Agents used in endophthalmectomy

Abstract

An object of the present invention is to find a means useful for removing the intraocular membrane. [Solution] The present invention relates to an agent used in endothelial ablation, which contains a solution containing a hydrogel-forming material and satisfies the following formula 1 in terms of dynamic viscoelasticity measured at a temperature of 25 to 40°C and a frequency of 1 Hz. TIFF2026042818000017.tif14170

Description

[Technical Field]

[0001] The present invention is in the field of endophthalmology. [Background technology]

[0002] Vitreous retinal surgery (hereinafter simply referred to as "vitreous surgery") is performed in various vitreous retinal diseases to remove bleeding or cloudy vitreous, membranous tissue or vitreous membranes that have formed on the retina, and proliferative membranes formed by cell proliferation within the eye.

[0003] From the viewpoint of preventing recurrence of disease and the induction of new diseases, more complete removal of the vitreous membrane and proliferative membrane remaining on the retina is desirable, but removal of these is often difficult.

[0004] In Non-Patent Document 1, a forceps equipped with a piece of polyvinyl alcohol (PVA) is used to remove the retina. A technique for removing the vitreous membrane by wiping it off has been described. However, this technique makes it difficult to efficiently remove the vitreous membrane. Furthermore, the retina is physically scraped, which may cause retinal damage.

[0005] Patent Document 1 describes a retinal cleaning instrument for cleaning the retina of the eye, which includes a retinal cleaning member. The retinal cleaning member is configured to remove the vitreous membrane by wiping the retina. The retinal cleaning member includes a polymer hydrogel, optionally a crosslinked polymer hydrogel. However, as with Non-Patent Document 1, this technique has difficulty in efficiently removing the vitreous membrane. Furthermore, since the retina is physically scraped, there is a possibility of damaging the retina.

[0006] Non-Patent Document 2 describes a technique in which a viscoelastic fluid is injected between the vitreous membrane or proliferative membrane and the retina, causing the vitreous membrane or proliferative membrane to float from the retina and then be excised. Sodium hyaluronate is used as the viscoelastic fluid. However, with such an extremely sophisticated surgical procedure, it is difficult to completely float the vitreous membrane or proliferative membrane from the retina, and it is necessary to cut off the portion that is not peeled off by the viscoelastic fluid. Therefore, the procedure is complicated and there is a possibility of damaging the retina.

[0007] Patent Document 2 describes a cross-linked glycosaminoglycan that is useful as a material for treating diseases that require treatment with a long-term residual tissue distension material, such as vesicoureteral reflux, etc. However, Patent Document 2 does not describe the use of such cross-linked glycosaminoglycan in endothelial ablation.

[0008] Patent Document 3 describes a crosslinked polymer composition comprising a first synthetic polymer containing multiple nucleophilic groups covalently bonded to a second synthetic polymer containing multiple electrophilic groups. The first synthetic polymer is preferably a synthetic polypeptide or polyethylene glycol modified to contain multiple nucleophilic groups, such as primary amino (-NH) or thiol (-SH) groups. The second synthetic polymer may be a hydrophilic or hydrophobic synthetic polymer containing, or derivatized to contain, two or more electrophilic groups, such as succinimidyl groups. The composition may further contain other components, such as naturally occurring polysaccharides or proteins (e.g., glycosaminoglycans or collagen), bioactive agents, etc. The crosslinked polymer composition is used as a bioadhesive for tissue adhesion, etc. However, Patent Document 3 does not describe the use of such a crosslinked polymer composition in endothelial ablation. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO2019 / 108061 [Patent Document 2] Japanese Patent Application Laid-Open No. 2016-172783 [Patent Document 3] International Publication No. 97 / 22371 Brochure [Non-patent literature]

[0010] [Non-Patent Document 1] Acta Ophthalmologica 2019: 97: e747-e752 [Non-patent document 2] Ophthalmology Clinic Journal, 85(9): 2408(1991) Summary of the Invention [Problem to be solved by the invention]

[0011] As described above, there is a need for a more efficient and simpler method for removing intraocular membranes, such as vitreous membranes and proliferative membranes remaining on the retina. Therefore, an object of the present invention is to find a means useful for removing intraocular membranes. [Means for solving the problem]

[0012] The present invention was made in consideration of the above-mentioned problems, and in order to solve the above-mentioned problems, the inventors have intensively studied means useful for peeling off the endophthalmic membrane. As a result, they have discovered a method in which a preparation is injected into the eye to form a hydrogel that adheres to the endophthalmic membrane, and the membrane is peeled off together with the hydrogel. They have also discovered materials suitable for such a method. Based on these findings, the present invention has been completed.

[0013] That is, the present invention relates to the following: [1] A solution containing a hydrogel-forming material, An agent used in endothelial cell removal, which satisfies the following formula 1 in terms of dynamic viscoelasticity measured at a temperature of 25 to 40°C and a frequency of 1 Hz: (Formula 1) 0 < V max <= 3 However, in Equation 1, V max (Pa / sec) is the maximum rate of change in storage modulus after the onset of gelation. [2] The agent according to [1], wherein the hydrogel-forming material comprises a polymer. [3] The agent according to [2], wherein the polymer comprises a compound selected from the group consisting of polysaccharide derivatives, polyalkylene glycol derivatives, collagen derivatives, polyvinyl alcohol derivatives, and fibrinogen. [4] The agent according to [2], wherein the polymer comprises a compound selected from the group consisting of polysaccharide derivatives and polyalkylene glycol derivatives. [5] The agent according to any one of [1] to [4], wherein the gelation is caused by a crosslinking reaction. [6] The agent according to any one of [1] to [5], wherein the hydrogel-forming material contains two or more compounds. [7] The agent according to [6], wherein the gelation is initiated by mixing the two or more compounds. [8] The agent according to any one of [1] to [7], wherein the intraocular membrane is at least one selected from the group consisting of a vitreous membrane and a proliferative membrane. [9] The tensile stress measured using a texture analyzer 3 minutes after the start of gelation was -3×10 -4 N / mm 2 The agent according to any one of [1] to [8], wherein

[10] The agent according to any one of [1] to [9], which contains a visualization agent.

[11] The agent according to any one of [1] to

[10] , which satisfies the following formula 2 in dynamic viscoelasticity measured at a temperature of 25 to 40°C and a frequency of 1 Hz: (Formula 2) 0.05 ≦ V max <= 2 However, in Equation 2, V max (Pa / sec) is the same as in Equation 1.

[12] V max is the maximum change in storage modulus from the start of gelation to 900 seconds. The agent according to any one of [1] to

[11] .

[13] The agent according to any one of [1] to

[12] , wherein the hydrogel-forming material comprises one selected from the group consisting of the following glycosaminoglycan derivative A, the following glycosaminoglycan derivative B, and compound C: (1) GAG derivative A, in which a SPAAC-type reactive group is introduced to the carboxyl group of glycosaminoglycan via an amide bond and a divalent spacer group; (2) Glycosaminoglycan derivative B in which a reactive group complementary to the reactive group of (1) is introduced into the carboxyl group via an amide bond and a divalent spacer group; (3) A compound C having at least two reactive groups complementary to the reactive groups of (1) and defined by the following structure:

[0014] [ka]

[0015] [wherein Y may be the same or different and is a reactive group complementary to the reactive group of (1); Z is an n-valent spacer group; and n is an integer of 2 or greater].

[14] A syringe filled with the agent according to any one of [1] to

[13] .

[15] A method for ablating the endothelium of the eye, comprising applying the agent according to any one of [1] to

[13] onto the endothelium of the eye of a patient.

[16] The method according to

[15] , wherein the endothelium is peeled off 10 seconds or more after the intraocular administration.

