Polysaccharide derivative, polysaccharide derivative-drug conjugate, and method for producing same

Abstract

To provide a novel polysaccharide derivative which can be used to form a conjugate with a drug and can be used as a medical material and the like, and a polysaccharide derivative-drug conjugate which uses the same.SOLUTION: A polysaccharide derivative obtained by introducing a group represented by formula (A) into a polysaccharide which is acidic, basic or amphoteric, and a polysaccharide derivative-drug conjugate of the polysaccharide derivative and a drug.

Description

[Technical Field]

[0001] The present invention relates to polysaccharide derivatives that can be used as DDS (Drug delivery system) carriers, medical materials such as pharmaceuticals, medical devices, and pharmaceutical additives, separation materials, and foods, supplements, and food additives, as well as polysaccharide derivative-drug conjugates using the same, and uses of these. [Background technology]

[0002] Polysaccharides such as hyaluronic acid and alginic acid generally exhibit excellent water solubility, water retention, moderate viscosity, adhesiveness, and biocompatibility, and are therefore widely used as medical materials, food additives, additives for cosmetics and daily necessities, and thickeners. For example, hyaluronic acid (HA) is a biodegradable and biocompatible natural polymer abundant in the body and is widely used in healthcare products such as orthopedic and ophthalmic pharmaceuticals, medical devices, cosmetics, and contact lenses. Alginic acid (AL), a natural polysaccharide found in brown algae, is highly biocompatible and can be crosslinked with polyvalent metal ions such as Ca. It is therefore used in pharmaceuticals, medical devices, and pharmaceutical additives, such as hemostatic agents and wound dressings, as well as in foods, supplements, and food additives.

[0003] In recent years, these polysaccharides have been investigated as potential drug carriers for drug delivery systems (DDS). HA specifically binds to the CD44 receptor, is overexpressed on the surface of various tumor cells, and can be easily derivatized using its hydroxyl and carboxyl groups, making it a promising drug carrier. For example, several HA-drug conjugates using Schiff bases have been reported (Non-Patent Document 1: Materials Science and Engineering: C, 2014, 36: 287-293; Non-Patent Document 2: Carbohydrate Polymers 134 (2015) 293-299; Non-Patent Document 3: Carbohydrate Polymers 189 (2018) 273-279; Non-Patent Document 4: Carbohydrate Polymers 216 (2019) 63-71). Schiff bases are imine-containing compounds formed by the condensation reaction of primary amines with reactive carbonyl compounds. Schiff bases exhibit reversible pH-responsive dissociation and are therefore expected to enable selective drug release. However, Schiff bases formed between ordinary aldehydes or ketones and amines undergo rapid reverse reactions in water, resulting in insufficient stability. As a result, drug conjugates using Schiff bases often exhibit rapid drug release rates and insufficient performance. On the other hand, by using hydrazide groups instead of amino groups to slow the hydrolysis rate, they have been used to create biocompatible in situ crosslinked hydrogels based on hyaluronic acid, alginate, and other compounds.

[0004] It has been reported that benzaldehyde, due to its aromatic structure, can form stable Schiff bases with the amine groups contained in various drugs (Non-Patent Document 5: Materials Chemistry Frontiers, 2018, 2(10): 1765-1778). Non-Patent Document 6 (J. Biomed. Nanotechnol. 2017, 13, 1647-1659) discloses a conjugate of carboxymethylchitosan and daunorubicin that utilizes Schiff base formation between the aldehyde group of triazolebenzylaldehyde and the amino group of daunorubicin. The conjugate formation in Non-Patent Document 6 involves multiple, complicated reaction steps: the drug is reacted with azidobenzylaldehyde, which is then reacted with alkyne-modified carboxymethylchitosan to form a triazole ring, thereby linking the drug and chitosan. In addition, the drug must first be reacted with azidobenzylaldehyde to form the conjugate. Furthermore, this reaction requires the use of a copper catalyst, which poses problems in terms of production and safety. In addition, Non-Patent Document 7 (Polymer Bulletin 31 (1993) 145-149) discloses dextran derivatives in which benzaldehyde has been introduced via an ester bond to the hydroxyl group of dextran. Because these polysaccharide derivatives use dextran, a neutral polysaccharide, it was difficult to introduce hydrophobic benzaldehyde at a high modification rate. Furthermore, the resulting polysaccharide derivatives also had poor water solubility, posing a problem in terms of application. This document also discloses derivatives in which benzaldehyde has been introduced into dextran that has been derivatized by the action of epihydrochlorin or epoxy. Although these derivatives are expected to have improved hydrophilicity due to the introduction of hydroxyl groups derived from epihydrochlorin or epoxy into the side chain, the synthesis of these derivatives requires multiple reaction steps, and there is room for improvement in terms of industrial production and the modification rate of benzaldehyde.

[0005] Numerous reports have also been published on Schiff base-free hyaluronic acid / drug conjugates, including HA protein conjugates (Non-Patent Document 8: Carbohydrate Polymers 92 (2013) 2163-2170), HA-IFNα2a conjugates (Non-Patent Document 9: Journal of Controlled Release 236 (2016) 79-89), and HA / pemetrexed conjugates (Non-Patent Document 10: European Journal of Pharmaceutical Sciences 138 (2019) 105008). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Materials Science and Engineering: C, 2014, 36: 287-293 [Non-patent document 2] Carbohydrate Polymers 134 (2015) 293-299 [Non-patent document 3] Carbohydrate Polymers 189 (2018) 273-279 [Non-patent document 4] Carbohydrate Polymers 216 (2019) 63-71 [Non-Patent Document 5] Materials Chemistry Frontiers 2 (2018) 1765-1778 [Non-patent document 6] J.Biomed.Nanotechnol. 13 (2017) 1647-1659 [Non-Patent Document 7] Polymer Bulletin 31 (1993) 145-149 [Non-patent document 8] Carbohydrate Polymers 92 (2013) 2163-2170 [Non-Patent Document 9] Journal of Controlled Release 236 (2016) 79-89 [Non-Patent Document 10] European Journal of Pharmaceutical Sciences 138 (2019) 105008 Summary of the Invention

[0007] Under these circumstances, there is a demand for new polysaccharide derivatives that can form conjugates with drugs.

[0008] The present invention is, for example, as follows. [1] A polysaccharide derivative in which a group represented by the following formula (A) is introduced into an acidic, basic, or amphoteric polysaccharide: [ka] (In the formula, R 1 is a hydrogen atom or C 1-4 represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring and the pyridine ring are optionally substituted with one or more substituents independently selected from a halogen atom, —CF3, —NO2, a carboxyl group, and —SO3H; Y is -NH-, -C(=O)-, -S-, -O-, C 1-6 Alkylene, -(CH2CH2O) n -, ... * indicates the linkage to the polysaccharide.) [2] The polysaccharide derivative according to [1], wherein the polysaccharide is selected from alginic acid, a derivative thereof or a salt thereof, hyaluronic acid, a derivative thereof or a salt thereof, carboxymethylcellulose, a derivative thereof or a salt thereof, carboxymethyldextran, a derivative thereof or a salt thereof, carboxymethylstarch, a derivative thereof or a salt thereof, heparin, a derivative thereof or a salt thereof, heparan sulfate, a derivative thereof or a salt thereof, chondroitin sulfate, a derivative thereof or a salt thereof, dermatan sulfate, a derivative thereof or a salt thereof, chitosan, a derivative thereof or a salt thereof, regenerated oxidized cellulose, a derivative thereof or a salt thereof, and pectinic acid, a derivative thereof or a salt thereof. [3] The polysaccharide derivative according to [1] or [2], wherein the group represented by formula (A) is a group selected from the group represented by formula (A-1) or (A-2): [ka] (In formulas (A-1) and (A-2), R 1 is a hydrogen atom or C 1-4 represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or the pyridine ring is optionally substituted with one or more substituents independently selected from a halogen atom, —CF3, —NO2, a carboxyl group, and —SO3H; L 1 is a single bond or -C(=O)-, -S-, -O-, C 1―6 Alkylene, -(CH2CH2O) n represents a divalent group selected from the group consisting of -, ... L 2 is a single bond or -NH-, -S-, -O-, C 1―6 Alkylene, -(CH2CH2O) n -, and any combination thereof. n is an integer from 1 to 9 * indicates the linkage to the polysaccharide.)

[0009] [4] The polysaccharide is a polysaccharide containing a carboxyl group and / or an amino group, The polysaccharide derivative according to any one of [1] to [3], wherein the group represented by formula (A) is introduced into the polysaccharide by forming an amide bond with a carboxyl group or an amino group of the polysaccharide. [5] The polysaccharide is a polysaccharide containing a carboxyl group, The group represented by the formula (A) is a group represented by the formula (A-1), The polysaccharide derivative according to any one of [1] to [4], wherein the group represented by formula (A-1) is introduced into the polysaccharide by substituting —OH of a carboxyl group of the polysaccharide to form an amide bond.

[0010] [6] The polysaccharide derivative according to any one of [1] to [5], which comprises at least one structural unit selected from the following formulae (c11), (c12), (c13), (c14) and (c15): [ka] (In the formula, R 11 , R 12 , R 13 , R 14 , R 21 , R 22 , R 23 , and R 24 are each independently a hydrogen atom, C 1-6 Alkyl, and -C(=O)-C 1-6 alkyl, R 31 , R 32 , and R 33 One to three of them are [ka] represents R 31 , R 32 , and R 33 The remainder are each independently a hydrogen atom, C 1-6 Alkyl, -C(=O)-C 1-6alkyl and -CH2COOH; R 41 , R 42 , and R 43 One to three of them are [ka] represents R 41 , R 42 , and R 43 The remainder are each independently a hydrogen atom, C 1-6 Alkyl, -C(=O)-C 1-6 alkyl and -CH2COOH; R 1 is a hydrogen atom or C 1-4 represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or the pyridine ring is optionally substituted with one or more substituents independently selected from a halogen atom, —CF3, —NO2, a carboxyl group, and —SO3H; Y is -L 1 represents -NH-, where L 1 is bonded to ring P, L 1 is a single bond, C 1-6 Alkylene, and -(CH2CH2O) n -, and n is an integer from 1 to 9.

[0011] [7] The polysaccharide is a polysaccharide containing an amino group, The group represented by the formula (A) is a group represented by the formula (A-2), The polysaccharide derivative according to any one of [1] to [4], wherein the group represented by formula (A-2) is introduced into the polysaccharide by substituting a hydrogen atom of an amino group of the polysaccharide to form an amide bond. [8] The polysaccharide derivative according to any one of [1] to [4] and [7], which contains a constitutional unit represented by the following formula (c16): [ka] (In the formula, R 81 and R 82 are each independently a hydrogen atom, C 1-6 Alkyl, and -C(=O)-C 1-6 alkyl, R 1 is a hydrogen atom or C 1-4 represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or the pyridine ring is optionally substituted with one or more substituents independently selected from a halogen atom, —CF3, —NO2, a carboxyl group, and —SO3H; Y is -L 2 represents -C(=O)-, where L 2 is bonded to ring P, L 2 is a single bond, C 1―6 Alkylene, -(CH2CH2O) n -, -(CH2) m1 -(CH2CH2O) n -(CH2) m2 - and -(CH2) m1 -O-(CH2CH2O) n -(CH2) m2 - selected from n is an integer from 1 to 9, m1 and m2 each independently represent an integer of 1 to 9.

[0012] [8-1] The polysaccharide derivative according to [3], wherein the group represented by formula (A) is a group represented by formula (A-1), the ring P is a phenyl ring, and the phenyl ring may be substituted with 1 to 4 substituents independently selected from a halogen atom (F, Cl, Br, and / or I), -CF3, -NO2, a carboxyl group, and -SO3H. [8-2] The polysaccharide derivative according to [3], wherein the group represented by formula (A) is a group represented by formula (A-1), and the group represented by formula (A-1) is a group selected from formulas (1), (2), and (3). [ka] (In formulas (1) to (3), *, R 1 and L 1 The definition of is as defined in [3], and R 51 , R 52 , R 53 , and R 54 are each independently selected from a hydrogen atom, a halogen atom, —CF3, —NO2, a carboxyl group, and —SO3H. [8-3] In the formulas (1) to (3), R 1 is a hydrogen atom, and L 1 is a single bond, and R 51 , R 52 , R 53 , and R 54 are all hydrogen atoms or selected from hydrogen atoms and halogen atoms (preferably F). [8-4] The polysaccharide derivative according to any one of [1] to [7], wherein the group represented by formula (A) is a group represented by the following formula: [ka] (In the formula, * represents the linkage to the polysaccharide.)

[0013] [8-5] The polysaccharide derivative according to any one of [1] to [7] and [8-1] to [8-4], wherein the polysaccharide is an acidic polysaccharide. [8-6] The polysaccharide derivative according to [1] to [4], [7], or [8-1], wherein the polysaccharide is a basic polysaccharide. [8-7] The polysaccharide derivative according to any one of [1] to [5] and [8-1] to [8-4], wherein the polysaccharide is an amphoteric polysaccharide. [8-8] The polysaccharide derivative according to any one of [1] to [7] and [8-1] to [8-4], wherein the polysaccharide is alginic acid, a derivative thereof, or a salt thereof. [8-9] The polysaccharide derivative according to any one of [1] to [7] and [8-1] to [8-4], wherein the polysaccharide is hyaluronic acid, a derivative thereof, or a salt thereof. [8-10] The polysaccharide derivative according to any one of [1] to [7] and [8-1] to [8-4], wherein the polysaccharide is carboxymethylcellulose, a derivative thereof, or a salt thereof. [8-11] The polysaccharide derivative according to any one of [1] to [7] and [8-1] to [8-4], wherein the polysaccharide is carboxymethyl starch, a derivative thereof, or a salt thereof. [8-12] The polysaccharide derivative according to [1] to [4], [7], or [8], wherein the polysaccharide is chitosan, a derivative thereof, or a salt thereof. [8-13] The polysaccharide is chitosan, a derivative thereof, or a salt thereof, and the group represented by formula (A) is a group represented by formula (A-2), The polysaccharide derivative according to any one of [1] to [4], [7], and [8], wherein the group represented by formula (A-2) is introduced into the polysaccharide by substituting a hydrogen atom of an amino group of the polysaccharide to form an amide bond.

[0014] [9] The polysaccharide derivative according to any one of [1] to [8] and [8-1] to [8-13], wherein the modification rate of the polysaccharide derivative with the group represented by the following formula (A) is 0.01 to 1:

[0015]

[10] A polysaccharide derivative-drug conjugate comprising a drug containing a primary amino group and the polysaccharide derivative according to any one of [1] to [9] and [8-1] to [8-13], A polysaccharide derivative-drug conjugate, in which a structure represented by the following formula (D) is formed between a primary amino group contained in the drug and a group represented by formula (A) contained in the polysaccharide: [ka] (wherein Drug represents the drug moiety excluding the primary amino group, Ring P, R 1 , and * are as defined in [1].)

[11] The polysaccharide derivative-drug conjugate according to

[10] , wherein the drug is released from the polysaccharide derivative-drug conjugate under low pH conditions.

[12] The polysaccharide derivative-drug conjugate according to

[10] or

[11] , wherein the drug is at least one selected from a low molecular weight compound, a medium molecular weight compound, a peptide, a nucleic acid, a nucleic acid derivative, an aptamer, a vitamin, a monoamine, an amino acid, a polyamine, an antibody, a fluorescent dye, and a contrast agent.

[0016]

[13] A crosslinked structure comprising the polysaccharide derivative according to any one of [1] to [9] and [8-1] to [8-13], wherein the polysaccharide derivative is crosslinked via a crosslinking group.

[14] A crosslinked structure comprising the polysaccharide derivative according to any one of [1] to [9], [8-1] to [8-13], and at least one of an amino group-containing polymer and an amino group-containing low molecular weight compound, each containing two or more primary amino groups, hydrazide groups, or aminooxy groups, wherein the primary amino groups, hydrazide groups, or aminooxy groups contained in the amino group-containing polymer and the amino group-containing low molecular weight compound are crosslinked via a covalent bond via a Schiff base to a group represented by formula (A) contained in the polysaccharide derivative.

[15] The crosslinked structure according to

[14] , wherein the amino group-containing polymer is at least one selected from the group consisting of linear, branched, or dendritic polyamines such as polyalkyleneimines (e.g., polyethyleneimine); polyalkylene glycols substituted with amino groups, hydrazide groups, or aminooxy groups; polyallylamine; polyvinylamine; polyacrylamine; amino group-containing polysaccharides such as chitosan; amino group-containing proteins such as gelatins, collagens, fibrinogen, and albumin; and polyamino acids such as polylysine.

[16] A crosslinked structure-drug conjugate of a drug containing a primary amino group and the crosslinked structure according to any one of

[13] to

[15] , wherein the primary amino group contained in the drug and the group represented by formula (A) contained in the crosslinked structure are covalently bonded via a Schiff base.

[0017]

[17] A composition comprising the polysaccharide derivative according to any one of [1] to [9], [8-1] to [8-13], the polysaccharide derivative-drug conjugate according to any one of

[10] to

[12] , the crosslinked structure according to any one of

[13] to

[15] , or the crosslinked structure-drug conjugate according to

[16] .

[18] A gel, sponge, film, or capsule comprising the polysaccharide derivative according to any one of [1] to [9], [8-1] to [8-13], the polysaccharide derivative-drug conjugate according to any one of

[10] to

[12] , the crosslinked structure according to any one of

[13] to

[15] , the crosslinked structure-drug conjugate according to

[16] , or the composition according to

[17] .

[19] A tissue adhesive material comprising the polysaccharide derivative according to any one of [1] to [9], [8-1] to [8-13], the polysaccharide derivative-drug conjugate according to any one of

[10] to

[12] , the crosslinked structure according to any one of

[13] to

[15] , or the crosslinked structure-drug conjugate according to

[16] .

[20] A drug transport carrier or separation material comprising the polysaccharide derivative according to any one of [1] to [9], [8-1] to [8-13] or the crosslinked structure according to any one of

[13] to

[15] .

[0018]

[21] A method for producing a polysaccharide derivative represented by the following formula (C1): A method comprising: subjecting a polysaccharide containing a carboxyl group to a condensation reaction with a compound (a1) represented by the following formula (a1) in an aqueous solvent: [ka] (In the formula, L 1 is a single bond, C 1-6 Alkylene, and -(CH2CH2O) n -, where n is an integer from 1 to 9; Rings P and R 1 is as defined in [1].)

[0019]

[22] The method according to

[15] , wherein the reaction between the compound (a1) and the polysaccharide is carried out under conditions of pH 5 to 10 (preferably pH 7.5 to 8.0).

[23] A method for producing the polysaccharide derivative-drug conjugate according to any one of [8] to

[10] , which comprises mixing the polysaccharide derivative according to any one of [1] to [9] and [8-1] to [8-13] with a drug containing a primary amino group in a solvent.

[0020] According to one aspect of the present invention, there is provided a novel polysaccharide derivative capable of forming a Schiff base with a primary amino group. The polysaccharide derivative can form a conjugate by forming a Schiff base with a drug having a primary amino group. According to one aspect of the present invention, there is provided a polysaccharide derivative-drug conjugate. The polysaccharide derivative-drug conjugate is capable of efficiently releasing a drug under low pH conditions. According to one aspect of the present invention, there is provided a crosslinked structure in which polysaccharide derivatives are crosslinked via crosslinking groups. According to one aspect of the present invention, there is provided a crosslinked structure comprising a polysaccharide derivative and an amino group-containing polymer containing two or more primary amino groups. In accordance with a further aspect of the present invention, there is provided a crosslinked structure-drug conjugate. In one embodiment of the present invention, the polysaccharide derivative and crosslinked structure can be used for a variety of purposes such as tissue adhesive materials, drug delivery carriers, and separation materials. In one embodiment of the present invention, the polysaccharide derivative, the polysaccharide derivative-drug conjugate, the crosslinked structure, and the crosslinked structure-drug conjugate can be suitably used as a medical material such as a drug release device or a tissue adhesive material. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1A shows the 1H NMR spectra of the benzaldehyde-modified alginic acid (AL-ABA) and alginic acid (AL) synthesized in Example I-1, and FIG. 1B shows the regions corresponding to peaks b to d shown in FIG. 1A. [Figure 2]FIG. 2 shows the ultraviolet-visible absorption spectra (UV-vis) of the benzaldehyde-modified alginic acid (AL-ABA), alginic acid (AL), and 4-aminobenzaldehyde (ABA) synthesized in Example I-1. [Figure 3] FIG. 3 shows the FT-IR spectra of the benzaldehyde-modified alginic acid (AL-ABA) and alginic acid (AL) synthesized in Example I-1. [Figure 4] FIG. 4 shows the cytotoxicity test results (WST assay results) of benzaldehyde-modified alginate (AL-ABA) and alginic acid (AL) synthesized in Example I-1 at different concentrations (0.01 mg / mL, 0.1 mg / mL, and 1 mg / mL) on MeT-5A (human mesothelial cell line), NIH / 3T3 (mouse embryonic fibroblasts), HUVEC (human umbilical vein endothelial cells), and RAW264.7 cells (mouse macrophage-like cell line). [Figure 5] FIG. 5 shows the 1H NMR spectrum of the AAP-modified alginic acid (AL-AAP) synthesized in Example I-3. [Figure 6] FIG. 6 shows the FT-IR spectrum of the AAP-modified alginic acid (AL-AAP) synthesized in Example I-3. [Figure 7] FIG. 7 shows the 1H NMR spectrum of the ADFBA-modified alginic acid (AL-ADFBA) synthesized in Example I-4. [Figure 8] FIG. 8 shows the FT-IR spectrum of the ADFBA-modified alginic acid (AL-ADFBA) synthesized in Example I-4. [Figure 9] FIG. 9 shows the 1H NMR spectrum of the AAP-modified alginic acid (AL-APCA) synthesized in Example I-5. [Figure 10] FIG. 10 shows the FT-IR spectrum of the AAP-modified alginic acid (AL-APCA) synthesized in Example I-5. [Figure 11] FIG. 11 shows the 1H NMR spectrum of the ANA-modified alginic acid (AL-ANA) synthesized in Example I-6. [Figure 12]FIG. 12 shows the FT-IR spectrum of the ANA-modified alginic acid (AL-ANA) synthesized in Example I-6. [Figure 13] Figure 13 shows the H NMR spectra of the benzaldehyde-modified alginate-vancomycin conjugate (AL-ABA-Van) synthesized in Example I-7, the benzaldehyde-modified alginate (AL-ABA) synthesized in Example I-1, vancomycin (Van), and alginate (AL). [Figure 14] Figure 14 shows the ultraviolet-visible absorption spectra (UV-vis) of the benzaldehyde-modified alginate-vancomycin conjugate (AL-ABA-Van) synthesized in Example I-7, the benzaldehyde-modified alginate (AL-ABA) synthesized in Example I-1, and vancomycin (Van). [Figure 15] Figure 15 shows the FT-IR spectra of the benzaldehyde-modified alginate-vancomycin conjugate (AL-ABA-Van) synthesized in Example I-7, the benzaldehyde-modified alginate (AL-ABA) synthesized in Example I-1, vancomycin (Van), and alginate (AL). [Figure 16A] Figure 16A shows the release behavior of vancomycin (Van) from the benzaldehyde-modified alginate-vancomycin conjugate (AL-ABA-Van) solution synthesized in Example I-7 at different pH values ​​(pH 5.0, 6.0, and 7.4). The cumulative release rate (%) of Van is shown on the vertical axis and was calculated from the absorbance of UV-vis spectroscopy. [Figure 16B] Figure 16B shows the release behavior of vancomycin from a mixed solution of sodium alginate and vancomycin (AL + Van) at different pH values ​​(pH = 5.0, 6.0, 7.4) (control experiment). The cumulative release rate (%) of vancomycin shown on the vertical axis was calculated from the absorbance measured by UV-vis spectroscopy. [Figure 16C]Figure 16C shows the Van release profile (control experiment) from vancomycin solutions (Van only) at different pH levels (pH = 5.0, 6.0, 7.4). The vertical axis shows the cumulative release rate (%) of Van, calculated from the absorbance of UV-vis spectroscopy. [Figure 17] Figure 17 shows confocal microscopy images of the FTSC-loaded AL-ABA capsules and FTSC-loaded AL capsules prepared in Example I-8. The top row shows a confocal microscopy image of the AL-capsules (Alg microcapsules) in FTSC-containing saline, the middle row shows a confocal microscopy image of the AL-ABA capsules (Alg-ABA microcapsules) in FTSC-containing saline, and the bottom row shows a confocal microscopy image of the AL-ABA capsules (Alg-ABA microcapsules) in FTSC-containing DMEM. In each image, the left side shows a transmitted image, the middle shows a fluorescent image (amine-fluorescein), and the right side shows a merged image of both. [Figure 18] Figure 18 shows the release profile of vancomycin (Van) from Van-loaded AL-ABA microcapsules (AL-ABA-Van capsules) prepared in Example I-9 and Van-loaded AL microcapsules (AL-Van capsules) as a control. Figure 18A shows the cumulative Van release rate (%) on the vertical axis, and Figure 18B shows the Van release rate (Vancomycin Release %) at each time point on the vertical axis. The Van release rate (Vancomycin Release %) was calculated from the absorbance measured by UV-vis spectroscopy. [Figure 19] Figure 19 shows photographs of Staphylococcus aureus growth around filter paper impregnated with a sustained-release solution from vancomycin-loaded capsules. (a) shows the results when a sustained-release solution from vancomycin-loaded AL capsules (AL-Van) was used, and (b) shows the results when a sustained-release solution from vancomycin-loaded AL-ABA capsules (AL-ABA-Van) was used. The upper panel shows the results immediately after the addition of the sustained-release solution, and the lower panel shows the results 24 hours after addition. [Figure 20] FIG. 20 shows a comparison (n=3) of the growth inhibition area of ​​Staphylococcus aureus by the sustained-release liquid at each sustained-release time between vancomycin-loaded AL capsules (AL-Van) and vancomycin-loaded AL-ABA capsules (AL-ABA-Van). [Figure 21] Figure 21 shows the ultraviolet-visible absorption spectra (UV-vis) of the benzaldehyde-modified alginate-bacitracin conjugate (AL-ABA-Bac) synthesized in Example I-10, the benzaldehyde-modified alginate (AL-ABA) synthesized in Example 1, and bacitracin (Bac). [Figure 22] FIG. 22 shows the FT-IR spectra of the benzaldehyde-modified alginate-bacitracin conjugate (AL-ABA-Bac) synthesized in Example I-10, bacitracin (Bac), the benzaldehyde-modified alginate (AL-ABA) synthesized in Example 1, and alginic acid (AL). [Figure 23] FIG. 23 shows the 1H NMR spectrum of the benzaldehyde-modified alginic acid-dopamine conjugate (AL-ABA-DOPA) synthesized in Example I-11. [Figure 24] FIG. 24 shows the FT-IR spectrum of the benzaldehyde-modified alginic acid-dopamine conjugate (AL-ABA-DOPA) synthesized in Example I-11. [Figure 25] FIG. 25 shows the 1H NMR spectrum of the benzaldehyde-modified alginic acid-serotonin conjugate (AL-ABA-Serotonin) synthesized in Example I-13. [Figure 26] FIG. 26 shows the FT-IR spectrum of the benzaldehyde-modified alginic acid-serotonin conjugate (AL-ABA-Serotonin) synthesized in Example I-13. [Figure 27] FIG. 27 shows the 1H NMR spectrum of the benzaldehyde-modified alginic acid-celecoxib conjugate (AL-ABA-Celecoxib) synthesized in Example I-14. [Figure 28]FIG. 28 shows the preparation method of AL-ABA-Apt and the experimental procedure for the sustained release experiment of AL-ABA-Apt in Example I-15. [Figure 29] Figure 29 shows the release behavior of the HGF aptamer (Apt) from the AL-ABA-HGF aptamer (AL-ABA-Apt) synthesized in Example I-15, a mixture of alginate and the HGF aptamer (ALG-Apt), and the HGF aptamer alone (Apt) under physiological pH conditions (pH 7.4). The vertical axis represents the cumulative release rate (%) of the HGF aptamer, calculated from the absorbance measured by UV-vis spectroscopy. [Figure 30] Figure 31 shows optical photographs of the benzaldehyde-modified alginate sponges prepared in Example I-16. Control indicates the AL sponge prepared as a control, while A, B, and C indicate AL / AL-ABA mixed sponges with AL and AL-ABA blend ratios (weight ratio) of 75:25, 50:50, and 25:75, respectively. [Figure 31] Figure 31 shows SEM photographs of the benzaldehyde-modified alginate sponges prepared in Example I-16. Control indicates the AL sponge prepared as a control, while A, B, and C indicate AL / AL-ABA mixed sponges with AL and AL-ABA blend ratios (weight ratio) of 75:25, 50:50, and 25:75, respectively. [Figure 32] Figure 32 shows the swelling and decomposition profile (change in hydrogel weight) of the Ca-crosslinked AL-ABA hydrogel. [Figure 33] Figure 33 shows an SEM photograph (magnification: ×500; 24 hours after the start of swelling) of a sponge obtained by freeze-drying the Ca-crosslinked AL-ABA hydrogel. The porous structure of the dried hydrogel can be confirmed. [Figure 34] FIG. 34 shows a schematic diagram of the crosslinked structure of AL-ABA and an amino group-containing polymer (DPI; dendritic polyethyleneimine) prepared in Example I-18. [Figure 35] FIG. 35 shows a photograph of a hydrogel consisting of a crosslinked structure of AL-ABA and DPI. [Figure 36A] FIG. 36A shows the 1H NMR spectrum of PEG-COOH prepared in Example I-19. [Figure 36B] Figure 36B shows the 1H NMR spectrum of PEGDH prepared in Example I-19. [Figure 36C] FIG. 36C shows a photograph of the hydrogel composed of a crosslinked structure of AL-ABA and PEGDH prepared in Example I-19. [Figure 37] FIG. 37 shows the results of dynamic viscoelasticity measurement of the hydrogel consisting of a crosslinked structure of AL-ABA and an amino group-containing polymer (PEGDH) prepared in Example I-19. [Figure 38] FIG. 38 shows a photograph of the hydrogel of AL-ABA and polyallylamine prepared in Example I-21. [Figure 39] Figure 39 shows the change in appearance over time when benzaldehyde-modified alginate (AL-ABA) and alginate (AL) calcium-crosslinked gels (AL-ABA gel and AL gel) were administered to the porcine esophagus and immersed in saline in Example I-22. In Figure 39, ALG (IL-6G) represents the AL gel, and ALG-ABA represents the AL-ABA gel. [Figure 40] Figure 40 shows the change in gel weight (adhesion rate, %) over time when calcium-crosslinked gels of benzaldehyde-modified alginate (AL-ABA) and alginate (AL) (AL-ABA gel, AL gel) were administered to the porcine esophagus and immersed in saline in Example I-22. In Figure 40, ALG (IL-6G) represents the AL gel, and ALG-ABA represents the AL-ABA gel. [Figure 41] FIG. 41 is a schematic diagram showing the binding of AL-ABA and AL to the esophageal mucosa and submucosa. [Figure 42] FIG. 42 shows the procedure for evaluating the adhesiveness of AL-ABA and AL to the esophageal mucosal layer and submucosal layer in Example I-23. [Figure 43]Figure 43 shows the change in appearance over time when gels (AL-ABA, AL) prepared by calcium cross-linking benzaldehyde-modified alginate (AL-ABA) and alginate (AL) at 50 mM and 100 mM Ca concentrations, respectively, were applied to the porcine esophageal mucosa (Control) and submucosa (ESD) and immersed in saline in Example I-23. The circles (dotted lines) in the figure indicate areas where the material remains. [Figure 44] Figure 44 shows the change in gel weight (adhesion rate, %) over time when gels (AL-ABA, AL) prepared by calcium cross-linking benzaldehyde-modified alginate (AL-ABA) and alginate (AL) at a Ca2+ concentration of 50 mM or 100 mM, respectively, were applied to the mucosa (Con) and submucosa (ESD) of the porcine esophagus and immersed in saline in Example I-23. [Figure 45] FIG. 45 is a schematic diagram showing the method of the tensile test in Example I-23. [Figure 46] FIG. 46 shows the results of a tensile test of Ca-crosslinked hydrogels of AL-ABA and AL adhered to the mucosa (Control) and submucosa (ESD) in Example I-23. [Figure 47] 47(A) and (B) are schematic diagrams showing the experimental procedure of the lap shear method in Example I-24. [Figure 48] Figure 48 shows the results of measuring the adhesion strength (Pa) of AL-ABA and AL Ca-crosslinked hydrogels and a conventional tissue adhesive adhered to submucosa and skin tissues, as described in Example I-24, using the lap shear method. [Figure 49] Figure 49(A) is a schematic diagram showing the apparatus used in the burst test in Example I-25, and Figure 49(B) is a schematic diagram showing the method of the burst test. [Figure 50]Figure 50 shows the results of measuring the burst pressure (mmHg) by burst test for the Ca-crosslinked hydrogels of AL-ABA and AL adhered to submucosa and skin tissues, as well as for a conventional tissue adhesive, in Example I-25. [Figure 51] FIG. 51 shows the 1H NMR spectrum of the benzaldehyde-modified hyaluronic acid (HA-ABA) synthesized in Example II-1. [Figure 52] FIG. 52 shows the ultraviolet-visible absorption spectra (UV-vis) of the benzaldehyde-modified hyaluronic acid (HA-ABA), hyaluronic acid (HA), and 4-aminobenzaldehyde (ABA) synthesized in Example II-1. [Figure 53] FIG. 53 shows the FT-IR spectra of the benzaldehyde-modified hyaluronic acid (HA-ABA), hyaluronic acid (HA), and 4-aminobenzaldehyde (ABA) synthesized in Example II-1. [Figure 54] Figure 54 shows the results of a cytotoxicity test (WST assay results) of the benzaldehyde-modified hyaluronic acid (HA-ABA) and hyaluronic acid (HA) synthesized in Example II-1 at different concentrations (0.01 mg / mL, 0.1 mg / mL, and 1 mg / mL) on MeT-5A (human mesothelial cell line), HUVEC (human umbilical vein endothelial cell), RAW264.7 cells (mouse macrophage-like cell line), NIH / 3T3 (mouse embryonic fibroblast) and AB22 cells (mouse mesothelioma cell). [Figure 55] FIG. 55 shows the H NMR spectra of the benzaldehyde-modified hyaluronic acid-pemetrexed conjugate (HA-ABA-PMX), benzaldehyde-modified hyaluronic acid (HA-ABA), and hyaluronic acid (HA) and pemetrexed (PMX) synthesized in Example II-2. [Figure 56]FIG. 56 shows the ultraviolet-visible absorption spectra (UV-vis) of the benzaldehyde-modified hyaluronic acid-pemetrexed conjugate (HA-ABA-PMX) and benzaldehyde-modified hyaluronic acid (HA-ABA) synthesized in Example II-2. [Figure 57] Figure 57 shows the release profiles of pemetrexed (PMX) from a benzaldehyde-modified hyaluronic acid-pemetrexed conjugate solution (HA-ABA-PMX) at different pH levels (pH 5.0, 6.0, 7.4), from a PMX solution (free PMX), and from a mixed solution of HA and PMX (free PMX mixed with HA). The vertical axis represents the cumulative drug release rate (%) of PMX, calculated from the absorbance measured by UV-vis spectroscopy. [Figure 58] Figures 58A and 58B show the cell growth inhibitory effects of the benzaldehyde-modified hyaluronic acid-pemetrexed conjugate (HA-ABA-PMX) synthesized in Example II-2, free pemetrexed (PMX), and HA-ADH-PMX, in which PMX is linked to HA via an irreversible amide bond, at different PMX concentrations (10 μg / mL, 10 μg / mL, 10 μg / mL, 1 μg / mL, and 10 μg / mL) on AB22 cells (mouse mesothelioma cells) and MeT-5A (human mesothelial cell line), respectively. The vertical axis represents cell viability (%). [Figure 59] Figure 59 shows the ultraviolet-visible absorption spectra (UV-vis) of the benzaldehyde-modified hyaluronic acid-doxorubicin conjugate (HA-ABA-DOX) synthesized in Example II-3, the benzaldehyde-modified hyaluronic acid (HA-ABA) synthesized in Example II-1, and doxorubicin (DOX). [Figure 60]Figure 60 shows the release behavior of doxorubicin (DOX) from benzaldehyde-modified hyaluronic acid-doxorubicin conjugate solution (HA-ABA-DOX) at different pH levels (pH 5.0, 6.0, 7.4). The vertical axis represents the cumulative drug release rate (%) of DOX, calculated from the absorbance measured by UV-vis spectroscopy. [Figure 61] Figure 61 shows the cell growth inhibitory effect of the benzaldehyde-modified hyaluronic acid-doxorubicin conjugate (HA-ABA-DOX) synthesized in Example II-3 and free doxorubicin (DOX) at different DOX concentrations (10 μg / mL, 10 μg / mL, 10 μg / mL, 10 μg / mL, and 100 μg / mL) on AB22 cells (mouse mesothelioma cells). The vertical axis represents cell viability (%). [Figure 62] Figure 62 shows the ultraviolet-visible absorption spectra (UV-vis) of the benzaldehyde-modified hyaluronic acid-gemcitabine conjugate (HA-ABA-GEM) synthesized in Example II-4, the benzaldehyde-modified hyaluronic acid (HA-ABA) synthesized in Example II-1, and gemcitabine (GEM). [Figure 63] FIG. 63 shows the 1H NMR spectrum of the benzaldehyde-modified hyaluronic acid-adenine conjugate (HA-ABA-Adenine) synthesized in Example II-5. [Figure 64] Figure 64 shows the 1H NMR spectrum of the benzaldehyde-modified hyaluronic acid-cytosine conjugate (HA-ABA-cytosine) synthesized in Example II-5. [Figure 65] Figure 65 shows the 1H NMR spectrum of the benzaldehyde-modified hyaluronic acid-guanine conjugate (HA-ABA-guanine) synthesized in Example II-5. [Figure 66]Figure 66 shows the FT-IR spectra of the benzaldehyde-modified hyaluronic acid-DNA nucleotide conjugates (HA-ABA-Adenine, HA-ABA-cytosine, and HA-ABA-guanine) synthesized in Example II-5 and the benzaldehyde-modified hyaluronic acid (HA-ABA) synthesized in Example II-1. [Figure 67] FIG. 67 shows the 1H NMR spectrum of the benzaldehyde-modified carboxymethyl cellulose (CMC-ABA) synthesized in Example III-1. [Figure 68] FIG. 68 shows the FT-IR spectra of the benzaldehyde-modified carboxymethyl cellulose (CMC-ABA) and carboxymethyl cellulose (CMC) synthesized in Example III-1. [Figure 69] FIG. 69 shows the 1H NMR spectrum of the benzaldehyde-modified carboxymethyl dextran (CMDX-ABA) synthesized in Example IV-1. [Figure 70] FIG. 70 shows the FT-IR spectra of the benzaldehyde-modified carboxymethyl dextran (CMD-ABA) and carboxymethyl dextran (CMD) synthesized in Example IV-1. [Figure 71] Figure 71 shows the FT-IR spectra of the benzaldehyde-modified carboxymethyl dextran-DNA nucleotide conjugates (CMD-ABA-Adenine, CMD-ABA-Cytosine, and CMD-ABA-Guanine) synthesized in Example IV-2, and the benzaldehyde-modified carboxymethyl dextran (CMD-ABA) and carboxymethyl dextran (CMD) synthesized in Example IV-1. [Figure 72] FIG. 72 shows the 1H NMR spectrum of the benzaldehyde-modified chitosan (Chitosan-CBA) synthesized in Example V-1. [Figure 73] FIG. 73 shows the FT-IR spectrum of the benzaldehyde-modified chitosan (Chitosan-CBA) synthesized in Example V-1. DETAILED DESCRIPTION OF THE INVENTION

