyl)maleimide, 2-maleimidoacetic acid, and N-(2-chloroethyl)maleimide.

[0066] (Solvent and reactant in reaction step B) As the solvent and the reactant in the reaction step B, the solvents and the reactants exemplified in the reaction step A can be used.

[0067] (Post-processing step B) The post-treatment step B includes a step of precipitating component B and a step of washing component B. As a method for precipitating component B, for example, a method of mixing an organic solvent such as ethanol with the reaction solution obtained after the reaction step B can be mentioned. As a method for washing component B, a method of washing a precipitate containing component B with an ethanol aqueous solution or the like can be mentioned.

[0068] <Medication> The gel-forming material may further contain a drug. The drug may be a water-soluble drug. Examples of the drug include peptides, proteins, and genes.

[0069] <Other ingredients> The gel-forming material may contain other components that are not component A, component B, and the drug. Examples of other components include water, ethanol, phosphates, inorganic salts such as sodium chloride and potassium chloride.

[0070] <Method for gelling gel-forming material> The gel is formed by mixing the gel-forming material of the present invention, namely, component A and component B. Examples of the gelation method include a method of mixing a solution containing component A and a solution containing component B, a method of mixing a powder containing component B in a solution containing component A, and a method of mixing a powder containing component A in a solution containing component B. From the viewpoint of suppressing the generation of lumps during gel formation, the gelation method is preferably a method of mixing a solution containing component A and a solution containing component B.

[0071] (Mass ratio of component B to component A) The ratio of the mass of component B to the mass of component A (mass of component B / mass of component A) can be greater than 0.01, greater than 0.05, greater than 0.06, greater than 0.07, greater than 0.08, greater than 0.09, or greater than 0.1, and can be less than 10.0, less than 9.0, less than 8.0, less than 7.0, less than 6.0, or less than 5.0.

[0072] (Gelation temperature conditions) The temperature (gelation temperature) at which the gel-forming material is gelled may be 0°C or higher, 10°C or higher, or 15°C or higher, or 40°C or lower, or 35°C or lower, or may be room temperature. In this specification, room temperature refers to 23°C.

[0073] <Method of administering the gel-forming material> As a method for administering the gel-forming material, administration by injection can be cited. When the gel-forming material is administered into the body by injection, a syringe having a syringe body and an injection needle capable of separately accommodating a first component containing at least component A and a second component containing at least component B can be used. When using this syringe, the first component and the second component are mixed before administration into the body (in the injection needle of the syringe) to form a gel composition at the administration site.

[0074] <Biocompatible gel formation> Since the gel-forming material forms a gel composition by the reaction of component A and component B, it can form a gel composition having biocompatibility.

[0075] <In situ gel forming material 1> The in situ gel-forming material of one embodiment includes the above-mentioned component A. The in situ gel-forming material including the component A can form a gel in situ by reacting with the above-mentioned component B.

[0076] <In situ gel forming material 2> The in situ gel-forming material of one embodiment includes the above-mentioned component B. The in situ gel-forming material including the component B can form a gel in situ by reacting with the above-mentioned component A.

[0077] <Gel Composition> The gel composition includes component A and component B, and the thiol group is bonded to the maleimide group. That is, the gel composition includes a partial structure represented by the following formula (II) formed by the reaction of the thiol group and the maleimide group. The gel composition is formed by component A and component B, and therefore has biocompatibility. [Chemical formula 6]

[0078] (Gel composition form) The specific form of the gel composition can be applied to the specific form of the gel-forming material described above. The gel composition can contain the above-mentioned drugs and other components. The gel composition does not exclude the inclusion of impurities inevitably contained in the preparation.

[0079] <Method for producing gel composition> The gel composition can be obtained by a method including the step of gelling the above-mentioned gel-forming material. The gelling step can be performed, for example, by a method including mixing the above-mentioned component A with the above-mentioned component B. The conditions for gelling can be as described above.

