Gel preparation kit, gel preparation material, medicinal drug, and medical device

Abstract

To provide a gel preparation kit, a gel preparation material, a medicinal drug, and a medical device that suppress a reaction with a medicine or a cell, and promote inclusion of a hydrophobic medicine into the gel or cell proliferation.SOLUTION: A gel preparation kit comprising a polymer (A) and a polymer (B), where the polymer (A) includes a hydrophilic polymer portion (A1) with 2 to 8 branches, a hydrophobic polymer portion (A2) that is bonded to the side chains or ends of each branch of the hydrophilic polymer portion (A1), and a click-reactive group (A3) that is bonded to at least one side chain or end of the hydrophobic polymer portion (A2), and polymer (B) includes a hydrophilic polymer portion (B1) with 2 to 8 branches, a hydrophobic polymer portion (B2) that is bonded to the side chains or ends of each branch of the hydrophilic polymer portion (B1), and a click-reactive group (B3) that is bonded to at least one side chain or end of the hydrophobic polymer portion (B2). The click-reactive groups (A3) and (B3) can be cross-linked through a click reaction.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a gel preparation kit, a gel preparation material, a pharmaceutical product, a medical device, a drug-releasing material, and a cell scaffold material. [Background technology]

[0002] In recent years, research and development has been conducted on materials (injectable gel materials) that are liquid outside the body but gel in response to stimuli (chemical reactions, temperature, pH, etc.) after injection into the body. As such a material, Patent Document 1 discloses a four-branched compound having four polyethylene glycol chains. The gel obtained using this compound is a hydrogel that does not dissolve and remains stable even in the presence of water, and is expected to be used in artificial cartilage and the like. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2010 / 070775 Summary of the Invention [Problem to be solved by the invention]

[0004] Injectable gel materials can be used in a variety of medical devices, as they can be injected into the body and gelate by incorporating drugs or cells. They can also be used as sustained-release materials or cell scaffolds for regenerative medicine, as they can be mixed with drugs and cells in a liquid state and form a gel encapsulating them at the administration site. For these medical devices, sustained-release materials, and cell scaffolds, the gel must be able to encapsulate drugs and cells without altering their physical properties. However, while drugs such as peptides and proteins, and the cell membrane surfaces of cells, contain numerous reactive functional groups such as amino and thiol groups, the gel material disclosed in Patent Document 1 utilizes amino group reactions to form gels. This can lead to problems such as changes in the chemical structure of drugs and cell membrane proteins, resulting in inactivation, or denaturation of the cell membrane structure, leading to cell death. Furthermore, the hydrophilic nature of polyethylene glycol chains makes it difficult to encapsulate hydrophobic drugs that are insoluble in water within the gel, and the gel does not function as a cell scaffold, making cell proliferation difficult.

[0005] The present invention has been made in consideration of the above-mentioned problems, and aims to provide a gel preparation kit, gel preparation materials, pharmaceuticals, and medical devices that suppress reactions with drugs and cells, and promote the inclusion of hydrophobic drugs in the gel and cell proliferation. [Means for solving the problem]

[0006] As a result of extensive investigations into the above-mentioned problems, the present inventors have found that the above-mentioned problems can be solved by using a polymer having a hydrophobic polymer moiety and a click-reactive group, and have thus completed the present invention. Specifically, the present invention provides the following.

[0007] [1] A kit for preparing a gel, comprising a polymer (A) and a polymer (B), the polymer (A) has a hydrophilic polymer segment (A1) having 2 to 8 branches, a hydrophobic polymer segment (A2) bonded to a side chain or an end of each branch of the hydrophilic polymer segment (A1), and a click reactive group (A3) bonded to a side chain or an end of at least one of the hydrophobic polymer segments (A2); the polymer (B) has a hydrophilic polymer segment (B1) having 2 to 8 branches, a hydrophobic polymer segment (B2) bonded to a side chain or an end of each branch of the hydrophilic polymer segment (B1), and a click reactive group (B3) bonded to a side chain or an end of at least one of the hydrophobic polymer segments (B2); The click reactive group (A3) and the click reactive group (B3) can be crosslinked by a click reaction; Gel preparation kit.

[0008] [2] The gel preparation kit according to [1], wherein the click reactive group (A3) and the click reactive group (B3) are Diels-Alder reactive groups.