[0022] In this specification, the term "halogen atom" refers to a fluorine atom (F), a chlorine atom (Cl), a bromine atom (Br), or an iodine atom (I). As used herein, "C 1-k "Alkyl" means a straight or branched chain alkyl group having 1 to k carbon atoms. As used herein, "C 1-k The term "alkylene" refers to a linear or branched alkylene group having 1 to k carbon atoms. As used herein, "polysaccharide" refers to a compound consisting of two or more monosaccharides linked together by glycosidic bonds, derivatives thereof (e.g., polysaccharides modified by esterification, maleimide modification, thiol modification, acrylate modification, aldehyde modification, disulfide modification (e.g., pyridyl disulfide), alkyne modification including cyclic alkyne, tetrazine modification, furan ring modification, etc.), salts thereof, and crosslinked products thereof. Although a compound formed by linking two to ten monosaccharides is sometimes called an oligosaccharide, in this specification, "polysaccharide" encompasses not only a "polysaccharide" in the narrow sense, in which ten or more monosaccharides are linked, but also an "oligosaccharide." As used herein, the term "polysaccharide derivative-drug conjugate" refers to a complex formed by linking a drug and a polysaccharide derivative via a bond.

[0023] 1. Polysaccharide derivatives One aspect of the present invention relates to a polysaccharide derivative (hereinafter also simply referred to as "polysaccharide derivative") in which a group represented by the following formula (A) (hereinafter also simply referred to as "modifying group (A)") is introduced into an acidic, basic, or amphoteric polysaccharide: [ka]

[0024] Polysaccharide derivatives can react with primary amino groups to form Schiff bases under neutral to basic pH conditions. The Schiff bases formed between polysaccharide derivatives and primary amino groups are stable under neutral to basic pH conditions but can dissociate under low pH conditions. This property can be utilized in drug delivery carriers, bioabsorbable materials, medical devices, and separation materials.

[0025] The polysaccharide derivative of this form has at least one of the following advantages. (1) The polysaccharide derivatives can form stable conjugates with various drugs having primary amino groups under neutral to basic pH conditions while releasing the drugs. In particular, the above-mentioned modifying group (A) can form stable Schiff bases with the amine groups contained in various drugs even in water due to its aromatic structure. (2) Because polysaccharide derivatives are based on acidic, basic, or amphoteric polysaccharides, the modifying group (A) can be introduced at a good modification rate, even if the modifying group (A) has a hydrophobic benzene ring or pyridyl ring. Furthermore, even if the modifying group (A) is introduced at a high modification rate, the resulting polysaccharide derivative has excellent water solubility due to the presence of unmodified acidic or basic functional groups (anionic or cationic functional groups). In particular, polysaccharides having anionic functional groups (such as carboxyl groups) can yield polysaccharide derivatives with particularly excellent water solubility, even when the modification rate of the modifying group (A) is high. (3) Because polysaccharides have acidic, basic, or both (amphoteric) functional groups (i.e., charged functional groups), they can be easily reacted with highly hydrophobic phenylaldehyde or pyridylaldehyde compounds in a single step under mild and safe conditions, allowing the modification group (A) to be easily introduced into the polysaccharide. (4) In some embodiments, stable polysaccharide derivative-drug conjugates can be easily obtained in one pot using a single-step reaction under mild and highly safe conditions. This eliminates the need for drug modification prior to conjugate formation, and also eliminates the need for complex experimental equipment or catalysts for conjugate formation, making in situ preparation of polysaccharide derivative-drug conjugates from the polysaccharide derivative and the drug possible. (5) In some embodiments, polysaccharide derivatives and conjugates of polysaccharide derivatives and drugs can be subjected to a crosslinking reaction using a crosslinking agent to form crosslinked structures of various shapes (e.g., tubular structures, fibrous structures, fibers, beads, gels, approximately spherical gels, capsules, sponges, and sheets). In some embodiments, polysaccharide derivatives and conjugates of polysaccharide derivatives and drugs can form crosslinked structures of the above-mentioned various forms between the polysaccharide derivatives and amino group-containing polymers.

[0026] (Modifying group (A): Group represented by formula (A)) In the above formula (A), R 1 is a hydrogen atom or C 1-4 Represents alkyl. R 1 is preferably a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a tert-butyl group, a sec-butyl group, an isobutyl group, or an n-butyl group, and more preferably a hydrogen atom or a methyl group. 1 is a hydrogen atom. In the above formula (A), * represents the linkage to the polysaccharide.

[0027] In the above formula (A), ring P is a phenyl ring or a pyridine ring, which may be substituted with one or more substituents independently selected from halogen atoms (F, Cl, Br, and / or I), —CF (trifluoromethyl group), —NO, carboxyl group, and —SOH. In one embodiment, ring P is a phenyl ring, which is optionally substituted with 1 to 4 substituents independently selected from halogen atoms (F, Cl, Br, and / or I), -CF3, -NO2, a carboxyl group, and -SO3H. In one embodiment, ring P is an unsubstituted phenyl ring. In one embodiment, ring P is a pyridine ring, which is optionally substituted with 1 to 3 substituents independently selected from a halogen atom (F, Cl, Br, and / or I), —CF 3 , —NO 2 , a carboxyl group, and —SO 3 H. In one embodiment, ring P is an unsubstituted pyridine ring.

[0028] In the above formula (A), Y is -NH-, -C(=O)-, -S-, -O-, alkylene, -(CH2CH2O) n - and any combination thereof, and n is an integer of 1 to 9 (preferably 1 to 4, more preferably 1 to 2). The number of carbon atoms in the alkylene group for Y is 1 to 6, preferably 1 to 4, more preferably 1 or 2, and even more preferably 1, from the viewpoint of hydrophilicity.

[0029] In some embodiments, Y is -L 1 -NH- or -L 2 -C(=O)-, wherein L 1 and L 2 is bonded to the ring P. That is, the modifying group (A) is selected from the following formula (A-1) or (A-2). [ka] In the above formulas (A-1) and (A-2), * represents a linking site to a polysaccharide. 1 The definition and preferred embodiments of ring P are as follows: 1 and the definition and preferred embodiments of ring P are the same as those of ring P.

[0030] In formula (A-1), L 1is a single bond or -C(=O)-, -S-, -O-, alkylene, -(CH2CH2O) n L represents a divalent group selected from the group consisting of -, -, and any combination thereof, and n is an integer of 1 to 9 (preferably 1 to 4, more preferably 1 to 2). 1 The alkylene group in the formula (I) has 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, more preferably 1 to 2 carbon atoms, and even more preferably 1 carbon atom, from the viewpoint of hydrophilicity. Among them, L 1 is a single bond, C 1―6 Alkylene, -(CH2CH2O) n -, -(CH2) m1 -(CH2CH2O) n -(CH2) m2 - and -(CH2) m1 -O-(CH2CH2O) n -(CH2) m2 -. n is an integer of 1 to 9 (preferably 1 to 4, more preferably 1 to 2). m1 and m2 are each independently an integer of 1 to 9 (preferably 1 to 4, more preferably 1 to 2). L 1 C in 1―6 The alkylene is preferably C 1―4 alkylene, more preferably C 1―2 It is preferably alkylene, and more preferably methylene. In one embodiment, L 1 is a single bond or C 1―2 In one embodiment, L 1 is a single bond.

[0031] In formula (A-2), L 2 is a single bond or -NH-, -S-, -O-, alkylene, -(CH2CH2O) n L represents a divalent group selected from the group consisting of -, -, and any combination thereof, and n is an integer of 1 to 9 (preferably 1 to 4, more preferably 1 to 2). 2The alkylene group in the formula (I) has 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, more preferably 1 to 2 carbon atoms, and even more preferably 1 carbon atom, from the viewpoint of hydrophilicity. Among them, L 2 is a single bond, C 1―6 Alkylene, -(CH2CH2O) n -, -(CH2) m1 -(CH2CH2O) n -(CH2) m2 - and -(CH2) m1 -O-(CH2CH2O) n -(CH2) m2 -. n is an integer of 1 to 9 (preferably 1 to 4, more preferably 1 to 2). m1 and m2 are each independently an integer of 1 to 9 (preferably 1 to 4, more preferably 1 to 2). L 2 C in 1―6 The alkylene is preferably C 1―4 alkylene, more preferably C 1―2 It is preferably alkylene, and more preferably methylene. In one embodiment, L 2 is a single bond or C 1―2 In one embodiment, L 2 is a single bond.

[0032] In some embodiments, the modifying group (A) is a group represented by the above formula (A-1), and the ring P is a phenyl ring, which may be substituted with 1 to 4 substituents independently selected from a halogen atom (F, Cl, Br, and / or I), -CF3, -NO2, a carboxyl group, and -SO3H.

[0033] For example, the modifying group (A) is selected from the following formulae (1), (2), and (3) (hereinafter sometimes referred to as "formulae (1) to (3)"). [ka] In the above formulas (1) to (3), * represents a linking site to a polysaccharide. 1The definition and preferred embodiments of R in the above formula (A) are as follows: 1 The definition and preferred embodiments of L are the same as those of 1 The definition and preferred embodiments of are as follows: 1 The definition and preferred embodiments are the same as those of the above. In the above formulas (1) to (3), R 51 , R 52 , R 53 , and R 54 are each independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), —CF, —NO, a carboxyl group, and —SOH. 51 , R 52 , R 53 , and R 54 are both hydrogen atoms. 51 , R 52 , R 53 , and R 54 are each independently selected from a hydrogen atom and a halogen atom (F, Cl, Br, I; preferably F). is.

[0034] In certain embodiments, in the above formulas (1) to (3), R 1 is a hydrogen atom, and L 1 is a single bond, and R 51 , R 52 , R 53 , and R 54 are each independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), —CF3, —NO2, a carboxyl group, and —SO3H. In one embodiment, in the above formulas (1) to (3), R 1 is a hydrogen atom, and L 1 is a single bond, and R 51 , R 52 , R 53 , and R 54 In one embodiment, in the above formulas (1) to (3), R 1 is a hydrogen atom, and L 1 is a single bond, and R 51 , R 52 , R53 , and R 54 are each independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0035] In certain embodiments, in the above formulas (1) to (3), R 1 is methyl and L 1 is a single bond, and R 51 , R 52 , R 53 , and R 54 are each independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), —CF3, —NO2, a carboxyl group, and —SO3H. In one embodiment, in the above formulas (1) to (3), R 1 is methyl and L 1 is a single bond, and R 51 , R 52 , R 53 , and R 54 In one embodiment, in the above formulas (1) to (3), R 1 is methyl and L 1 is a single bond, and R 51 , R 52 , R 53 , and R 54 are each independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0036] In certain embodiments, in the above formulas (1) to (3), R 1 is a hydrogen atom, and L 1 is methylene (-CH2-) or ethylene (-C2H4-), and R 51 , R 52 , R 53 , and R 54 are each independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), —CF3, —NO2, a carboxyl group, and —SO3H. In one embodiment, in the above formulas (1) to (3), R 1 is a hydrogen atom, and L 1 is methylene (-CH2-) or ethylene (-C2H4-), and R 51 , R52 , R 53 , and R 54 In one embodiment, in the above formulas (1) to (3), R 1 is a hydrogen atom, and L 1 is methylene (-CH2-) or ethylene (-C2H4-), and R 51 , R 52 , R 53 , and R 54 are each independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0037] In certain embodiments, in the above formulas (1) to (3), R 1 is methyl and L 1 is methylene (-CH2-) or ethylene (-C2H4-), and R 51 , R 52 , R 53 , and R 54 are each independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), —CF3, —NO2, a carboxyl group, and —SO3H. In one embodiment, in the above formulas (1) to (3), R 1 is methyl and L 1 is methylene (-CH2-) or ethylene (-C2H4-), and R 51 , R 52 , R 53 , and R 54 In one embodiment, in the above formulas (1) to (3), R 1 is methyl and L 1 is methylene (-CH2-) or ethylene (-C2H4-), and R 51 , R 52 , R 53 , and R 54 are each independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0038] In one embodiment, the modifying group (A) is selected from groups represented by the formula: [ka] (In the formula, * represents the linkage to the polysaccharide.) In one embodiment, the modifying group (A) is a group represented by the formula: [ka] (In the formula, * represents the linkage to the polysaccharide.)

[0039] In some embodiments, the modifying group (A) is a group represented by the above formula (A-1), and the ring P is a pyridine ring, which may be substituted with 1 to 3 substituents independently selected from a halogen atom (F, Cl, Br, I), -CF3, -NO2, a carboxyl group, and -SO3H. For example, the modifying group (A) is selected from the following formulas (4), (5), (6), (7), (8), (9), (10), (11), (12), and (13) (hereinafter sometimes referred to as "formulas (4) to (13)"). [ka] In the above formulas (4) to (13), * represents a linking site to a polysaccharide. 1 The definition and preferred embodiments of R in the above formula (A) are as follows: 1 The definition and preferred embodiments of L are the same as those of 1 The definition and preferred embodiments of are as follows: 1 The definition and preferred embodiments are the same as those of the above. In the above formulas (4) to (13), R 61 , R 62 , and R 63 are each independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), —CF, —NO, a carboxyl group, and —SOH. 61 , R 62 , and R 63 are both hydrogen atoms. 61 , R 62 , and R 63are each independently selected from a hydrogen atom and a halogen atom (F, Cl, Br, I).

[0040] In certain embodiments, in the above formulas (4) to (13), R 1 is a hydrogen atom, and L 1 is a single bond, and R 61 , R 62 , and R 63 are each independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), —CF3, —NO2, a carboxyl group, and —SO3H. In one embodiment, in the above formulas (4) to (13), R 1 is a hydrogen atom, and L 1 is a single bond, and R 61 , R 62 , and R 63 In one embodiment, in the above formulas (4) to (13), R 1 is a hydrogen atom, and L 1 is a single bond, and R 61 , R 62 , and R 63 are each independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0041] In certain embodiments, in the above formulas (4) to (13), R 1 is methyl and L 1 is a single bond, and R 61 , R 62 , and R 63 are each independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), —CF3, —NO2, a carboxyl group, and —SO3H. In one embodiment, in the above formulas (4) to (13), R 1 is methyl and L 1 is a single bond, and R 61 , R 62 , and R 63 In one embodiment, in the above formulas (4) to (13), R 1 is methyl and L 1 is a single bond, and R 61 , R62 , and R 63 are each independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0042] In certain embodiments, in the above formulas (4) to (13), R 1 is a hydrogen atom, and L 1 is methylene (-CH2-) or ethylene (-C2H4-), and R 61 , R 62 , and R 63 are each independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), —CF3, —NO2, a carboxyl group, and —SO3H. In one embodiment, in the above formulas (4) to (13), R 1 is a hydrogen atom, and L 1 is methylene (-CH2-) or ethylene (-C2H4-), and R 61 , R 62 , and R 63 In one embodiment, in the above formulas (4) to (13), R 1 is a hydrogen atom, and L 1 is methylene (-CH2-) or ethylene (-C2H4-), and R 61 , R 62 , and R 63 are each independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0043] In certain embodiments, in the above formulas (4) to (13), R 1 is methyl and L 1 is methylene (-CH2-) or ethylene (-C2H4-), and R 61 , R 62 , and R 63 are each independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), —CF3, —NO2, a carboxyl group, and —SO3H. In one embodiment, in the above formulas (1) to (3), R 1 is methyl and L 1 is methylene (-CH2-) or ethylene (-C2H4-), and R61 , R 62 , and R 63 In one embodiment, in the above formulas (1) to (3), R 1 is methyl and L 1 is methylene (-CH2-) or ethylene (-C2H4-), and R 61 , R 62 , and R 63 are each independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0044] In one embodiment, the modifying group (A) is selected from groups represented by the formula: [ka] (In the formula, * represents the linkage to the polysaccharide.)

[0045] In some embodiments, the modifying group (A) is a group represented by the above formula (A-2), and the ring P is a phenyl ring, which may be substituted with 1 to 4 substituents independently selected from a halogen atom (F, Cl, Br, and / or I), -CF3, -NO2, a carboxyl group, and -SO3H.

[0046] For example, the modifying group (A) is selected from the following formulae (14), (15), and (16) (hereinafter sometimes referred to as "formulae (14) to (16)"). [ka] In the above formulas (14) to (16), * represents a linking site to a polysaccharide. 1 The definition and preferred embodiments of R in the above formula (A) are as follows: 1 The definition and preferred embodiments of L are the same as those of 2 The definition and preferred embodiments of are as follows: 2 The definition and preferred embodiments are the same as those of the above. In the above formulas (1) to (3), R 71 , R72 , R 73 , and R 74 are each independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), —CF, —NO, a carboxyl group, and —SOH. 71 , R 72 , R 73 , and R 74 are both hydrogen atoms. 71 , R 72 , R 73 , and R 74 are each independently selected from a hydrogen atom and a halogen atom (F, Cl, Br, I; preferably F). is.

[0047] In certain embodiments, in the above formulas (14) to (16), R 1 is a hydrogen atom, and L 2 is a single bond, and R 71 , R 72 , R 73 , and R 74 are each independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), —CF3, —NO2, a carboxyl group, and —SO3H. In one embodiment, in the above formulas (14) to (16), R 1 is a hydrogen atom, and L 2 is a single bond, and R 71 , R 72 , R 73 , and R 744 In one embodiment, in the above formulae (14) to (16), R 1 is a hydrogen atom, and L 2 is a single bond, and R 71 , R 72 , R 73 , and R 74 are each independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0048] In certain embodiments, in the above formulas (14) to (16), R 1 is methyl and L 2is a single bond, and R 71 , R 72 , R 73 , and R 74 are each independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), —CF3, —NO2, a carboxyl group, and —SO3H. In one embodiment, in the above formulas (14) to (16), R 1 is methyl and L 2 is a single bond, and R 71 , R 72 , R 73 , and R 74 In one embodiment, in the above formulae (14) to (16), R 1 is methyl and L 2 is a single bond, and R 71 , R 72 , R 73 , and R 74 are each independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0049] In certain embodiments, in the above formulas (14) to (16), R 1 is a hydrogen atom, and L 2 is methylene (-CH2-) or ethylene (-C2H4-), and R 71 , R 72 , R 73 , and R 74 are each independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), —CF3, —NO2, a carboxyl group, and —SO3H. In one embodiment, in the above formulas (14) to (16), R 1 is a hydrogen atom, and L 2 is methylene (-CH2-) or ethylene (-C2H4-), and R 71 , R 72 , R 73 , and R 74 In one embodiment, in the above formulae (14) to (16), R 1 is a hydrogen atom, and L 2 is methylene (-CH2-) or ethylene (-C2H4-), and R 71 , R72 , R 73 , and R 74 are each independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0050] In certain embodiments, in the above formulas (14) to (16), R 1 is methyl and L 2 is methylene (-CH2-) or ethylene (-C2H4-), and R 71 , R 72 , R 73 , and R 74 are each independently selected from a hydrogen atom, a halogen atom (F, Cl, Br, I), —CF3, —NO2, a carboxyl group, and —SO3H. In one embodiment, in the above formulas (14) to (16), R 1 is methyl and L 2 is methylene (-CH2-) or ethylene (-C2H4-), and R 71 , R 72 , R 73 , and R 74 In one embodiment, in the above formulae (14) to (16), R 1 is methyl and L 2 is methylene (-CH2-) or ethylene (-C2H4-), and R 71 , R 72 , R 73 , and R 74 are each independently a hydrogen atom or a halogen atom (F, Cl, Br, I; preferably F).