[0080] <Application of gel composition> Examples of uses of the gel composition include local sustained-release materials for drugs, anti-adhesion materials, hemostatic materials (e.g., hemostatic materials for endoscopic surgery), wound dressings, scaffold materials for cell culture, skin filling materials, and joint injection materials.

[0081] <Sustained-release preparations> The sustained-release preparation comprises: a first component comprising component A; a second component comprising component B; and a drug. The drug may be contained in either or both of the first and second components. The above-mentioned other components may be contained in either or both of the first and second components. The sustained-release preparation contains the first and second components in a state before mixing. By mixing the first and second components before or at the same time as administration to the body, a gel composition containing the drug is formed at a target site in the body. When the target site is in the body, the gel composition containing the drug is hydrolyzed in the body, and the drug is gradually released by the disintegration of the gel composition.

[0082] <Use of Component A and / or Component B for Production of Gel-Forming Material> As one embodiment of the present invention, the use (application) of component A and component B for producing a gel-forming material is provided. As one embodiment of the present invention, the use of component A for producing an in situ gel-forming material and the use of component B for producing an in situ gel-forming material are provided. In these embodiments, the above-mentioned forms of the gel-forming material can be applied without limitation.

[0083] <Component A and / or Component B for Gel Formation> As one embodiment of the present invention, there are provided component A for gel formation and component B. As one embodiment of the present invention, there are provided component A for in situ gel formation and component B for in situ gel formation. In these embodiments, the above-mentioned forms of the gel-forming material can be applied without limitation. [Example]

[0084] Hereinafter, the present invention will be described in more detail based on Examples etc. However, the present invention is not limited to the following Examples.

[0085] [Example 1] <Method for producing modified hyaluronic acid having a thiol group> 0.5 g of hyaluronic acid (Hyabest (S) LF-P manufactured by Kewpie Co., Ltd., average molecular weight of about 300,000) was dissolved in 50 mL of pure water to obtain a hyaluronic acid solution. 106 mg of 1-hydroxybenzotriazole (HOBT), 120 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 141 mg of cystamine dihydrochloride were added to the hyaluronic acid solution and reacted overnight. 150 mL of ethanol was slowly added to the obtained reaction solution to precipitate the modified hyaluronic acid, and washed 3 times with 50 mL of 80% ethanol to obtain a precursor of modified hyaluronic acid with a thiol group. 0.5 g of the obtained precursor was dissolved in 50 mL of pure water, 180 mg of tris(2-carboxyethyl)phosphine (TCEP) was added, and reacted overnight. To the obtained reaction solution, 150 mL of ethanol was slowly added to precipitate the modified hyaluronic acid, which was then washed three times with 100 mL of 80% ethanol to obtain a modified hyaluronic acid having a thiol group (thiol group-containing modified HA).

[0086] [Example 2] <Method for producing modified hyaluronic acid having a thiol group> 0.5 g of hyaluronic acid (Hyabest (S) LF-P manufactured by Kewpie Co., Ltd., average molecular weight of about 300,000) was dissolved in 50 mL of pure water to obtain a hyaluronic acid solution. 53 mg of 1-hydroxybenzotriazole (HOBT), 60 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 71 mg of cystamine dihydrochloride were added to the hyaluronic acid solution and reacted overnight. 150 mL of ethanol was slowly added to the obtained reaction solution to precipitate the modified hyaluronic acid, and washed 3 times with 50 mL of 80% ethanol to obtain a precursor of modified hyaluronic acid with a thiol group. 0.25 g of the obtained precursor was dissolved in 25 mL of pure water, 180 mg of tris(2-carboxyethyl)phosphine (TCEP) was added, and reacted overnight. To the obtained reaction solution, 75 mL of ethanol was slowly added to precipitate the modified hyaluronic acid, which was then washed three times with 50 mL of 80% ethanol to obtain a modified hyaluronic acid having a thiol group (thiol group-containing modified HA).