[0009] [3] The gel preparation kit according to [2], wherein one of the click reactive group (A3) and the click reactive group (B3) is a group containing a norbornene skeleton, a trans-cyclooctene skeleton, or a cyclooctyne skeleton, and the other is a group containing a tetrazine skeleton or a triazine skeleton.

[0010] [4] A gel preparation kit according to any one of [1] to [3], wherein the hydrophilic polymer portion (A1) and the hydrophilic polymer portion (B1) each comprise a central structure having 2 to 8 branches and a polyalkylene glycol bonded to each branch of the central structure.

[0011] [5] The gel preparation kit according to any one of [1] to [4], wherein the hydrophobic polymer portion (A2) and the hydrophobic polymer portion (B2) are made of a polyester or a polypeptide.

[0012] [6] The gel preparation kit according to any one of [1] to [5], wherein the hydrophobic polymer portion (A2) and the hydrophobic polymer portion (B2) have 8 or more carbon atoms in their main chains.

[0013] [7] A gel-preparing material containing either polymer (A) or polymer (B) and used to form a gel by mixing with the other polymer, the polymer (A) has a hydrophilic polymer segment (A1) having 2 to 8 branches, a hydrophobic polymer segment (A2) bonded to a side chain or an end of each branch of the hydrophilic polymer segment (A1), and a click reactive group (A3) bonded to a side chain or an end of at least one of the hydrophobic polymer segments (A2); the polymer (B) has a hydrophilic polymer segment (B1) having 2 to 8 branches, a hydrophobic polymer segment (B2) bonded to a side chain or an end of each branch of the hydrophilic polymer segment (B1), and a click reactive group (B3) bonded to a side chain or an end of at least one of the hydrophobic polymer segments (B2); The click reactive group (A3) and the click reactive group (B3) can be crosslinked by a click reaction; Materials for gel preparation.

[0014] [8] A pharmaceutical product comprising the gel preparation kit according to any one of [1] to [6].

[0015] [9] A medical device comprising the gel preparation kit according to any one of [1] to [6].

[0016]

[10] A sustained-release drug material comprising the gel preparation kit according to any one of [1] to [6].

[0017]

[11] A cell scaffold material comprising the gel preparation kit according to any one of [1] to [6]. [Effects of the Invention]

[0018] According to the present invention, it is possible to provide a gel preparation kit, gel preparation materials, pharmaceuticals, and medical devices that suppress reactions with drugs and cells, and promote the inclusion of hydrophobic drugs in the gel and cell proliferation. [Brief explanation of the drawings]

[0019] [Figure 1]FIG. 1 shows the results of drug tests in the examples. [Figure 2] FIG. 1 shows the results of a cell test in an example. DETAILED DESCRIPTION OF THE INVENTION

[0020] Hereinafter, embodiments of the present invention will be described, but the present invention is not limited to these.

[0021] <Gel preparation kit> The gel preparation kit includes a polymer (A) and a polymer (B). The polymer (A) includes a hydrophilic polymer segment (A1) having 2 to 8 branches, a hydrophobic polymer segment (A2) bonded to a side chain or terminal of each branch of the hydrophilic polymer segment (A1), and a click-reactive group (A3) bonded to a side chain or terminal of at least one of the hydrophobic polymer segments (A2). The polymer (B) includes a hydrophilic polymer segment (B1) having 2 to 8 branches, a hydrophobic polymer segment (B2) bonded to a side chain or terminal of each branch of the hydrophilic polymer segment (B1), and a click-reactive group (B3) bonded to a side chain or terminal of at least one of the hydrophobic polymer segments (B2). The click-reactive group (A3) and the click-reactive group (B3) can be crosslinked by a click reaction.

[0022] As used herein, the term "click reactive group" refers to a group that participates in a click reaction. Polymer (A) and polymer (B) have groups that participate in a click reaction, which is a highly selective reaction, and therefore can suppress reactions with drugs or cells, thereby preventing drug inactivation and cell death. Furthermore, since polymer (A) and polymer (B) have a hydrophobic polymer portion in addition to a hydrophilic polymer portion, they can be used to encapsulate hydrophobic drugs within the gel by emulsification, or to serve as a scaffold for cells to grow.