[0051] In one embodiment, the modifying group (A) is a group represented by the formula: [ka] (In the formula, * represents the linkage to the polysaccharide.)

[0052] In some embodiments, Y is -L 3 -S- or -L 4 - selected from, in which L 3 and L4 is bonded to the ring P. That is, the modifying group (A) is selected from the following formula (A-3) or (A-4). [ka] In the above formulas (A-3) and (A-4), * represents a linking site to a polysaccharide. 1 The definition and preferred embodiments of ring P are as follows: 1 and the definition and preferred embodiments of ring P are the same as those of ring P.

[0053] In formula (A-3), L 3 is a single bond or -NH-, -C(=O)-, -O-, alkylene, -(CH2CH2O) n L represents a divalent group selected from the group consisting of -, -, and any combination thereof, and n is an integer of 1 to 9 (preferably 1 to 4, more preferably 1 to 2). 3 The alkylene group in the formula (I) has 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, more preferably 1 to 2 carbon atoms, and even more preferably 1 carbon atom, from the viewpoint of hydrophilicity. Among them, L 3 is a single bond, C 1―6 Alkylene, -(CH2CH2O) n -, -(CH2) m1 -(CH2CH2O) n -(CH2) m2 - and -(CH2) m1 -O-(CH2CH2O) n -(CH2) m2 -. n is an integer of 1 to 9 (preferably 1 to 4, more preferably 1 to 2). m1 and m2 are each independently an integer of 1 to 9 (preferably 1 to 4, more preferably 1 to 2). L 3 C in 1―6 The alkylene is preferably C 1―4 alkylene, more preferably C 1―2 It is preferably alkylene, and more preferably methylene. In one embodiment, L 3is a single bond or C 1―2 In one embodiment, L 3 is a single bond.

[0054] In formula (A-4), L 4 is a single bond or -NH-, -C(=O)-, -S-, -O-, alkylene, -(CH2CH2O) n L represents a divalent group selected from the group consisting of -, -, and any combination thereof, and n is an integer of 1 to 9 (preferably 1 to 4, more preferably 1 to 2). 4 The alkylene group in the formula (I) has 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, more preferably 1 to 2 carbon atoms, and even more preferably 1 carbon atom, from the viewpoint of hydrophilicity. Among them, L 4 is a single bond, C 1―6 Alkylene, -(CH2CH2O) n -, -(CH2) m1 -(CH2CH2O) n -(CH2) m2 - and -(CH2) m1 -O-(CH2CH2O) n -(CH2) m2 -. n is an integer of 1 to 9 (preferably 1 to 4, more preferably 1 to 2). m1 and m2 are each independently an integer of 1 to 9 (preferably 1 to 4, more preferably 1 to 2). L 4 C in 1―6 The alkylene is preferably C 1―4 alkylene, more preferably C 1―2 It is preferably alkylene, and more preferably methylene. In one embodiment, L 4 is C 1―2 It is alkylene.

[0055] The modifying group (A) has a -C(=O)R 1 (aldehyde or ketone group), and this -C(=O)R 1 can react with a primary amino group to form a Schiff base. 1can form a hyperconjugated structure with the adjacent phenyl ring or pyridine ring, which can stabilize the formed Schiff base and improve the bond stability with the amino group.

[0056] (polysaccharide) The polysaccharide is not particularly limited as long as it is an acidic, basic, or amphoteric polysaccharide and the above-mentioned modifying group (A) can be introduced thereinto. The polysaccharide may be a polysaccharide extracted or isolated from a natural plant or animal, a polysaccharide produced by a genetically engineered microorganism, or a chemically synthesized polysaccharide.

[0057] Acidic polysaccharides refer to polysaccharides having anionic functional groups (e.g., carboxyl groups, sulfate groups, phosphate groups, etc.) within their structure. Examples of acidic polysaccharides include polysaccharides having uronic acids (e.g., guluronic acid, mannuronic acid, glucuronic acid, iduronic acid, galacturonic acid, etc.), polysaccharides having sulfate groups or phosphate groups in part of their structure, or polysaccharides having both. Acidic polysaccharides include not only polysaccharides that inherently have anionic functional groups within their structure, but also polysaccharides that do not inherently have anionic functional groups (e.g., neutral polysaccharides) in which anionic functional groups have been introduced by substituting anionic functional groups such as carboxyalkyl groups for some or all of the hydrogen atoms of the hydroxyl groups. Specific examples of acidic polysaccharides include alginic acid, hyaluronic acid, carboxymethylcellulose, carboxymethyldextran, carboxymethylstarch, heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, regenerated oxidized cellulose, pectinic acid, gellan gum, gum arabic, xanthan gum, agar, agaropectin, carrageenan, and derivatives or salts thereof.

[0058] A basic polysaccharide refers to a polysaccharide having a cationic functional group (e.g., an amino group) in its structure. Basic polysaccharides include not only polysaccharides that inherently have a cationic functional group in their structure, but also polysaccharides that do not inherently have a cationic functional group (e.g., neutral polysaccharides) to which a cationic functional group has been introduced. Specific examples of basic polysaccharides include chitosan, its derivatives, and salts thereof.

[0059] Amphoteric polysaccharides refer to polysaccharides that have both anionic functional groups (e.g., carboxyl groups, sulfate groups, phosphate groups, etc.) and cationic functional groups (e.g., amino groups, etc.) in their structure. Specific examples of amphoteric polysaccharides include anion-modified basic polysaccharides such as succinic acid-modified chitosan; deacetylated polysaccharides such as deacetylated hyaluronic acid; amine-modified acidic polysaccharides such as cation-modified alginic acid and cation-modified hyaluronic acid; phosphocholine-modified polysaccharides such as phosphocholine-modified hyaluronic acid; carbobetaine-modified polysaccharides; and sulfobetaine-modified polysaccharides.

[0060] By using an acidic, basic, or amphoteric polysaccharide, the modifying group (A) can be introduced at a good modification rate, even if the modifying group (A) has a hydrophobic benzene ring or pyridyl ring. Furthermore, even when the hydrophobic modifying group (A) is introduced at a high modification rate, the presence of unmodified acidic or basic functional groups (anionic or cationic functional groups) allows the production of polysaccharide derivatives with excellent water solubility.

[0061] In some embodiments, the polysaccharide is selected from alginic acid, a derivative thereof or a salt thereof, hyaluronic acid, a derivative thereof or a salt thereof, carboxymethylcellulose, a derivative thereof or a salt thereof, carboxymethyldextran, a derivative thereof or a salt thereof, carboxymethylstarch, a derivative thereof or a salt thereof, heparin, a derivative thereof or a salt thereof, heparan sulfate, a derivative thereof or a salt thereof, chondroitin sulfate, a derivative thereof or a salt thereof, dermatan sulfate, a derivative thereof or a salt thereof, chitosan, a derivative thereof or a salt thereof, regenerated oxidized cellulose, a derivative thereof or a salt thereof, and pectinic acid, a derivative thereof or a salt thereof.

[0062] In the present invention, polysaccharide derivatives may be polysaccharides that have been subjected to any modification. Examples include polysaccharides modified with esterification, maleimide modification, thiol modification, acrylate modification, aldehyde modification, disulfide modification (e.g., pyridyl disulfide), alkyne modification including cyclic alkynes, tetrazine modification, and furan ring modification. These modification methods are known to those skilled in the art, and polysaccharide modification can be performed using conventional methods such as carbodiimide reaction. For example, a maleimide modification method is described in International Publication WO 2019 / 189330. A method for alkyne modification including cyclic alkynes can be described, for example, in Akira Takahashi et al., Biomacromolecules, 2013, 14(10), 3581-3588. For example, the method described in Vianney Delplace et al., Nonswelling, Ultralow Content Inverse Electron-Demand Diels-Alder Hyaluronan Hydrogels with Tunable Gelation Time: Synthesis and In Vitro Evaluation, Advanced Functional Materials, 2020, 30(14) can be used for tetrazine modification. For example, the method described in Nimmo, Chelsea M. et al., BIOMACROMOLECULES, 2011, 12(3) 824-830; RSC Adv., 2018, 8, 11036-11042 can be used for furan ring modification. These modifications can also be combined with copper-free click reactions (e.g., between cyclic alkynes and azides), inverse electron-demand Diels-Alder reactions (e.g., between tetrazines and cyclic alkenes), and Diels-Alder reactions (e.g., between furans and maleimides) to introduce a second drug (an additional drug of a different type) or crosslink the polysaccharide derivatives.

[0063] In some embodiments, the polysaccharide is an acidic polysaccharide. When a polysaccharide has an anionic functional group, even if the modification rate of the modifying group (A) is high, a polysaccharide derivative having particularly excellent water solubility can be obtained.

[0064] In some embodiments, the polysaccharide is a polysaccharide containing a carboxyl group. The carboxyl group of the polysaccharide can be reacted with an amino group to form an amide bond, thereby introducing the modifying group (A) into the polysaccharide. In a specific embodiment, the polysaccharide is a polysaccharide containing a carboxyl group, and the group represented by formula (A) is a group represented by formula (A-1) above, which is introduced into the polysaccharide by substituting -OH of the carboxyl group of the polysaccharide to form an amide bond. By forming the amide bond, a polysaccharide derivative with excellent stability can be obtained.

[0065] The polysaccharide having a carboxyl group may be any polysaccharide having at least one unmodified carboxyl group. The polysaccharide having a carboxyl group is a polysaccharide having a carboxyl group in its structure, or a derivative thereof, or a salt thereof (e.g., alginic acid, a derivative thereof, or a salt thereof, hyaluronic acid, a derivative thereof, or a salt thereof, carboxymethylcellulose, a derivative thereof, or a salt thereof, carboxymethyldextran, a derivative thereof, or a salt thereof, carboxymethylstarch, a derivative thereof, or a salt thereof, heparin, a derivative thereof, or a salt thereof, heparan sulfate, a derivative thereof, or a salt thereof, chondroitin sulfate, a derivative thereof, or a salt thereof, dermatan sulfate, a derivative thereof, or a salt thereof, pectinic acid, a derivative thereof, or a salt thereof, regenerated oxidized cellulose, a derivative thereof, or a salt thereof, gellan gum, a derivative thereof, or a salt thereof, gum arabic, a derivative thereof, or a salt thereof, xanthan gum, a derivative thereof, or a salt thereof, agarose, a derivative thereof, or a salt thereof, agaropectin, a derivative thereof, or a salt thereof, etc.; preferably, alginic acid, a derivative thereof, or a salt thereof, hyaluronic acid, a derivative thereof, or a salt thereof, carboxymethylcellulose, The term "polysaccharide" refers to polysaccharides having no carboxyl groups (e.g., chitosan, derivatives or salts thereof, curdlan, derivatives or salts thereof, agar, derivatives or salts thereof, carrageenan, derivatives or salts thereof, guar gum, derivatives or salts thereof, roasted gum bean gum, derivatives or salts thereof, tamarind seed gum, derivatives or salts thereof; preferably chitosan, derivatives or salts thereof, etc.), in which carboxyl groups have been introduced by substituting carboxyalkyl groups for some or all of the hydrogen atoms of the hydroxyl groups.

[0066] In certain embodiments, the polysaccharide having a carboxyl group is selected from alginic acid or a salt thereof, hyaluronic acid or a salt thereof, carboxymethylcellulose or a salt thereof, carboxymethyldextran or a salt thereof, carboxymethylstarch or a salt thereof, heparin or a salt thereof, heparan sulfate or a salt thereof, chondroitin sulfate or a salt thereof, dermatan sulfate or a salt thereof, pectinic acid or a salt thereof, and regenerated oxidized cellulose or a salt thereof. In certain embodiments, the polysaccharide is selected from alginic acid, a derivative thereof or a salt thereof, hyaluronic acid, a derivative thereof or a salt thereof, carboxymethylcellulose, a derivative thereof or a salt thereof, and carboxymethyldextran, a derivative thereof or a salt thereof. In certain embodiments, the polysaccharide is selected from alginic acid or a salt thereof, hyaluronic acid or a salt thereof, carboxymethylcellulose or a salt thereof, and carboxymethyldextran or a salt thereof. In certain embodiments, the polysaccharide is alginic acid, a derivative thereof, or a salt thereof, or hyaluronic acid, a derivative thereof, or a salt thereof. In certain embodiments, the polysaccharide is alginic acid or a salt thereof, or hyaluronic acid or a salt thereof.

[0067] (Alginic acids) In one embodiment, the polysaccharide is alginic acid, a derivative thereof, or a salt thereof (hereinafter also referred to as "alginates"). Alginate is a biodegradable and biocompatible polymer composed of two uronic acids, D-mannuronic acid (M) and L-guluronic acid (G), in a linear chain. More specifically, it is a block copolymer consisting of a homopolymer fraction of D-mannuronic acid (MM fraction), a homopolymer fraction of L-guluronic acid (GG fraction), and a randomly arranged fraction of D-mannuronic acid and L-guluronic acid (MG fraction). The ratio of D-mannuronic acid to L-guluronic acid (M / G ratio) in alginate varies widely, ranging from a high G ratio of approximately 0.2 to a high M ratio of approximately 5, depending primarily on the species of alginate (e.g., seaweed) from which it is derived, as well as on the habitat and season of the organism.

[0068] The alginic acid derivative is not particularly limited and may be any modified alginic acid or a salt thereof. Examples of the modifications made to alginic acid include esterification, sulfation, maleimide modification, thiol modification, acrylate modification, aldehyde modification, disulfide modification (e.g., pyridyl disulfide), and cation modification (e.g., heparin-bound alginic acid). In one embodiment, the alginic acid derivative is alginate ester, propylene glycol alginate, sulfated alginic acid in which the hydroxyl group is sulfated, maleimide-modified alginic acid, thiol-modified alginic acid, acrylate-modified alginic acid, or cation-modified alginic acid (e.g., the heparin-bound alginic acid described in JP 2001-233786 A). These modifications of alginic acid can be carried out by known methods or methods equivalent thereto.

[0069] Examples of salts of alginic acid or its derivatives include metal salts of alginic acid or its derivatives. For example, a salt of alginic acid or its derivatives can be prepared by replacing the hydrogen atom (hydrogen ion) of the carboxylic acid at the 6-position of alginic acid or its derivatives with a monovalent metal ion (e.g., Na + YaK +Examples of suitable salts include water-soluble salts prepared by ion exchange with an alkali metal ion (e.g., Mg). Specific examples include sodium alginate, potassium alginate, sodium salts of alginic acid derivatives, and potassium salts of alginic acid derivatives. In a specific embodiment, the salt of alginic acid or a derivative thereof is sodium alginate or a sodium salt of an alginic acid derivative. A solution of a monovalent metal salt of alginic acid or a derivative thereof forms a gel when mixed with a crosslinker. Alternatively, a metal salt of alginic acid or a derivative thereof may be prepared by ion exchange of the hydrogen atom (hydrogen ion) of the carboxylic acid at the 6-position of alginic acid or a derivative thereof with a divalent metal ion (e.g., Mg 2+ , Ca 2+ It may also be a salt (crosslinked product) made by ion exchange with an alkaline earth metal ion such as

[0070] When alginic acid is first extracted from brown algae, it has a high molecular weight and a high viscosity. However, the molecular weight decreases and the viscosity decreases during processes such as thermal drying, freeze-drying, and purification. Therefore, alginic acids with different molecular weights can be produced by appropriately controlling the temperature at each production step. By controlling the temperature at each production step to be lower, alginic acids with higher molecular weights can be obtained, and the higher the temperature, the lower the molecular weight of alginic acids can be obtained. Furthermore, alginic acids with different molecular weights can be produced by appropriately selecting the brown algae used as raw material or by fractionating the alginic acid by molecular weight during the production process. Furthermore, after measuring the molecular weight or viscosity of alginic acids produced by each method, it is possible to obtain alginic acids with the desired molecular weight by mixing them with other lots of alginic acids with different molecular weights or viscosities.

[0071] The viscosity of the alginic acids used is not particularly limited, but when measured as a 1 w / w% aqueous solution of alginic acids, it is preferably 10 mPa·s to 1000 mPa·s, and more preferably 50 mPa·s to 800 mPa·s.

[0072] The viscosity of an aqueous solution of alginic acids can be measured according to a conventional method. For example, the viscosity can be measured using a rotational viscometer such as a coaxial double cylinder rotational viscometer, a single cylinder rotational viscometer (Brookfield type viscometer), or a cone-plate rotational viscometer (cone-plate type viscometer). Preferably, the viscosity is measured according to the viscosity measurement method of the Japanese Pharmacopoeia (16th edition). More preferably, a cone-plate type viscometer is used.

[0073] The position of the modifying group (A) introduced into alginic acids is not particularly limited, but it is preferable that it be introduced into the 6-position carboxyl group of D-mannuronic acid (M) and L-guluronic acid (G) (uronic acid) that constitute alginic acids. In a particular embodiment, the group represented by the above formula (A-1) is introduced into the polysaccharide by substituting the —OH of the carboxyl group of alginic acid.

[0074] In one embodiment, the polysaccharide derivative comprises constitutional units represented by the following formula (c11) and / or (c12): [ka] In formulas (c11) and (c12), R 1 The definition and preferred embodiments of ring P are as follows: 1 and the definition and preferred embodiments of ring P are the same as those of ring P. In formulas (c11) and (c12), Y is -L 1 represents -NH-, where L 1 is bonded to ring P. 1 The definition and preferred embodiments of L in formula (A-1) are as follows: 1 The definition and preferred embodiments are the same as those of the above. In formulas (c11) and (c12), R 11 , R 12 , R 13 , and R 14 are each independently a hydrogen atom, C 1-6 Alkyl, and -C(=O)-C 1-6 In one embodiment, R11 , R 12 , R 13 , and R 14 are all hydrogen atoms.

[0075] (Hyaluronic acids) In one embodiment, the polysaccharide is hyaluronic acid, a derivative thereof, or a salt thereof (hereinafter also referred to as "hyaluronic acids"). Hyaluronic acid is a biodegradable and biocompatible polymer with a repeating structure in which the disaccharide units D-glucuronic acid and N-acetyl-D-glucosamine are linked in a linear chain.

[0076] The derivative of hyaluronic acid is not particularly limited, and may be any modified hyaluronic acid or its salt.The optional modifications to hyaluronic acid include, for example, esterification, sulfation, maleimide modification, thiol modification, acrylate modification, aldehyde modification, and disulfide modification (e.g., pyridyl disulfide).In one embodiment, the derivative of hyaluronic acid is hyaluronic acid ester, propylene glycol hyaluronate, sulfated hyaluronic acid in which hydroxyl groups are sulfated, maleimide-modified hyaluronic acid, thiol-modified hyaluronic acid, or acrylate-modified hyaluronic acid.These modifications to hyaluronic acid can be carried out by known methods or methods based thereon.

[0077] Examples of salts of hyaluronic acid or its derivatives include metal salts of hyaluronic acid or its derivatives. For example, a salt of hyaluronic acid or its derivatives can be prepared by substituting a monovalent metal ion (e.g., Na) for the hydrogen atom (hydrogen ion) of the carboxylic acid at the 6-position of hyaluronic acid or its derivatives. + YaK +Examples of suitable salts include water-soluble salts prepared by ion exchange with alkali metal ions (e.g., Mg). Specific examples include sodium hyaluronate, potassium hyaluronate, sodium salts of hyaluronic acid derivatives, and potassium salts of hyaluronic acid derivatives. In a specific embodiment, the salt of hyaluronic acid or a derivative thereof is sodium hyaluronate or a sodium salt of a hyaluronic acid derivative. A solution of a monovalent metal salt of hyaluronic acid or a derivative thereof forms a gel when mixed with a crosslinking agent. Alternatively, a metal salt of hyaluronic acid or a derivative thereof may be prepared by exchanging the hydrogen atom (hydrogen ion) of the carboxylic acid at the 6-position of hyaluronic acid or a derivative thereof with a divalent metal ion (e.g., Mg 2+ , Ca 2+ The crosslinked product may be a salt (crosslinked product) prepared by ion exchange with an alkaline earth metal ion such as methyl methacrylate.

[0078] The position of the modifying group (A) to be introduced into the hyaluronic acid is not particularly limited, but it is preferably introduced into the position of the carboxyl group of D-glucuronic acid that constitutes the hyaluronic acid. In a particular embodiment, the group represented by the above formula (A-1) is introduced into the polysaccharide by substituting the —OH of the carboxyl group of hyaluronic acid.

[0079] In one embodiment, the polysaccharide derivative comprises a constitutional unit represented by the following formula (c13): [ka] In formula (c13), R 1 The definition and preferred embodiments of ring P are as follows: 1 and the definition and preferred embodiments of ring P are the same as those of ring P. In formula (c13), Y is -L 1 represents -NH-, where L 1 is bonded to ring P. 1 The definition and preferred embodiments of L in formula (A-1) are as follows: 1 The definition and preferred embodiments are the same as those of the above. In formula (c13), R 21 , R22 , R 23 , and R 24 are each independently a hydrogen atom, C 1-6 Alkyl, and -C(=O)-C 1-6 In one embodiment, R 21 , R 22 , R 23 , and R 24 are all hydrogen atoms.

[0080] (Carboxymethylcellulose) In one embodiment, the polysaccharide is carboxymethylcellulose, a derivative thereof, or a salt thereof (hereinafter also referred to as "carboxymethylcelluloses"). Carboxymethyl cellulose is a cellulose derivative that has been solubilized by introducing a carboxymethyl group into cellulose, and has excellent thickening properties, water absorption properties, and water retention properties.

[0081] The derivative of carboxymethylcellulose is not particularly limited, and may be any modified carboxymethylcellulose or its salt. The optional modifications to carboxymethylcellulose include, for example, esterification, sulfation, maleimide modification, thiol modification, acrylate modification, aldehyde modification, and disulfide modification (e.g., pyridyl disulfide). These modifications to carboxymethylcellulose can be carried out by known methods or methods similar thereto.

[0082] Examples of salts of carboxymethyl cellulose or its derivatives include metal salts of carboxymethyl cellulose or its derivatives. For example, a metal salt of carboxymethyl cellulose or its derivatives can be obtained by converting a hydrogen atom (hydrogen ion) of a carboxylic acid contained in carboxymethyl cellulose or its derivatives to a monovalent metal ion (e.g., Na + YaK +Examples of suitable salts include water-soluble salts prepared by ion exchange with alkali metal ions (e.g., sodium carboxymethylcellulose, potassium carboxymethylcellulose, sodium salts of carboxymethylcellulose derivatives, or potassium salts of carboxymethylcellulose derivatives). Specific examples include sodium carboxymethylcellulose, potassium carboxymethylcellulose, sodium salts of carboxymethylcellulose derivatives, and potassium salts of carboxymethylcellulose derivatives. In a specific embodiment, the salt of carboxymethylcellulose or a derivative thereof is sodium carboxymethylcellulose or a sodium salt of a carboxymethylcellulose derivative. A solution of a monovalent metal salt of carboxymethylcellulose or a derivative thereof forms a gel when mixed with a crosslinking agent. Alternatively, a metal salt of carboxymethylcellulose or a derivative thereof may be prepared by exchanging the hydrogen atom (hydrogen ion) of the carboxylic acid contained in carboxymethylcellulose or a derivative thereof with a divalent metal ion (e.g., Mg 2+ , Ca 2+ The crosslinked product may be a salt (crosslinked product) prepared by ion exchange with an alkaline earth metal ion such as methyl methacrylate.

[0083] The position of the modifying group (A) to be introduced into the carboxymethyl cellulose is not particularly limited, but it is preferable to introduce it into the position of the carboxyl group contained in the carboxymethyl cellulose. In a specific embodiment, the group represented by the above formula (A-1) is introduced into the polysaccharide by substituting the —OH of the carboxyl group of carboxymethylcellulose.

[0084] In one embodiment, the polysaccharide derivative comprises a constitutional unit represented by the following formula (c14): [ka] In formula (c14), R 31 , R 32 , and R 33 One to three of these represent a group represented by the following formula (i). [ka] In the above formula (i), R 1The definition and preferred embodiments of ring P are as follows: 1 and the definition and preferred embodiments of ring P are the same as those of ring P. In addition, in the above formula (i), Y is -L 1 represents -NH-, where L 1 is bonded to ring P. 1 The definition and preferred embodiments of L in formula (A-1) are as follows: 1 The definition and preferred embodiments are the same as those of the above. R 31 , R 32 , and R 33 The remainder (substituents other than the group represented by formula (i)) are each independently a hydrogen atom, C 1-6 Alkyl, -C(=O)-C 1-6 It is selected from alkyl and -CH2COOH, preferably selected from a hydrogen atom and -CH2COOH, more preferably a hydrogen atom. In one embodiment, R 31 is a group represented by the above formula (i), and R 32 and R 33 is a hydrogen atom.

[0085] (Carboxymethyl dextrans) In one embodiment, the polysaccharide is carboxymethyldextran, a derivative thereof, or a salt thereof (hereinafter also referred to as "carboxymethyldextrans"). Carboxymethyl dextran is the carboxymethyl ether of dextran.

[0086] The derivative of carboxymethyldextran is not particularly limited and may be any modified carboxymethyldextran or a salt thereof. The optional modifications to carboxymethyldextran include, for example, esterification, sulfation, maleimide modification, thiol modification, acrylate modification, aldehyde modification, and disulfide modification (e.g., pyridyl disulfide). These modifications to carboxymethyldextran can be carried out by known methods or methods based thereon.

[0087] Examples of salts of carboxymethyldextran or its derivatives include metal salts of carboxymethyldextran or its derivatives. For example, a metal salt of carboxymethyldextran or its derivatives can be obtained by converting a hydrogen atom (hydrogen ion) of a carboxylic acid contained in carboxymethyldextran or its derivatives to a monovalent metal ion (e.g., Na + YaK + Examples of suitable salts include water-soluble salts prepared by ion exchange with alkali metal ions (e.g., carboxymethyl dextran sodium, carboxymethyl dextran potassium, sodium salts of carboxymethyl dextran derivatives, or potassium salts of carboxymethyl dextran derivatives). Specific examples include sodium carboxymethyl dextran, potassium carboxymethyl dextran, and the like. In a specific embodiment, the salt of carboxymethyl dextran or a derivative thereof is the sodium salt of carboxymethyl dextran or a carboxymethyl dextran derivative. A solution of a monovalent metal salt of carboxymethyl dextran or a derivative thereof forms a gel when mixed with a crosslinker. Alternatively, a metal salt of carboxymethyl dextran or a derivative thereof may be prepared by ion exchange of a hydrogen atom (hydrogen ion) of a carboxylic acid contained in carboxymethyl dextran or a derivative thereof with a divalent metal ion (e.g., Mg 2+ , Ca 2+ The crosslinked product may be a salt (crosslinked product) prepared by ion exchange with an alkaline earth metal ion such as methyl methacrylate.

[0088] The position of the modifying group (A) to be introduced into the carboxymethyldextrans is not particularly limited, but it is preferably introduced into the position of the carboxyl group contained in the carboxymethyldextran. In a specific embodiment, the group represented by the above formula (A-1) is introduced into the polysaccharide by substituting the —OH of the carboxyl group of carboxymethyldextran.

[0089] In one embodiment, the polysaccharide derivative comprises a constitutional unit represented by the following formula (c15): [ka] In formula (c15), R 41, R 42 , and R 43 One to three of these represent a group represented by the following formula (i). [ka] In the above formula (i), R 1 The definition and preferred embodiments of ring P are as follows: 1 and the definition and preferred embodiments of ring P are the same as those of ring P. In addition, in the above formula (i), Y is -L 1 represents -NH-, where L 1 is bonded to ring P. 1 The definition and preferred embodiments of L in formula (A-1) are as follows: 1 The definition and preferred embodiments are the same as those of the above. R 41 , R 42 , and R 43 The remainder (substituents other than the group represented by formula (i)) are each independently a hydrogen atom, C 1-6 Alkyl, -C(=O)-C 1-6 It is selected from alkyl and -CH2COOH, preferably selected from a hydrogen atom and -CH2COOH, more preferably a hydrogen atom. In one embodiment, R 41 is a group represented by the above formula (i), and R 42 and R 43 is a hydrogen atom.

[0090] The polysaccharide may be heparin, a derivative thereof or a salt thereof, heparan sulfate, a derivative thereof or a salt thereof, chondroitin sulfate, a derivative thereof or a salt thereof, dermatan sulfate, a derivative thereof or a salt thereof, regenerated oxidized cellulose, a derivative thereof or a salt thereof, or pectinic acid, a derivative thereof or a salt thereof. In one embodiment, the polysaccharide derivative is heparin, a derivative thereof or a salt thereof, heparan sulfate, a derivative thereof or a salt thereof, chondroitin sulfate, a derivative thereof or a salt thereof, dermatan sulfate, a derivative thereof or a salt thereof, regenerated oxidized cellulose, a derivative thereof or a salt thereof, or pectinic acid, a derivative thereof or a salt thereof, and the group represented by formula (A-1) above has been introduced by substituting -OH of a carboxyl group contained in the polysaccharide.

[0091] In some embodiments, the polysaccharide is an amino group-containing polysaccharide, which can be introduced into the polysaccharide by forming an amide bond through a reaction between the amino group and a carboxyl group, thereby introducing the modifying group (A) into the polysaccharide. In a specific embodiment, the polysaccharide is an amino group-containing polysaccharide, and the group represented by formula (A) is a group represented by formula (A-2) above, which is introduced into the polysaccharide by substituting a hydrogen atom of an amino group of the polysaccharide to form an amide bond. By forming the amide bond, a polysaccharide derivative with excellent stability can be obtained. The term "polysaccharides having amino groups" refers to polysaccharides that inherently have amino groups in their structure, or derivatives thereof, or salts thereof (e.g., chitosan, its derivatives, or salts thereof), as well as polysaccharides that do not have amino groups but have amino groups introduced into some of them.