[17] A drug containing a solution containing a hydrogel-forming material, which is injected into a patient's eye to form a hydrogel that adheres to the intraocular membrane, and which is used to peel off the membrane together with the hydrogel. [Effects of the Invention]

[0016] The agent used in the endothelial membrane ablation procedure of the present invention (hereinafter also referred to as "the agent of the present invention") and the method for ablation of the endothelial membrane are excellent in the effect of removing the endothelial membrane. Furthermore, the agent and the ablation method of the present invention are also superior in simplicity compared to conventional highly sophisticated surgical procedures. [Brief explanation of the drawings]

[0017] [Figure 1] Figure 1 is a schematic diagram showing one embodiment of the method for exfoliating the endophthalmic membrane of the present invention. Figure 1A shows the endophthalmic membrane adhering to the internal limiting membrane. Figure 1B shows the injection of a hydrogel-forming material onto the endophthalmic membrane. Figure 1C shows the formation of a hydrogel on the endophthalmic membrane. Figure 1D shows the exfoliation of the hydrogel that has adhered to the endophthalmic membrane and gelled. [Figure 2] FIG. 2 is a graph showing the change over time in storage modulus G′ after mixing HA-DBCO and HA-AEA. [Figure 3] Figure 3 shows the results of an evaluation of the hydrogel's ability to peel off the vitreous membrane in pig eyes (photographs substituted for drawings). Figure 3A shows the time of TA layer formation, Figure 3B shows the time immediately after the release of the hydrogel-forming material mixture, and Figure 3C shows the time of removal of the hydrogel after 5 minutes in place. [Figure 4] FIG. 4 is a photograph (a drawing substitute) showing the results of an evaluation of the vitreous membrane peeling performance of the hydrogel in pig eyes, and is a photograph after the hydrogel was removed. [Figure 5] FIG. 5 is a photograph (a drawing substitute) showing the results of an evaluation of the hydrogel's ability to peel off the vitreous membrane and proliferative membrane in a proliferative vitreoretinopathy model animal, and is a photograph taken when the hydrogel was added. [Figure 6] FIG. 6 is a photograph (a drawing substitute) showing the results of an evaluation of the hydrogel's ability to peel off the vitreous membrane and proliferative membrane in a proliferative vitreoretinopathy animal model, showing the hydrogel during removal. [Figure 7] FIG. 7 is a photograph (a drawing substitute) showing the results of an evaluation of the hydrogel's ability to peel off the vitreous membrane and proliferative membrane in a proliferative vitreoretinopathy model animal, and is a photograph after the hydrogel was partially removed. DETAILED DESCRIPTION OF THE INVENTION

[0018] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to the following embodiments.

[0019] <Agents used in endophthalmectomy> One aspect of the present invention includes a solution containing a hydrogel-forming material (hereinafter, sometimes referred to as "the hydrogel-forming material of the present invention"); The present invention relates to an agent used in endothelial cell removal, which satisfies the following formula 1 in terms of dynamic viscoelasticity measured at a temperature of 25 to 40°C and a frequency of 1 Hz. (Formula 1) 0 < V max <= 3 However, in Equation 1, V max (Pa / sec) is the maximum rate of change in storage modulus after the onset of gelation.

[0020] As used herein, a hydrogel-forming material is a material capable of forming a hydrogel. Furthermore, as used herein, a hydrogel is a gel that contains water and is poorly soluble or insoluble in water. As used herein, "poorly soluble in water" means that when immersed in water for one hour or more, the material shows little dissolution and maintains its shape prior to immersion. For example, the solubility of the hydrogel in water at 20°C may be 1 g / L or less.

[0021] As described above, the present inventors, after extensive research, have come up with a method for adhering the endophthalmic membrane, which is problematic in vitreoretinal surgery if it remains, to a viscoelastic substance and then peeling off the endophthalmic membrane together with the viscoelastic substance. Furthermore, the present inventors have come up with the idea of ​​using a hydrogel that satisfies the above-mentioned specific range of maximum change rate of storage modulus as a viscoelastic substance suitable for such a method, and have completed the present invention.

[0022] One embodiment of the method for removing intraocular membranes of the present invention will be described with reference to FIG. 1. FIG. 1A schematically illustrates the adhesion of intraocular membranes, such as the vitreous membrane and proliferative membrane remaining after vitreoretinal surgery, to the internal limiting membrane, which is the surface of the retina. The remaining vitreous membrane and the proliferative membrane formed by cell proliferation are prone to contraction, which may cause retinal detachment or other problems. Therefore, it is necessary to peel and remove the intraocular membranes, such as the remaining vitreous membrane and proliferative membrane. In one embodiment of the present invention, as shown in FIG. 1B, a hydrogel-forming material contained in the agent of the present invention is injected onto the intraocular membrane. Because the hydrogel-forming material is in the form of a solution, it can be easily injected into the vitreous cavity. As shown in FIG. 1C, a hydrogel is formed on the intraocular membrane. As shown in FIG. 1D, the gelled hydrogel that has adhered to the vitreous membrane is grasped and peeled off. In this way, the intraocular membrane can be efficiently and easily peeled off. As described above, the present invention effectively assists the surgeon's technique in intraocular membrane peeling surgery, which is performed in the narrow intraocular space.

[0023] <Maximum rate of change in storage modulus> The agent of the present invention comprises a solution containing a specific hydrogel-forming material, and after gelation begins, the agent of the present invention satisfies the above-mentioned specific range of maximum change rate of storage modulus and forms a hydrogel suitable for a method of exfoliating the intraocular membrane.

[0024] Adhesion to the endophthalmic membrane, ease of grasping and peeling of the hydrogel integrated with the endophthalmic membrane, and the ability to be injected into the eye From the viewpoint of ease of handling, such as being able to pass through a needle or the like for administering the agent and quickly gelling, the maximum rate of change in storage modulus V after the start of gelation of the agent according to the present invention is max is more than 0 Pa / sec and 3 Pa / sec or less. Preferably, for example, V max may be 0.01 Pa / sec or more, 0.05 Pa / sec or more, 0.3 Pa / sec or more, 0.4 Pa / sec or more, or 0.6 Pa / sec or more, and may be 2 Pa / sec or less, 1.5 Pa / sec or less, 1.0 Pa / sec or less, or 0.8 Pa / sec or less, or any consistent combination thereof.

[0025] The rate of change in storage modulus referred to in the present invention is the rate of change (Pa / sec) per unit time (sec) of storage modulus G' (Pa) obtained from the results of measuring dynamic viscoelasticity.

[0026] The maximum rate of change in storage modulus referred to in the present invention refers to the maximum rate of change in storage modulus within the time range from when the storage modulus increases to when it plateaus or decreases. max is the maximum value of the slope (dG' / dt) of the tangent to a time-change curve plotted with the storage modulus G' on the vertical axis and time t on the horizontal axis. For example, in a crosslinking reaction, the storage modulus of a crosslinked substance increases in proportion to the crosslink density, and the maximum change in storage modulus can be regarded as a value representing the change in storage modulus within a predetermined time range after the start of gelation due to the crosslinking reaction.

[0027] From the viewpoint that the agent of the present invention is suitably used in vitreous surgery, the above-mentioned time range may be, for example, from the start of gelation (more than 0 seconds after the start of gelation) to 900 seconds, 600 seconds, 300 seconds, 180 seconds, 100 seconds, or 60 seconds, or any compatible combination of these.

[0028] The storage modulus G' (Pa) in the present invention is a value measured based on a conventional method for measuring dynamic viscoelasticity. Specifically, for example, the value can be measured using a rheometer equipped with parallel plates spaced 0.5 mm apart as a dynamic viscoelasticity measuring device, using the endothelial cell removal agent after the start of gelation as a sample, at a frequency of 1 Hz and at a temperature of 25 to 40°C (preferably 25°C).

[0029] The maximum rate of change in storage modulus of the agent according to the present invention can be controlled, for example, by adjusting the concentration of the hydrogel-forming material in a solution containing the hydrogel-forming material, or, when a crosslinking agent is used, by adjusting the ratio of the hydrogel-forming material to the crosslinking agent, etc. More specifically, the maximum rate of change in storage modulus can be increased, for example, by increasing the concentration of the hydrogel-forming material in a solution containing the hydrogel-forming material.

[0030] <Tensile stress> The agent according to the present invention preferably has a suitable ductility from the viewpoints of adhesion to the endophthalmium, ease of grasping and peeling of the hydrogel integrated with the endophthalmium, etc. In one embodiment, the ductility of the agent according to the present invention is, for example, −3×10 as a tensile stress 3 minutes after the start of gelation. -4 N / mm 2 That's it, -2×10 -4 N / mm 2 or more, or -1.5×10 -4 N / mm 2 and may be greater than or equal to -0.1 x 10 -4 N / mm 2 Below, -0.5×10 -4 N / mm 2 , or -0.6×10 -4 N / mm 2 The following may be used:

[0031] The tensile stress in the present invention is a value measured based on a conventional tensile stress measurement method. Specifically, for example, a measurement method based on Ippei Watanabe et al., Chem. Pharm. Bull. 67 (3), 277-283 (2019) can be used. More specifically, for example, a texture analyzer can be used as a tensile stress tester, and the endothelial cell removal agent 3 minutes after the start of gelation can be used as a sample liquid, and the value measured at a temperature of 20°C to 25°C (preferably 25°C) can be used.