[0023] <Polymer (A)> (Hydrophilic polymer portion (A1)) The hydrophilic polymer constituting the hydrophilic polymer segment (A1) is not particularly limited as long as it is a polymer capable of forming a hydrogel by a crosslinking reaction described below, and examples thereof include polyalkylene glycol, polysaccharide, poly(meth)acrylate, poly(meth)acrylamide, etc. Among these, the hydrophilic polymer constituting the hydrophilic polymer segment (A1) is preferably polyalkylene glycol.

[0024] The hydrophilic polymer segment (A1) preferably comprises a central structure having 2 to 8 branches and a hydrophilic polymer bonded to each branch of the central structure. Examples of the central structure include structures derived from polyhydric alcohols, polycarboxylic acids, polyamines, etc., having 2 to 8 hydroxy groups, carboxy groups, amino groups, etc., which can be bonded to hydrophilic polymers. The polyhydric alcohol is preferably an aliphatic polyhydric alcohol, such as glycerin, pentaerythritol, dipentaerythritol, or tripentaerythritol. The central structure preferably has 1 to 30 carbon atoms, more preferably 1 to 10 carbon atoms, and even more preferably 1 to 5 carbon atoms.

[0025] The number of carbon atoms in the alkylene group in the polyalkylene glycol is preferably 1 to 5, more preferably 2 or 3, and even more preferably 2. That is, the polyalkylene glycol is preferably polyethylene glycol.

[0026] The number of repeats of the oxyalkylene group in each branch of the polyalkylene glycol is preferably 5 to 600, more preferably 25 to 250, and even more preferably 25 to 60. The number of repeats of the oxyalkylene group in each branch may be the same or different, but the closer the number of repeats, the more uniform the three-dimensional structure can be, and the more likely it is that a structurally strong gel will be obtained. Therefore, from the viewpoint of obtaining a structurally strong gel, it is preferable that the number of repeats of the oxyalkylene group in each branch is the same.

[0027] The hydrophilic polymer segment (A1) has 2 to 8 branches, and the number of branches in the hydrophilic polymer segment (A1) is preferably 4.

[0028] (Hydrophobic polymer portion (A2)) The hydrophobic polymer segment (A2) is bonded to a side chain or terminal of each branch of the hydrophilic polymer segment (A1), and is preferably bonded to the terminal of each branch.

[0029] Examples of hydrophobic polymers constituting the hydrophobic polymer segment (A2) include polyesters, polypeptides, polyolefins, etc. Among these, the hydrophobic polymer segment (A2) is preferably composed of polyesters or polypeptides, more preferably composed of polypeptides.

[0030] Examples of polyesters include polyesters having a constitutional unit derived from a dicarboxylic acid and a constitutional unit derived from a diol. Examples of dicarboxylic acids include aliphatic dicarboxylic acids and aromatic group-containing dicarboxylic acids. Examples of the diol include an aliphatic diol and an aromatic group-containing diol.

[0031] The number of carbon atoms in the aliphatic diol is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3. Examples of the aliphatic diol include 1,2-ethanediol (ethylene glycol), 1,2-propanediol (propylene glycol), 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 2-methyl-1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 2,2-dimethyl-1,3-propanediol (neopentyl glycol), 2,2-diethyl-1,3-propanediol (3,3-dimethylolpentane), 2-n-butyl-2-ethyl-1,3propanediol (3,3-dimethylolheptane), 3-methyl 1,5-pentanediol, 1,6-hexanediol, 2,2,4-trimethyl-1,3-pentanediol, 2-ethyl-1,3-hexanediol, 2-methyl-1,8-octanediol, 1,9-nonanediol, 1,10-decanediol, 1,12-octadecanediol, 1,4-cyclohexanedimethanol, hydrogenated bisphenol A, 1,2-, 1,3- or 1,4-cyclohexanediol, cyclododecanediol, dimer diol, hydrogenated dimer diol, diethylene glycol, dipropylene glycol, triethylene glycol, and the like.

[0032] The number of carbon atoms in the aromatic group-containing diol is preferably 6 to 20, more preferably 6 to 15. Examples of the aromatic group-containing diol include bisphenol A, 1,2-hydroxybenzene, 1,3-hydroxybenzene, 1,4-hydroxybenzene, and 1,4-benzenedimethanol.