[0092] For example, examples of polysaccharide derivatives in which a group represented by the above formula (A-2) has been introduced into a polysaccharide having an amino group are shown below. [ka] In the above formula, R 1The definition and preferred embodiments of ring P are as follows: 1 and the definition and preferred embodiments of ring P are the same as those of ring P. In addition, in the above formula, L 2 The definition and preferred embodiments of L in formula (A-2) are as follows: 2 The definition and preferred embodiments are the same as those of the above.

[0093] (Chitosan) The polysaccharide may be chitosan, its derivatives, or salts thereof (also referred to as "chitosans"). Chitosan is a polysaccharide in which D-glucosamine units and N-acetyl-D-glucosamine units are linked by β-(1-4) glycosidic bonds. Chitosan can be obtained by partial deacetylation of the polysaccharide chitin. The degree of deacetylation is not particularly limited. In some embodiments, chitosans are deacetylated to a degree of more than about 50% (more typically, more than about 75%). Chitosan derivatives are not particularly limited and include chitosan modified with any desired modification or their salts. Modifications of chitosan include, for example, esterification, sulfation, maleimide modification, thiol modification, acrylate modification, aldehyde modification, and disulfide modification (e.g., pyridyl disulfide). Examples of chitosan derivatives include carboxymethylchitosan, hydroxybutylchitin, N-acylchitosan, O-acylchitosan, N-alkylchitosan, O-alkylchitosan, N-alkylidenechitosan, O-sulfonylchitosan, sulfated chitosan, phosphorylated chitosan, nitrated chitosan, alkali chitosan, and metal chelates of chitosan. These modifications of chitosan can be carried out by known methods or methods similar thereto. Commercially available chitosans can also be used.

[0094] Chitosan has a primary amino group. In one embodiment of the polysaccharide derivative, the polysaccharide is chitosan, a derivative thereof, or a salt thereof, and the group represented by the above formula (A-2) is introduced by substituting a hydrogen atom of an amino group contained in the polysaccharide. The position of the modifying group (A) introduced into chitosan is not particularly limited, but it is preferably introduced into the amino group position of the D-glucosamine unit constituting the chitosan. In a specific embodiment, the group represented by the above formula (A-2) is introduced into the polysaccharide by substituting the hydrogen atom of the amino group of chitosan.

[0095] In one embodiment, the polysaccharide derivative comprises a constitutional unit represented by the following formula (c16): [ka] In formula (c16), R 1 The definition and preferred embodiments of ring P are as follows: 1 and the definition and preferred embodiments of ring P are the same as those of ring P. In formula (c16), Y is -L 2 represents -C(=O)-, where L 2 is bonded to ring P. 2 The definition and preferred embodiments of L in formula (A-2) are as follows: 2 The definition and preferred embodiments are the same as those of the above. In formula (c16), R 81 and R 82 are each independently a hydrogen atom, C 1-6 Alkyl, and -C(=O)-C 1-6 In one embodiment, R 81 and R 82 are all hydrogen atoms.

[0096] In certain embodiments, the polysaccharide is a polysaccharide having an ethylenic double bond group. The ethylenic double bond group of the polysaccharide can be reacted with a thiol group (-SH) to form a sulfide bond (-S-), thereby introducing the modifying group (A) into the polysaccharide. In certain embodiments, the polysaccharide is a polysaccharide having an ethylenic double bond group, and the group represented by formula (A) is a group represented by formula (A-3) above, and the group represented by formula (A) is introduced into the polysaccharide by reacting with the ethylenic double bond group of the polysaccharide.

[0097] The polysaccharide having an ethylenic double bond is not particularly limited, but examples thereof include maleimide-modified or acrylate-modified polysaccharides (e.g., alginic acid, hyaluronic acid, carboxymethyl cellulose, carboxymethyl dextran, carboxymethyl starch, pectin, regenerated oxidized cellulose, chitosan), and salts thereof. For example, examples of polysaccharide derivatives in which a group represented by the above formula (A-3) has been introduced into a maleimide-modified polysaccharide are shown below. [ka] In the above formula, R 1 The definition and preferred embodiments of ring P are as follows: 1 and the definition and preferred embodiments of ring P are the same as those of ring P. In addition, in the above formula, L 3 The definition and preferred embodiments of L in formula (A-3) are as follows: 3 The definition and preferred embodiments are the same as those of the above.

[0098] The polysaccharide derivative may have a structure in which a modifying group (A) is introduced into the position of a hydroxyl group contained in the polysaccharide. In a specific embodiment, the group represented by formula (A-4) is introduced by substituting a hydrogen atom of a hydroxyl group contained in the polysaccharide. In this embodiment, as shown below, the polysaccharide derivative has an ether bond formed by the oxygen atom derived from the hydroxyl group contained in the polysaccharide. [ka]

[0099] (Molecular weight and viscosity of polysaccharide derivative) Although it is generally difficult to accurately determine the molecular weight of high molecular weight polysaccharides, they generally have a weight average molecular weight in the range of 1,000 to 10,000,000.

[0100] For example, the molecular weight of the polysaccharide derivative is preferably 1,000 to 5,000,000, more preferably 1,000 to 3,000,000, as weight average molecular weight (Mw) measured by gel filtration chromatography (GFC). The preferred range of the molecular weight of the polysaccharide derivative varies depending on the type of polysaccharide constituting the polysaccharide derivative (presence or absence of decomposition enzymes, presence or absence of inflammatory response, etc.) and the use of the polysaccharide derivative. For example, when used in DDS applications, although it depends on the charge of the polysaccharide derivative, the renal excretion limit is generally said to be 40,000 to 50,000. Therefore, polysaccharide derivatives composed of (non-biodegradable) polysaccharides (dextran, cellulose derivatives, alginic acid, salts of these, or derivatives of these, etc.) for which there are no degrading enzymes in the body, generally preferably have a weight-average molecular weight (Mw) in the range of 1,000 to 50,000 or 1,000 to 40,000. However, although there are no enzymes for decomposing alginic acid in the body, alginic acid has almost no immunogenicity, and therefore alginic acid with a larger weight-average molecular weight (for example, Mw of 1,000 to 5,000,000, or 1,000 to 2,000,000) can be used. Hyaluronic acid with a large weight-average molecular weight (for example, Mw of up to 2,000,000 or up to 5,000,000) can be used because differentiation enzymes are abundant in the body. However, hyaluronic acid with a low molecular weight (approximately 100,000) is known to induce inflammation, so hyaluronic acid with a molecular weight of 500,000 or more is preferred. Typically, when the molecular weight of a high molecular weight polysaccharide is calculated by gel filtration chromatography, a measurement error of 10 to 20% or more can occur. For example, if the molecular weight is 400,000, the value can vary within a range of 320,000 to 480,000; if the molecular weight is 500,000, the value can vary within a range of 400,000 to 600,000; and if the molecular weight is 1,000,000, the value can vary within a range of 800,000 to 1,200,000. Therefore, the preferred weight-average molecular weight range for a polysaccharide is at least 1,000. Polysaccharides with molecular weights that are too high are difficult to produce and can cause problems such as excessive viscosity when made into an aqueous solution, reduced solubility, and difficulty in maintaining physical properties during long-term storage. Therefore, the weight-average molecular weight is preferably 5,000,000 or less, and more preferably 3,000,000 or less. The weight-average molecular weight of the polysaccharide derivative into which the modifying group (A) has been introduced is increased by the introduction of the modifying group (A) compared to the molecular weight of the polysaccharide before the introduction of the modifying group (A). By appropriately selecting the molecular weight of the raw material polysaccharide, a polysaccharide derivative having the desired molecular weight can be obtained.

[0101] Generally, polymeric substances derived from natural products do not have a single molecular weight, but are an aggregate of molecules with various molecular weights, and are therefore measured as a molecular weight distribution with a certain range. A typical measurement method is gel filtration chromatography. Typical information on molecular weight distribution obtained by gel filtration chromatography includes the weight average molecular weight (Mw), number average molecular weight (Mn), and polydispersity ratio (Mw / Mn). The weight average molecular weight is determined by taking into account the contribution of large molecular weight polymers to the average molecular weight, and is expressed by the following formula. Mw=Σ(WiMi) / W=Σ(HiMi) / Σ(Hi) The number average molecular weight is calculated by dividing the total weight of the polymers by the total number of polymers. Mn=W / ΣNi=Σ(MiNi) / ΣNi=Σ(Hi) / Σ(Hi / Mi) Here, W is the total weight of polymers, Wi is the weight of the i-th polymer, Mi is the molecular weight at the i-th elution time, Ni is the number of molecules with molecular weight Mi, and Hi is the height at the i-th elution time.

[0102] It is known that when measuring the molecular weight of polymeric substances derived from natural products, the values ​​can vary depending on the measurement method (for example, hyaluronic acid: Chikako YOMOTA et al., Bull. Natl. Health Sci., Vol. 117, pp. 135-139 (1999), Chikako YOMOTA et al., Bull. Natl. Inst. Health Sci., Vol. 121, pp. 30-33 (2003)). Regarding the measurement of the molecular weight of alginate, there are literatures that describe a method of calculating from intrinsic viscosity and a method of calculating using SEC-MALLS (Size Exclusion Chromatography with Multiple Angle Laser Light Scattering Detection) (ASTM F2064-00 (2006), published by ASTM International). This document recommends that when measuring molecular weight by size exclusion chromatography (gel filtration chromatography), a calibration curve using pullulan as a standard substance should be used in conjunction with a multi-angle light scattering (MALS) detector (measurement by SEC-MALS). SEC-MALS is described in ASTM F2065-16. Furthermore, there are cases where the molecular weight determined by SEC-MALS is used as the standard value in the catalog for alginates (FMC Biopolymer, PRONOVA, etc.). TM sodium alginates catalog). In the present invention, when the molecular weight of a polysaccharide derivative is specified, unless otherwise specified, it is the weight average molecular weight calculated by gel filtration chromatography.

[0103] Typical conditions for gel filtration chromatography include the use of a calibration curve using pullulan as a standard substance. It is preferable to use pullulan with molecular weights of at least 1.6 million, 788,000, 404,000, 212,000, and 112,000 as the standard substance. Other factors, such as the eluent (200 mM sodium nitrate solution) and column conditions, can also be specified. Regarding column conditions, it is preferable to use at least one to three columns with a polymethacrylate resin-based packing material and an exclusion limit molecular weight of 10 million or more. A typical column is TSKgel GMPW. x1 (7.8mm diameter x 300mm) and G2500PW XL (Diameter 7.8mm x 300mm) (manufactured by Tosoh Corporation).

[0104] (qualification rate) The modification rate refers to the number of modifying groups (A) per monosaccharide structural unit contained in the polysaccharide derivative.

number

[0105] The modification rate of the polysaccharide derivative with the modifying group (A) can be, for example, in the range of 0.01 to 1, 0.02 to 0.6, 0.05 to 0.2, or 0.1 to 0.2. When the polysaccharide derivative is used as a drug delivery carrier, a modification rate in such a range may be used. In another embodiment, the modification rate of the polysaccharide derivative with the modifying group (A) can be, for example, in the range of 0.1 to 1, or 0.1 to 0.8, or 0.15 to 0.6. When the polysaccharide derivative is used in the form of a hydrogel, a modification rate in such a range may be used. In another embodiment, the modification rate of the polysaccharide derivative with the modifying group (A) can be, for example, in the range of 0.00001 to 0.01.

[0106] For example, when the polysaccharide is alginic acid, it has one carboxyl group per monosaccharide unit, and therefore when the modifying group (A) is introduced into the carboxyl group, the modification rate of the modifying group (A) in the polysaccharide derivative is a maximum of 1, and can be in the range of, for example, 0.01 to 1, 0.01 to 0.6, 0.05 to 0.2, or 0.00001 to 0.01. For example, when the polysaccharide is hyaluronic acid, which is composed of a monosaccharide unit (D-glucuronic acid) having one carboxyl group and a monosaccharide unit (N-acetyl-D-glucosamine) having no carboxyl group, the modification rate of the modifying group (A) in the polysaccharide derivative when the modifying group (A) is introduced into the carboxyl group is a maximum of 0.5, and can be in the range of, for example, 0.01 to 0.5, 0.05 to 0.2, or 0.00001 to 0.01. For example, when the polysaccharide is a carboxymethylcellulose, which has one to three carboxyl groups in the monosaccharide constituent unit, and when the modifying group (A) is introduced into the carboxyl group, the modification rate of the modifying group (A) in the polysaccharide derivative is a maximum of 3 (when carboxymethyl substitution is performed at all three positions), and can be, for example, in the range of 0.01 to 3, 0.05 to 1.5, 0.05 to 1, or 0.00001 to 0.01. For example, when the polysaccharide is a carboxymethyldextrans, which have one to three carboxyl groups in the monosaccharide constituent unit, and when the modifying group (A) is introduced into the carboxyl group, the modification rate of the modifying group (A) in the polysaccharide derivative is a maximum of 3 (when carboxymethyl substitution is performed at all three positions), and can be in the range of, for example, 0.01 to 3, 0.05 to 1.5, 0.05 to 1, or 0.00001 to 0.01. The modification rate is 1 It can be calculated by H NMR measurement (DO). The modification rate can also be calculated by colorimetric aldehyde assay. 1 The repair rate calculated by H NMR measurement and colorimetric aldehyde assay does not completely agree, and there may be discrepancies between them. However, in the present invention, 1 The repair rate calculated by either 1 H NMR measurement or colorimetric aldehyde assay may be within the above range.

[0107] A spacer may be further used when introducing the modifying group (A). Usable spacers may be those commonly used in the art (e.g., spacers described in Greg T. Hermanson, Bioconjugate Techniques, Third Edition (2013)), and more specifically, include polyethylene glycol, polyamides such as peptides, and hydrocarbons.

[0108] 2. Method for producing polysaccharide derivatives The method for producing a polysaccharide derivative is not particularly limited, but the polysaccharide derivative can be produced, for example, by a method comprising reacting a compound (a) represented by the following formula (a) with a polysaccharide having a functional group capable of reacting with the -X group of the compound (a). This results in a polysaccharide derivative (C) in which a modifying group (A) corresponding to the compound (a) (the group represented by the formula (A) described above) has been introduced into the polysaccharide. [ka]

[0109] In the above formula (a), X is selected according to Y in formula (C). Y is -L 1 -NH-(L 1 is attached to ring P), X is -L 1 -NH2. Y is -L 2 -C(=O)-(L 2 is attached to ring P), X is -L 2 -C(=O)OH. Y is -L 3 -S-(This time L 3is attached to ring P), X is -L 3 -SH. Y is -L 4 -(At this time L 4 is attached to ring P), X is -L 4 -R L and R L is selected from halogen atoms (F, Cl, Br, or I). The rings P and R in the above formulae (a) and (C) 1 The definition and preferred embodiments of Y in formula (C) are the same as those in formula (A) and formulas (A-1), (A-2), (A-3), and (A-4) above.

[0110] In this production method, the polysaccharide has an acidic, basic, or both (amphoteric) functional group (i.e., a functional group having a charge), and thus can react with a compound (a) represented by formula (a) having a highly hydrophobic phenyl ring or pyridyl ring under mild and highly safe conditions in a single step to introduce a modifying group (A) into the polysaccharide.

[0111] In a specific embodiment, a method for producing a polysaccharide derivative represented by formula (C) comprises reacting an acidic, basic, or amphoteric polysaccharide with a compound (a) represented by the above formula (a) by adding the compound (a) to a solution containing the polysaccharide in an aqueous solvent, a polar solvent, or a higher alcohol. By such a reaction, the modifying group (A) can be introduced into the polysaccharide at a high modification rate.

[0112] Below, Y is -L 1 -NH-(L 1 is bonded to ring P), and X is -L 1 The method for producing a polysaccharide derivative will be described below using the case where Y is -NH2 as an example. 1 In the case of a group other than —NH—, the polysaccharide derivative represented by the above formula (C) can be produced by a method known in the literature.

[0113] One aspect of the present invention relates to a method for producing a polysaccharide derivative represented by the following formula (C1): The polysaccharide derivative represented by the following formula (C1) can be obtained by a condensation reaction between the carboxyl group of a polysaccharide and the amino group of a compound (a1) represented by the following formula (a1): This reaction forms an amide bond between the amino group of compound (a1) and the carboxyl group of the polysaccharide, thereby obtaining a polysaccharide derivative represented by the following formula (C1) in which a modifying group (A) corresponding to compound (a1) (the group represented by formula (A-1) described above) has been introduced into the polysaccharide. [ka]

[0114] The rings P and R in the above formulae (a1) and (C1) 1 , and L in formula (C1) 1 The definitions and preferred embodiments of are the same as those in the formula (A) and formula (A-1) above.

[0115] The method for the condensation reaction is not particularly limited, and can be carried out in accordance with known methods, such as those described in "Experimental Chemistry Lectures, 5th Edition, 16, Synthesis of Organic Compounds IV, Carboxylic Acids and Derivatives, Esters, pp. 35-70, Acid Amides and Acid Imides, pp. 118-154, Amino Acids and Peptides, pp. 258-283, 2007, Maruzen." The reaction is usually carried out in a solvent.

[0116] In a specific embodiment, a method for producing a polysaccharide derivative represented by formula (C1) comprises subjecting a polysaccharide containing a carboxyl group and a compound (a1) represented by the following formula (a1) to a condensation reaction in a solvent (e.g., an aqueous solvent): In one embodiment, the compound (a1) is dissolved or dispersed in an aqueous solvent, a polar solvent, or a higher alcohol solvent, and then added to a solution containing a polysaccharide to react with the polysaccharide, thereby allowing the modifying group (A) to be introduced into the polysaccharide at a high modification rate. Compound (a1) represented by formula (a1) generally has low solubility in aqueous solvents. Therefore, compound (a1) can be added to an aqueous solvent containing a polysaccharide and reacted with the polysaccharide under stirring, allowing the reaction to proceed with compound (a1) in a suspended state. In one embodiment, compound (a1) is dissolved in a polar solvent (preferably DMSO) or a higher alcohol solvent, and the solution of compound (a1) is added to an aqueous solvent containing a polysaccharide, thereby increasing the modification rate of compound (a1). It was surprising that the modification rate was dramatically improved by the simple method of dissolving or dispersing compound (a1) in a specific solvent and adding it to the polysaccharide. Although the details of the improvement in the modification rate are unknown, the inventors speculate that the excess carboxyl groups relative to compound (a1) resulted in the consumption of the less soluble (hydrophobic) compound (a1) in the reaction under such conditions, which promoted the dissolution of the subsequent compound (a1), leading to the improvement in the modification rate. In addition, they speculate that the reaction between the carboxyl groups of the polysaccharide and the amino groups of compound (a) proceeds more efficiently than the reaction between the amino groups of compound (a) and the aldehyde groups of compound (a) (Schiff base formation reaction), which is another factor that led to the improvement in the modification rate. However, the production method of this embodiment is not limited to this mechanism.

[0117] Examples of polar solvents include N,N-dimethylformamide, dimethyl sulfoxide (DMSO), dioxane, and N-methyl-2-pyrrolidone (NMP). Examples of aqueous solvents include water, and mixed solvents containing water and a solvent selected from ether solvents such as tetrahydrofuran (THF) and 1,4-dioxane, alcohol solvents such as methanol, ethanol, and 2-propanol, and polar solvents such as N,N-dimethylformamide, dimethyl sulfoxide (DMSO), dioxane, and N-methyl-2-pyrrolidone (NMP), to the extent that the polysaccharide does not precipitate.

[0118] The condensation reaction of compound (a1) with polysaccharide is preferably carried out under conditions of pH 5 to 10 (more preferably pH 5.5 to 8.5, and even more preferably pH 7.5 to 8.0). By allowing the reaction to proceed while adjusting the pH within this range, the modification rate with the modifying group (A) can be further improved. To adjust the pH range, it is preferable to use an aqueous sodium hydroxide solution or an aqueous hydrochloric acid solution. The reaction can also be carried out using a buffer solution.

[0119] The condensation reaction of compound (a1) with a polysaccharide is preferably carried out in the presence of a condensing agent selected from 1,3-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSC·HCl), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent), bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BOP-Cl), 2-chloro-1,3-dimethylimidazolinium hexafluorophosphate (CIP), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), or the like, in the presence or absence of an inorganic base such as sodium bicarbonate or sodium carbonate, or an organic base such as triethylamine or pyridine. Such a condensation reaction can be carried out, for example, in the range of 0°C to 50°C. A preferred temperature range is 15°C to 40°C. By carrying out the reaction at a temperature around room temperature, the reaction time can be shortened while suppressing a decrease in the molecular weight of the polysaccharide. Furthermore, by using such a temperature range, a side reaction (Schiff base formation reaction) between the amino group of compound (a) and the aldehyde group of compound (a) can also be suppressed. In order to suppress the production of by-products, an additive such as 1-hydroxy-1H-benzotriazole (HOBt) or 1-hydroxy-7-azabenzotriazole (HOAt) can be added to the condensation reaction.

[0120] After the reaction, purification may be carried out by filtration and / or dialysis.

[0121] One aspect of the present invention relates to a method for producing a polysaccharide derivative represented by the following formula (C2): In some embodiments, the method for producing the polysaccharide derivative represented by formula (C2) comprises a condensation reaction of a polysaccharide containing an amino group with a compound (a2) represented by the following formula (a2): The polysaccharide derivative represented by the following formula (C2) can be obtained by a condensation reaction between the amino group of a polysaccharide and the carboxyl group of compound (a2) represented by the following formula (a2): This reaction forms an amide bond between the carboxyl group of compound (a2) and the amino group of the polysaccharide, thereby obtaining a polysaccharide derivative represented by the following formula (C2) in which a modifying group (A) corresponding to compound (a2) (the group represented by formula (A-2) described above) has been introduced into the polysaccharide. [ka]

[0122] The rings P and R in the above formulas (a2) and (C2) 1 , and L in formula (C2) 2 The definitions and preferred embodiments of are the same as those in the formula (A) and formula (A-2) above.

[0123] The method of the condensation reaction is not particularly limited, and can be carried out by a conventionally known method such as a method using a carbodiimide-based condensing agent. The condensation reaction of compound (a2) with polysaccharide is preferably carried out under conditions of pH 4 to 7 (more preferably pH 5 to 6). By proceeding with the reaction while adjusting the pH within this range, precipitation of the reaction product can be suppressed and the reaction can proceed efficiently. To adjust the pH range, it is preferable to use an aqueous sodium hydroxide solution or an aqueous hydrochloric acid solution. The reaction can also be carried out using a buffer solution. It is also preferable to react compound (a2) with a polysaccharide in such a manner that the molar ratio of carboxyl groups in compound (a2) to amino groups in the polysaccharide is in the range of 0.8: 1 to 1.2: 1. In this case, precipitation of the reaction product can be suppressed, and the reaction can proceed efficiently.

[0124] 3. Polysaccharide derivative-drug conjugates One aspect of the present invention relates to a polysaccharide derivative-drug conjugate of a drug containing a primary amino group and the polysaccharide derivative of the above-mentioned form. The polysaccharide derivative can form a conjugate by forming a Schiff base with the drug having a primary amino group. Specifically, the polysaccharide derivative can form a conjugate by forming a Schiff base with the aldehyde or ketone group (-CR 1 (=O)) reacts with a primary amino group on a drug to form a Schiff base. Therefore, in this form of polysaccharide derivative-drug conjugate, the drug and the group represented by formula (A) (modifying group (A)) contained in the polysaccharide are covalently bonded via a Schiff base to form a structure represented by the following formula (D): [ka] In the above formula (D), Drug represents the drug moiety excluding the primary amino group. In the above formula (D), R 1 The definitions and preferred embodiments of rings P and Y are as follows: 1 and the definition and preferred embodiments of ring P are the same as those of ring P.

[0125] (drugs with primary amino groups) The drug is not particularly limited as long as it has one or more primary amino groups in the molecule. The drug may be a synthetic or natural product. For example, the drug may be at least one selected from low molecular weight compounds, medium molecular weight compounds, peptides, nucleic acids, nucleic acid derivatives, aptamers, vitamins, monoamines, amino acids, polyamines, antibodies, fluorescent dyes, and contrast agents. The primary amino group may be a hydrazide group (-C(=O)-NH-NH2). The hydrazide group may be substituted with an aldehyde or ketone group (-CR 1 (=O)) to form a hydrazone bond (-C(=O)-NH-N=CH-). The hydrazone bond is more stable than a normal Schiff base, and delayed release can be expected.

[0126] Examples of low molecular weight compounds (e.g., molecular weight of 500 or less) and medium molecular weight compounds (e.g., molecular weight of 500 to 2000) having a primary amino group include doxorubicin, gemcitabine, aminosalicylic acid, pemetrexed, methotrexate, andronate, eribulin, memantine, remdesivir, ampicillin, amoxicillin, aztreonam, tigemonam, vancomycin, cephalosporin C, gentamicin, trimethoprim, sulfamethoxazole, oseltamivir, mexiletine, and mitomycin. These include dodrine, levodopa, tenofovir, darunavir, procaine, ethyl aminobenzoate, procainamide, fluvoxamine, milnacipran, baclofen, benserazide, carbidopa, droxidopa, gusperimus, ubenimex, fingolimod, amfenac, sulfamine, triamterene, meticiletine, amlodipine, azelnidipine, methyldopa, hydralazine, mosapride, sitagliptin phosphate hydrate, valacyclovir, acyclovir, and celecoxib. The peptide is not particularly limited as long as it has a primary amino group at the N-terminus, etc. Examples of peptides include methionine enkephalin, leucine enkephalin, dynorphin A, β-endorphin, bacitracin, daptomycin, colistin, elcatonin, and oxytocin. The nucleic acid bases adenine, thymine, guanine, and cytosine have a primary amino group, and nucleic acids and nucleic acid derivatives containing these nucleic acid bases have a primary amino group and can be used in the present invention. Examples of nucleic acid derivatives include cytarabine, cladribine, and fludarabine. As the aptamer, a nucleic acid (DNA / RNA) aptamer or a peptide aptamer can be used. Examples of vitamins having a primary amino group include folic acid, vitamin B1 (thiamine), vitamin B6 (pyridoxamine), vitamin B12 (cyanocobalamin), and nicotinamide. Examples of monoamines having a primary amino group include dopamine, noradrenaline, serotonin, histamine, thiamine, and octobamine. The amino acid may be any of various amino acids having a primary amino group in the molecule, and may be a natural amino acid or an artificial amino acid such as tranexamic acid. Polyamines include molecules that exist in living organisms and have multiple primary amines in the molecule, such as spermine, spermidine, and putrescine. Fluorescent dyes include fluorescein-5-thiosemicarbazide (FTSC).

[0127] The drug may be used alone or in combination of two or more. The drug may be water-soluble or water-insoluble. As long as the drug has a primary amino group, it may be in the form of a salt, hydrate, or solvate.

[0128] (drug introduction rate) The drug incorporation rate of the modifying group (A) in the polysaccharide derivative-drug conjugate is not particularly limited, but is, for example, a percentage of the modifying group (A) introduced into the polysaccharide derivative that is bonded to the drug (Schiff base). The drug incorporation rate is not particularly limited, but can be, for example, 1 to 100%.

[0129] (sustained release properties) Aldehyde or ketone groups (-CR 1 The Schiff base formed between (=O)) and the primary amino group of a drug is stable under neutral to basic pH conditions, but dissociates at low pH conditions, releasing the drug. As used herein, "low pH conditions" refers to a pH of less than 7, but typically refers to a pH of 3.5 to 7.0. Although some drug release occurs in a neutral to alkaline (high pH) environment (e.g., pH 7.4 or higher), the release is slow, whereas under low pH conditions, the drug release rate is rapid and a large amount of drug is released. While physiological pH, such as in blood, is around pH 7.4, inflamed tissues and tumor tissues (pH 6.5-7.2) and the interior of lysosomes and endosomes (pH 4.5-5.5) are known to be low pH environments. Because this polysaccharide derivative-drug conjugate can release drugs in response to low pH, it is possible to suppress drug release at pH 7.4, which corresponds to the pH of blood, and stably retain the drug, enabling efficient drug release at target sites in a low pH environment. [ka] In the above formula, Drug represents the drug moiety excluding the primary amino group. In the above formula, R 1 The definitions and preferred embodiments of rings P and Y are as follows: 1 and the definition and preferred embodiments of ring P are the same as those of ring P.

[0130] (Manufacturing method) A further aspect of the present invention relates to a method for producing a polysaccharide derivative-drug conjugate. This production method comprises mixing the above-mentioned polysaccharide derivative with a drug containing a primary amino group in a solvent. By mixing, the aldehyde or ketone group (-CR 1 (=O)) reacts with the amino group of the drug to form a Schiff base, resulting in a polysaccharide derivative-drug conjugate. This production method allows stable polysaccharide derivative-drug conjugates to be obtained easily and in one pot using mild, safe, single-step reaction conditions. This production method does not require modification of the drug prior to conjugate formation, nor does it require complex experimental equipment or catalysts for conjugate formation, making it possible to prepare polysaccharide derivative-drug conjugates in situ from the polysaccharide derivative and drug.