[0032] The tensile stress of the agent according to the present invention can be controlled, for example, by selecting the type of hydrogel-forming material.

[0033] <Hydrogel-forming materials> The hydrogel-forming material can be any material capable of forming a hydrogel satisfying the maximum rate of change in storage modulus within the above-mentioned specific range. In one embodiment, the hydrogel-forming material contains a polymer. The polymer is not particularly limited, but examples include derivatives of compounds selected from the group consisting of polysaccharides, polyalkylene glycols, collagen, and polyvinyl alcohol, etc., in which reactive functional groups have been introduced into the basic skeleton, as well as fibrinogen. Polysaccharides or polyalkylene glycols are preferred. One or more types of hydrogel-forming materials can be used in combination. From the viewpoint of ease of use in vitreous surgery, the hydrogel-forming material preferably contains a polysaccharide, and more preferably contains a glycosaminoglycan (GAG), as described below.

[0034] Examples of polysaccharides include, but are not limited to, glycosaminoglycans (GAGs) such as hyaluronic acid, alginic acid, celluloses, dextrans, chitosan, and medically acceptable salts thereof.

[0035] Glycosaminoglycans are acidic polysaccharides that have a repeating disaccharide unit consisting of an amino sugar (glucosamine, galactosamine) and uronic acid or galactose. Examples of such GAGs include hyaluronic acid, heparin, heparan sulfate, and keratan sulfate. Among these, hyaluronic acid is particularly preferred in the present invention.

[0036] In the present invention, the origin of alginic acid is not particularly limited.

[0037] Known celluloses can be used, such as cellulose, carboxymethyl cellulose, etc. In the present invention, the origin of the celluloses is not particularly limited. Known dextrans can be used, for example, carboxymethyl dextran, etc. In the present invention, the origin of the dextrans is not particularly limited.

[0038] In the present invention, the origin of chitosan is not particularly limited. In the present invention, the degree of deacetylation of chitosan is not particularly limited, but may be, for example, 70 to 100%.

[0039] Examples of pharmaceutically acceptable salts include alkali metal salts such as sodium salts and potassium salts, and alkaline earth metal salts such as magnesium salts and calcium salts.

[0040] The polyalkylene glycol is not limited, but examples thereof include those in which the number of carbon atoms in the alkylene group, which is a constituent unit of the polyalkylene glycol, is, for example, 2 to 4, preferably 2 or 3, and more preferably 2. Specific examples include polyethylene glycol, polypropylene glycol, polybutylene glycol, etc. A preferred example of the polyalkylene glycol is polyethylene glycol. Furthermore, the polyalkylene glycol may also be one having a multi-branched polyalkylene glycol structure.

[0041] Collagen is a protein that mainly constitutes the dermis, ligaments, tendons, bones, cartilage, etc. of vertebrates, and includes types I to XIX, etc., and any of these can be used. In the present invention, the origin of collagen is not particularly limited.

[0042] Examples of polyvinyl alcohol include, but are not limited to, polyvinyl alcohol acetate, polyvinyl alcohol formate, polyvinyl alcohol benzoate, polyvinyl alcohol stearate, polyvinyl alcohol chloroacetate, polyvinyl alcohol fluoroacetate, and polyvinyl alcohol propionate. Polyvinyl alcohol is preferably polyvinyl alcohol acetate. Polyvinyl alcohols having a hyperbranched polyvinyl alcohol structure can also be used.

[0043] Fibrinogen is a protein involved in blood clotting. The enzyme thrombin acts on fibrinogen to form a paste-like clot, which is used in a commercially available preparation (fibrin glue) for sealing tissue, gluing damaged organs, and stopping bleeding.

[0044] The type of fibrinogen is not particularly limited, and may be derived from blood or produced by recombinant technology. Commercially available fibrin glues can also be used. Examples of commercially available fibrin glues that can be used in the present invention include, but are not limited to, Bolheal (KM Biologics), Tiseal (Baxter), and Veriplast (CSL Behring).

[0045] The derivatives herein may be derivatives that have been derivatized so as to be capable of forming a hydrogel. The hydrogel-forming material of the present invention may form a hydrogel using reactive functional groups that the material originally has, or may be a derivative into which reactive functional groups capable of forming a hydrogel have been introduced. Derivatives derivatized to form hydrogels may include, but are not limited to, those having a reactive functional group. The reactive functional group is not particularly limited as long as it is capable of gelation. Examples include electrophilic functional groups and nucleophilic functional groups such as carbodiimide groups, carbonylimidazole groups, sulfonyl chloride groups, chlorocarbonate groups, N-hydroxysuccinimidyl ester groups, succinimidyl ester groups, sulfasuccinimidyl ester groups, N-hydroxyethoxylated succinimide ester groups, methane diisocyanate groups, methylene-bis(4-cyclohexylisocyanate) groups, isocyanate groups, diisocyanate groups, hexamethylene diisocyanate groups, maleimide groups, alkynyl groups, alkynylene groups, vinyl groups, acryloyl groups, methacryloyl groups, amino groups, hydroxy groups, carboxy groups, thiol groups, azide groups, and hydrazide groups. The reactive functional group can be selected, introduced, etc., by a conventionally known method. For example, the degree of substitution with the reactive functional group in the polysaccharide derivative (the number of substituents relative to the number of repeating units expressed as a percentage) is usually 1 to 60%, preferably 5 to 40%.

[0046] The molecular weight of the hydrogel-forming material used in the present invention is not particularly limited as long as it can form a hydrogel that satisfies the maximum rate of change in storage modulus within the above-mentioned specific range, but for example, a material with a weight-average molecular weight of approximately 500 to 10,000,000 can be used. Note that the molecular weight of the hydrogel-forming material in this specification is a value determined using gel permeation chromatography (GPC).

[0047] The agent of the present invention includes both of the following forms: (1) a form in which the hydrogel-forming material is contained in a container in the form of a solution beforehand, and (2) a form in which the hydrogel-forming material is contained in a container in a dry state and is dissolved in a solvent when used. The solvent used in the solution containing the hydrogel-forming material is not particularly limited, and examples thereof include water, ocular irrigation solution (BSS), and phosphate buffered saline (PBS).

[0048] The concentration of the hydrogel-forming material in the solution containing the hydrogel-forming material is not particularly limited, and may be, for example, 0.2% by weight or more, 0.8% by weight or more, 0.9% by weight or more, or 1.0% by weight or more, and may be 10% by weight or less, 3% by weight or less, 2% by weight or less, or 1.5% by weight or less, and may be any combination that is not contradictory thereto.

[0049] ≪Gelation≫ Generally, the starting point of gelation is recognized as the point at which the relationship between the storage modulus G’ (Pa) and the loss modulus G” (Pa) in the measurement of dynamic viscoelasticity changes from G’ ≦ G” to G” < G’. The method for forming a hydrogel using a hydrogel-forming material is not limited, and for example, a three-dimensional network structure may be formed and gelled by crosslinking formed between hydrogel-forming materials. In a preferred embodiment of the present invention, a hydrogel-forming material that is gelled by a crosslinking reaction is used.

[0050] The agent according to the present invention may include a solution containing a compound that is one type of hydrogel-forming material, or may include a plurality of solutions each containing any combination of compounds that are two or more types of hydrogel-forming materials. In one embodiment, in the case of a combination of compounds that are two types of hydrogel-forming materials, it may include a solution (1) containing one of the combinations and a solution (2) containing the other of the combinations.

[0051] In one embodiment, the combination is a combination of compounds into which functional groups that form a crosslinked structure in pairs (hereinafter sometimes referred to as “reactive functional groups and reactive functional groups complementary to the reactive functional groups”) are separately introduced, and a hydrogel is formed by a crosslinking reaction between the functional groups. In one embodiment, a hydrogel-forming material having a reactive functional group and a compound having a reactive functional group complementary to the reactive functional group are contained in separate containers, and in a more specific embodiment, the compound having the complementary reactive functional group is also a hydrogel-forming material.

[0052] When two or more compounds are used in the agent of the present invention, the agent may, for example, include a combination of a hydrogel-forming material having a reactive functional group with a hydrogel-forming material having a reactive functional group complementary to the reactive functional group, or may include a combination of a hydrogel-forming material having a reactive functional group with a crosslinker. When two or more compounds are used as the hydrogel-forming material, for example, gelation may be initiated by mixing the two or more compounds.