[0087] [Example 3] <Method for producing modified hyaluronic acid having a maleimide group> 0.474 g of hyaluronic acid (Hyalo-Oligo manufactured by Kewpie Co., Ltd., average molecular weight 6000) was dissolved in 25 mL of pure water to obtain a hyaluronic acid solution. 211 mg of 1-hydroxybenzotriazole (HOBT), 240 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 221 mg of N-(2-aminoethyl)maleimide hydrochloride were added to the hyaluronic acid solution and reacted overnight. 100 mL of ethanol was slowly added to the obtained reaction solution to precipitate the modified hyaluronic acid, and 50 mL of 85% ethanol was used to wash 3 times to obtain a modified hyaluronic acid having a maleimide group (modified HA containing a maleimide group).

[0088] [Examples 4 to 11] <Method for producing modified hyaluronic acid having a thiol group> Thiol group-containing modified HA of Examples 4 to 11 were prepared in the same manner as in Example 1 or Example 2 except that the average molecular weight of hyaluronic acid (raw material HA) used as a raw material and the amounts of HOBT, EDC·HCl and cystamine dihydrochloride were changed to the conditions shown in Table 1. [Table 1] Example Average molecular weight of raw material HA HOBT(mg) EDC·HCI(mg) Cystamine dihydrochloride (mg) 4 300,000 14 15 18 5 300,000 27 30 36 6 300,000 424 480 564 7 1.6 million 106 120 141 8 800,000 106 120 141 9 80,000 106 120 141 10 10,000 106 120 141 11 6000 106 120 141

[0089] The raw material HA having average molecular weights of 300,000, 1,600,000, 800,000, 80,000, 10,000, and 6,000 used in the production of Examples 4 to 11 were hyaluronic acids shown below. Average molecular weight 300,000: Hyabest(S)LF-P manufactured by Kewpie Corporation Average molecular weight 1.6 million: Hyaluronsan HA-LQH manufactured by Kewpie Corporation Average molecular weight 800,000: Hyabest (J) manufactured by Kewpie Corporation Average molecular weight 80,000: Hyaluronsan HA-LQL manufactured by Kewpie Corporation Average molecular weight 10,000: Hyaluronsan HA-LF5-A manufactured by Kewpie Corporation Average molecular weight 6000: Hyalo-Oligo manufactured by Kewpie Co., Ltd.

[0090] [Examples 12 to 15] <Method for producing modified hyaluronic acid having a maleimide group> The maleimide group-containing modified HA of Examples 12 to 15 were prepared in the same manner as in Example 3 except that the average molecular weight of the hyaluronic acid (raw material HA) used as a raw material and the amounts of HOBT, EDC·HCl and cystamine dihydrochloride were changed to the conditions shown in Table 2. [Table 2] Example Average molecular weight of raw material HA HOBT(mg) EDC·HCI(mg) Maleimide hydrochloride (mg) 12 6000 422 480 442 13 6000 211 76 70 14 6000 26 30 26 15 10,000 211 240 221

[0091] The raw material HA having an average molecular weight of 10,000 and 6,000 used in the production of Examples 12 to 15 was hyaluronic acid shown below. Average molecular weight 10,000: Hyaluronsan HA-LF5-A manufactured by Kewpie Co., Ltd. Average molecular weight 6,000: Hyalo-Oligo manufactured by Kewpie Co., Ltd.

[0092] <Determination of Modification Ratio> (Sample preparation) 7 mg of a sample and 1 mg of an internal standard substance, 4,4-dimethyl-4-silapentane sodium sulfonate (DSS), were dissolved in 0.7 mL of heavy water, and the mixture was transferred to an NMR sample tube and sealed.