[0131] The solvent is not particularly limited, and may be selected appropriately from those capable of dissolving the polysaccharide to be used. For example, it is preferable to use an aqueous solvent or a polar solvent such as dimethyl sulfoxide (DMSO) because of their excellent solubility for polysaccharides. Examples of aqueous solvents include water, and mixed solvents containing water and a solvent selected from ether solvents such as tetrahydrofuran (THF) and 1,4-dioxane, alcohol solvents such as methanol, ethanol, and 2-propanol, and polar solvents such as N,N-dimethylformamide and dimethyl sulfoxide (DMSO), to the extent that the polysaccharide does not precipitate. The temperature during mixing is not particularly limited, and can be, for example, in the range of 0° C. to 50° C. A preferred temperature range is 15° C. to 40° C. By carrying out the reaction at a temperature around room temperature, it is possible to achieve both an appropriate reaction rate and prevention of a decrease in the amount of the reaction mixture.

[0132] The pH of the reaction between the polysaccharide derivative and the drug may be any pH at which the drug dissolves but does not denature; from the viewpoint of reaction rate, a pH of 4.0 to 11.0 (more preferably, 6.0 to 9.0, and even more preferably, 7.0 to 8.0) is preferred. By allowing the reaction to proceed while adjusting the pH within this range, the rate of introduction of the drug into the modifying group (A) can be further improved. To adjust the pH range, it is preferable to use hydrochloric acid or sodium hydroxide. Furthermore, the reaction between the polysaccharide derivative and the drug is preferably carried out under light-shielded conditions in the case of a drug with poor photostability. The reaction time is not particularly limited, but is about 1 minute to 24 hours, preferably 10 minutes to 2 hours. The mixing ratio of the polysaccharide derivative and the drug is appropriately set depending on the number of modifying groups (A) introduced into the polysaccharide derivative, the desired drug incorporation rate, and the solubility of the resulting polysaccharide derivative-drug conjugate. For example, 0.001 to 10 moles, preferably 0.05 to 3 moles, and more preferably 0.1 to 1 mole of the drug is mixed per mole of the modifying group (A) introduced into the polysaccharide derivative. The method for mixing the polysaccharide derivative and the drug is not particularly limited as long as both components can be mixed uniformly.

[0133] The polysaccharide derivative-drug conjugate (reaction solution) obtained by mixing may be used as is without purification. After the conjugate is formed, purification treatments such as dialysis, salting out, gel filtration, ion exchange chromatography, and electrophoresis, or freeze-drying may be performed.

[0134] 4. Composition A further aspect of the present invention relates to a composition comprising the polysaccharide derivative of the above form or the polysaccharide derivative-drug conjugate of the above form. In some embodiments, a composition is provided comprising the polysaccharide derivative or polysaccharide derivative-drug conjugate in the above form and an aqueous solvent, wherein the weight ratio of the polysaccharide derivative or conjugate to the aqueous solvent is, for example, 0.0001:1 to 0.3:1, preferably 0.001:1 to 0.1:1. In some embodiments, a composition is provided comprising the polysaccharide derivative of the above form and a drug having a primary amino group, wherein the molar ratio of the polysaccharide derivative to the drug, for example, the ratio of A to the drug, is in the range of 1:0.001 to 1:10, preferably 1:0.05 to 1:3, and more preferably 1:0.1 to 1:1. In some embodiments, a composition is provided that contains the polysaccharide derivative of the above form, a drug having a primary amino group, and an aqueous solvent. In the composition, the ratio of the total weight of the polysaccharide derivative and drug to the weight of the aqueous solvent is, for example, 0.0001:1 to 0.3:1, preferably 0.001:1 to 0.1:1. In the composition, the molar ratio of the polysaccharide derivative to the drug, for example, the ratio of A to drug, is in the range of 1:0.001 to 1:10, preferably 1:0.05 to 1:3, and more preferably 1:0.1 to 1:1. In some embodiments, a composition is provided that includes a crosslinked structure of a polysaccharide derivative described below and an aqueous solvent, wherein the weight ratio of the crosslinked structure to the aqueous solvent in the composition is, for example, 0.0001:1 to 0.3:1, and preferably 0.001:1 to 0.1:1. In some embodiments, a composition is provided that includes a crosslinked structure of a polysaccharide derivative-drug conjugate (crosslinked structure-drug conjugate) described below and an aqueous solvent, wherein the weight ratio of the crosslinked structure to the aqueous solvent in the composition is, for example, 0.0001:1 to 0.3:1, and preferably 0.001:1 to 0.1:1. As the aqueous solvent, those mentioned in the sections on the method for producing a polysaccharide derivative and the method for producing a conjugate can be used in the same way.

[0135] The polysaccharide derivative or polysaccharide derivative-drug conjugate of the present invention can be prepared into a pharmaceutical composition together with additives such as pharmaceutical carriers and diluents known per se, and can be administered parenterally or orally to mammals, including humans, or non-mammals. Examples of mammals include, but are not limited to, humans, chimpanzees, apes, monkeys, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, reptiles, etc. One embodiment provides a pharmaceutical composition comprising the polysaccharide derivative of the above form, a drug, and a pharmaceutically acceptable excipient. One embodiment provides a pharmaceutical composition comprising a polysaccharide derivative-drug conjugate of the above form and a pharmaceutically acceptable excipient. Pharmaceutically acceptable additives include excipients, fillers, bulking agents, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, solubilizing agents, antiseptics, flavoring agents, soothing agents, stabilizers, and isotonic agents that are commonly used in drug production. Pharmaceutical compositions can be prepared by conventional methods using appropriate additives.

[0136] The polysaccharide derivative or polysaccharide derivative-drug conjugate of the present invention can be formulated into a food composition by blending it with additives commonly used in the food industry (for example, at least one component such as various nutritional supplements (amino acids, vitamins, minerals, etc.), sugars, dairy products, sweeteners, flavorings, fragrances, antioxidants, preservatives, colorants, emulsifying aids, pH adjusters, organic acids, buffers, fruit juice, etc.).

[0137] 5.Crosslinked structure One aspect of the present invention relates to a crosslinked structure comprising a polysaccharide derivative.

[0138] (1) Crosslinked structure of polysaccharide derivatives via a crosslinking agent In some embodiments, the crosslinked structure is a crosslinked structure of a polysaccharide derivative or a crosslinked structure of a polysaccharide derivative-drug conjugate (also referred to as a "crosslinked structure-drug conjugate"). The crosslinked structure of a polysaccharide derivative is formed by crosslinking polysaccharide derivatives via crosslinking groups to form a three-dimensional network structure. The crosslinked structure of a polysaccharide derivative-drug conjugate (crosslinked structure-drug conjugate) is formed by crosslinking polysaccharide derivative-drug conjugates via crosslinking groups to form a three-dimensional network structure. The crosslinked structure of a polysaccharide derivative can be obtained by subjecting a polysaccharide derivative having a crosslinking group to a crosslinking reaction using a crosslinking agent. The crosslinked structure of a polysaccharide derivative-drug conjugate (crosslinked structure-drug conjugate) can typically be obtained by a method of subjecting a polysaccharide derivative-drug conjugate having a crosslinking group to a crosslinking reaction using a crosslinking agent, or by a method of producing a crosslinked structure of a polysaccharide derivative and then bonding the crosslinked structure to a drug via a Schiff base.

[0139] For example, unmodified carboxyl groups can function as crosslinking groups to form crosslinked structures via crosslinking agents such as divalent metal ions. Crosslinked structures can be formed by reacting a composition containing a polysaccharide derivative or a polysaccharide derivative-drug conjugate having unmodified carboxyl groups in a solution containing a crosslinking agent. The shape of these crosslinked structures is not particularly limited, and examples thereof include tubular structures, fibrous structures, fibers, beads, gels, approximately spherical gels, capsules, sponges, sheets, and films. In certain embodiments, there are provided gels, capsules, sponges, beads, fibers, tubes, sheets, or films comprising the polysaccharide derivatives, polysaccharide derivative-drug conjugates, crosslinked structures, or crosslinked structure-drug conjugates in the above-described forms.

[0140] Specifically, a solution containing a polysaccharide derivative having unmodified carboxyl groups can be partially crosslinked by dropping it into a solution containing a divalent metal ion as a crosslinking agent, to obtain a crosslinked structure.Also, a solution containing a polysaccharide derivative-drug conjugate having unmodified carboxyl groups can be partially crosslinked by dropping it into a solution containing a divalent metal ion, to obtain a crosslinked structure. The crosslinked structure produced by such a method may have the shape of, for example, a capsule, a bead, a fiber, a tube, or a film.

[0141] Alternatively, a solution containing a polysaccharide derivative containing unmodified carboxyl groups and a solution containing a divalent metal ion as a crosslinking agent can be applied to a substrate to partially crosslink the polysaccharide derivative, thereby obtaining a crosslinked structure. The crosslinked structure produced by this method can be in the form of a gel (hydrogel).

[0142] The gel is freeze-dried to obtain a cross-linked structure in the shape of a sponge.

[0143] After forming the crosslinked structure, the drug can be bound to the crosslinked structure via a Schiff base by a method similar to the method for producing a polysaccharide derivative-drug conjugate. For example, a method including mixing the crosslinked structure with a drug containing a primary amino group in a solvent after forming the crosslinked structure can be used. By mixing, the aldehyde group or ketone group (-CR 1 (=O)) reacts with the amino group of the drug to form a Schiff base, resulting in a crosslinked structure-drug conjugate.

[0144] A drug-loaded crosslinked structure of a polysaccharide derivative can also be obtained by chemically bonding or physically attaching a drug to the crosslinked structure of a polysaccharide derivative. For example, a crosslinked structure of a polysaccharide derivative carrying a drug can be obtained by applying a solution containing a drug to the crosslinked structure of a polysaccharide derivative, immersing the crosslinked structure of a polysaccharide derivative in the solution, or printing a drug onto the crosslinked structure of a polysaccharide derivative.

[0145] Specific examples of divalent metal ions used as crosslinking agents in the crosslinking reaction include calcium ions, magnesium ions, barium ions, strontium ions, and zinc ions, with calcium ions being preferred. More specifically, divalent metal ion compounds that can be used include CaCl2, MgCl2, CaSO4, ZnCl2, BaCl2, and SrCl2 (preferably CaCl2, CaSO4, ZnCl2, SrCl2, and BaCl2).

[0146] In addition to the above divalent metal ions (divalent metal ion compounds), the crosslinking agent also contains Fe 3+ Alternatively, a trivalent metal ion compound (FeCl3) such as methyltrimethylsilylsilane or a crosslinking reagent having 2 to 4 amino groups in the molecule may be used. Examples of crosslinking reagents having 2 to 4 amino groups in the molecule include diaminoalkanes that may have a lysyl group (-COCH(NH2)-(CH2)4-NH2) on the nitrogen atom, i.e., diaminoalkanes and derivatives in which the amino group is substituted with a lysyl group to form a lysylamino group, such as diaminoethane, diaminopropane, and N-(lysyl)-diaminoethane.

[0147] In addition to the above, when a drug has multiple primary amino groups, the drug itself introduced into the polysaccharide may function as a crosslinking group. For example, polysaccharide derivatives can be crosslinked via a medium-molecular compound (peptide) having multiple primary amino groups (e.g., bacitracin) to form a three-dimensional network structure.

[0148] The amount of crosslinking agent used is desirably adjusted appropriately depending on the amount and molecular weight of the polysaccharide derivative used, the type of polysaccharide constituting the polysaccharide derivative, etc. For example, when calcium ions are used as the crosslinking agent, the calcium ion concentration of the solution containing calcium ions is not particularly limited, but may be, for example, 1 mM to 1 M, preferably 5 mM to 500 mM, and more preferably 10 mM to 300 mM. The degree of crosslinking can be adjusted by adjusting the amount of crosslinking agent used.

[0149] (2) Crosslinked structure of polysaccharide derivative and amino group-containing polymer In some embodiments, the crosslinked structure comprises a polysaccharide derivative and at least one of an amino group-containing polymer and an amino group-containing low molecular weight compound containing two or more primary amino groups, hydrazide groups, or aminooxy groups, and is crosslinked by a covalent bond via a Schiff base between the primary amino groups, hydrazide groups, or aminooxy groups contained in the amino group-containing polymer and the amino group-containing low molecular weight compound and a group represented by formula (A) (modifying group (A)) contained in the polysaccharide derivative. Specifically, the aldehyde group or ketone group (-CR 1 (=O)) reacts with the primary amino group, hydrazide group, or aminooxy group of the amino group-containing polymer or amino group-containing low molecular weight compound to form a Schiff base (-CR 1 =N-). The hydrazide group is represented by -C(=O)-NH-NH2, and reacts with the aldehyde or ketone group of the modifying group (A) to form a hydrazone bond. The aminooxy group is represented by -O-NH2, and the terminal -NH2 reacts with the aldehyde or ketone group of the modifying group (A) to form an oxime bond. An amino group-containing polymer refers to a substance that contains two or more primary amino groups, hydrazide groups, or aminooxy groups and has a molecular weight of 1,000 or more. In this specification, the term "amino group-containing polymer" also encompasses molecules generally called oligomers, which have a molecular weight of about 1,000 to 10,000. An amino group-containing low molecular weight compound is a substance with a molecular weight of less than 1,000 that contains two or more primary amino groups, hydrazide groups, or aminooxy groups. The amino group-containing polymer and amino group-containing low molecular weight compound may be synthetically produced or may be naturally occurring.

[0150] The amino group-containing polymer (also simply referred to as "amino group-containing polymer") containing a primary amino group, a hydrazide group, or an aminooxy group is not particularly limited as long as it contains a total of two or more primary amino groups, hydrazide groups, or aminooxy groups, each of which is at least one of the primary amino groups, hydrazide groups, or aminooxy groups. The primary amino groups, hydrazide groups, or aminooxy groups may be present at the terminals of the polymer, in the side chains of the polymer, or in pendant groups within the polymer.

[0151] The amino group-containing polymer may be at least one selected from polyamines, polyalkylene glycols substituted with amino groups, aminooxy groups, or hydrazide groups, polyallylamine, polyvinylamine, polyacrylamine, amino group-containing polysaccharides, amino group-containing proteins, and polyamino acids. These may be commercially available products or may be synthesized by a conventionally known method.

[0152] Polyamines can be linear, branched, or dendritic. "Dendron" means that the polyamine has a dendritic, hyperbranched morphology, a polymer with multiple arms of equal or unequal length. The polyamine is not particularly limited, but examples thereof include linear, branched or dendritic polyalkyleneimines, branched or dendritic polyetheramines, and the like.

[0153] Polyalkyleneimine refers to a polymer containing an alkyleneimine structure as a repeating unit and having a primary amino group at its terminal. The alkylene moiety and imine moiety in the alkyleneimine structure may each be substituted. A linear polyalkyleneimine contains an alkyleneimine structure having a secondary amino group. A branched polyalkyleneimine and a dendritic polyalkyleneimine contain an alkyleneimine structure containing a primary amino group, a secondary amino group, or a tertiary amino group. The polyalkyleneimine preferably has a lower (e.g., C1-C6, C1-C3) alkyleneimine structure, and specific examples include, but are not limited to, polyethyleneimine and polypropyleneimine.

[0154] Branched or dendritic polyetheramines refer to polymers containing alkylene oxide repeating units and terminal primary amino groups. Examples include, but are not limited to, amino-terminated star polyethylene oxides, amino-terminated dendritic polyethylene oxides, amino-terminated comb polyethylene oxides, amino-terminated star polypropylene oxides, amino-terminated dendritic polypropylene oxides, amino-terminated comb polypropylene oxides, amino-terminated star polyethylene oxide-polypropylene oxide copolymers, amino-terminated dendritic polyethylene oxide-polypropylene oxide copolymers, and amino-terminated comb polyethylene oxide-polypropylene oxide copolymers. Examples of amino-terminated star polymers include polymers with 3, 4, 6, or 8 arms terminated with primary amines. For example, amino-terminated star polyethylene glycols include star polyethylene glycols with 3, 4, 6, or 8 arms terminated with primary amines (3-, 4-, 6-, or 8-arm star PEG amines). Examples of amino-terminated star polymers include, but are not limited to, star polyethylene glycols with 3, 4, 6, or 8 arms terminated with primary amines (3-, 4-, 6-, or 8-arm star PEG amines). Branched or dendritic polyetheramines can be commercially available, such as polyoxyalkylene triamines sold under the trade name Jeffamine® triamines by Huntsman LLC. (Houston, TX).

[0155] The polyalkylene glycol substituted with an amino group, a hydrazide group, or an aminooxy group includes polyethylene glycol (PEG), polypropylene glycol, or polyethylene oxide-polypropylene oxide copolymer substituted with a plurality (two or more) of amino groups, hydrazide groups, or aminooxy groups.

[0156] Polyallylamine refers to a polymer containing an allylamine structure as a repeating unit. The allyl moiety in the allylamine structure may be substituted. In addition to the repeating unit having the allylamine structure, polyallylamine may further contain other repeating units as copolymerization components. Polyvinylamine refers to a polymer containing a vinylamine structure as a repeating unit, and the vinyl moiety in the vinylamine structure may be substituted. Polyvinylamine may further contain other repeating units as copolymerization components in addition to the repeating unit having the vinylamine structure. Polyacrylamine refers to a polymer containing an acrylic structure with a side chain containing a primary amino group, and the vinyl moiety in the acrylic structure may be substituted. For example, an acrylic polymer containing an amine structure with a polyalkyleneimine grafted to the side chain may be mentioned. In addition to the repeating unit having the acrylic structure with a side chain containing a primary amino group, polyacrylamine may also contain other repeating units as copolymerization components. Commercially available products such as aminoethylated acrylic polymer sold under the trade name Polyment (registered trademark) (Nippon Shokubai Co., Ltd.) can be used.

[0157] Examples of amino group-containing polysaccharides include polysaccharides containing amino groups, such as chitosan. Furthermore, amino group-containing polysaccharides may be those obtained by modifying polysaccharides that do not have amino groups to introduce amino groups (aminated polysaccharides). Examples of aminated polysaccharides include those obtained by introducing amino groups into aminodextran, etc. Examples of amino group-containing proteins include fibrinogen, albumin, gelatins, and collagens. Examples of polyamino acids (polypeptides) include polylysine, polyarginine, polyglutamic acid, and polyaspartic acid.

[0158] The lower limit of the weight average molecular weight of the amino group-containing polymer is not particularly limited, but is generally 1.00 or more. The upper limit of the weight average molecular weight of the amino group-containing polymer is not particularly limited, but is generally 1,000,000 or less.

[0159] The amino group-containing low molecular weight compound is not particularly limited as long as it contains a total of two or more of at least one of primary amino groups, hydrazide groups, and aminooxy groups. Examples include hydrazide crosslinking agents such as adipic acid dihydrazide, sebacic acid dihydrazide, dodecanediohydrazide, and isophthalic acid dihydrazide.

[0160] The amino group in the amino group-containing polymer or amino group-containing low molecular weight compound may form a salt. Examples of the salt of the amino group-containing polymer or amino group-containing low molecular weight compound include halide salts (e.g., hydrochloride), phosphate, phosphite, carbonate, bicarbonate, sulfate, hydrogensulfate, hydroxide, nitrate, persulfate, sulfite, acetate, ascorbate, citrate, oxalate, succinate, tartrate, taurocholate, and cholate.

[0161] Figure 34 shows a schematic diagram of a crosslinked structure formed from a polysaccharide derivative (AL-ABA) and an amino group-containing polymer (DPI; polyethyleneimine), which was prepared in Example I-17 described below. As shown in Figure 34, the crosslinked structure has a crosslinked structure formed by a covalent bond between a primary amino group contained in the amino group-containing polymer, polyethyleneimine, and a benzaldehyde group contained in the polysaccharide derivative via a Schiff base (-C=N-).

[0162] The crosslinked structure of the polysaccharide derivative and the amino group-containing polymer or the amino group-containing low molecular weight compound is typically produced by mixing the polysaccharide derivative and the amino group-containing polymer or the amino group-containing low molecular weight compound in a solvent. From the viewpoint of biocompatibility, the solvent is preferably an aqueous solvent (e.g., water or physiological saline).

[0163] After obtaining a crosslinked structure between a polysaccharide derivative and an amino group-containing polymer or an amino group-containing low molecular weight compound, the crosslinked structure may be further crosslinked via the crosslinking groups (e.g., unmodified carboxyl groups) contained in the crosslinked structure using a crosslinking agent (e.g., a divalent metal ion) different from the amino group-containing low molecular weight compound. Specifically, the polysaccharide derivative and the amino group-containing polymer or the amino group-containing low molecular weight compound are mixed in a solvent, and then a solution containing a divalent metal ion as a crosslinking agent is added dropwise to the solution containing the crosslinked structure; alternatively, the polysaccharide derivative and the amino group-containing polymer or the amino group-containing low molecular weight compound are mixed in a solvent, and then the solution containing the crosslinked structure is added dropwise to a solution containing a divalent metal ion as a crosslinking agent, thereby partially crosslinking the crosslinked structure to obtain a crosslinked structure. The type of crosslinking agent and the amount of crosslinking agent used are not particularly limited, but the type of crosslinking agent and the amount of crosslinking agent used in the crosslinking reaction of the polysaccharide derivative using the crosslinking agent can be similarly used.

[0164] The shape of the crosslinked structure of the polysaccharide derivative and the amino group-containing polymer or amino group-containing low molecular weight compound is not particularly limited, and examples include a tubular structure, a fibrous structure, a fiber, a bead, a gel, a substantially spherical gel, a capsule, a sponge, a sheet, and a film. In one embodiment, the crosslinked structure of the polysaccharide derivative and the amino group-containing polymer or amino group-containing low molecular weight compound may be a gel (hydrogel). The gel can be freeze-dried to obtain a sponge-shaped crosslinked structure.

[0165] 6. Uses of polysaccharide derivatives, polysaccharide derivative-drug conjugates, cross-linked structures, cross-linked structure-drug conjugates, and compositions The polysaccharide derivatives, polysaccharide derivative-drug conjugates, crosslinked structures, crosslinked structure-drug conjugates, or compositions can be used in a wide range of fields, including food, medicine, cosmetics, daily necessities, fibers, and papermaking. The shape of the polysaccharide derivatives, polysaccharide derivative-drug conjugates, crosslinked structures, crosslinked structure-drug conjugates, and compositions is not particularly limited and can be selected depending on the application. Examples include tubular structures, fibrous structures, fibers, beads, gels, approximately spherical gels, capsules, sponges, sheets, and films. In one embodiment, the shape of the polysaccharide derivatives, polysaccharide derivative-drug conjugates, crosslinked structures, crosslinked structure-drug conjugates, or compositions can be a gel, sponge, film, or capsule.

[0166] The polysaccharide derivatives and crosslinked structures can be used in place of conventional polysaccharide materials in a wide range of fields, including food, medicine, cosmetics, daily necessities, textiles, and papermaking.

[0167] The polysaccharide derivative, polysaccharide derivative-drug conjugate, crosslinked structure, crosslinked structure-drug conjugate, or composition has excellent biodegradability and biocompatibility, and can therefore be suitably used as a medical material.

[0168] When used as a medical material, examples include drug release devices, sutures, materials for hemostasis, adhesion, and adhesion prevention in the body, tissue adhesive materials, wound dressings, substrates for cell culture, substrates for cell transplantation, regenerative engineering materials, antithrombotic materials, diagnostic agents, dialysis carriers, etc. Medical materials can be used for both mammals and non-mammals. In particular, polysaccharide derivatives can bind to drugs having primary amino groups under neutral and high pH conditions and release them under low pH conditions (pH-responsive drug release), making them suitable for use as drug transport carriers (drug delivery carriers). One aspect of the present invention provides a drug delivery device comprising the above-mentioned polysaccharide derivative, polysaccharide derivative-drug conjugate, crosslinked structure, crosslinked structure-drug conjugate, or a composition containing any of these. The drug delivery device can be used as a means for selectively and efficiently introducing a drug carried by the polysaccharide derivative into a target tissue. Another aspect provides a method for releasing or controlling the release of an encapsulated desired drug in a target tissue.

[0169] When used as a medical material, the shape of the crosslinked structure may be tubular, fibrous, fiber, bead, capsule, gel, approximately spherical gel, sponge, sheet, film, etc., and is preferably in the form of beads, capsules, gel, or approximately spherical gel or sponge, and more preferably in the form of capsules, hydrogel, or sponge.

[0170] In some embodiments, a tissue adhesive material is provided, comprising the polysaccharide derivative, polysaccharide derivative-drug conjugate, crosslinked structure, or crosslinked structure-drug conjugate. In certain embodiments, the tissue adhesive material is a biological tissue adhesive or sealant used to adhere biological tissues.

[0171] In some embodiments, a tissue adhesive material is provided that includes the polysaccharide derivative or crosslinked structure. The polysaccharide derivative or crosslinked structure has an aldehyde group or a ketone group (—C(═O)R ) of the modifying group (A) introduced therein. 1 ) can adhere to biological tissue by interacting with the functional groups of the biological tissue. In a specific embodiment, the polysaccharide derivative or crosslinked structure has an aldehyde group or a ketone group (—C(═O)R 1 ) reacts with primary amino groups in biological tissue to form a Schiff base, thereby adhering to biological tissue.

[0172] In some embodiments, the tissue adhesive material comprises a polysaccharide derivative-drug conjugate or a crosslinked structure-drug conjugate. Preferably, the polysaccharide derivative-drug conjugate or the crosslinked structure-drug conjugate has a drug having a primary amino group bound to a portion of the modifying group (A) and has unreacted modifying groups (A) to which no drug is bound. According to this embodiment, the aldehyde group or ketone group (-C(=O)R of the unmodified modifying group (A) is converted to a hydroxy group (-C(=O)R) while delivering the drug to the adhesion site. 1 ) reacts with primary amino groups in biological tissue to form a Schiff base, thereby adhering to biological tissue.

[0173] The shape of the tissue adhesive material is not particularly limited, and examples thereof include tubular, fibrous, beaded, capsule, gel, approximately spherical gel, sponge, sheet, and film. Beads, capsules, gel, or approximately spherical gel or sponge are preferred, capsules, hydrogel, or sponge are more preferred, and hydrogel is even more preferred. Hydrogels can be useful as tissue adhesives or sealants for medical applications requiring rapid degradation, such as preventing undesirable tissue-tissue adhesions due to trauma or surgery.

[0174] The method of using the tissue adhesive material is not particularly limited. In some embodiments, the tissue adhesive material is a tissue adhesive used to adhere two biological tissues together. In some embodiments, two biological tissues are adhered together by applying a tissue adhesive containing the polysaccharide derivative or crosslinked structure to at least one adhesion site of biological tissues and bringing the two or more sites into contact. In some embodiments, two biological tissues are adhered by applying a tissue adhesive containing the polysaccharide derivative or crosslinked structure to at least one adhesion site of biological tissues, then applying a crosslinking agent (e.g., a solution containing divalent metal ions) to the adhesion site, and bringing the two or more sites into contact. In some embodiments, two biological tissues are adhered by applying a crosslinking agent (e.g., a solution containing divalent metal ions) to at least one adhesion site of biological tissues, then applying a tissue adhesive containing the polysaccharide derivative or crosslinked structure to the adhesion site, and bringing the two or more sites into contact. Application of a crosslinking agent can form a hydrogel or increase the degree of crosslinking of the hydrogel.

[0175] In some embodiments, the tissue adhesive material is a sealant used to seal air / fluid leaks in living tissue or to seal or fill small cavities or defects in living tissue. In some embodiments, a sealant containing the above-mentioned polysaccharide derivative or crosslinked structure is applied to biological tissue and allowed to stand, thereby sealing air / fluid leaks in the biological tissue or sealing or filling small cavities or defects in the biological tissue. In some embodiments, a sealant containing the polysaccharide derivative or crosslinked structure is applied to biological tissue, and then a crosslinker (e.g., a solution containing divalent metal ions) is applied to the application site and allowed to stand, thereby sealing air / fluid leaks in the biological tissue or sealing or filling small cavities or defects in the biological tissue. In some embodiments, a crosslinker (e.g., a solution containing divalent metal ions) is applied to biological tissue, and then a sealant containing the polysaccharide derivative or crosslinked structure is applied to the application site and allowed to stand, thereby sealing air / fluid leaks in the biological tissue or sealing or filling small cavities or defects in the biological tissue. Application of a crosslinker can form a hydrogel or increase the degree of crosslinking of the hydrogel. In some embodiments, the mounting medium may be combined with a tissue fragment derived from a living tissue, such as a skin flap or periosteum, and the tissue fragment combined with the mounting medium may be attached to the tissue. In some embodiments, the sealant may be combined with biodegradable or non-biodegradable nonwoven fabrics, sheets, films, etc., and can also be used to fix or adhere these nonwoven fabrics, sheets, and films. Examples of biodegradable nonwoven fabrics, sheets, and films include those made from polyglycolic acid, L-lactide-ε-caprolactone polymer, polylactic acid, glycolic acid / lactic acid polyester, and sodium alginate, while examples of non-degradable nonwoven fabrics, sheets, and films include those made from polytetrafluoroethylene (PTFE). Nonwoven fabrics, sheets, and films combined with these sealants can be attached to tissue.

[0176] In certain embodiments, the tissue adhesive material (tissue glue, sealant) is applied to the biological tissue in the form of an aqueous solution or dispersion. In certain embodiments, the tissue adhesive material (tissue glue, sealant) is applied to the living tissue in the form of a gel, sponge, sheet, or film.

[0177] The tissue adhesive material may further contain various additives depending on the intended use, such as at least one additive selected from a pH adjuster, an antibacterial agent, a colorant, and a surfactant.