[0053] When two or more types of compounds are used, the amount of each compound can be appropriately selected depending on the type of reactive functional group, the performance of the desired hydrogel, etc., but the reactive functional group and the reactive functional group complementary to the reactive functional group can be blended in a molar ratio of 4:1 to 1:4, 2:1 to 1:2, or 1:1.

[0054] The solution (1) contains one of the combinations, and the solution (2) contains the other of the combinations, but each solution may further contain a different combination of materials that form a hydrogel. That is, in addition to a one-solution, one-solute form in which one solution contains one material, a one-solution, multi-solute form in which one solution contains two or more solutes can also be used as a solution in the present invention, as long as no undesirable phenomena occur in the single solution.

[0055] In this embodiment, a solution (1) containing one of the combinations of at least two compounds that form a hydrogel and a solution (2) containing the other compound are used, but this does not mean that only these two types of solutions are used. Therefore, as long as the formation of the hydrogel is not impaired, Additional solutions containing different combinations of hydrogel-forming compounds may also be utilized. That is, the combination of solutions may be one or more, and this aspect also includes an embodiment in which a solution other than solution (1) and solution (2) containing a combination of compounds other than the combination contained in solution (1) and solution (2) is used together with solution (1) and solution (2).

[0056] The two different functional groups that form a pair to form a crosslinked structure are not limited, but a preferred example is a combination of functional groups that undergo a click reaction, such as a combination of an azide and an alkyne (azide-alkyne cycloaddition (Huisgen cycloaddition)). .

[0057] One example of a hydrogel that can be used in the present invention and has a combination of functional groups that can undergo such a click reaction is a GAG crosslinker described in Japanese Patent Application Laid-Open No. 2016-172783, which is obtained by subjecting a derivative in which a SPAAC (strain-promoted azide-alkyne cycloaddition) reactive (functional) group has been introduced into a GAG such as hyaluronic acid to an SPAAC reaction. Following the teachings of Japanese Patent Application Laid-Open No. 2016-172783, gelation can be achieved by adjusting the properties of solutions containing each derivative and mixing them.

[0058] More specifically, one of the hydrogels that can be used in the present invention is made of the following crosslinked product: Between the carboxyl group of the first GAG molecule and the carboxyl group of the second GAG molecule, there is provided the following group: -CONH-R 1 -XR 2 -NHCO-, or -CONH-R 3 -XR 4 -X'-R 5 -NHCO- [In the formula, -CONH and NHCO- at both ends represent amide bonds via the carboxyl groups of the GAG ​​molecules. R 1 , R2 , R 3 , R 4 , and R 5 are the same or different and are an alkylene group, an alkenylene group, group or an alkynylene group, and -CH2- in the group is >C=O (i.e., -C(= -O)-, -CONH-, arylene, -O-, or -S-; X and X′ may be the same or different and represent the formula:

[0059] [ka]

[0060] The structure shown here is that the A and B bonds can be oriented in either direction; where A and B represent binding sites; Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , and Y 6 are the same or different, -CR 6 R 6’ -, -C(-R 6 )=, -NR 7 represents -, ═N-, -O-, or -S-, and -NR 7 -, ═N-, -O-, and -S- cannot be adjacent; R 6 and R 6’ are the same or different and represent a hydrogen atom, a halogen atom, a hydroxyl group, an amino group which may be mono- or di-substituted with alkyl, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group or a carboxyl group, or are taken together to form an oxo group. In the alkyl group, alkenyl group, alkynyl group, or alkoxy group, -CH2- is replaced by >C=O, -CONH-, arylene, -O-, or -S-. Also well; R 7represents an alkyl group, an alkenyl group, or an alkynyl group, and -CH2- in the alkyl group, alkenyl group, or alkynyl group is not a C=O, -CONH-, arylene, -O - or -S-; or Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , and Y 6 In two adjacent groups among 6 and R 6’ can form a saturated or unsaturated 3- to 6-membered ring together with the ring atoms to which it is bonded, and the bond B can also be bonded to the 3- to 6-membered ring. A GAG crosslinked body in which the first GAG molecule and the second GAG molecule are linked via an amide bond (the first GAG molecule and the second GAG molecule may be the same molecule).

[0061] One embodiment of the GAG ​​crosslinked product is a product of the formula:

[0062] [ka]

[0063] [In the formula, R 8 and R 9 represents a hydrogen atom or a hydroxyl group; 8 If is a hydroxyl group, R 9 is a hydrogen atom, and R 8 If is a hydrogen atom, R 9 is a hydroxyl group; R 10 is R 11 or represents a bridge as described above; R 11 is ONa or OH], The basic skeleton is a repeating structure of the structural unit represented by R 8 is a hydroxyl group In such a case, preferred examples of crosslinked GAGs include those in which at least one of the hydroxyl groups in the structural unit is -OSO3Na or -OSO3H.

[0064] The agent of the present invention may contain, as a hydrogel-forming material, one selected from the group consisting of (1) GAG derivative A, (2) GAG derivative B, and (3) compound C below. (1) GAG derivative A, in which a SPAAC-type reactive group was introduced into the carboxyl group of GAG via an amide bond and a divalent spacer group; (2) GAG derivative B in which a reactive group complementary to the reactive group of (1) is introduced into the carboxyl group via an amide bond and a divalent spacer group; (3) A compound C having at least two reactive groups complementary to the reactive groups of (1) and defined by the following structure:

[0065] [ka]

[0066] [In the formula, Y is the same or different and is a reactive group complementary to the reactive group of (1); Z is an n-valent spacer group, and n is an integer of 2 or more. That is, the agent according to one embodiment of the present invention is The combination includes (1) GAG derivative A and (2) GAG derivative B; or (1) GAG derivative A and (3) compound C.

[0067] In the GAG ​​derivatives A and B constituting the agent of the present invention, the SPAAC-type reactive group or a reactive group complementary to the reactive group is amide-bonded to the carboxyl group of the GAG ​​via a divalent spacer group. Any chain-like group can be used as the divalent spacer group as long as it does not inhibit the reaction between the SPAAC-type reactive group and the reactive group complementary to the reactive group. As such a divalent spacer group, the group R used in the above-mentioned GAG crosslinked product can be used. 1 , R 2 , R 3 , R 4 , and R 5, i.e., alkylene group, alkenylene A group or an alkynylene group can be used, and -CH2- in the group can be >C=O, -C It may be replaced by ONH-, arylene, -O-, or -S-.

[0068] The n-valent spacer contained in the compound C constituting the agent according to the present invention is the same as the divalent spacer, and an n-valent group derived from an alkyl group, an alkenyl group, or an alkynyl group can be used, and -CH2- in the group can be >C=O, -CONH-, arylene, -O The arylene may be substituted with - or -S-. Here, the arylene may be a phenylene group such as 1,2-, 1,3-, or 1,4-phenylene, and among these, a 1,4-phenylene group is preferably used. Here, n is an integer of 2 or more, preferably 2.

[0069] In the agent of the present invention, GAG derivatives A and B, and compound C, have an SPAAC-type reactive group or a reactive group complementary to the reactive group. The SPAAC-type reaction refers to a click reaction in which an azide group reacts with an alkyne group to form a 1,2,3-triazole ring, using a cycloalkynylenyl group as the alkyne group. The click reaction can rapidly, easily, and efficiently form a 1,2,3-triazole ring without producing undesired by-products. By using a cycloalkynylenyl group as the alkyne group, a copper catalyst is not required, and the crosslinking reaction proceeds quickly and highly selectively due to the distortion of the ring structure. The GAG ​​derivatives A and B, and compound C, which can be used in the agent of the present invention, can have any SPAAC-type reactive group or a reactive group complementary to the reactive group. Specifically, the SPAAC reactive group or a reactive group complementary to the reactive group can be a combination of a group derived from a cycloalkynylenyl group having 7 to 9 carbon atoms, preferably 7 or 8 carbon atoms, more preferably 8 carbon atoms, and an azide group. The cycloalkynylenyl group can be a group represented by the formula:

[0070] [ka]

[0071] [In the formula, where B represents a binding site to a spacer group (e.g., a divalent spacer group); Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , and Y 6 are the same or different, -CR 6 R 6’ -, -C(-R 6 )=, -NR 7 represents -, ═N-, -O-, or -S-, and -NR 7 -, ═N-, -O-, and -S- cannot be adjacent; R 6 and R 6’ are the same or different and represent a hydrogen atom, a halogen atom, a hydroxyl group, an amino group which may be mono- or di-substituted with alkyl, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group or a carboxyl group, or are taken together to form an oxo group, and -CH in the alkyl group, alkenyl group, alkynyl group or alkoxy group 2- is replaced by >C=O, -CONH-, arylene, -O-, or -S-. It's okay; R 7 represents an alkyl group, an alkenyl group, or an alkynyl group, and -CH2- in the alkyl group, alkenyl group, or alkynyl group is not a C=O, -CONH-, arylene, -O - or -S-; or Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , and Y 6 In two adjacent groups among 6 and R 6’can form a saturated or unsaturated 3- to 6-membered ring together with the ring atoms to which it is bonded, and the bond B can also be bonded to the 3- to 6-membered ring. Examples of such 3- to 6-membered rings include 3- to 6-membered cycloalkyl rings, phenyl rings, and 5- or 6-membered heteroaryl rings.