[0033] The number of carbon atoms in the aliphatic dicarboxylic acid is preferably 2 to 15, more preferably 2 to 10. Examples of aliphatic dicarboxylic acids include oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, 1,10-decanedicarboxylic acid, 1,4-cyclohexanedicarboxylic acid, dimer acid, maleic acid, and fumaric acid. Alkyl esters and acid halides of these may also be used.

[0034] The number of carbon atoms in the aromatic group-containing dicarboxylic acid is preferably 8 to 20, more preferably 8 to 15, and even more preferably 8 to 12. Examples of aromatic dicarboxylic acids include terephthalic acid, isophthalic acid, phthalic acid, phenylmalonic acid, homophthalic acid, phenylsuccinic acid, β-phenylglutaric acid, α-phenyladipic acid, β-phenyladipic acid, biphenyl-2,2'-dicarboxylic acid, biphenyl-4,4'-dicarboxylic acid, and naphthalenedicarboxylic acid. Alkyl esters and acid halides of these may also be used.

[0035] When the hydrophobic polymer segment (A2) is composed of a polyester having structural units derived from a dicarboxylic acid and structural units derived from a diol, the total number of structural units derived from a dicarboxylic acid and the total number of structural units derived from a diol in each hydrophobic polymer segment (A2) is preferably 2 to 30, more preferably 2 to 20, and even more preferably 2 to 10.

[0036] Examples of polypeptides include hydrophobic peptides composed mostly of hydrophobic amino acid residues. The ratio of the number of hydrophobic amino acid residues to the number of amino acid residues constituting the hydrophobic peptide may be, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100%. Examples of hydrophobic amino acid residues include glycine, alanine, valine, leucine, proline, phenylalanine, tryptophan, and isoleucine residues.

[0037] When the hydrophobic polymer segment (A2) consists of a polypeptide, the number of amino acid residues of the polypeptide in each hydrophobic polymer segment (A2) is preferably 2 to 30, more preferably 2 to 20, and even more preferably 2 to 10.

[0038] The main chain of the hydrophobic polymer segment (A2) preferably has 5 or more carbon atoms, more preferably 8 or more carbon atoms, even more preferably 8 to 150 carbon atoms, particularly preferably 8 to 100 carbon atoms, and most preferably 8 to 50 carbon atoms. In this specification, when the main chain of the hydrophobic polymer portion contains a ring structure, the number of carbon atoms in the main chain of the hydrophobic polymer portion includes the number of carbon atoms constituting the ring structure.

[0039] (Click reactive group (A3)) The click reactive group (A3) is bonded to a side chain or terminal of at least one hydrophobic polymer segment (A2), and is preferably bonded to the terminal of at least one hydrophobic polymer segment (A2).

[0040] The click reaction involving the click reactive group (A3) is not particularly limited as long as it is a reaction conventionally known as a click reaction, and examples thereof include Diels-Alder reactions such as the strain-promoted inverse electron demand Diels-Alder (SPIEDAC) reaction, Staudinger ligation reaction, and azide-alkyne reactions such as the strain-promoted azide-alkyne cycloaddition (SPAAC) reaction. Among these, the Diels-Alder reaction is preferred, and the SPIEDAC reaction is more preferred.

[0041] The SPIEDAC reaction utilizes the high reactivity of highly electron-deficient heterocycles such as tetrazines and triazines with strained C-C multiple bond compounds such as norbornene, trans-cyclooctene, and cyclooctyne.

[0042] It is preferable that either the click reactive group (A3) or the click reactive group (B3) described later is a group containing a norbornene skeleton, a trans-cyclooctene skeleton, or a cyclooctyne skeleton, and the other is a group containing a tetrazine skeleton or a triazine skeleton.

[0043] The polymer (A) is preferably a compound represented by the following formula (a-1). [ka] (In formula (a-1), each m independently represents an integer of 5 to 600. R a1 represents the hydrophobic polymer segment (A2). a2 represents a hydrogen atom or a click reactive group (A3), where R a2 At least one of the groups is a click-reactive group (A3).

[0044] In formula (a-1), "C2H4O" represents an ethyleneoxy group (CH2CH2O). The same applies to other formulas.

[0045] Preferred embodiments of the hydrophobic polymer segment (A2) and the click reactive group (A3) in formula (a-1) are as described above.