[0178] The biological tissue to which the tissue adhesive material can be applied is not particularly limited and includes, for example, skin, oral cavity, esophagus, stomach, small intestine, large intestine (rectum), bone, nerve, exon, cartilage, blood vessel, cornea, muscle, fascia, brain, prostate, breast, endometrium, lung, spleen, liver, testis, ovary, cervix, lymph node, bone marrow, and kidney, etc. Among these, preferred are skin, mucosa of the digestive tract (oral cavity, esophagus, stomach, small intestine, large intestine), submucosa of the digestive tract (site of mucosal damage), etc. In a preferred embodiment, the biological tissue to which the tissue adhesive material is applied is a biological tissue having an amino group exposed on the surface. In such a case, the aldehyde group or ketone group (-C(=O)R 1A covalent bond is formed between the amino group and the Schiff base, thereby improving adhesive strength. Examples of biological tissues with exposed amino groups on the surface include skin (particularly areas where collagen is exposed after surgery) and the submucosa of the digestive tract (areas where mucosa is damaged).

[0179] There are no particular limitations on the specific site of the biological tissue, and sites where tissue adhesive materials have conventionally been used can be suitably employed, such as suture sites after surgical operations, bleeding sites, fixation sites for fracture fragments, anastomosis sites for peripheral nerves or microvessels, sites affected by tendon adhesion or tendon suture, and adhesion sites for organ wounds.

[0180] According to some embodiments, there is also provided a kit comprising a tissue adhesive material to be applied to biological tissue to adhere said biological tissue, and instructions for use of the tissue adhesive material. According to some embodiments, there is also provided a kit comprising a precursor of a tissue adhesive material to be applied to biological tissue to adhere said biological tissue, and instructions for use of the tissue adhesive material. In one embodiment, the precursor of the tissue adhesive material comprises an uncrosslinked polysaccharide derivative or a partially crosslinked crosslinked structure, or a conjugate of either of them with a drug, and a crosslinking agent (e.g., a solution containing a divalent metal ion).

[0181] According to some embodiments, there is provided an adhesion preventing material comprising the polysaccharide derivative, polysaccharide derivative-drug conjugate, crosslinked structure, or crosslinked structure-drug conjugate.

[0182] Polysaccharide derivatives, polysaccharide derivative-drug conjugates, crosslinked structures, and crosslinked structure-drug conjugates have excellent biocompatibility and can therefore be used as bioabsorbable materials and medical devices.

[0183] The polysaccharide derivatives and crosslinked structures can be used as separation materials, such as chromatographic carriers, nonwoven fabrics, and membrane materials, because they bind to drugs having primary amino groups.

[0184] The polysaccharide derivatives, polysaccharide derivative-drug conjugates, crosslinked structures, and crosslinked structure-drug conjugates can be used as foods, supplements, and food additives.

[0185] All publications cited in this specification, such as prior art documents, published patent applications, patent publications, and other patent documents, are incorporated herein by reference. This specification also includes the disclosures of the claims, specification, and drawings of Japanese Patent Application No. 2020-137010 (filed August 14, 2020), which is the basis for the priority claim of this application.

[0186] Furthermore, the objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description in this specification, and those skilled in the art will be able to easily implement the present invention from the description in this specification. The best mode for carrying out the invention and specific examples show preferred embodiments of the present invention and are shown for the purpose of illustration and explanation, and are not intended to limit the present invention thereto. It will be apparent to those skilled in the art that various modifications can be made based on the description in this specification within the spirit and scope of the present invention disclosed in this specification. [Example]

[0187] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to these examples. In this specification, "room temperature" generally refers to about 10° C. to about 35° C. "%" refers to percent by weight unless otherwise specified. As used herein, the term "about" can mean ±10%. Nuclear magnetic resonance spectrum ( 1 For the measurement of 1 H NMR, an ALPHA FT-NMR spectrometer (500 MHz) JEOL JNM-A500 type (JEOL) and a deuterated solvent: D2O were used.

[0188] The abbreviations used in the examples are conventional abbreviations well known to those skilled in the art. Some abbreviations are listed below. AL: Alginate (sodium) Alg: Alginate (sodium) ABA: 4-aminobenzaldehyde AL-ABA: benzaldehyde-modified alginate HOBt: 1-hydroxybenzotriazole WSCD / HCl,EDC·HCl:1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride THF: tetrahydrofuran DMSO: dimethyl sulfoxide NaCl: Sodium chloride eq: equivalent Van: Vancomycin PBS: phosphate buffered saline FTSC: Fluorescein-5-thiosemicarbazide FBS: fetal bovine serum DMEM: Dulbecco's modified Eagle's medium Bac:Bacitracin HA: Hyaluronic acid PMX: Pemetrexed CMC: Carboxymethyl cellulose DCC: N,N'-dicyclohexylcarbodiimide CMD, CMDX: carboxymethyl dextran Mw: Weight average molecular weight In the synthesis schemes and reaction formulas shown in the following examples, the polysaccharides (AL, HA, CMC, CMDX) are shown in their free forms (forms having carboxyl groups), but the carboxyl groups (-COOH) in the polysaccharides (AL, HA, CMC, CMDX) shown in the synthesis schemes and reaction formulas are in their ionized states (-COO - ) or salt form (-COOX). For example, in the following Example 1, sodium alginate, which is a sodium salt, is used as AL, and in Synthesis Scheme 1, the carboxyl group (-COOH) in AL is in an ionized state (-COO -or in the sodium salt form (-COONa).

[0189] I. Alginate derivatives 1. AL-ABA [Example I-1] Benzaldehyde-modified alginic acid (AL-ABA) <Synthesis of AL-ABA(1)> [Chemical formula] In Scheme 1 above, for convenience, the reaction in which a modifying group derived from 4-aminobenzaldehyde (ABA) is introduced into the carboxyl group of the guluronic acid unit (the left monosaccharide unit) is shown. However, the modifying group derived from ABA may be introduced into the carboxyl group of the mannuronic acid unit (the right monosaccharide unit). That is, in Scheme 1, AL-ABA has modifying groups derived from ABA introduced into both the carboxyl group of the guluronic acid unit (the left monosaccharide unit) and the carboxyl group of the mannuronic acid unit (the right monosaccharide unit).

[0190] According to the above synthesis scheme, AL-ABA(1) was synthesized from the following steps (1)-(6) by an amidation reaction via carbodiimide. (Synthesis procedure) (1) 200 mg (1 mmol) of sodium alginate (AL) (IL-6G manufactured by Kimika Co., Ltd., viscosity 50-80 mPa·s (1%), weight average molecular weight: Mw = 750,000 - 850,000) was dissolved in 50 mL of pure water and stirred overnight to prepare an alginic acid solution. (2) 242.9 mg (2 mmol, 1 eq) of ABA was dissolved in 20 mL of THF and stirred overnight to prepare an ABA solution. (3) 270.3 mg (2 mmol, 2 eq) of 1-hydroxybenzotriazole (HOBt) was dissolved in 10 mL of DMSO and dropped dropwise into the alginic acid solution prepared in (1) above. Next, 383.7 mg (2 mmol, 2 eq) of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCD / HCl) was dissolved in 5 mL of pure water and dropped dropwise into the alginic acid solution. (4) The ABA solution prepared in (2) above was added dropwise to the alginic acid solution obtained in (3) above. (5) The solution obtained in (4) above was adjusted to pH 5.5, stirred for 3 to 4 hours, and then diluted with 100 mL of pure water. (6) The solution obtained in (5) above was filtered under reduced pressure, dialyzed against NaCl for 2 days and against pure water for 2 days, frozen in liquid nitrogen, and lyophilized for 3 days to obtain AL-ABA (1).

[0191] < 1 H NMR spectroscopy, ultraviolet-visible absorption spectroscopy (UV-vis), and FT-IR spectroscopy Regarding AL-ABA(1), 1 H NMR spectroscopy, ultraviolet-visible absorption spectroscopy (UV-vis), and Fourier transform infrared spectroscopy (FT-IR) were performed, and the results are shown in Figures 1 to 3. 1 In the H NMR spectrum (Figure 1), peaks derived from ABA (peaks b and c derived from the benzene ring and peak d derived from the aldehyde) were observed in the modified AL-ABA. In addition, characteristic peaks derived from ABA (peaks at approximately 235 nm and approximately 330 nm) were observed in the modified AL-ABA in UV-vis (Figure 2). In the FT-IR spectrum (Figure 3), absorption derived from the C=O of the amide, which is the binding point of ABA, (approximately 1740 cm) was observed in the modified AL-ABA. -1 From these results, the synthesis of benzaldehyde-modified alginic acid (AL-ABA) was confirmed, in which the amino group of 4-aminobenzaldehyde was linked to the carboxyl group of alginic acid. 1 From the 1 H NMR spectrum, the ABA modification ratio for the carboxyl group (-COOH) of alginic acid was calculated to be 0.069.

[0192] <Evaluation of cytotoxicity of benzaldehyde-modified alginate (AL-ABA) (WST assay)> (Experimental Procedure) MeT-5A (human mesothelial cell line), NIH / 3T3 (mouse embryonic fibroblast), HUVEC (human umbilical vein endothelial cell), and RAW264.7 cells (mouse macrophage-like cell line) were seeded in 24-well plates and cultured for 24 hours. Then, the medium was replaced with a medium containing AL-ABA and AL dissolved at different concentrations (0.01 mg / mL, 0.1 mg / mL, 1 mg / mL). The cell viability (%) 48 hours after adding the samples was measured using the WST-8 assay (Cell Counting Kit-8, Dojindo). AL-ABA used was AL-ABA(1) synthesized in Example I-1. (Results) The results are shown in Figure 4. AL-ABA showed cell viability equivalent to that of AL. It was confirmed that AL-ABA has low cytotoxicity and high biocompatibility.

[0193] [Example I-2] Benzaldehyde-Modified Alginate (AL-ABA) AL-ABA(2) to AL-ABA(3) were synthesized by the alternative method described below.

[0194] <Synthesis of AL-ABA(2)> 1 g (0.0046 mol) of AL-500 (manufactured by Mita Pharmaceutical Co., Ltd., sodium alginate, viscosity: 400 - 600 mPa·s) was dissolved in 300 ml of distilled water and stirred for 6 - 8 hours. 2.81 g (0.020 mol) of HOBt was dissolved in 10 ml of DMSO and added dropwise to the AL-500 solution. Subsequently, 3.97 g (0.020 mol) of WSCD / HCl was dissolved in 10 ml of distilled water and added dropwise to the AL-500 solution. The AL-500 solution was further stirred for 10 minutes to obtain an alginic acid solution. 0.84 g (0.0039 mol) of 4-aminobenzaldehyde (ABA) in 10 ml of DMSO was added dropwise to the alginic acid solution. The pH of the reaction mixture was maintained at pH 7.5 with 1N NaOH and stirred at room temperature for 16 - 20 hours. The reaction mixture was transferred to a 50 ml centrifuge tube and centrifuged at 3000 rpm for 10 minutes at room temperature, and the supernatant was collected. Then, using a dialysis tube (MWCO: 6 - 8 kDa) (Spectra / Pro (registered trademark)), the solution was dialyzed against deionized water for 72 hours and then lyophilized to obtain AL-ABA(2).

[0195] <Synthesis of AL-ABA(3)> Except that the amount of HOBt used was changed to 1.04 g (0.007 mol), the amount of WSCD / HCl used was changed to 1.98 g (0.010 mol), and the pH of the reaction mixture was changed to 5.5, AL-ABA(3) was obtained in the same manner as AL-ABA(2).

[0196] < 1 <H NMR Spectrum Measurement and FT-IR Spectrum Measurement> For AL-ABA(2) - AL-ABA(3), 1 H NMR spectrum measurement and FT-IR spectrum measurement were performed (not shown). From these results, the synthesis of ABA-modified alginic acid (AL-ABA) in which the amino group of ABA and the carboxyl group of alginic acid were bonded was confirmed. Also, 1The ABA modification ratios of the carboxyl groups (-COOH) of alginic acid in AL-ABA(2) to AL-ABA(3) were calculated from the H NMR spectra. The results are shown in Table 1 below, along with the reaction conditions for AL-ABA(2) to AL-ABA(3).

[0197] <Quantitative determination of aldehydes using colorimetric aldehyde assay> The aldehydes in AL-ABA(2) and AL-ABA(3) were quantified using a colorimetric aldehyde assay kit, Blue (MAK140), and the ABA modification rate relative to the carboxyl group (-COOH) of alginic acid was calculated. Assay Procedure (1) Dilute 10 μl of the 10 mM standard solution with 990 μl of assay buffer to prepare a 100 μM standard solution. Further, serially dilute the 100 μM standard solution two-fold with assay buffer. Add 50 μL of the diluted standard solution to a 96-well plate to generate 0 (blank), 1.56, 3.125, 6.25, 12.5, 25, 50, and 100 μM standards. (2) Add 50 μL of Master Reaction Mix to each well. Mix well using a horizontal shaker or by pipetting, and incubate the reaction at room temperature for 20–30 minutes. Protect the plate from light during incubation. (3) Add 50 μL of Blue Enhancer to each well. (4) The reaction is incubated for an additional 20 minutes at room temperature, protected from light. (5) After the incubation period, measure the absorbance at 620-660 nm. (result) The ABA modification rate of the carboxyl group (-COOH) of alginic acid, calculated from the amount of aldehyde, was 0.56 or 0.64 for AL-ABA(2) and 0.33 for AL-ABA(3). The results are shown in Table 1 below. [Table 1] In Table 1, DS stands for degree of substitution, and corresponds to the percentage of ABA modification rate (ABA modification rate x 100). 1 1 H NMR spectrum, and DS Colorimetric is a value calculated from a colorimetric aldehyde assay. AL-ABA(2) *1 and *2 are AL-ABA prepared in different batches. In the following examples, *1 may be referred to as AL-ABA(2)(Low DS) and *2 as AL-ABA(2)(High DS).

[0198] 2.AL-AAP, AL-ADFBA, AL-APCA, AL-ANA Alginic acid modified with 4-aminoacetophenone (AAP), 4-amino-2,6-difluorobenzaldehyde (ADFBA), 2-amino-3-pyridinecarboxaldehyde (APCA), or 6-aminonicotinaldehyde (ANA) was obtained in the same manner as in Example I-1. For convenience, the following schemes show the introduction of modifying groups derived from AAP, ADFBA, APCA, or ANA to the carboxyl group of the guluronic acid unit (the monosaccharide unit on the left). However, these modifying groups can also be introduced to the carboxyl group of the mannuronic acid unit (the monosaccharide unit on the right). That is, in AL-AAP, AL-ADFBA, AL-APCA, and AL-ANA, modifying groups derived from AAP, ADFBA, APCA, or ANA are introduced to both the carboxyl group of the guluronic acid unit (the monosaccharide unit on the left) and the carboxyl group of the mannuronic acid unit (the monosaccharide unit on the right).

[0199] [Example I-3] AAP-modified alginate (AL-AAP) [ka]

[0200] 0.5 g (0.0023 mol) of AL-500 (Mochida Pharmaceutical Co., Ltd., sodium alginate, viscosity: 400–600 mPa s) was dissolved in 100 ml of distilled water and stirred for 6–8 hours. 1.4 g (0.010 mol) of HOBt was dissolved in 10 ml of DMSO and added dropwise to the AL-500 solution. 1.97 g (0.010 mol) of WSCD / HCl was then dissolved in 10 ml of distilled water and added dropwise to the AL-500 solution. The AL-500 solution was stirred for another 10 minutes to obtain an alginate solution. 0.310 g (0.0023 mol) of 4'-aminoacetophenone (AAP) in 10 ml of DMSO was added dropwise to the alginate solution. The pH of the reaction mixture was maintained at 5.5 with 1 N NaOH and stirred at room temperature for 16–20 hours. The reaction mixture was transferred to a 50 ml centrifuge tube and centrifuged at 3,000 rpm for 10 minutes at room temperature, and the supernatant was collected. The solution was then dialyzed against deionized water for 72 hours using a dialysis tube (MWCO: 6-8 kDa) (Spectra / Pro®) and then lyophilized to obtain AL-AAP.

[0201] < 1 H NMR spectrum measurement, FT-IR spectrum measurement About AL-AAP 1 H NMR and FT-IR spectroscopy were performed. The results are shown in Figures 5 and 6. These results confirmed the synthesis of AAP-modified alginic acid (AL-AAP), in which the amino group of AAP was linked to the carboxyl group of alginic acid. Also, 1 From the 1 H NMR spectrum, the AAP modification ratio for the carboxyl group (-COOH) of alginic acid was calculated to be 0.31. <Quantitative determination of aldehydes using colorimetric aldehyde assay> The AAP modification rate for the carboxyl group (-COOH) of alginic acid calculated from the amount of aldehyde measured using a colorimetric aldehyde assay kit, Blue (MAK140), was 0.34.

[0202] [Example I-4] ADFBA-modified alginate (AL-ADFBA) [ka]

[0203] An alginate solution was prepared in the same manner as in Example I-3. 0.361 g (0.0023 mol) of 4'-4-amino-2,6-difluorobenzaldehyde (ADFBA) in 10 ml of DMSO was added dropwise to the alginate solution. The pH of the reaction mixture was maintained at 5.5 with 1N NaOH and stirred at room temperature for 16 to 20 hours. The reaction mixture was transferred to a 50 ml centrifuge tube and centrifuged at 3,000 rpm for 10 minutes at room temperature. The supernatant was collected. The solution was then dialyzed against deionized water for 72 hours using a dialysis tube (MWCO: 6-8 kDa) (Spectra / Pro®) and lyophilized to obtain AL-ADFBA.

[0204] < 1 H NMR spectrum measurement, FT-IR spectrum measurement About AL-ADFBA 1 H NMR spectroscopy and FT-IR spectroscopy were performed. The results are shown in Figures 7 and 8. These results confirmed the synthesis of ADFBA-modified alginic acid (AL-ADFBA), in which the amino group of ADFBA was bonded to the carboxyl group of alginic acid. Also, 1 From the 1 H NMR spectrum, the ADFBA modification ratio for the carboxyl group (-COOH) of alginic acid was calculated to be 0.12.

[0205] <Quantitative determination of aldehydes using colorimetric aldehyde assay> The ADFBA modification rate relative to the carboxyl group (-COOH) of alginic acid calculated from the amount of aldehyde measured using a colorimetric aldehyde assay kit, Blue (MAK140), was 0.18.

[0206] [Example I-5] APCA-modified alginate (AL-APCA) [ka]

[0207] An alginate solution was prepared in the same manner as in Example I-3. 0.28 g (0.0023 mol) of 2-amino-3-pyridinecarboxaldehyde (APCA) in 10 ml of DMSO was added dropwise to the alginate solution. The pH of the reaction mixture was maintained at 5.5 with 1N NaOH and stirred at room temperature for 16 to 20 hours. The reaction mixture was transferred to a 50 ml centrifuge tube and centrifuged at 3,000 rpm for 10 minutes at room temperature. The supernatant was collected. The solution was then dialyzed against deionized water for 72 hours using a dialysis tube (MWCO: 6-8 kDa) (Spectra / Pro®) and lyophilized to obtain AL-APCA.

[0208] < 1 H NMR spectrum measurement, FT-IR spectrum measurement About AL-APCA 1 H NMR spectroscopy and FT-IR spectroscopy were performed. The results are shown in Figures 9 and 10. These results confirmed the synthesis of APCA-modified alginic acid (AL-APCA), in which the amino group of APCA was bonded to the carboxyl group of alginic acid. Also, 1 From the 1 H NMR spectrum, the APCA modification ratio for the carboxyl group (-COOH) of alginic acid was calculated to be 0.41.

[0209] <Quantitative determination of aldehydes using colorimetric aldehyde assay> The APCA modification rate for the carboxyl group (-COOH) of alginic acid calculated from the amount of aldehyde measured using a colorimetric aldehyde assay kit, Blue (MAK140), was 0.33.

[0210] [Example I-6] ANA-modified alginate (AL-ANA) [ka]

[0211] An alginate solution was prepared in the same manner as in Example I-3. 0.280 g (0.0023 mol) of 6-aminonicotinaldehyde (ANA) in 10 ml of DMSO was added dropwise to the alginate solution. The pH of the final reaction mixture was maintained at 5.5 with 1N NaOH and stirred at room temperature for 16-20 hours. The reaction mixture was transferred to a 50 ml centrifuge tube and centrifuged at 3000 rpm for 10 minutes at room temperature. The supernatant was collected. The solution was then dialyzed against deionized water for 72 hours using a dialysis tube (MWCO: 6-8 kDa) (Spectra / Pro®) and lyophilized to obtain AL-ANA.

[0212] < 1 H NMR spectrum measurement, FT-IR spectrum measurement About AL-ANA 1 H NMR spectroscopy and FT-IR spectroscopy were performed. The results are shown in Figures 11 and 12. These results confirmed the synthesis of ANA-modified alginic acid (AL-ANA), in which the amino group of ANA was bonded to the carboxyl group of alginic acid. Also, 1 From the 1 H NMR spectrum, the ANA modification ratio for the carboxyl group (-COOH) of alginic acid was calculated to be 0.17.

[0213] <Quantitative determination of aldehydes using colorimetric aldehyde assay> The ANA modification rate for the carboxyl group (-COOH) of alginic acid calculated from the amount of aldehyde measured using a colorimetric aldehyde assay kit, Blue (MAK140), was 0.26.

[0214] In the table below, 1 The modification rates of the modifying group (A) determined from 1 H NMR spectra (NMR) and colorimetric aldehyde assays (Colorimetric) are summarized. [Table 2]

[0215] 3. AL-ABA drug conjugates [Example I-7] Benzaldehyde-modified alginic acid-vancomycin conjugate (AL-ABA-Van) [Synthesis of AL-ABA-Van] [Chemical formula]

[0216] Vancomycin (Van) is an antibiotic having a primary amino group. According to the above synthesis scheme, a conjugate (AL-ABA-Van) in which Van is bound to AL-ABA was synthesized by the Schiff base reaction between the aldehyde group of AL-ABA and the primary amino group of Van according to the following procedure. Specifically, 50 mg of AL-ABA (1) obtained in Example I-1 was dissolved in 70 mL of pure water and stirred for 1 hour or more. 60 mg of vancomycin hydrochloride was dissolved in 60 mL of pure water and added to the AL-ABA solution. 0.1 M NaOH was added dropwise to adjust the pH to around 7.0, and the reaction was carried out with stirring at room temperature overnight under light-shielded conditions. After dilution with 150 mL of pure water, dialysis was performed with pure water for 2 days (apparatus: Spectra / Pro (registered trademark) 1 Dialysis Membrane Standard RC Tubing MWCO: 6 - 8 kD), and freeze-drying was carried out for 3 days to recover 86.8 mg of white AL-ABA-Van. (Yield: 78.9%)

[0217] 1 1H NMR spectrum measurement, ultraviolet-visible absorption spectrum (UV-vis) measurement, and FT-IR spectrum measurement For the obtained AL-ABA-Van, 1 1H NMR spectrum measurement, ultraviolet-visible absorption spectrum (UV-vis) measurement, and FT-IR spectrum measurement were performed. The results are shown in Figures 13 to 15. 1 ​In the H NMR spectrum (Fig. 13), peaks derived from ABA and vancomycin (Van) were observed in AL-ABA-Van. Furthermore, in the UV-vis spectrum (Fig. 14), peaks derived from ABA and vancomycin (Van) (indicated by arrows in the figure; shifts to lower wavelengths were observed) were observed in AL-ABA-Van. In the FT-IR spectrum (Fig. 15), absorption (approximately 1740 cm) derived from the C=O of the amide bond between AL and ABA was observed in AL-ABA-Van. -1 A peak of 1000 s was observed. These results confirmed that a conjugate between AL-ABA and Van was formed. 1 The conjugation rate of Van as a drug to the carboxyl groups (-COOH) of alginic acid was 5.9%, indicating that 82.4% of the ABA in AL-ABA reacted with Van. These results demonstrate that conjugation is completed instantly by simply mixing AL-ABA and Van in an aqueous solvent. Furthermore, the reaction has the advantage of producing only water, eliminating the need for subsequent purification. This reaction is applicable to a variety of primary amine-containing drugs, suggesting that AL-ABA may be a convenient and versatile platform for developing alginate-drug conjugates.

[0218] <Release experiment of vancomycin from benzaldehyde-modified alginate-vancomycin conjugate (AL-ABA-Van)> (Experimental Procedure) The release behavior of Vancomycin from AL-ABA-Van solutions at different pH levels was investigated using dialysis membranes (pore size: 50 kDa). Specifically, 1 mL of the AL-ABA-Van solution (prepared in Example I-7 above, dissolved in purified water at 3 mg / mL) was injected into a 9 cm-long dialysis membrane, and both ends of the membrane were tied with string. This was placed in an 80 mL security container, and 79 mL of PBS solution was added. The solution was then stirred at maximum speed using a 1 cm diameter stirrer tip in a 37°C incubator. 1.0 mL of the solution was sampled from the security container at predetermined time points (1, 2, 3, 4, 6, 8, 12, 24, and 48 hours). UV-vis spectroscopy of the sampled solutions was performed, and the drug concentration in the solution was quantified from the absorbance, and the release rate of Vancomycin (Vancomycin Release %) was calculated. The above measurement was carried out for each of the PBS solutions with pH values ​​of 5.0, 6.0, and 7.4 (n=4 for each pH sample).

[0219] For comparison, as a control experiment, the above release experiment was performed using a mixed solution of sodium alginate (AL) (IL-6G, manufactured by Kimika Co., Ltd., viscosity 50-80 mPa·s (1%)) and 8.6 mg / mL vancomycin (Van) dissolved in pure water (AL+Van), instead of the AL-ABA-Van solution, and a solution of 8.6 mg / mL vancomycin (Van) dissolved in pure water (Van only).

[0220] (result) The results are shown in Figure 16. Figure 16A shows the release profile of Van from the AL-ABA-Van solution (cumulative release rate of Van, %), Figure 16B shows the release profile of Van from the AL+Van mixed solution (cumulative release rate of Van, %), and Figure 16C shows the release profile of Van from the Van solution (cumulative release rate of Van, %). After 10 hours, over 80% of Van was released from the AL-ABA-Van solution at pH 5.0, whereas just under 50% remained unreleased at pH 7.4. This result indicates that Van is released more rapidly with decreasing pH. This is likely due to the fact that the imine bond between AL-ABA and Van is easily dissociated at low pH. On the other hand, in the AL+Van solution and the Van solution (control experiment), most of the Van was released within the first 10 hours. Furthermore, no difference in release behavior was observed depending on the pH. These results suggest that the AL-ABA-Van conjugate enables sustained release of Van compared with the control groups (AL+Van, Van only), and further demonstrates that the drug is selectively released at low pH, allowing the Van release rate to be modified in a pH-dependent manner. [ka]

[0221] [Example I-8] FTSC-loaded AL-ABA microcapsules (AL-ABA-FTSC capsules) (Capsule production) [ka] Fluorescein-5-thiosemicarbazide (FTSC) is a fluorescent dye containing a primary amino group. FTSC-loaded AL-ABA capsules were prepared. Specifically, AL-ABA (1) prepared in Example I-1 was dissolved in pure water to prepare a 2 wt% AL-ABA solution. This was added dropwise to a 50 mM CaCl2 aqueous solution to obtain capsules (AL-ABA capsules). FTSC was dissolved in DMEM (Dulbecco's modified Eagle's medium) containing physiological saline and 10% FBS (fetal bovine serum), and the obtained AL-ABA capsules were immersed in this solution to load FTSC into the capsules by reactive diffusion. For comparison, as a control experiment, capsules (AL capsules) were obtained in the same manner as above, except that AL-ABA was replaced with sodium alginate (AL) (IL-6G, manufactured by Kimika Co., Ltd., viscosity 50-80 mPa s (1%)). The obtained AL-capsules were similarly immersed in saline containing FTSC to confirm whether FTSC was loaded.

[0222] (result) The AL and AL-ABA capsules were immersed in the FTSC solution and observed using a confocal microscope. The resulting transmitted image, fluorescent image (Amine-Fluorescein), and merged image are shown in Figure 17. From Figure 17, it can be seen that when AL-ABA was used, microscale capsules were produced, just as with AL. Furthermore, while almost no fluorescence derived from FTSC was detected in the AL capsules, fluorescence derived from FTSC was clearly detected in the AL-ABA capsules, indicating that FTSC was immobilized in the capsules in situ. This indicates that benzaldehyde-modified alginate (AL-ABA), like alginate (AL), can effectively absorb Ca. 2+ It was confirmed that the capsules could be easily prepared while retaining the crosslinking ability, and that the amine compounds could be loaded simply by immersing them.

[0223] [Example I-9] Van-loaded AL-ABA microcapsules (AL-ABA-Van capsules) (Capsule production) AL-ABA (1) prepared in Example I-1 was dissolved in pure water to prepare a 2 wt% AL-ABA solution. 15 mg of vancomycin (Van) was added to this solution to obtain a mixed solution of AL-ABA and Van. This mixed solution of AL-ABA and Van was added dropwise to a 50 mM aqueous solution of CaCl to obtain capsules (AL-ABA-Van capsules). For comparison, as a control experiment, capsules (AL-Van capsules) were obtained in the same manner as above, except that AL-ABA was replaced with sodium alginate (AL) (IL-6G, manufactured by Kimika Co., Ltd., viscosity 50–80 mPa·s (1%)).

[0224] (Result) Even when using a mixed solution of AL-ABA and Van, benzaldehyde-modified alginic acid (AL-ABA) retains the ability to crosslink with Ca 2+ and it was confirmed that, similar to alginic acid (AL), capsules can be easily prepared.

[0225] ><Van Release Experiment from AL-ABA-Van Capsules> (Experimental Procedure) The AL-ABA-Van capsules prepared above were collected and added to a 10 mM CaCl2 solution in 1 mL of physiological saline. The external solution (sustained release solution) was collected at predetermined time points (3, 24, and 48 hours), and the total amount of the external solution was exchanged with a 10 mM CaCl2 solution in fresh 1 mL of physiological saline. UV-vis spectrum measurement was performed on the external solution (sustained release solution) collected at predetermined time points, the drug concentration in the solution was quantified from the absorbance, and the release rate of the drug Van (Vancomycin Release %) was calculated.