[0072] Preferred examples of such SPAAC-type reactive groups or reactive groups complementary to such reactive groups include combinations of reactive groups having the following skeletons with azide groups.

[0073] [ka]

[0074] Even more preferred examples include combinations of the following groups with an azide group:

[0075] [ka]

[0076] Of the GAG ​​derivatives A and B, the GAG ​​derivative having a cycloalkynylenyl group as the SPAAC-type reactive group is preferably the following:

[0077] [ka]

[0078] Examples of the GAG ​​derivative include a cyclooctyne derivative selected from the above, in which the amino group of the amine is bonded to the carboxyl group of the GAG ​​via an amide bond.

[0079] Of the GAG ​​derivatives A and B, the GAG ​​derivative having an azide group as the SPAAC-type reactive group is preferably the following: N3-CH2-CH2-NH2; N3-CH2-CH2-CH2-NH2; N3-CH2-CH2-CH2-CH2-NH2; N3-CH2-C(=O)-NH-CH2-CH2-NH2; N3-CH2-CH2-O-CH2-CH2-NH2; N3-CH2-[CH2-O-CH2] 2-10 -CH2-NH2; Examples of GAG derivatives include those obtained by condensation reaction of the amino group of an azidoamine selected from the group consisting of the above with the carboxyl group of GAG.

[0080] As the compound C, the following compounds can be preferably mentioned.

[0081] [ka]

[0082] In the agent of the present invention, the SPAAC-type reactive groups possessed by GAG derivatives A, B and compound C and the reactive groups complementary to said reactive groups are present in a molar ratio of 1:1 to 1:4, preferably a molar ratio of 1:1.

[0083] Another hydrogel that can be used in the present invention is a hydrogel that is composed of a first synthetic polymer containing multiple nucleophilic groups, such as primary amino (-NH2) or thiol (-SH) groups, and a second synthetic polymer containing multiple electrophilic groups, such as succinimidyl groups, as described in International Publication No. WO 97 / 22371. According to the teachings of WO 97 / 22371, gelation can be achieved by adjusting the properties of solutions containing each derivative and mixing them.

[0084] Another hydrogel that can be used in the present invention is a hydrogel consisting of a combination of a multi-armed polyalkylene glycol (multi-arm-PEG) derivative, preferably a multi-armed polyethylene glycol (PEG) derivative containing an N-hydroxysuccinimidyl ester group, and a compound containing a reactive functional group complementary to the N-hydroxysuccinimidyl ester group. A multi-armed PEG derivative is a compound in which multiple PEG derivatives containing reactive functional groups are bonded to the end opposite the reactive functional group. The multi-armed polyalkylene glycol derivative and the compound containing a reactive functional group complementary to the N-hydroxysuccinimidyl ester group can be, for example, commercially available products.

[0085] There are no particular limitations on the number of branches and the type of substituents on PEG. Therefore, structures not found in commercially available multi-branched PEG derivatives, such as 8-branched PEG derivatives having maleimide groups or 6-branched PEG derivatives having thiol groups, can be prepared according to the synthetic methods of commercially available multi-branched PEG derivatives, and such multi-branched PEG derivatives can also be used in the present invention.

[0086] Another type of hydrogel that can be used in the present invention is a hydrogel that is composed of a combination of a hyaluronic acid derivative containing a thiol group and a compound containing a reactive functional group complementary to the thiol group.The hyaluronic acid derivative containing a thiol group and the compound containing a reactive functional group complementary to the thiol group can be, for example, commercially available products.

[0087] <Target> The agents of the present invention can be used in any animals that can suffer from eye diseases, such as humans and non-human animals (for example, dogs, cats, rabbits, rats, mice, etc.).

[0088] Vitreous surgery in the present invention refers to surgery to remove the endophthalmium for the purpose of treating, preventing, or preventing recurrence of eye diseases. Here, the endophthalmium includes, for example, the vitreous membrane or the proliferative membrane.

[0089] Examples of eye diseases include, but are not limited to, retinal detachment, diabetic retinopathy, proliferative vitreoretinopathy, proliferative diabetic retinopathy, macular diseases (macular hole, premacular membrane, vitreomacular traction syndrome, macular edema, age-related macular degeneration), etc.

[0090] The dosage of the agent according to the present invention varies depending on the symptoms of the patient to be administered, particularly the condition of the eyes, age, administration method, etc., but it is sufficient that the amount of the agent for endophthalmectomy administered into the vitreous cavity allows the endophthalmium to be visualized. For example, the amount of the agent used for endophthalmectomy during vitreous surgery is typically 1 to 100 mg, but is not limited thereto.

[0091] <Visualization agent> In addition to the above components, the agent according to the present invention may further contain a vitreous visualization agent, which is an auxiliary agent during vitreous surgery. The visualization agent aids in the peeling of the endothelium by visualizing the vitreous in the method for peeling the endothelium using the agent according to the present invention.

[0092] The visualization agent is not particularly limited as long as it is used as an auxiliary agent in vitreous surgery, and examples thereof include dyes such as brilliant blue G, triamcillonone acetonide, etc. Regarding application, etc., the method used in vitreous surgery can be referred to.

[0093] <Syringe> One aspect of the present invention is a drug for use in endothelial cell removal according to the present invention, which is injectable after gelation has begun. Another aspect of the present invention relates to a syringe filled with the agent of the present invention (hereinafter, sometimes referred to as the "syringe of the present invention"). Note that all of the matters described above regarding the agent used in endophthalmectomy also apply to the description of the syringe of the present invention.

[0094] The agent of the present invention is administered into the vitreous cavity during vitreous surgery and is preferably used for endophthalmectomy, and therefore its administration form is preferably injection, particularly local administration by injection.

[0095] For example, the syringe of the present invention contains the agent of the present invention in a syringe chamber, and a plunger is connected to the base end of the syringe chamber. The syringe chamber has a discharge port for discharging the agent for endophthalmectomy at the other base end. The discharge port may be provided with a connector for connecting an injection needle.

[0096] The material of the syringe is not limited as long as it can stably hold the solution containing the hydrogel-forming material, but examples that can be used include glass, which has excellent visibility, polyolefin-based plastics such as polyethylene and polypropylene, and plastics such as polyethylene terephthalate and polycarbonate.

[0097] When gelation is initiated by mixing two or more compounds, the syringe of the present invention is preferably a syringe having two or more chambers in the syringe, each chamber containing an agent of the present invention, and capable of mixing the two or more compounds to form a hydrogel when the plunger is advanced. In one embodiment, a solution containing a hydrogel-forming material having a reactive functional group and a solution containing a compound having a reactive functional group complementary to the reactive functional group are contained in separate syringe chambers. In a more specific embodiment, the compound having the complementary reactive functional group is also a hydrogel-forming material. In a further embodiment, two or more solutions contained in the separate syringe chambers are mixed at an outlet provided on one base end side of the syringe chamber, and gelation is initiated by this mixing.

[0098] The injection needle is not limited as long as it can eject the hydrogel, but for example, an injection needle of 25G to 27G can be used.

[0099] <Method of endophthalmic removal> One aspect of the present invention relates to a method for ablating the endophthalmic lining of a patient (hereinafter, sometimes referred to as the "method for ablating the endophthalmic lining of the present invention"), which comprises applying an agent according to the present invention to the endophthalmic lining of a patient. Note that all of the matters described above regarding the agent and syringe used in the endophthalmic ablating procedure also apply to the description of the method for ablating the endophthalmic lining of the present invention.