[0046] m is preferably an integer of 5 to 600, more preferably an integer of 25 to 250, and even more preferably an integer of 25 to 60. m in each branch may be the same or different, but the closer m is, the more uniform the three-dimensional structure can be, and the more likely it is that a structurally strong gel will be obtained. Therefore, from the viewpoint of obtaining a structurally strong gel, it is preferable that m is the same.

[0047] R a2 are preferably all click reactive groups (A3).

[0048] Polymer (A) can be produced by known organic synthesis methods. For example, when the hydrophilic polymer segment (A1) is composed of a four-branched polyethylene glycol, as described in Patent Document 1, ethylene oxide is first added to pentaerythritol to form a polyethylene glycol chain. Next, a monomer constituting a hydrophobic polymer such as polyester or polypeptide is polymerized to the hydroxy group at the end of the polyethylene glycol chain to form a hydrophobic polymer segment. Then, a compound having a click-reactive group and a group capable of binding to the end of the hydrophobic polymer segment is reacted with the end of the hydrophobic polymer segment. Polymer (B) can be produced by a similar method.

[0049] <Polymer (B)> Preferred embodiments of the hydrophilic polymer portion (B1), the hydrophobic polymer portion (B2), and the click reactive group (B3) are the same as those of the hydrophilic polymer portion (A1), the hydrophobic polymer portion (A2), and the click reactive group (A3) in the polymer (A), respectively.

[0050] The polymer (B) is preferably a compound represented by the following formula (b-1). [ka] (In formula (b-1), n's each independently represent an integer of 5 to 600. R b1 represents the hydrophobic polymer segment (B2). b2 represents a hydrogen atom or a click reactive group (B3), where R b2 At least one of the groups is a click-reactive group (B3).

[0051] m is preferably an integer of 5 to 600, more preferably an integer of 25 to 250, and even more preferably an integer of 25 to 60.

[0052] R b2 are preferably all click reactive groups (B3).

[0053] The sum of the number of branches of the hydrophilic polymer portion (A1) in the polymer (A) and the number of branches of the hydrophilic polymer portion (B1) in the polymer (B) is preferably 5 or more, since this facilitates the formation of a three-dimensional network structure and gelation. The total number of click reactive groups (A3) in polymer (A) and the number of click reactive groups (B3) in polymer (B) is preferably 5 or more, since this facilitates the formation of a three-dimensional network structure and gelation.

[0054] The gel preparation kit may contain polymer (A) as a solution of polymer (A) (polymer solution (A-1)), or may contain polymer (B) as a solution of polymer (B) (polymer solution (B-1)).

[0055] The concentrations of the polymer (A) and the polymer (B) in the polymer solution (A-1) and the polymer solution (B-1) are preferably 1 to 40 mass%, more preferably 3 to 25 mass%, and even more preferably 5 to 15 mass%.

[0056] Examples of the solvent in the polymer solution (A-1) and the polymer solution (B-1) include water and alcohols such as ethanol, etc. Among these, water is preferred.

[0057] The volumes of the polymer solution (A-1) and the polymer solution (B-1) in the gel preparation kit can be appropriately set depending on the application.

[0058] <Gel preparation materials> The gel-forming material contains either polymer (A) or polymer (B) and is used to form a gel by mixing with the other.

[0059] Preferred embodiments of the polymer (A) and the polymer (B) are the same as those of the polymer (A) and the polymer (B) in the gel preparation kit.

[0060] Gel By injecting a solution containing polymer (A) and polymer (B) into a living body, the click reactive group (A3) and the click reactive group (B3) are crosslinked by a click reaction, and a gel containing a polymer compound having a constitutional unit derived from polymer (A) and a constitutional unit derived from polymer (B) is obtained.

[0061] In the solution containing polymer (A) and polymer (B), the contents of polymer (A) and polymer (B) are each preferably adjusted to 0.5 to 15 mass%, more preferably 1.5 to 10 mass%, and even more preferably 2.5 to 7.5 mass%.

[0062] In the solution containing the polymer (A) and the polymer (B), the mass ratio of the polymer (A) to the polymer (B) is preferably (A):(B)=20:80 to 80:20, more preferably 30:70 to 70:30, and even more preferably 40:60 to 60:40.