[0226] [[ID=二十]] For comparison, as a control experiment, the above release experiment was performed using AL-Van capsules instead of AL-ABA-Van capsules.

[0227] (Result) The results are shown in Fig. 18. Fig. 18A shows the cumulative release rate (%) of Van from each capsule, and Fig. 1 B shows the transition of the release rate (%) of Van at each time point from each capsule. From Fig. 18A and Fig. 18B, it was confirmed that both AL-Van capsules and AL-ABA-Van capsules gradually released vancomycin, and about 60% of Van was released at the 48-hour time point. There was no significant difference between the two in the cumulative amount of sustained release (Fig. 18A), which is probably because the rate of release of Van from the microcapsules by diffusion was slower than the rate of release of Van due to the dissociation of the Schiff base with ABA, making the effect of the Schiff base-mediated loading less visible.

[0228] <Bacterial growth inhibition experiment> To verify the antibacterial effect of vancomycin-loaded AL-ABA microcapsules, the release solutions collected at each time point in the Van release experiment conducted above were used to evaluate the growth inhibitory effect of each release solution in a halo test using Staphylococcus aureus. The specific experimental procedure is as follows. (Experimental Procedure) (1) Inoculation of Staphylococcus aureus onto agar medium 1.1 15 g of agar was added to 1 L of Mueller-Hinton medium (BD), and the mixture was dissolved and sterilized by autoclaving. 15 mL of the mixture was then added to each 100 mm dish and cooled at room temperature to prepare an agar medium. 1.2 700 μl of Mueller-Hinton medium was added to a 1.5 mL microtube (Azunol sterilized tube), and the Staphylococcus aureus stock was inoculated into the medium using a toothpick, followed by incubation overnight in an incubator at 37°C. 1.3 Bacterial solution 1.0 x 10 7 The cells were diluted to 100 μl / ml, and 100 μl of the diluted solution was added to the agar medium and spread evenly with a Conrad rod. (2) Impregnation of filter paper with the slow-release solution and application to the agar medium 2.1 1 mL of each sustained-release solution collected in the release experiment was filter-sterilized using a syringe filter (0.22 μm). 2.2 Filter paper (AS ONE: MFWG4780) was cut into a circle with a diameter of 16 mm. 2.3 Place the cut filter paper in the center of the agar plate inoculated with Staphylococcus aureus. 2.4 40 μl of the sustained release solution was dropped evenly onto the filter paper and allowed to soak. 2.5 The dish was transferred to an incubator and cultured at 37°C for 24 hours. 2.6 The dish was removed from the incubator, and the state of the growing bacteria was photographed with a camera. The area of ​​the growth-inhibited area was measured by image analysis using Image J (provided by NIH).

[0229] (result) The results of observing the growth of Staphylococcus aureus on agar medium under each condition are shown in Fig. 19. For both Van-loaded AL capsules (AL-Van) and Van-loaded AL-ABA capsules (AL-ABA-Van), circular regions where no bacteria grew were observed around the filter paper, confirming a growth inhibitory effect. Furthermore, the area where growth was inhibited was calculated by image analysis, and the results of comparison for each condition are shown in Fig. 20. In the case of AL capsules (AL-Van), the growth inhibition area decreased as time passed at 3 hours, 24 hours, and 48 hours after the start of sustained release. In contrast, for AL-ABA capsules (AL-ABA-Van), this decrease was suppressed, showing a tendency that the difference in area between 3 hours and 48 hours was small. From these results, it was suggested that in AL-Van capsules, a large amount of vancomycin was released initially, and the release amount decreased with time, resulting in a decrease in the antibacterial effect. In contrast, in AL-ABA-Van capsules, the initial release of vancomycin was suppressed, and it was possible that the effective concentration was maintained over a longer period.

[0230] [Example I-10] Benzaldehyde-modified alginic acid-bacitracin conjugate (AL-ABA-Bac) <Synthesis of AL-ABA-Bac>

Chemical formula

[0231] <Ultraviolet-visible absorption spectrum (UV-vis) measurement and FT-IR spectrum measurement> The obtained AL-ABA-Bac was subjected to ultraviolet-visible absorption spectroscopy (UV-vis) and FT-IR spectroscopy, and the results are shown in Figures 21 and 22. In the UV-vis spectrum (Fig. 21), peaks derived from bacitracin (Bac) and ABA (indicated by arrows in the figure; shifts to lower wavelengths were observed) were also observed in AL-ABA-Bac. In the FT-IR spectrum (Fig. 16), absorption (approximately 1740 cm) derived from the C=O of the amide, which is the bonding point between AL and ABA, was observed in AL-ABA-Bac. -1 A peak of 1000 s was observed. These results confirmed that a conjugate between AL-ABA and Bac was formed.

[0232] The recovered AL-ABA-Bac formed a hydrogel. It is thought that the two amino groups in Bac formed bonds with the aldehyde groups in ABA, causing Bac itself to function as a cross-linking agent (gelling agent) and form a gel. This gel structure is expected to enable sustained release over a long period of time. In addition to bacitracin (Bac), there are many polypeptide antibacterial drugs that have two or more amino groups (for example, daptomycin, colistin, ercatonin, etc.). In these drugs, the drug itself is expected to function as a cross-linking agent and form a gel structure.

[0233] [Example I-11] Benzaldehyde-modified alginic acid-dopamine conjugate (AL-ABA-DOPA) <Synthesis of AL-ABA-DOPA> [Chemical formula] Dopamine (DOPA) is a neurotransmitter that plays an important role in the brain and the body and is an organic chemical substance belonging to the catecholamine and phenethylamine families. DOPA has a primary amino group. The benzaldehyde-modified alginic acid-dopamine conjugate (AL-ABA-DOPA) was synthesized according to the following procedure. Dopamine (DOPA) is a neurotransmitter that plays an important role in the brain and the body and has a primary amino group. The benzaldehyde-modified alginic acid-dopamine conjugate (AL-ABA-DOPA) was synthesized according to the following procedure.

[0234] 0.25 g (0.00115 mol) of AL-ABA(2) (Low DS; modification rate (Colorimetric): 0.56) prepared in Example I-2 was dissolved in 100 ml of PBS (pH 7.4) and stirred for 6 to 8 hours. 0.43 g (0.0023 mol) of dopamine hydrochloride (Sigma Aldrich) was dissolved in 10 ml of PBS (pH: 7.4) and added dropwise to the AL-ABA solution. The solution was dialyzed against deionized water for 72 hours using a dialysis tube (MWCO: 6-8 kDa) (Spectra / Pro (registered trademark)) to obtain an AL-ABA-DOPA solution, which was then lyophilized.

[0235] [[ID=​​​​​​H NMR spectroscopy and FT-IR spectroscopy confirmed the formation of a conjugate of AL-ABA and dopamine. The results are shown in Figures 23 and 24. 1 From the H NMR spectrum, the conjugation rate of DOPA as a drug to ABA in AL-ABA was 47% (DOPA modification rate to ABA: 0.47). 1 Because the modification rate (Colorimetric) calculated by the colorimetric aldehyde assay is a more accurate value than the modification rate (NMR) calculated by the H NMR spectrum, the conjugation rate of DOPA was calculated from the modification rate (Colorimetric) value obtained by the colorimetric aldehyde assay. In the following examples, when the modification rate (Colorimetric) was measured, the conjugation rate of the drug to ABA was calculated based on the modification rate (Colorimetric) value.

[0236] [Example I-12] Sponge carrying AL-ABA-DOPA (AL-ABA-DOPA sponge) (Production procedure) AL-ABA (modification ratio (NMR): 0.60) was synthesized under the same conditions as for AL-ABA (2) in Example I-2. AL-ABA was dissolved in PBS (pH 7.4) to prepare a 0.5% AL-ABA solution. Two times the molar equivalent of ABA, dopamine (DOPA), was added, and the solution was stirred at room temperature for 16 to 20 minutes. The solution was then dialyzed against pure water for 72 hours and subsequently lyophilized to obtain AL-ABA-DOPA. AL-ABA-DOPA was dissolved in PBS to obtain a 1.0 wt% AL-ABA-DOPA solution. The AL-ABA-DOPA solution was mixed with 1.0 ml of 10 mM CaCl2 aqueous solution in a 35 mm Petri dish. After hydrogel formation, the mixture was frozen at -20°C overnight and then lyophilized. AL-ABA-DOPA sponges were obtained by lyophilization. Cross-linking with calcium ions enabled the formation of hydrogels and sponges of the polysaccharide derivative-drug conjugate AL-ABA-DOPA.

[0237] [Example I-13] Benzaldehyde-modified alginic acid-serotonin conjugate (AL-ABA-Serotonin) <Synthesis of AL-ABA-Serotonin> [Chemical formula] Serotonin (5-hydroxytryptamine) is a monoamine neurotransmitter and has a primary amino group. The benzaldehyde-modified alginic acid-serotonin conjugate (AL-ABA-Serotonin) was synthesized according to the following procedure. 0.25 g (0.00115 mol) of AL-ABA(2) (Low DS, modification rate (Colorimetric): 0.56) prepared in Example I-2 was dissolved in 100 ml of PBS (pH 7.4) and stirred for 6 - 8 hours. 0.489 g (0.0023 mol) of serotonin hydrochloride (Sigma Aldrich) was dissolved in 10 ml of PBS (pH: 7.4) and added dropwise to the AL-ABA solution. The solution was dialyzed against deionized water for 72 hours using a dialysis tube (MWCO: 6 - 8 kDa) (Spectra / Pro (registered trademark)) and then lyophilized.

[0238] < 1 1H NMR spectrum measurement and FT-IR spectrum measurement For AL-ABA-Serotonin, 1 1H NMR spectrum measurement and FT-IR spectrum measurement were performed to confirm that the conjugate of AL-ABA and serotonin was formed. The results are shown in Figures 25 - 26. 1 From the 1H NMR spectrum, the conjugation rate of serotonin as a drug to ABA in AL-ABA was 46% (serotonin modification rate to ABA: 0.46).

[0239] [Example I-14] Benzaldehyde-modified alginic acid-celecoxib conjugate (AL-ABA-Celecoxib) <Synthesis of AL-ABA-Celecoxib>

Chemical formula

[0240] < 1 1H NMR spectrum measurement and FT-IR spectrum measurement> For AL-ABA-Celecoxib, 1 1H NMR spectrum measurement and FT-IR spectrum measurement (not shown) were performed to confirm that the conjugate of AL-ABA and celecoxib was formed. 1 The results of 1H NMR spectrum measurement are shown in Figure 27. 1 From the 1H NMR spectrum, the conjugation rate of celecoxib as a drug to ABA of AL-ABA was 11% (celecoxib modification rate to ABA: 0.11).

[0241] [Example I-15] Benzaldehyde-modified alginic acid-HGF aptamer conjugate (AL-ABA-Apt) For the purpose of developing a novel anti-adhesion hydrogel having a sustained release function of HGF aptamer (Lot: 1649399-3, manufactured by Eurofins Genomics K.K.) with a promoting effect on mesothelial cell proliferation, synthesis of benzaldehyde-modified alginic acid-HGF aptamer conjugate (AL-ABA-Apt) and a sustained release experiment of HGF aptamer from AL-ABA-Apt were conducted. The experimental procedure is shown in Fig. 28.

[0242] (Experiment) <Synthesis of AL-ABA-Apt> Under the same conditions as AL-ABA (1) in Example I-1, AL-ABA (modification rate (NMR) 4.0%) was synthesized. AL-ABA was dissolved in phosphate buffered saline (PBS, pH = 7.4) to obtain a 0.4 wt% AL-ABA solution. An HGF aptamer containing 1.0 equivalent amino groups with respect to the aldehyde groups of AL-ABA was added to the AL-ABA solution and stirred for 1 hour. Thereafter, dialysis was performed with pure water for 3 days to remove unreacted HGF aptamer, and then lyophilized. Ultraviolet-visible absorption spectrum (UV-vis) measurements were performed for each of AL-ABA-HGF aptamer conjugate (AL-ABA-Apt) and HGF aptamer, and the presence or absence of loading of HGF aptamer (Apt) by AL-ABA was evaluated from the peak value at 256 nm of the UV-vis spectrum.

[0243] <Release experiment of Apt from AL-ABA-Apt> 1.0 wt% of AL-ABA was dissolved in PBS (pH = 7.4). Subsequently, an HGF aptamer containing 1.0 equivalent of amino groups relative to the aldehyde groups of AL-ABA was added to the AL-ABA aqueous solution. By stirring this for 1 hour, an AL-ABA-HGF aptamer conjugate (AL-ABA-Apt) was synthesized. Then, the AL-ABA-HGF aptamer was placed in a dialysis membrane (MWCO = 50 kDa) and stirred in PBS (pH = 7.4). At each time point from the start of stirring, 1 mL of the external solution was collected and the cumulative release rate (%) of the HGF aptamer was calculated by UV-vis measurement. As a control experiment, a mixture of normal alginic acid and the HGF aptamer (ALG-Apt) and the HGF aptamer alone (Apt) were each dissolved in PBS, placed in a dialysis membrane, and the same sustained release experiment was conducted to compare the release rates.

[0244] <AL-ABA-HGF aptamer / Ca 2+ Preparation of gel> The AL-ABA-HGF aptamer (AL-ABA-APt) synthesized above was dissolved in pure water at a concentration of 1.0 wt% to obtain an AL-ABA-HGF aptamer aqueous solution. The presence or absence of gelation was examined by mixing 300 μL each of the AL-ABA-HGF aptamer aqueous solution and a 100 mM CaCl2 aqueous solution in a microtube.

[0245] (Results) <Synthesis of AL-ABA-HGF aptamer> In the UV-vis spectrum of the synthesized AL-ABA-HGF aptamer (not shown), a peak derived from the HGF aptamer was confirmed near 256 nm, indicating that the synthesis of the AL-ABA-HGF aptamer (AL-ABA-Apt) was successful.

[0246] <Sustained release of HGF aptamer from AL-ABA-HGF aptamer> The results of the sustained release experiment of the HGF aptamer (Apt) from AL-ABA-HGF aptamer (AL-ABA-Apt), a mixture of alginate and HGF aptamer (ALG-Apt), and the HGF aptamer alone (Apt) are shown in Figure 29. The release of HGF aptamer (Apt) was delayed in the AL-ABA-Apt group compared to the ALG-Apt and Apt groups. This indicates that conjugation of the HGF aptamer with AL-ABA can achieve sustained release of the HGF aptamer under physiological pH conditions.

[0247] <AL-ABA-HGFアプタマー / Ca 2+ Preparation of gel Ca 2+ The AL-ABA-HGF aptamer gelled instantly upon crosslinking, and the gel was successfully prepared. As described later in Examples I-16 and I-17, AL-ABA acts as a Ca 2+ It was confirmed that cross-linking resulted in instantaneous gelation, suggesting that conjugation of the HGF aptamer to AL-ABA had little effect on the gelation ability of AL-ABA. We have successfully synthesized the AL-ABA-HGF aptamer carrying the HGF aptamer, which has the effect of promoting mesothelial cell proliferation, and demonstrated that the HGF aptamer can be sustainedly released from the AL-ABA-HGF aptamer. 2+ We successfully fabricated an AL-ABA-HGF aptamer (AL-ABA-Apt) gel by crosslinking, suggesting its potential use as an adhesion barrier.

[0248] 4. AL-ABA sponge and hydrogel [Example I-16] Calcium-crosslinked benzaldehyde-modified alginate sponge (AL-ABA sponge) (Production procedure) A total of 20 mg of sodium alginate (AL) (IL-6G, manufactured by Kimika Co., Ltd., viscosity 50-80 mPa·s (1%)) and AL-ABA (1) obtained in Example I-1 were dissolved in 2 ml of purified water at weight ratios of 75:25, 50:50, and 25:75, respectively, to prepare AL-ABA aqueous solutions. This was mixed with 2 ml of 10 mM CaCl2 aqueous solution in a dish to obtain an AL-ABA hydrogel. The solution was then frozen overnight in a -20°C freezer and then lyophilized for 3 days in a freeze dryer to obtain an AL-ABA sponge. For comparison, as a control experiment, a sponge (AL sponge) was obtained in the same manner as above, except that AL-ABA was replaced with AL alone (AL 100% by weight).

[0249] (result) Optical photographs of the AL-ABA sponge obtained in the above example and the AL sponge as a control are shown in FIG. 30, and SEM photographs are shown in FIG. It was confirmed that a benzaldehyde-modified alginate sponge (AL-ABA sponge) can be produced using the same simple method as AL. Figure 31 confirms that the AL-ABA sponge has a porous structure similar to that of the AL sponge. It is believed that the unmodified carboxyl groups of AL and AL-ABA are crosslinked via calcium (Ca) ions to form a crosslinked structure. By mixing AL and AL-ABA, it is possible to control the content of benzaldehyde (ABA), the modifying group contained in the sponge obtained, as well as the crosslink density.

[0250] [Example I-17] Calcium-crosslinked benzaldehyde-modified alginate hydrogel (Ca-crosslinked AL-ABA hydrogel) A calcium-crosslinked hydrogel (calcium-crosslinked AL-ABA hydrogel) was prepared by crosslinking AL-ABA(2) (High DS, colorimetric modification ratio: 0.64) prepared in Example I-2 with calcium. Specifically, 20 mg of AL-ABA(2) (High DS, colorimetric modification ratio: 0.64) prepared in Example I-2 was dissolved in 1 ml of pure water to prepare an AL-ABA aqueous solution. This was mixed with 1 ml of a 100 mM CaCl2 aqueous solution on a dish to obtain a calcium-crosslinked AL-ABA hydrogel.

[0251] <In vitro swelling and decomposition test> The Ca-crosslinked AL-ABA hydrogel obtained above was swollen in phosphate-buffered saline (PBS) at pH 7.4. Specifically, four freshly prepared hydrogel samples were prepared, weighed, and placed in 25 ml of PBS at 37 °C for 72 hours. After 72 hours of swelling, the degradation of each sample was monitored. The weight of the hydrogel samples was measured every 24 hours for two weeks. The PBS was replaced weekly with fresh PBS equilibrated at 37 °C. The fully swollen Ca-crosslinked AL-ABA hydrogels were freeze-dried and their morphology was observed using a SEM.

[0252] (result) The swelling and decomposition profile (weight change of the hydrogel) of the Ca-crosslinked AL-ABA hydrogel is shown in Figure 32. The weight change value is the average value of four samples. Figure 33 shows an SEM photograph (magnification ×500; 24 hours after the start of swelling) of a cross section cut with a scalpel from a sponge obtained by freeze-drying the Ca-crosslinked AL-ABA hydrogel. The porous structure of the dried hydrogel can be confirmed.

[0253] 5. Hydrogel containing AL-ABA and amino group-containing polymer [Example I-18] Hydrogel of AL-ABA and DPI (AL-ABA-DPI) [ka] AL-ABA(2) (Low DS, Colorimetric modification rate: 0.56) prepared in Example I-2 was dissolved in physiological saline as a solvent to prepare a 2% w / v AL-ABA solution. Dendritic poly(ethyleneimine) (DPI) (manufactured by BASF, product name: PS, weight-average molecular weight M: 750,000) was dissolved in water to prepare a 10% w / v DPI solution. Two solutions were mixed so that the aldehyde groups of the AL-ABA solution and the primary amino groups (-NH2) of the DPI solution were equimolar (1:1). Specifically, 5 mL of 2% w / v AL-ABA solution (aldehyde groups: 2.59×10 -4 mol) was mechanically mixed with 250 μL of 10% w / v DPI solution (amino groups: 2.59×10 -4 mol) with a pipette. A hydrogel was formed within 20 - 30 seconds (Figure 35). The hydrogel was stable in water for two weeks.

[0254] [Example I-19] Hydrogel of AL-ABA and PEG(4k)-dihydrazide (AL-ABA-PEGDH) <Synthesis of PEGDH> [Chemical formula] For the preparation of PEGDH, a two-step reaction was carried out. First, 10 g of PEG (Mw 4000) (0.0025 mol, 162 - 09115, Wako) was dissolved at room temperature in a 300 - ml eggplant - shaped flask containing 100 ml of dichloromethane (DCM) solvent. After 2.0 g (0.02 mol) of succinic anhydride, 2.57 g (0.0125 mol) of N,N’ - dicyclohexylcarbodiimide and 1.83 g (0.015 mol) of 4 - dimethylaminopyridine were added to the PEG solution. The flask was closed with a rubber septum and stirred at room temperature for 24 hours. Then, the reaction mixture was filtered through filter paper, and the filtrate was evaporated at 50 °C using a rotary evaporator. After completely evaporating DCM, a viscous reaction mixture was left in the flask. Then, 100 ml of distilled water was added and stirred for another 1 hour. Then, the solution was dialyzed against pure water for 48 hours using a dialysis tube (MWCO 1 kDa) (Spectra / Pro (registered trademark)). After dialysis, the solution was lyophilized, 1 characterized by 1H NMR (using D2O solvent). The yield was about 73%, and the conversion rate from PEG to PEG - COOH was about 100%. The second step is a carbodiimide reaction. 4 g (0.00097 mol) of newly prepared PEG - COOH was taken in a 200 - ml Erlenmeyer flask and dissolved in 50 ml of distilled water for 2 - 4 hours. 1.05 g (0.0070 mol) of HOBt was dissolved in 10 ml of DMSO (Wako), then 1.49 g (0.0078 mol) of WSCD.HCl (Peptide Institute) was dissolved in 10 ml of distilled water and added dropwise to the PEG - COOH solution. The solution was stirred for an additional 10 minutes. 3.399 g (0.019 mol) of adipic dihydrazide was dissolved in 15 ml of pure water and added to the PEG - COOH solution. The pH of the final reaction mixture was maintained between 7.5 pH with 1N NaOH and stirred at room temperature for 16 - 20 hours. Then, using a dialysis tube (MWCO 1 kDa) (Spectra / Pro (registered trademark)), the solution was thoroughly dialyzed against deionized water for 48 hours and then lyophilized. 1 The binding of PEGDH was confirmed using 1H NMR. The degree of substitution was about 91%.

[0255] <Synthesis of AL - ABA - PEGDH> AL-ABA (2) (High DS, colorimetric modification rate: 0.64) prepared in Example I-2 was dissolved in physiological saline as a solvent to prepare a 1.5% w / v AL-ABA solution (2). The PEGDH prepared above was dissolved in water to prepare a 10% w / v PEGDH solution. The two solutions were mixed so that the aldehyde groups in the AL-ABA (2) solution and the hydrazide groups in the PEGDH solution were equimolar (1:1). Specifically, 1 mL of a 1.5% w / v AL-ABA solution (aldehyde groups: 6.9 × 10 -5 mol) in 0.5 mL of 10% w / v PEGDH solution (hydrazide groups: 2.27 × 10 -5 mol) was mechanically mixed with a pipette. A hydrogel formed within 30 seconds (Figure 36).

[0256] <Dynamic viscoelasticity measurement> The storage modulus G' and loss modulus G" were measured using a rheometer. The results are shown in FIG. 37. Within the measurement frequency range, G'>G" was confirmed for AL-ABA-PEGDH, confirming the formation of a hydrogel.

[0257] [Example I-20] Benzaldehyde-modified alginate films (uncrosslinked and crosslinked AL-ABA films) <Film Preparation> (1) Uncrosslinked AL-ABA film Uncrosslinked AL-ABA films were prepared by solution casting and vacuum drying. Specifically, AL-ABA (2) (High DS, colorimetric modification ratio: 0.64) prepared in Example I-2 was dissolved in distilled water to prepare a 1.5% w / v AL-ABA solution. After complete dissolution, air bubbles were removed by sonication for 15 minutes. The solution was then poured into a 60 mm Petri dish, and the Petri dish was placed in a vacuum oven at 50°C for 40-48 hours. The film was then peeled off and stored at 4°C. The resulting film was nonporous, transparent, and approximately 63 μm thick.

[0258] (2) Cross-linked AL-ABA film The uncrosslinked AL-ABA film obtained in (1) above was crosslinked with PEGDH to obtain a crosslinked AL-ABA film. Specifically, the PEGDH synthesized in Example I-19 above was dissolved in an aqueous butanol solution (butanol:water = 9:1), and the film obtained in (1) above was immersed in the solution and maintained at room temperature for 6 to 8 hours. The film was removed from the PEGDH solution, dried in the air, and stored at 4°C. The resulting crosslinked AL-ABA film was nonporous and transparent.

[0259] <Film swelling and dissolution test> (Experimental Procedure) The uncrosslinked AL-ABA film obtained in (1) above, the crosslinked AL-ABA film obtained in (2) above, and the AL film were immersed in PBS (pH = 7.4) for 2 to 4 weeks, and the swelling and dissolution of the films were observed. The AL film was prepared by the method described in (1) above, except that AL-500 (Mochida Pharmaceutical Co., Ltd., sodium alginate, viscosity: 400 to 600 mPa s) was used instead of the AL-ABA (2) prepared in Example I-2. (result) The results are shown in the table below. [Table 3]

[0260] [Example I-21] Hydrogels of AL-ABA and polyallylamine (AL-ABA-PAA1, AL-ABA-PAA2, AL-ABA-PAA3, AL-ABA-PAA4) [ka] (i) PAA1: Product name: Allylamine maleic acid copolymer (product number: PAA-1151, Nittobo Medical Co., Ltd., viscosity 20 wt% 20 cp at 20 degrees) (ii) PAA2: Diallyldimethylammonium chloride acrylamide copolymer (product number: PAS-J-81, Nittobo Medical Co., Ltd., viscosity 25 wt% 900 cp at 20°C), weight average molecular weight Mw: 180,000 (iii) PAA3: Allylamine hydrochloride dimethylallylamine salt copolymer (product number: PAA-1112CL, Nittobo Medical Co., Ltd., viscosity 15 wt% 5 cp at 20°C) (iv) PAA4: allylamine hydrochloride polymer (product number: PAA-HCl-10L, Nittobo Medical Co., Ltd., viscosity 1500 cp at 40 wt% (20°C)), weight-average molecular weight Mw: 150,000 The polyallylamine (PAA1 to PAA4) described above in (i) to (iv) was dissolved in water to prepare 10% w / v PAA solutions (PAA1 solution, PAA2 solution, PAA3 solution, PAA4 solution). AL-ABA (2) (low DS, colorimetric modification ratio: 0.56) prepared in Example I-2 was dissolved in physiological saline as a solvent to prepare a 2% w / v AL-ABA solution. The two solutions were mixed so that the aldehyde groups in the AL-ABA solution and the primary amino groups (-NH2) in the PAA solution were equimolar (1:1). Specifically, 5 mL of the 2% w / v AL-ABA solution (aldehyde groups: 2.59 × 10 -4 mol), 10% w / v each PAA solution (PAA1 solution, PAA2 solution, PAA3 solution, PAA4 solution) (amino group in each solution: 2.59 × 10 -4 mol) were mechanically mixed with a pipette. In both cases, hydrogels formed within 20 to 30 seconds (Figure 38).

[0261] 6. Tissue adhesive material using AL-ABA [Example I-22] Benzaldehyde-modified alginate (AL-ABA) as a tissue adhesive material (adhesion to the submucosa) <Evaluation of adhesive behavior to mucosa> The adhesiveness of AL-ABA to the submucosal layer was examined. The submucosal layer is rich in collagen and therefore contains a large number of amino groups. The following experiment confirmed that a cross-linked structure formed by a Schiff base between the amino groups in the submucosal layer and the aldehyde groups in AL-ABA can improve adhesiveness. The specific experimental procedure is as follows:

[0262] (Experimental Procedure) 1. A pig esophagus was incised longitudinally and cut into 2 cm x 2 cm sections. The inner mucosal layer of the esophagus was excised to expose the submucosal layer. 25 mL of saline containing 0.1% sodium benzoate was placed in an 80 mL security container. Each esophageal section with the exposed submucosal layer was immersed in the container and placed on a shaker in a 37°C incubator and shaken overnight. After shaking, the weight of the esophageal section was measured. (Esophageal sections: n=4) 2. AL-ABA and unmodified AL (IL-6G) for comparative studies were each dissolved in pure water at 2 w / v%. AL-ABA (1) prepared in Example I-1 was used. 3. The alginate solution (AL-ABA aqueous solution or AL aqueous solution) prepared in step 2 above was placed in one side of a double syringe, and a 50 mM CaCl2 aqueous solution was placed in the other side. 4. The slice prepared in step 1 above was removed from the security container and placed on a petri dish. 0.5 mL of the alginate solution prepared in step 3 and 0.5 mL of 50 mM CaCl2 aqueous solution were simultaneously sprayed onto the slice using a double syringe and nitrogen gas. The nitrogen gas flow rate was 2 L / min. The slice was left to stand at room temperature (25°C) for 10 minutes for gelation. This treatment resulted in the benzaldehyde-modified alginate (AL-ABA) or alginate (AL) converting to calcium (Ca 2+ ) to form hydrogels (AL-ABA gel, AL gel). 5. The gel on the dish was washed away with pure water, and the esophageal section was wiped dry and then weighed. The weight of the esophageal section measured in 1 above was subtracted from this weight to calculate the weight of the gel on the esophageal section. This was the weight of the gel after 0 hours. 6. The saline solution containing 0.1% sodium benzoate in the security container was replaced with fresh saline solution. The esophageal section was immersed in the new solution with the gel side facing downwards. This was then placed on a shaker and shaken in a 37°C incubator. 7. The weight of the esophageal section was measured after a specified time (1, 2, 3, 4, 6, 8, 12, 24, and 48 hours), and the weight of the esophageal section measured in 1 above was subtracted to calculate the weight of the remaining gel. The remaining gel percentage at each time point was calculated from the gel weight at 0 hours and the weight of the remaining gel [(remaining gel weight) / (gel weight at 0 hours) × 100]. The appearance of the gel at each time point was also observed. The above procedure was carried out for four samples each of AL and AL-ABA, and the average value of the remaining gel percentage at each time point was taken as the gel adhesion rate (%).