[0100] Another preferred embodiment of the present invention is a method for exfoliating the endophthalmic lining, which further comprises exfoliating the endophthalmic lining. The method for exfoliating the endophthalmic lining can be the same as the conventionally known endophthalmic lining exfoliation techniques used in vitreous surgery, except for the use of a hydrogel. However, by using the agent of the present invention, the endophthalmic lining to which the hydrogel has adhered can be exfoliated, which makes it possible to exfoliate the endophthalmic lining more efficiently than when exfoliating the endophthalmic lining alone.

[0101] From the viewpoint of operability, etc., the agent according to the present invention may be administered intraocularly, for example, but not limited to, in a manner similar to that described above. The endothelial membrane can be peeled off after 0 seconds or more, 20 seconds or more, or 30 seconds or more have elapsed. The endothelial membrane can be peeled off, for example, within 1 hour, within 30 minutes, or within 15 minutes after the intraocular administration of the agent of the present invention. [Example]

[0102] Hereinafter, preferred embodiments of the present invention will be described in more detail using examples, but the technical scope of the present invention is not limited to the following examples.

[0103] Example 1 1-1. Preparation of hydrogel-forming materials A two-liquid mixed hydrogel-forming material was prepared according to the method described in JP 2016-172783 A. The specific method is shown below.

[0104] A 50% (v / v) aqueous ethanol solution of sodium hyaluronate (weight-average molecular weight: approximately 300,000) was prepared (final sodium hyaluronate concentration: 5 mg / mL, HA reaction solution).

[0105] Dibenzocyclooctyne-amine (DBCO-amine) in 0.5M hydrochloric acid / ethanol A 1:1 (v / v) mixture of DBCO-amine and HA was prepared (final DBCO-amine concentration: 0.12 mmol / g, DBCO-amine solution). 1 mL of DBCO-amine solution was added to the HA reaction solution (60 mL) in the presence of a condensing agent (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride; DMT-MM). After stirring overnight at 25 °C, the reaction was stopped by adjusting the pH to 11 or higher with aqueous sodium hydroxide. The pH of the reaction solution was then adjusted to 6–7 with aqueous acetic acid. 1.5 g of sodium chloride and 90 mL of ethanol were added to the reaction solution to precipitate the product. After removing the supernatant, the precipitate was washed three times with ethanol. The resulting precipitate was dried to obtain HA-DBCO (hydrogel-forming material 1).

[0106] A 50% (v / v) aqueous solution of 2-azidoethylamine hydrochloride (AEA) in ethanol was prepared (final AEA concentration: 0.12 mmol / g, AEA solution). In the presence of a condensing agent (DMT-MM), 1 mL of the AEA solution was added to the HA reaction solution (60 mL). The same process as for preparing HA-DBCO was then carried out to obtain HA-AEA (hydrogel-forming material 2).

[0107] 1-2. Measurement of dynamic viscoelasticity HA-DBCO and HA-AEA solutions were prepared at various concentrations using BSS Plus 500 Ocular Irrigation Solution 0.0184% (BSS, Alcon). As an indicator of gelation of the hydrogel-forming materials at various concentrations, the HA-DBCO and HA-AEA solutions were mixed at equal weights, and then the dynamic viscoelasticity was measured immediately. Brilliant Blue G (final concentration: 0.27 mg / mL, BBG) was added as a visualization agent to the test samples used for dynamic viscoelasticity measurements when the HA-DBCO and HA-AEA solutions were mixed.

[0108] (Measuring Instruments) Rheometer: Modular Compact Rheometer MCR302 (Anton Paar) Probe: PPT25-SN38699 [Distance between the rheometer sample stage surface and the probe surface: 0.5 mm] Sample volume: 280 μL Measurement temperature: 25℃ Frequency: 1Hz Measurement time: 900 seconds Interval: 1 second Analysis software: RHEOPLUS / 32 V3.62.

[0109] 1-3.In vitro evaluation of peeling performance Using a triamcinolone acetonide (TA) formulation (MacuAid®, Wakamoto Pharmaceutical Co., Ltd.), which is used as a visualization agent during vitreous surgery, as a model, the peeling performance of hydrogel-forming materials of various concentrations was evaluated. 40 mg of Macuaid® was suspended in BSS at a concentration of 10 mg / mL to obtain a TA mother solution. BSS was dispensed into a 24-well plate at 2 mL / well. 200 μL of the vortexed TA mother solution was dispensed into each well (2 mg TA / well) to form a TA layer on the bottom of the well. 350 μL of a mixture of HA-DBCO and HA-AEA (prepared by the same method as in 1-2 above, 5–10 seconds after stirring) was quickly dispensed to cover the entire surface of the TA layer. After allowing the mixture to remain in the well for a desired period of time to form a hydrogel, the hydrogel was removed from the well using tweezers. The TA remaining in the well was collected in a microcentrifuge tube and centrifuged (>9,200 × g, 5 minutes) to remove the supernatant. The precipitated TA was suspended in water, and the turbidity (OD 660 nm) was measured using a spectrophotometer. The turbidity of a 1 mg / mL TA / BSS suspension was used as a control, and the amount of TA remaining in each well was determined. The detachment rate was calculated from the amount of TA dispensed into the well and the amount of TA remaining.

[0110] Peeling rate (%) = (2 mg - remaining amount of TA) / 2 mg x 100

[0111] 1-4.Results The results for a 5-minute retention time are shown in Figure 2. Gelation began immediately after mixing HA-DBCO and HA-AEA. As shown in Figure 2, the change in storage modulus of each test sample exhibited a sigmoid curve-like shape. From the results of dynamic viscoelasticity measurements, the maximum rate of change (V) of storage modulus (G' (Pa)) per unit time (seconds) was max (Pa / sec) was calculated.

[0112] The results are shown in Table 1. The concentration of the hydrogel-forming material refers to the total amount of HA-DBCO and HA-AEA. The retention time indicates the time the hydrogel-forming material was retained. The detachment rate is the average value of the detachment performance evaluation performed in duplicate.

[0113] [Table 1]

[0114] V after gelation begins max It was suggested that the intraocular membrane can be efficiently peeled off by using a solution containing a hydrogel-forming material in which the value is greater than 0 and less than 3.

[0115] The storage modulus of OPEGANHI (registered trademark), a commercially available cataract surgery / intraocular lens insertion aid (1% sodium hyaluronate preparation), did not change over time (V max was 0.00 (Pa / sec), and TA could not be removed.

[0116] Example 2 2-1.Ex vivo evaluation of peeling performance Fresh pig eyes were cut into anterior and posterior sections at the equator of the eyeball, and the vitreous was carefully peeled off and removed, leaving the vitreous membrane on the retina. 0.05 mL of Kenacort (registered trademark, 40 mg / mL TA suspension, Bristol-Myers Squibb Co.) was applied to the vitreous membrane to form a TA layer. Then, 0.1 mL of an equal weight mixture of HA-DBCO and HA-AEA (1.20 wt.% total weight of HA-DBCO and HA-AEA), prepared as described in 1-2 above, was rapidly released to cover the entire TA layer. After allowing the hydrogel to form for 5 minutes, the hydrogel was removed from the pig eye using tweezers.

[0117] 2-2.Results The pig's eye was observed under a stereomicroscope (magnification: 8x) during the formation of the TA layer (Figure 3A), immediately after the mixed solution was released (Figure 3B), and during the hydrogel removal process after 3 minutes of indwelling (Figure 3C). The pig's eye after the hydrogel was removed was also observed under an optical microscope (magnification: 32x, Figure 4). The white area in the center of Figure 3A is the formed TA layer. The dark area in the center of Figure 3B is the released mixed solution. The arrows in Figure 3C and Figure 4 indicate the boundary where the hydrogel was formed. As shown in Figure 3C and Figure 4, by removing the hydrogel, the vitreous membrane could be cleanly peeled off together with the TA layer.

[0118] Example 3 3-1.In vivo evaluation of peeling performance The hydrogel-forming material was evaluated for its ability to remove vitreous and proliferative membranes using rabbits. Endophthalmic membrane removal surgery was performed at the Kyushu University Animal Center and its affiliated operating room.