[0063] Examples of the solvent for the solution containing the polymer (A) and the polymer (B) include water and alcohols such as ethanol, etc. Among these, water is preferred.

[0064] The solution containing the polymer (A) and the polymer (B) may contain the drugs and cells described below.

[0065] <Pharmaceuticals and medical devices> The above-described gel preparation kit and gel preparation material can be applied to various pharmaceuticals and medical devices as an injectable gel material that gels in response to stimuli such as the pH of body fluids after being injected into a living body.

[0066] Specifically, a gel preparation kit or gel preparation material can be combined with a drug such as a hydrophobic drug, and a mixed solution of polymer (A), polymer (B), and the drug can be injected into the area where the drug is to be applied and gelled, thereby allowing the drug to be released in a sustained manner, and therefore the product can be used as a drug sustained-release material.

[0067] The drug is not particularly limited as long as it has physiological activity, and examples thereof include proteins, peptides, low molecular weight compounds, nucleic acids, and sugar chains.

[0068] As described above, polymer (A) and polymer (B) have a hydrophilic polymer portion and a hydrophobic polymer portion, and therefore a hydrophobic drug can be encapsulated in the gel and released sustainedly. Therefore, the drug may be a hydrophobic drug.

[0069] As used herein, a hydrophobic drug refers to a drug whose solubility in water (20°C) is 3.3 g / 100 g or less. The solubility of a drug in water (20°C) can be confirmed by the method described in General Rule 29 of the Japanese Pharmacopoeia, 15th Edition. Specifically, drug powder is placed in water (20°C) and vigorously shaken for 30 seconds every 5 minutes. The mass that dissolves within 30 minutes is confirmed.

[0070] Examples of hydrophobic drugs include phenolic compounds such as thymol; carbamate compounds such as 3-iodo-2-propargyl butylcarbamate; and paclitaxel.

[0071] Furthermore, by combining cells such as stem cells with a gel preparation kit or gel preparation material, and injecting a mixed solution of polymer (A), polymer (B), and cells into the area where tissue regeneration is desired and allowing it to gel, tissue can be regenerated, and therefore the product can be used as a cell scaffold material for tissue regeneration.

[0072] Examples of the cells include animal cells from humans, mice, rats, pigs, cattle, monkeys, etc. Examples of the cells also include embryonic stem cells, mesenchymal stem cells, adipose-derived stem cells, endothelial stem cells, dental pulp stem cells, tumor cells, chondrocytes, osteoblasts, skin fibroblasts, myofibroblasts, myofibroblasts, hepatocytes, smooth muscle cells, endothelial cells, epithelial cells, adipose tissue, adipocytes, cardiac cells, etc.

[0073] Before injection into a living body, a solution containing polymer (A) and a solution containing polymer (B) are mixed. The mixing method is not particularly limited and can be appropriately determined depending on the application. For example, the two solutions may be mixed in advance and then injected using an injection port or the like, or the two solutions may be transported separately to the injection site and mixed using a static mixer or the like at the time of injection. [Example]

[0074] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0075] As the polymer (a1), a compound represented by the following formula was used: In the formula, m was 56 and ma was 4. R a1 (hydrophobic polymer portion) and R a2 (Click reactive groups) were all groups in the formula, where * represents a bond between themselves and ** represents a bond to the oxyethylene group. [ka]

[0076] As polymer (a2), a compound represented by the following formula was used: m in the formula was 56. R a2 (Click reactive groups) were all groups in the formula, where * represents a bond. [ka]

[0077] As the polymer (b1), a compound represented by the following formula was used: In the formula, n was 56 and na was 4. R b1 (hydrophobic polymer portion) and R b2 (Click reactive groups) were all groups in the formula, where * represents a bond between themselves and ** represents a bond to the oxyethylene group. [ka]

[0078] As the polymer (b2), a compound represented by the following formula was used, where n was 56. b2 (Click reactive groups) were all groups in the formula, where * represents a bond. [ka]

[0079] As the polymer (c), in the polymer (a1), R a2 A compound in which the above was changed to a group having a succinimidyl group at the end was used.