[0093] (Measurement conditions) Apparatus: Varian NMR system 400NB (Varian Technologies Japan Ltd.) Observation frequency: 400MHz Temperature: 30℃ Standard: DSS (0ppm) Total times: 64 times

[0094] (Analysis method) right 1 The peak derived from CH3 of N-acetyl group (2.0 ppm) of hyaluronic acid, the peak derived from CH2 proton of cysteamine (2.7 ppm), and the peak derived from -CH=CH- proton of maleimide (6.9 ppm) of H-NMR spectrum were integrated. The modification rate was calculated by the following formula from the integrated values. Thiol group modification rate (%) = (integral value of the peak value of protons derived from CH2 at 2.7 ppm / 2) / (integral value of the peak value of protons derived from CH3 at 2.0 ppm / 3) × 100 Maleimide group modification rate (%) = (integrated value of the peak value of protons derived from -CH=CH- at 6.9 ppm / 2) / (integrated value of the peak value of protons derived from CH3 at 2.0 ppm / 3) × 100

[0095] <Method for measuring average molecular weight> The modified HA containing a thiol group or the modified HA containing a maleimide group was dissolved in a mobile phase at a concentration of 0.1%, and the average molecular weight was relatively determined by the following liquid chromatography analysis. A plurality of (purified) hyaluronic acids with known average molecular weights were used as standard substances, and a standard curve was prepared based on the retention time of these, and the average molecular weight of the modified HA containing a thiol group or the modified HA containing a maleimide group was calculated.

[0096] [Test example] <Method for producing gel composition> The gel composition was prepared by mixing the aqueous solution of modified HA containing a thiol group of Examples 1, 2 and 4 to 11 with the aqueous solution of modified HA containing a maleimide group of Examples 3 and 12 to 15. The amount of modified HA containing a thiol group shown in Table 3 was dissolved in phosphate buffered saline (PBS) or water to obtain an aqueous solution of modified HA containing a thiol group. The amount of modified HA containing a maleimide group shown in Table 3 was dissolved in PBS or water to obtain an aqueous solution of modified HA containing a maleimide group. In Test Example 25, the modified HA containing a maleimide group was not dissolved in PBS or water, but the powder was used directly. In Test Examples 1 to 9, 12 to 23 and 25, when the aqueous solution of modified HA containing a maleimide group or the powder of modified HA containing a maleimide group was dropped into the aqueous solution of modified HA containing a thiol group, gelation occurred immediately. In this case, gelation is performed immediately after the modified HA aqueous solution containing a thiol group and the modified HA aqueous solution containing a maleimide group are mixed. The temperature during mixing refers to the temperature of the modified HA aqueous solution containing a thiol group and the modified HA aqueous solution containing a maleimide group or the powder of the modified HA containing a maleimide group when the two are mixed. The room temperature shown in Table 3 is 23°C. [Table 3]

Claims

1. A gel-forming material, characterized in that: The invention comprises: at least one component A selected from the group consisting of modified hyaluronic acid having a thiol group and a salt thereof; and At least one component B selected from the group consisting of modified hyaluronic acid having a maleimide group and salts thereof.

2. The gel-forming material according to claim 1, characterized in that The thiol group modification rate of the component A is 5% or more and 50% or less.

3. The gel-forming material according to claim 1 or 2, characterized in that The average molecular weight of the component A is 10,000 or more and 600,000 or less.

4. The gel-forming material according to claim 1 or 2, characterized in that The maleimide group modification rate of the component B is 1% or more and 30% or less.

5. A material for in-situ gel formation, characterized in that: The present invention contains at least one component selected from the group consisting of modified hyaluronic acid having a thiol group and salts thereof.

6. A material for in-situ gel formation, characterized in that: The present invention contains at least one component selected from the group consisting of modified hyaluronic acid having a maleimide group and salts thereof.

7. A gel composition, characterized in that: The invention comprises: at least one component A selected from the group consisting of modified hyaluronic acid having a thiol group and a salt thereof; and at least one component B selected from the group consisting of modified hyaluronic acid having a maleimide group and salts thereof, The thiol group is bonded to the maleimide group.

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