[0119] The surgical microscope used for endophthalmectomy was Zeiss OPMI, and the vitreous surgical instrument was Alcon Aculus, and the procedure was performed under the same settings as in normal clinical surgery. The posterior vitreous model animal was prepared by intravitreous injection of 0.1 mL of clean room air into a domestic rabbit (adult Dutch strain, male) two weeks before endophthalmectomy. To create the proliferative vitreoretinopathy model, cultured pigment epithelial cells were injected intravitreally into the posterior vitreous model prepared as described above. After cell injection, the rabbits were raised for 28 days, and after confirming the formation of a proliferative membrane by fundus examination, intraocular membrane peeling was performed.

[0120] For endothelial membrane peeling, four trocars were attached to the sclera. Two of the four trocars were equipped with a chandelier lighting system, one with an irrigation system, and the remaining one was used as a port. Next, a vitrectomy was performed to expose the posterior vitreous body and proliferative membrane (in the case of proliferative vitreoretinopathy model animals). Kenacort® was then injected into the vitreous cavity, and excess floating Kenacort was irrigated and aspirated. A 0.1 mL mixture of equal weights of HA-DBCO and HA-AEA (1.15 wt. % total of HA-DBCO and HA-AEA), prepared using the same method as in 1-2 above, was dripped onto the Kenacort using a syringe and 27G needle within 30 seconds of mixing (Figure 5; the area containing the hydrogel-forming material is indicated within the dotted line). After 3 minutes of microscopic observation, the posterior vitreous membrane and proliferative membrane (in the case of proliferative vitreoretinopathy model animals) were removed using a vitreous surgical pliers, and then excised and aspirated using a vitreous cutter. After the experiment, the rabbits were euthanized and the eyeballs were removed for pathological evaluation.

[0121] 3-2.Results The test sample injected into the rabbit eye became a hydrogel within 3 minutes and hardened to a firmness that allowed it to be grasped with vitreous surgery pliers. The hydrogel gelled together with the posterior hyaloid membrane and proliferative membrane. By grasping and peeling the hydrogel, the posterior hyaloid membrane could be split and peeled off. Similarly, the proliferative membrane could be removed by grasping the hydrogel (Figure 6, the area within the dotted line shows the area where the hydrogel formed). The peeled vitreous membrane and hydrogel could be easily excised and aspirated with a vitreous cutter, which is thought to be effective in improving the efficiency of vitreous surgery (Figure 7, the area within the dotted line shows the area where the hydrogel formed).

[0122] Example 4 4-1. Safety evaluation The safety of the hydrogel-forming material was evaluated in vivo. A Zeiss OPMI surgical microscope was used. The test sample was an equal weight mixture of HA-DBCO and HA-AEA prepared using the same method as in 3-1 above.

[0123] After creating a side port and adjusting the intraocular pressure during surgery, 0.1 mL of the test sample was injected into the vitreous cavity using a syringe and a 27G needle. The fundus was observed immediately after surgery, and the test sample was confirmed. One, three, five, and seven days after surgery, intraocular pressure was measured using a contact tonometer (iCare), and the anterior segment was confirmed. After instilling a mydriatic (one drop of Mydrin P eye drops), the fundus was observed.

[0124] 4-2.Results No corneal edema or pathological changes in the vitreous or retina were observed throughout the test period. A slight increase within the normal range of intraocular pressure was observed 1 to 3 days after surgery, but normalized by the 7th day. The test sample adhered to the vitreous immediately after injection, but gradually swelled and dissolved, floating in the vitreous cavity by the 5th day and disappearing by the 7th day. After the observation period, pathological observations were performed using light and electron microscopes, but no obvious pathological changes were observed.

[0125] Example 5 5-1. Evaluation of peeling performance using commercially available hydrogel-forming materials (1) We evaluated the applicability of polyethylene glycol derivatives as hydrogel-forming materials for endothelial membrane ablation using a commercially available dural sealant kit (DuraSeal; Covidien Inc.) containing N-hydroxysuccinimide ester-polyethylene glycol (NHS-PEG) and trilysine amine solution (crosslinker).

[0126] The NHS-PEG and crosslinker included in the kit were each diluted 4-fold with BSS. The diluted NHS-PEG and diluted crosslinker were mixed at a volume ratio of 1:1 or 1:0.9 to prepare test samples. Gelation began immediately after mixing the diluted NHS-PEG and diluted crosslinker.

[0127] Measurement of dynamic viscoelasticity and evaluation of peeling performance in vitro were carried out in accordance with the above 1-2 and 1-3.

[0128] 5-2.Results After mixing NHS-PEG and the crosslinker, gelation began immediately. From the results of dynamic viscoelasticity measurements, the maximum rate of change (V max (Pa / sec) was calculated. The results are shown in Table 2.

[0129] [Table 2]

[0130] Even when a polyethylene glycol derivative is used as a hydrogel-forming material, the V max It was shown that by adjusting the value so that it is greater than 0 and less than 3, it can be used as an agent for endothelial cell removal.

[0131] On the other hand, when the hydrogel-forming material consisted only of polyethylene glycol derivatives, the ductility was high when peeled off, and it was shown that formulations containing glycosaminoglycan derivatives were superior in terms of maneuverability in the narrow intraocular space.

[0132] Example 6 6-1. Evaluation of peeling performance using commercially available hydrogel-forming materials (2) We evaluated the applicability of a commercially available hyaluronic acid-based scaffold for three-dimensional cell culture as a hydrogel-forming material for endothelial ablation. A commercially available three-dimensional cell culture scaffold kit (HyStem; Advanced BioMatrix) containing thiol-modified hyaluronic acid (Glycosil) and thiol-reactive polyethylene glycol diacrylate (Extralink) was used for the study. Dilute the Glycosil included in the kit with BSS to make a 2 wt% Glycosil solution. In addition, Extralink was diluted with BSS to prepare 1 wt%, 2 wt%, or 3 wt% Extralink solutions. The Extralink solution and Glycosil solution were mixed in a 1:2 (volume ratio) ratio to prepare test samples. Gelation began immediately after mixing the Extralink solution and Glycosil solution. Dynamic viscoelasticity was measured according to 1-2 above. Furthermore, according to 1-3 above, an in vitro peeling performance evaluation (retention time 5 minutes) was performed using a mixture of 3 wt% Extralink solution and Glycosil solution as a test sample.

[0133] 6-2.Results From the results of dynamic viscoelasticity measurements, the maximum rate of change (V max (Pa / sec) was calculated. The results are shown in Table 3.

[0134] [Table 3]

[0135] By adjusting the dilution or reaction volume, the V max It was shown that by adjusting the value so that it is greater than 0 and less than 3, it can be used as an agent for endothelial cell removal.

[0136] Example 7 7-1. Ductility measurement The ductility of the formulations was measured using test samples: an equal weight mixture of HA-DBCO and HA-AEA (1.1 wt% total amount of HA-DBCO and HA-AEA, glycosaminoglycan derivative 1) prepared by the same method as in 1-2 above; a 4-fold diluted commercial dura sealant (diluted NHS-PEG:diluted crosslinker = 1:1 (volume ratio), polyethylene glycol derivative) described in 5-1 above; and a cell scaffold (2 wt% Glycosil solution: 3 wt% Extralink solution = 2:1 (volume ratio), glycosaminoglycan derivative + polyethylene glycol derivative) described in 6-1 above. Ductility was measured using a cylindrical probe made of polyoxymethylene (contact surface area: 78. 5mm 2 The texture was measured using a texture analyzer (TA.XT plus, Stable Micro Systems, UK) equipped with a 3D scanner according to the method of Watanabe et al. (Reference: Ippei Watanabe et al., Chem. Pharm. Bull. 67 (3), 277-283 (2019)) compression test method. Measurements were performed at 20°C to 25°C. Specifically, a sterile polystyrene petri dish (outer diameter: 90 mm) was fixed to the measurement table, and the test sample (0.1 mL) was dispensed into the center of it. After leaving it in place for 3 minutes, the probe was moved downward at 0.5 mm / sec to adhere to the test sample, and then immediately moved upward at 0.5 mm / sec for 8 seconds. The force at the point when the ductility of each test sample broke (the test sample itself was torn off, or the test sample was peeled off from the contact surface with the probe or petri dish) was measured. The tensile stress (N / mm 2 ) was calculated by dividing the measured value by the unit area of ​​the probe.

[0137] 7-2.Results The ductility measurement results are shown in Table 4.