[0080] <Drug testing> Lysozyme was added to an aqueous solution of polymer (a1) and dissolved uniformly to prepare a lysozyme-containing aqueous solution of polymer (a1) (polymer (a1): 15% by mass). Equal amounts of this lysozyme-containing aqueous solution of polymer (a1) and an aqueous solution of polymer (b1) (15% by mass) were mixed to prepare 10 mL of a mixed solution (lysozyme: 100 μg / mL). The mixed solution was allowed to stand at 37°C for 1 hour to gel. The gel was crushed and dispersed in 10 mL of phosphate-buffered saline (PBS(-)). The dispersion was centrifuged, and the lysozyme concentration in the recovered supernatant was measured by the BCA method. The results are shown in Figure 1A. Similarly, an aqueous solution of polymer (c) containing lysozyme (polymer (c): 15% by mass) was prepared. Equal amounts of this aqueous solution of polymer (c) containing lysozyme and an aqueous solution of ε-polylysine (15% by mass) were mixed to prepare 10 mL of a mixed solution (lysozyme: 100 μg / mL). The mixed solution was allowed to stand at 37°C for 1 hour to gel. The gel was crushed and dispersed in 10 mL of phosphate-buffered saline (PBS(-)). The dispersion was centrifuged, and the lysozyme concentration in the recovered supernatant was measured by the BCA method. The results are shown in Figure 1B. PBS(-) containing lysozyme (lysozyme: 100 μg / mL) was left to stand at 37°C for 1 hour, and the lysozyme concentration was measured by the BCA method. The results are shown in Figure 1C.

[0081] Drugs such as peptides and proteins often contain amino groups, which can cause reactions with materials that gel using amino group reactions. This can change the structure of the drug, reducing its solubility, or trapping the drug in the cross-linked structure within the gel. Therefore, the effect of gelation on the drug can be evaluated by measuring the drug concentration before and after gelation.

[0082] As shown in Figure 1, in the case of A, which was gelled using polymer (a1) and polymer (b1), the lysozyme concentration did not decrease substantially, but in the case of B, which was gelled using polymer (c) and polylysine, the lysozyme concentration decreased. This is thought to be because the polymer used in A does not have a functional group that reacts with lysozyme, while the polymer used in B has a succinimidyl group that reacts with lysozyme. That is, it is clear that when polymer (a1) and polymer (b1) are used, the reaction with the drug can be suppressed.

[0083] <Solubilization test of hydrophobic drugs> 2 mg of paclitaxel, a model hydrophobic drug, was mixed with 1 mL of an aqueous solution (15% by mass) of polymer (a1) and stirred overnight at room temperature. The mixture was then centrifuged and visually inspected for solubilization. A homogeneous solution without precipitate was evaluated as "homogeneous," while a solution with precipitate was evaluated as "heterogeneous." The precipitated paclitaxel was separated after centrifugation and its mass was measured. The same test was repeated three times. Similarly, tests were carried out by changing the polymer (a1) to the polymer (a2), the polymer (b1), or the polymer (b2). The results are shown in Table 1.

[0084] [Table 1]

[0085] As shown in Table 1, it was found that the hydrophobic drug paclitaxel could be solubilized in aqueous solutions of polymer (a1) and polymer (b1), whereas it could not be solubilized in aqueous solutions of polymer (a2) and polymer (b2). Therefore, it is clear that when such polymer (a1) and polymer (b1) are used to form a gel, a hydrophobic drug can be encapsulated in the gel.

[0086] <Cell test> L929 fibroblasts were grown at a cell count of 1.0 × 10 6 A cell suspension was prepared by diluting it with serum-containing medium (RPMI1640) to a concentration of 1.0 × 10 cells / mL. The cell suspension was mixed with an aqueous solution of polymer (a1) to prepare a polymer (a1) solution containing cells (polymer (a1): 15% by mass). Equal amounts of the polymer (a1) solution containing cells and an aqueous solution of polymer (b1) (15% by mass) were mixed to prepare a mixed solution (cell count: 1.0 × 10 cells). 4 The mixed solution was added to a 96-well plate (polystyrene plate for tissue culture) to create a gel containing cells. The cells were stained at predetermined intervals, and the number of viable cells was measured using a hemocytometer. The number of viable cells (cell viability) (%) at predetermined intervals, with the number of cells added to the plate taken as 100%, is shown in Figure 2A. Similarly, the cell suspension prepared above was mixed with an aqueous solution of polymer (c) to prepare a polymer (c) solution containing cells (polymer (c): 15% by mass). The polymer (c) solution containing cells and an aqueous solution of ε-polylysine (15% by mass) were mixed in equal amounts to prepare a mixed solution (cell count: 1.0 × 10 4 The mixed solution was added to a 96-well plate (polystyrene plate for tissue culture) to prepare a gel containing cells. As above, the number of live cells at each time point, assuming the number of cells added to the plate as 100%, is shown in Figure 2B. L929 fibroblasts were grown at a cell count of 1.0 × 10 4A cell suspension was prepared by diluting it with serum-containing medium (RPMI 1640) to a concentration of 100 cells / mL. The cell suspension was added to a 96-well plate (polystyrene plate for tissue culture). As described above, the number of live cells added to the plate was defined as 100%. Figure 2C shows the number of live cells at each time point.

[0087] As shown in Figure 2, in the case of A, which was gelled using polymer (a1) and polymer (b1), the number of viable cells was not much different from that of C, whereas in the case of B, which was gelled using polymer (c) and polylysine, the number of viable cells was significantly reduced. That is, it is clear that when polymer (a1) and polymer (b1) are used, cell death can be suppressed and cell proliferation can be promoted.

Claims

1. A kit for preparing a gel, comprising a polymer (A) and a polymer (B), the polymer (A) has a hydrophilic polymer segment (A1) having 2 to 8 branches, a hydrophobic polymer segment (A2) bonded to a side chain or an end of each branch of the hydrophilic polymer segment (A1), and a click-reactive group (A3) bonded to a side chain or an end of at least one of the hydrophobic polymer segments (A2); the polymer (B) has a hydrophilic polymer segment (B1) having 2 to 8 branches, a hydrophobic polymer segment (B2) bonded to a side chain or an end of each branch of the hydrophilic polymer segment (B1), and a click-reactive group (B3) bonded to a side chain or an end of at least one of the hydrophobic polymer segments (B2); The click reactive group (A3) and the click reactive group (B3) can be crosslinked by a click reaction; Gel preparation kit.

2. The gel preparation kit according to claim 1, wherein the click reactive group (A3) and the click reactive group (B3) are Diels-Alder reactive groups.

3. 3. The gel preparation kit according to claim 2, wherein one of the click reactive group (A3) and the click reactive group (B3) is a group containing a norbornene skeleton, a trans-cyclooctene skeleton, or a cyclooctyne skeleton, and the other is a group containing a tetrazine skeleton or a triazine skeleton.

4. 2. The gel preparation kit according to claim 1, wherein the hydrophilic polymer portion (A1) and the hydrophilic polymer portion (B1) each comprise a central structure having 2 to 8 branches and a polyalkylene glycol bonded to each branch of the central structure.

5. 2. The gel preparation kit according to claim 1, wherein the hydrophobic polymer portion (A2) and the hydrophobic polymer portion (B2) are composed of a polyester or a polypeptide.

6. 2. The gel preparation kit according to claim 1, wherein the number of carbon atoms in the main chain of the hydrophobic polymer segment (A2) and the hydrophobic polymer segment (B2) is 8 or more.

7. A gel-forming material comprising either polymer (A) or polymer (B), which is mixed with the other to form a gel, the polymer (A) has a hydrophilic polymer segment (A1) having 2 to 8 branches, a hydrophobic polymer segment (A2) bonded to a side chain or an end of each branch of the hydrophilic polymer segment (A1), and a click-reactive group (A3) bonded to a side chain or an end of at least one of the hydrophobic polymer segments (A2); the polymer (B) has a hydrophilic polymer segment (B1) having 2 to 8 branches, a hydrophobic polymer segment (B2) bonded to a side chain or an end of each branch of the hydrophilic polymer segment (B1), and a click-reactive group (B3) bonded to a side chain or an end of at least one of the hydrophobic polymer segments (B2); The click reactive group (A3) and the click reactive group (B3) can be crosslinked by a click reaction; Materials for gel preparation.

8. A pharmaceutical product comprising the gel preparation kit according to any one of claims 1 to 6.

9. A medical device comprising the gel preparation kit according to any one of claims 1 to 6.

10. A drug sustained-release material comprising the gel preparation kit according to any one of claims 1 to 6.

11. A cell scaffold material comprising the gel preparation kit according to any one of claims 1 to 6.

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