[0138] [Table 4]

[0139] When only polyethylene glycol derivative was used, no fracture was observed during the measurement period, and the tensile stress was -1.34 × 10 -3 N / mm 2 It was smaller. The tensile stress of the endothelial cell membrane removal agent containing glycosaminoglycan derivatives was -3×10 -4 N / mm 2 That was all.

[0140] The tensile stress 3 minutes after the start of gelation of the formulation was -3×10 -4 N / mm 2The above results indicate that the composition is particularly suitable for use in endothelial cell removal.

[0141] Example 8 8-1. Preparation of hydrogel-forming materials HA-DBCO (hydrogel-forming material 3) was obtained in the same manner as in Example 1, except that the amount of DBCO-amine solution added to the HA reaction solution (60 mL) was changed to 3 mL. HA-AEA (hydrogel-forming material 4) was obtained in the same manner as in Example 1, except that the amount of AEA solution added to the HA reaction solution (60 mL) was changed to 3 mL. Dynamic viscoelasticity was measured according to 1-2 above. In addition, in vitro peeling performance was evaluated according to 1-3 above (detention time 5 minutes, concentration of hydrogel-forming material = 0.7 8% by weight was carried out.

[0142] 8-2. Degree of substitution of reactive functional groups Using the HA-DBCO prepared in Examples 1 and 8, 1 The degree of substitution (DS) of reactive functional groups (cycloalkynylenyl groups) per HA disaccharide unit was determined by H-NMR analysis. Specifically, approximately 10 mg of HA-DBCO was dissolved in 1 mL of heavy water (DO) and lyophilized. The lyophilized powder was redissolved in 0.7 mL of DO and transferred to an NMR tube. Analysis was performed using a Bruker AVANCE III 500 500 MHz NMR system. 1 H-NMR data was acquired and analyzed. 1 In H-NMR analysis, a chemical shift at 7-8 ppm was detected due to the aromatic protons, which are the partial structure of the cycloalkynylenyl group. For the HA-AEA prepared in Examples 1 and 8, the substitution degree (DS) of the reactive functional group (azide group) per hyaluronic acid disaccharide unit was determined by the following procedure. Specifically, HA-AEA was subjected to a click reaction with an excess amount of DBCO-Amine to convert the AEA residue of HA-AEA into the following structure (in the following structure, * indicates the bonding site with the ethylene group). The product of the click reaction was analyzed in the same way as for HA-DBCO.1 H-NMR data was obtained. 1 In H-NMR analysis, a chemical shift due to aromatic protons was detected at 7-8 ppm.

[0143] [ka]

[0144] The DS (the number of substituents relative to the number of hyaluronic acid disaccharide repeating units, expressed as a percentage) was calculated by calculating the ratio of the relative peak area (integral value) of the aromatic protons and the protons (1.9-2.1 ppm) derived from the N-acetyl group, which is a partial structure of hyaluronic acid disaccharide (Equations 3-5 below). Note that the number of aromatic protons in Equation 3 was set to 8, and the number of N-acetyl group-derived protons in Equation 4 was set to 3 as a constant. The results are shown in Table 5. (Equation 3) Value A = [total value of each peak area of ​​aromatic protons] / [number of aromatic protons] (Equation 4) Value B = [total value of peak areas of N-acetyl group-derived protons] / [number of N-acetyl group-derived protons] (Equation 5) DS [%] = (value A / value B) × 100

[0145] [Table 5]

[0146] 8-3.Results After mixing HA-DBCO and HA-AEA, gelation started immediately (V max The peeling rate was 0.70 (Pa / sec) and the peeling rate was 79%. From the above, by increasing the degree of substitution of reactive functional groups, V max It was shown that the

[0147] Example 9 9-1. Evaluation of peeling performance using commercially available hydrogel-forming materials (3) A commercially available fibrinogen preparation (fibrin glue) was diluted and used as a hydrogel-forming material to evaluate its applicability as a drug for endothelial ablation. A commercially available plasma fraction kit containing thrombin (Beriplast P Combicet for Tissue Adhesion; CSL Behring) was used. Dynamic viscoelasticity and ductility were measured in accordance with 1-2 and 7-1 above. A TA layer was formed on the bottom of the wells of a 24-well plate according to 1-3 above. The fibrinogen-containing solution (combination A) and thrombin-containing solution (combination B) included in the kit were each diluted 10-fold with BSS. A mixture of 175 μL of the diluted fibrinogen-containing solution and 10 μL of BBG solution (10 mg / mL) was layered on the TA layer. 175 μL of the diluted thrombin-containing solution was then layered on top. Gelation began immediately after the diluted thrombin-containing solution was layered on top of the diluted fibrinogen-containing solution. The detachment rate was determined after leaving the plate in place for 5 minutes according to 1-3 above.

[0148] 9-2.Results From the results of dynamic viscoelasticity measurements, the maximum rate of change (V max (Pa / sec) was calculated. Also, from the ductility measurement results, the tensile stress (N / mm 2 ) was calculated. The results are shown in Table 6.

[0149] [Table 6]

[0150] As described above, it was demonstrated that the compound can be used as an agent for endothelial cell removal. [Industrial Applicability]

[0151] The agent used for endophthalmic membrane peeling and the method for peeling the endophthalmic membrane of the present invention can peel the endophthalmic membrane simply and efficiently, and are therefore useful as an agent for peeling the endophthalmic membrane in vitreoretinal surgery and the like.

[0152] While the present invention has been described with reference to specific examples and various embodiments, it will be readily apparent to those skilled in the art that many modifications and adaptations of the embodiments described herein are possible without departing from the spirit and scope of the invention. This application claims priority based on Japanese Patent Application No. 2020-54641, filed with the Japan Patent Office on March 25, 2020, the contents of which are incorporated herein by reference in their entirety.

Claims

1. The hydrogel-forming material includes a combination of a hyaluronic acid derivative having a reactive functional group and a hyaluronic acid derivative having a reactive functional group complementary to the reactive functional group, the combination is a combination of functional groups that undergo a click reaction, the hydrogel-forming material is administered to a patient in the form of a solution containing 0.2% by weight or more and 3% by weight or less of the hydrogel-forming material; The dynamic viscoelasticity measured at a temperature of 25°C and a frequency of 1 Hz satisfies the following formula 1: (Equation 1) 0 < V ｍａｘ ≦ 3 However, in formula 1, V ｍａｘ (Pa / sec) is the maximum rate of change in storage modulus after the onset of gelation, The agent, wherein the gelation is due to a crosslinking reaction.

2. The agent according to claim 1 , wherein the hydrogel-forming material is contained in advance in the form of a solution in a container.

3. The agent according to claim 1 , wherein the hydrogel-forming material is stored in a container in a dry state and is dissolved in a solvent when used.

4. The agent according to any one of claims 1 to 3, wherein the degree of substitution of the reactive functional group and the reactive functional group complementary to the reactive functional group is 1 to 60%.

5. The agent according to any one of claims 1 to 4, which is administered locally by injection.

6. The agent according to any one of claims 1 to 5, which is applied to the intraocular membrane that is adhered to the internal limiting membrane.

7. The agent according to claim 6, wherein the intraocular membrane is at least one selected from the group consisting of a vitreous membrane and a proliferative membrane.

8. The tensile stress measured using a texture analyzer 3 minutes after the start of gelation was −3×10 －４ N / mm ２ The agent according to any one of claims 1 to 7.

9. The agent according to any one of claims 1 to 8, which comprises a visualization agent.

10. The agent according to any one of claims 1 to 9, wherein the dynamic viscoelasticity measured at a temperature of 25°C and a frequency of 1 Hz satisfies the following formula 2: (Equation 2) 0.05 ≦ V ｍａｘ ≦ 2 However, in formula 2, V ｍａｘ (Pa / sec) is the same as in Equation 1.

11. The V ｍａｘ The agent according to any one of claims 1 to 10, wherein is the maximum rate of change in storage modulus from the start of gelation to 900 seconds.

12. The agent according to any one of claims 1 to 11, wherein the hydrogel-forming material comprises the following hyaluronic acid derivative A and the following hyaluronic acid derivative B: (1) Hyaluronic acid derivative A in which an SPAAC-type reactive group is introduced into the carboxyl group of hyaluronic acid via an amide bond and a divalent spacer group; (2) Hyaluronic acid derivative B in which a reactive group complementary to the reactive group of (1) is introduced into the carboxyl group via an amide bond and a divalent spacer group.

13. A syringe filled with the agent according to any one of claims 1 to 12.

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