[0263] (result) The results are shown in Figures 39 and 40. Figure 39 shows the appearance of the AL gel and AL-ABA gel at each time point. Figure 40 shows the adhesion rate (%) of the AL gel and AL-ABA gel at each time point. As shown in Figure 39, in the AL-sprayed groups (ALG(IL-6G) and AL gel), the gel peeled off in three of the four samples after 1 hour. The remaining gel also peeled off after 2 hours. In the AL-ABA-sprayed groups (ALG-ABA and AL-ABA gel), the gel gradually peeled off after 2 hours, and a small amount of gel remained visible even after 12 hours. Figure 40 also shows that the gel adhesion rate (%) in the AL-sprayed groups (ALG(IL-6G) and AL gel) reached zero after 3 hours, whereas the AL-ABA-sprayed groups (ALG-ABA and AL-ABA gel) maintained approximately 40% gel adhesion. These results suggest that modifying AL with ABA improves adhesion.

[0264] [Example I-23] AL-ABA as a tissue adhesive material (evaluation of adhesion to the esophageal mucosal layer and submucosa and Ca 2+ Concentration effect assessment (37℃)

[0265] <Adhesion experiments to the mucosal layer and submucosal layer> The adhesiveness of AL-ABA and AL to the esophageal mucosal layer and submucosal layer was examined in a 37°C environment. The sample with the mucosal layer was used as the control, and the sample with the mucosal layer peeled off and the submucosal layer exposed was used as the ESD sample. 2+ Two concentrations, 50 mM and 100 mM, were examined. 2+ The change in adhesiveness due to the change in concentration was investigated. The experimental procedure is shown in Figure 42. The specific procedure is as follows.

[0266] (material) AL-ABA: AL-ABA (modification ratio (NMR): 0.052) prepared under the same conditions as AL-ABA (1) in Example I-1 was dissolved in pure water at 2 w / v %. AL:IL-6G (IL-6G manufactured by Kimika Co., Ltd., viscosity 50-80 mPa·s (1%)) was dissolved in pure water at 2 w / v%. CaCl2 aqueous solution (for cross-linking): 50 mM or 100 mM Esophageal section (Control: with mucosal layer; ESD: mucosal layer peeled off, submucosal layer exposed)

[0267] (Experimental Procedure) 1. The porcine esophagus was incised longitudinally and cut into 2 cm x 2 cm sections. In the submucosal group (ESD), the mucosal layer was peeled off with scissors. 2. The masses of the cut porcine esophageal sections (control) and sections with the submucosal layer exposed (ESD) were measured. 3. AL or AL-ABA was dissolved in pure water at 2 w / v%. 4. The solution prepared in step 3 and CaCl2 solution were placed in a 2.5 mL syringe and set into a double syringe (0.5 mL of alginate solution + 0.5 mL of CaCl2 solution per sample). 5. A spray tip was attached to a double syringe, and the esophageal sections were sprayed with nitrogen gas at 4 L / min (n=4 per sample). 6. The mixture was left for 10 minutes until gelation occurred. This treatment resulted in the formation of calcium (Ca) in benzaldehyde-modified alginate (AL-ABA) or alginate (AL). 2+ The esophageal sections were then cross-linked with AL-ABA to form hydrogels (AL-ABA gel, AL gel). The mass of the esophageal sections was then measured. 7. 25 mL of physiological saline containing 0.1% methyl benzoate and 1.25 mM CaCl2 was placed in a 6 cm diameter glass dish, and the esophageal section was immersed in the solution and shaken with a shaker. 2+ is the Ca contained in saliva 2+ It is an imitation of the 8. The mass of the esophageal section was measured after a specified time (1, 2, 3, 4, 6, 8, 12, 24, 48, and 72 hours) to determine the mass of the remaining gel. The remaining gel percentage at each time point was calculated from the gel weight at 0 hours and the remaining gel weight [(remaining gel weight) / (gel weight at 0 hours) × 100]. The appearance of the gel at each time point was also observed. The above procedure was repeated for esophageal sections (control) and exposed submucosal sections (ESD). 2+ The concentration was set to 50 mM or 100 mM, and four samples of AL and AL-ABA were tested. The average value of the remaining percentage of gel at each time point was taken as the adhesion rate (%) of the gel.

[0268] (result) The appearance of the gel at each time point (2, 4, 12, 24, 48, and 72 hours) is shown in Figure 43. The remaining rate of gel at each time point (gel adhesion rate; %) is shown in Figure 44. The groups sprayed with AL-ABA on the submucosal layer (AL-ABA ESD) and the groups sprayed with AL-ABA on the mucosal layer (AL-ABA Control; AL-ABA Con) were shown to be able to maintain gel adhesion for a longer period of time than the groups sprayed with AL (AL ESD and AL Control (Con)). It was confirmed that modifying AL with ABA improved adhesion. In particular, the adhesive strength was further improved when AL-ABA was sprayed onto the submucosal layer (AL-ABA ESD) compared with when AL-ABA was sprayed onto the mucosal layer (AL-ABA Control; AL-ABA Con). It is presumed that strong adhesion was achieved by ABA binding to the amino groups in the submucosal layer. Furthermore, it was shown that when the CaCl2 concentration was high (100 mM), the gel's adhesion rate could be maintained for a longer period of time. It is thought that an increase in the amount of Ca ions improved the cross-linking density, thereby lengthening the gel's mechanical strength and degradation time. Since cohesive failure occurred at the maximum breaking strength in the tensile test, it is thought that the adhesive strength increased due to the increased mechanical strength of the gel.

[0269] <Adhesion evaluation by tensile test> In the above adhesion experiment, both gel peeling and gel dissolution occurred simultaneously. A tensile test was performed to verify the adhesive properties of AL-ABA alone. The procedure for the tensile test is shown in Figure 45. (material) AL-ABA: AL-ABA (modification ratio (NMR): 0.052) prepared in the same manner as in Example I-1 was dissolved in pure water at 2 w / v %. AL:IL-6G (IL-6G manufactured by Kimika Co., Ltd., viscosity 50-80 mPa·s (1%)) was dissolved in pure water at 2 w / v%. CaCl2 aqueous solution (for cross-linking): 50 mM or 100 mM Esophageal section (Control: with mucosal layer; ESD: mucosal layer peeled off, submucosal layer exposed) (Device) Rheometer for measuring physical properties (Shiro Sangyo Co., Ltd., M993R-3000S) (Experimental Procedure) 1. The porcine esophagus was incised longitudinally and cut into 1 cm × 3 cm sections. The sections were used as they were in the mucosal group (control). In the submucosal group (ESD), the mucosal layer of the sections was removed (n = 3). 2. Alginate solution (AL-ABA or AL) was added to an area of ​​1 cm x 1 cm on one end of each section. 3. CaCl2 solution was added on top of the alginate solution to cause gelation. 4. A weight (30 mL of water in an 80 mL container) was placed on top to remove any air. The mixture was left to stand in a thermostatic bath at 5.37°C for 1 hour. 6. The breaking strength (N) was measured using a rheometer. 7. The breaking strength (N) and the area (m 2 ) and the rupture pressure (Pressure, Pa) was calculated.

[0270] (result) The results are shown in Figure 46. It was confirmed that the AL-ABA hydrogel had significantly greater adhesive strength to the mucosa and submucosa than the AL hydrogel. 2+ It was confirmed that the adhesive strength changes depending on the concentration of ions.

[0271] [Example I-24] AL-ABA as a tissue adhesive material (evaluation of adhesion to submucosal layer and skin tissue using lap shear method) The adhesive strength of AL-ABA, AL, and conventional tissue adhesives to biological tissue was evaluated using a lap shear test. Specifically, the following pregel solutions were used, and the adhesive strength of each material to biological tissue was evaluated using porcine submucosa and porcine skin tissue as the biological tissues, according to the following procedure. The experimental procedure for the lap shear test is shown in Figure 47. (Pregel solution) AL-ABA: 4 w / v% aqueous solution of AL-ABA (2) (High DS; colorimetric modification rate: 0.64) prepared in Example I-2 AL500: 4 w / v% AL-500 (Mochida Pharmaceutical Co., Ltd., sodium alginate, viscosity: 400-600 mPa·s) Fibrin Glue: Fibrin glue (Beriplast P, manufactured by CSL Behring Co., Ltd.) Hydrofit® (urethane-based hemostatic agent; Terumo Corporation) Dermabond® (cyanoacrylate skin surface adhesive; Johnson & Johnson) The gelation of the pre-gel solution was carried out as follows. For AL-ABA and AL500, the pregel solution (AL-ABA or AL500) was applied and the biological tissue was superimposed, and then 200 μL of 50 mM or 100 mM CaCl2 aqueous solution was applied to both sides of the biological tissue (200 μL x 2). Fibrin Glue was gelled using a double syringe. Hydrofit® is a one-component product and was applied as is. Dermabond® is a one-part product and was applied as is. (biological tissue) Submucosa: The porcine esophagus was cut into pieces 40 mm long x 10 mm wide, and the mucosal layer inside the cut esophagus was removed to expose the submucosa. Skin tissue: Pig skin tissue cut into a size of 40 mm length x 10 mm width was prepared. (Device) Dynamic Mechanical Analyzer (DMA) CR3000-EX manufactured by Sun Scientific Co., Ltd.

[0272] (Lapsed shear method experiment procedure) 0.5 mL of pregel solution was applied to the overlapping area (10 mm long x 10 mm wide) at the ends of two pieces of biological tissue (40 mm long x 10 mm wide), overlapped, and then allowed to gel. After 20 minutes of gelation, a lap shear test was performed at a rate of 5 mm / min using a dynamic mechanical analyzer (DMA). The adhesive energy was measured by a 180° peel test. The adhesive strength was calculated using the following formula: Adhesive strength = force (N / m 2 ) / overlapped area (m 2 )

[0273] (result) The results are shown in Figure 48. It was shown that AL-ABA hydrogels can be used as tissue adhesives for biological tissues such as submucosa and skin tissue.

[0274] [Example I-25] AL-ABA as a tissue adhesive material (tissue sealant) (Evaluation of its effectiveness as a sealant for submucosal and skin tissues by burst test) To investigate its effectiveness as a tissue sealant, a burst test was performed using a device designed with slight modifications to the device reported by Lei Zhou et al., "Injectable Self-Healing Natural Biopolymer-Based Hydrogel Adhesive with Thermoresponsive Reversible Adhesion for Minimally Invasive Surgery," ADVANCED FUNCTIONAL MATERIALS, Volume 31, Issue 14, 2021, 2007-457. Specifically, the burst test was performed using the following pregel solution and porcine submucosa and porcine skin tissues as biological tissues, according to the following burst test procedure. The device and burst test procedure used are shown in Figure 49. (Pregel solution) AL-ABA: 4 w / v% aqueous solution of AL-ABA (2) (High DS; colorimetric modification rate: 0.64) prepared in Example I-2 AL500: 4 w / v% AL-500 (Mochida Pharmaceutical Co., Ltd., sodium alginate, viscosity: 400-600 mPa·s) Fibrin Glue: Fibrin glue (Beriplast P, manufactured by CSL Behring Co., Ltd.) Hydrofit® (urethane-based hemostatic agent; Terumo Corporation) Dermabond® (cyanoacrylate skin surface adhesive; Johnson & Johnson) The gelation of the pre-gel solution was carried out as follows. For AL-ABA and AL500, the pregel solution (AL-ABA or AL500) was injected into the puncture site, and 500 μL of 100 mM CaCl 2 aqueous solution was added dropwise from above, followed by crosslinking for 5 to 10 minutes. Fibrin Glue was gelled using a double syringe. Hydrofit® is a one-component product and was applied as is. Dermabond® is a one-part product and was applied as is. (biological tissue) Submucosa: The porcine esophagus was cut into pieces 50 mm long x 50 mm wide, and the mucosal layer inside the cut esophagus was removed to expose the submucosa. Skin tissue: Pig skin tissue cut into a size of 50 mm length x 50 mm width was prepared. (Burst test procedure) The biological tissue was fixed on the top surface of the burst pressure device and punctured to create a 2 mm diameter hole in the center of the tissue. 2.5 to 3 mL of pregel solution was injected into the 2 mm diameter hole (puncture site) and allowed to gel. The measuring device was connected to a syringe pump filled with PBS solution. Finally, the PBS solution was injected into the device, and the maximum burst pressure (in mmHg) was recorded using a digital pressure gauge. The experiment was repeated 5 times.

[0275] (Results) The results are shown in the following table and Figure 50.

Table 3

[0276] II. Hyaluronic acid derivatives [Example II-1] Benzaldehyde-modified hyaluronic acid (HA-ABA) <Synthesis of HA-ABA>

Chemical formula

[0277] According to the above synthesis scheme, HA-ABA was synthesized from the following procedure by amidation reaction via carbodiimide. (Synthesis procedure) A 100 ml aqueous solution of HA (Mw: 890 kDa) (2 mg / ml) was dissolved in 25 mL of THF. It was mixed with 121 mg of 4-amino-benzaldehyde (ABA) (equivalent to 2 equivalents of ABA relative to the carboxyl group of HA). 2 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide monohydrochloride (EDC·HCl) and 2 equivalents of 1-hydroxybenzotriazole (HOBt) were added, the pH was adjusted to 5.5, and the mixture was stirred overnight at room temperature. Then, it was purified by dialysis and lyophilized to obtain HA-ABA.

[0278] <Measurement of molecular weight> The weight-average molecular weight (Mw) of HA was measured by gel filtration chromatography (GPC) under the following conditions. (Measurement procedure) The molecular weight distribution of the polymer was measured by gel filtration chromatography (GPC). A high-performance liquid chromatograph pump (LC-10ADVP, Shimadzu Corporation) was used. An intelligent refractive index detector (830-RI, JASCO Corporation) was used. Separation was performed at room temperature using TSK-GEL GMPWXL (molecular weight cutoff: 104-106, Tosoh) and TSK-GEL-3000 (molecular weight cutoff: 103-105, Tosoh) columns. The flow rate was set to 0.5 mL / min, and the eluent was the above-mentioned phosphate buffer (pH 6.7). A calibration curve was prepared using dextran (8000 Da, 15000 Da, 40000 Da, 70000 Da, 500000 Da, EXTRASYNTHESE). The day before the measurement, phosphate buffer was run overnight at 0.2 mL / min to exchange the solvent. The calibration curve solution and sample solution were each filtered through a 0.22 μm filter. Before measurement, the RI value was confirmed to be stable, and then the measurement was started. After use, pure water was run overnight at 0.2 mL / min to perform solvent replacement again.

[0279] < 1 H NMR spectroscopy, ultraviolet-visible absorption spectroscopy (UV-vis), and FT-IR spectroscopy About HA-ABA 1 H NMR spectroscopy, ultraviolet-visible absorption spectroscopy (UV-vis), and FT-IR spectroscopy were performed, and the results are shown in Figures 51 to 53. 1 In the H NMR spectrum (Figure 51), peaks derived from ABA (peak a derived from the methyl group of HA, peaks b and c derived from the benzene ring, and peak d derived from the aldehyde) were observed in HA-ABA. In addition, in the UV-vis spectrum (Figure 52), a characteristic peak derived from the benzyl group of ABA (peak at approximately 250 nm) was observed in HA-ABA. In the FT-IR spectrum (Figure 53), an absorption derived from ABA (approximately 2950 cm) was observed in HA-ABA. -1A peak) was observed. From these results, the synthesis of benzaldehyde-modified hyaluronic acid (HA-ABA), in which the amino group of 4-aminobenzaldehyde was bonded to the carboxyl group of hyaluronic acid, was confirmed. Furthermore, 1 By using the peaks at 1.95 ppm (a: -CH3 of the acetamide moiety of N-acetyl-D-glucosamine), 6.75 ppm (b: -CH of the benzyl ring), 7.65 ppm (c: -CH of the benzyl ring), and 9.44 ppm (d: -CH peak of the aldehyde) in the 1H NMR spectrum, the modification rate of ABA to the carboxyl group (-COOH) of hyaluronic acid was calculated to be 0.16.

[0280] <Cytotoxicity evaluation of benzaldehyde-modified hyaluronic acid (HA-ABA) (WST assay)> (Experimental procedure) MeT-5A (human mesothelial cell line), HUVEC (human umbilical vein endothelial cells), RAW264.7 cells (mouse macrophage-like cell line), NIH / 3T3 (mouse fetal fibroblast cells), and AB22 cells (mouse mesothelioma cells) were seeded in 24-well plates and cultured for 24 hours. Then, the medium was replaced with a medium containing HA-ABA and HA dissolved at different concentrations (0.01 mg / mL, 0.1 mg / mL, 1 mg / mL). The cell viability (Cell viability (%)) 48 hours after adding the samples was measured using the WST-8 assay (Cell Counting Kit-8, Dojindo). (Results) The results are shown in Fig. 54. HA-ABA showed a high cell viability comparable to that of HA in most cells. In the fibroblast cell line NIH / 3T3, the viability decreased to 20% with 1 mg / mL of HA-ABA. This may be related to the different expression of CD44 depending on the cell type.

[0281] [Example II-2] Benzaldehyde-modified hyaluronic acid-pemetrexed conjugate (HA-ABA-PMX) <Synthesis of HA-ABA-PMX> [Chemical formula]

[0282] Pemetrexed (PMX), an anticancer drug with a primary amino group, was conjugated to HA-ABA via a Schiff base reaction between the aldehyde group of HA-ABA and the amino group of PMX according to the synthetic scheme described above. Specifically, HA-ABA obtained in Example II-1 was dissolved in pure water at a concentration of 1 mg / ml, and 1.5 equivalents of PMX dissolved in DMSO was added dropwise. The mixture was reacted overnight at room temperature in the dark. The mixture was then dialyzed and lyophilized to obtain HA-ABA-PMX.

[0283] < 1 H NMR spectroscopy and ultraviolet-visible absorption spectroscopy (UV-vis) Regarding the obtained HA-ABA-PMX, 1 H NMR spectroscopy and ultraviolet-visible absorption spectroscopy (UV-vis) were performed, and the results are shown in Figures 55 and 56. 1 In the H NMR spectrum (Figure 55), peaks derived from HA-ABA and PMX were observed in HA-ABA-PMX. Furthermore, in the UV-vis spectrum (Figure 56), an increase in the characteristic PMX peak (225 nm) was observed in HA-ABA-PMX. These results confirmed the formation of a conjugate of HA-ABA and PMX. 1 The conjugation rate of the drug PMX to the carboxyl groups (-COOH) of HA was 11.5% from the H NMR spectrum, indicating that 71.8% of the ABA in HA-ABA had reacted with PMX. These results demonstrate that PMX can be conjugated to HA-ABA simply by mixing HA-ABA and PMX in an aqueous solvent. This reaction can be applied to various primary amine-containing drugs, and HA-ABA is expected to become a convenient and versatile platform for developing hyaluronan-drug conjugates.

[0284] <Release experiment of PMX from benzaldehyde-modified hyaluronic acid-pemetrexed conjugate (HA-ABA-PMX)> (Experimental Procedure) The release of PMX from HA-ABA-PMX at different pH levels was investigated using dialysis tubing (MWCO: 1 kDa). Specifically, 1 ml of the HA-ABA-PMX (1 mg / ml) aqueous solution obtained in Example II-2 above was placed in the dialysis tubing and incubated in 79 ml of PBS (37°C, pH = 5.0, 6.0, 7.4) with shaking at 50 rpm. The external solution (sustained release solution) was collected at predetermined time points and completely replaced with fresh PBS. The drug concentration in the collected external solution (sustained release solution) was quantified by UV-vis, and the release rate of the drug PMX was calculated.

[0285] For comparison, as control experiments, the above release experiments were performed using an aqueous solution of PMX (0.12 mg / ml) (Free PMX), a mixed solution of hyaluronic acid (HA) (1 mg / ml) and PMX (0.12 mg / ml) dissolved in water (Free PMX mixed with HA), and an aqueous solution of HA-ADH-PMX in which PMX was linked to HA via an irreversible amide bond, instead of the HA-ABA-PMX solution. HA-ADH-PMX has the following structure: HA-ADH-PMX was synthesized by the method described in Amano Y. European Journal of Pharmaceutical Sciences, 2019, 138: 105008. [ka]

[0286] (result) The results are shown in Figure 57. Figure 57 shows the release profiles of pemetrexed (PMX) from a benzaldehyde-modified hyaluronic acid-pemetrexed conjugate solution (HA-ABA-PMX), from a PMX solution (free PMX), and from a mixed solution of HA and PMX (free PMX mixed with HA) at different pH levels (pH 5.0, 6.0, and 7.4). The vertical axis represents the cumulative drug release rate (%) of PMX. In the case of HA-ABA-PMX, after 24 hours, more than 80% of PMX was released at pH 5.0, whereas 40% remained unreleased at pH 7.4. This result indicated that PMX was released more rapidly with decreasing pH. This is thought to be due to the fact that the imine bond between HA-ABA and PMX is easily dissociated at low pH. On the other hand, in the free PMX solution (Free PMX) and the mixed solution of free PMX and HA (Free PMX mixed with HA), almost all of the free PMX was rapidly released within 2 hours without any sustained release effect. In the HA-ADH-PMX solution, which has an irreversible amide bond, almost no PMX was released from HA-ADH-PMX. These results suggest that HA-ABA-PMX is capable of pH-responsive sustained drug release through Schiff base formation. These results suggest that the HA-ABA-PMX conjugate enables sustained release of PMX compared to the control group containing free PMX. Furthermore, the drug is selectively released at low pH, and the PMX release rate can be changed in a pH-dependent manner. [ka]

[0287] <Cytotoxicity evaluation of benzaldehyde-modified hyaluronic acid-pemetrexed conjugate (HA-ABA-PMX) (WST assay)> (Experimental Procedure) The cytotoxicity of HA-ABA-PMX against MeT-5A (human mesothelial cell line) and AB22 cells (mouse mesothelioma cells) was evaluated (WST assay) in the same manner as for HA-ABA. Specifically, MeT-5A (human mesothelial cell line) and AB22 cells (mouse mesothelioma cells) were seeded in a 24-well plate and cultured for 24 hours. Then, the medium was changed to different concentrations of PMX (10 -4 μg / mL, 10 -3 μg / mL, 10 -2 μg / mL, 10 -1 The medium was replaced with HA-ABA-PMX (1 μg / mL, 1 μg / mL, 10 μg / mL). Cell viability (%) was measured 48 hours after sample addition using the WST-8 assay (Cell Counting Kit-8, Dojindo). For comparison, as control experiments, the above cytotoxicity evaluation was performed using free PMX and HA-ADH-PMX instead of HA-ABA-PMX.

[0288] (result) The results are shown in Figure 58. Figure 58A and Figure 58B show the effect of different PMX concentrations (10 -4 μg / mL, 10 -3 μg / mL, 10 -2 μg / mL, 10 -1 The figure shows the cell growth inhibitory effects (vertical axis: cell viability, %) of HA-ABA-PMX with concentrations of 1 μg / mL, 1 μg / mL, and 10 μg / mL), free pemetrexed (PMX), and HA-ADH-PMX, in which PMX is linked to HA via an irreversible amide bond. Both HA-ABA-PMX and free PMX showed a dose-dependent cell growth inhibitory effect against AB22 and MeT-5A. In addition, HA-ABA-PMX showed a decrease in cell viability at a lower concentration compared to free PMX and HA-ADH-PMX in which PMX was conjugated to HA via an irreversible amide bond. From these results, a high cell growth inhibitory effect of HA-ABA-PMX was suggested. This is presumably due to the promotion of cell uptake by HA-CD44 interaction and the reversible dissociation of the conjugated PMX.

[0289] [Example II-3] Benzaldehyde-modified hyaluronic acid-doxorubicin conjugate (HA-ABA-DOX) <Synthesis of HA-ABA-DOX>

Chemical formula

[0290] Doxorubicin (DOX) is an anticancer agent having a primary amino group. Benzaldehyde-modified hyaluronic acid-doxorubicin conjugate (HA-ABA-DOX) was synthesized in the same manner as in Example II-2. Specifically, HA-ABA obtained in Example II-1 was dissolved in pure water at a concentration of 1 mg / ml, and 1.5 equivalents of DOX hydrochloride dissolved in pure water was added dropwise. The reaction was carried out overnight at room temperature under light-shielded conditions. Then, this was dialyzed and freeze-dried to obtain HA-ABA-DOX.

[0291] <Ultraviolet-visible absorption spectrum (UV-vis) measurement> The obtained HA-ABA-DOX was subjected to ultraviolet-visible absorption spectrum (UV-vis) measurement. The results are shown in Fig. 59. In the UV-vis (Fig. 59), a peak derived from doxorubicin (DOX) was observed in HA-ABA-DOX, and it was confirmed that a conjugate of HA-ABA and DOX was formed.

[0292] <DOX release experiment from benzaldehyde-modified hyaluronic acid-doxorubicin conjugate (HA-ABA-DOX)> (Experimental Procedure) The release of DOX from HA-ABA-DOX at different pH levels was investigated using the same method as in Example II-2. Specifically, 1 ml of the HA-ABA-DOX (1 mg / ml) aqueous solution obtained in Example II-3 above was placed in a dialysis tube (MWCO: 1 kDa) and incubated in 79 ml of PBS (37°C, pH = 5.0, 6.0, 7.4) while shaking at 50 rpm. The external solution (sustained release solution) was collected at predetermined time points and completely replaced with fresh PBS. The drug concentration in the collected external solution (sustained release solution) was quantified by UV-vis, and the release rate of the drug DOX was calculated.

[0293] (result) The results are shown in Figure 60. Figure 60 shows the release behavior of doxorubicin (DOX) from benzaldehyde-modified hyaluronic acid-doxorubicin conjugate (HA-ABA-DOX) solutions at different pH levels (pH 5.0, 6.0, 7.4). The vertical axis represents the cumulative drug release rate (%) of DOX. The HA-ABA-DOX conjugate was shown to be capable of selective drug release at low pH, and the DOX release rate was shown to change in a pH-dependent manner.

[0294] <Cytotoxicity evaluation of benzaldehyde-modified hyaluronic acid-doxorubicin conjugate (HA-ABA-DOX) (WST assay)> (Experimental Procedure) The cytotoxicity of HA-ABA-DOX against AB22 cells (mouse mesothelioma cells) was evaluated (WST assay) in the same manner as for HA-ABA. Specifically, AB22 cells (mouse mesothelioma cells) were seeded in a 24-well plate and cultured for 24 hours. Then, the medium was changed to a solution containing different DOX concentrations (10 -3 μg / mL, 10 -2 μg / mL, 10 -1The medium was replaced with a medium in which HA-ABA-PMX (0 μg / mL, 1 μg / mL, 10 μg / mL, 100 μg / mL) was dissolved. The cell viability (%) 48 hours after adding the sample was measured using the WST-8 assay (Cell Counting Kit-8, Dojindo). For comparison, as a control experiment, the above cytotoxicity evaluation was performed using free DOX instead of HA-ABA-DOX.

[0295] (Results) The results are shown in Fig. 61. From Fig. 61, both HA-ABA-DOX and free DOX showed a dose-dependent cell growth inhibitory effect on AB22. HA-ABA-DOX showed a decrease in cell viability at a lower concentration compared to free DOX. These results suggest a high cell growth inhibitory effect of HA-ABA-DOX.

[0296] [Example II-4] Benzaldehyde-modified hyaluronic acid-gemcitabine conjugate (HA-ABA-GEM) <Synthesis of HA-ABA-GEM> [Chemical formula]

[0297] Gemcitabine (GEM) is an anticancer agent having a primary amino group. Benzaldehyde-modified hyaluronic acid-gemcitabine conjugate (HA-ABA-GEM) was synthesized in the same manner as in Example II-##**#**#**. Specifically, HA-ABA obtained in Example II-1 was dissolved in pure water at a concentration of 1 mg / ml, and 1.5 equivalents of GEM dissolved in pure water was added dropwise. The reaction was carried out overnight at room temperature under light-shielded conditions. Then, this was dialyzed and freeze-dried to obtain HA-ABA-GEM.

[0298] [Ultraviolet-visible absorption spectrum (UV-vis) measurement] The obtained HA-ABA-GEM was subjected to ultraviolet-visible absorption spectrum (UV-vis) measurement. The results are shown in Fig. 62. In UV-vis (Figure 62), a peak derived from gemcitabine (GEM) was observed in AL-ABA-GEM, confirming that a conjugate of HA-ABA and GEM was formed.

[0299] [Example II-5] Benzaldehyde-modified hyaluronic acid-DNA nucleotide conjugates (HA-ABA-Adenine, HA-ABA-cytosine, HA-ABA-guanine) <Synthesis of HA-ABA-Adenine, HA-ABA-cytosine, and HA-ABA-guanine>

Chemical formula

[0300] Conjugates (HA-ABA-Adenine, HA-ABA-cytosine, HA-ABA-guanine) of adenine, cytosine, and guanine, which are DNA nucleotides, and benzaldehyde-modified hyaluronic acid were synthesized in the same manner as in Example II-2. Specifically, the HA-ABA obtained in Example 8 was dissolved in pure water at a concentration of 1 mg / ml, and 2.5 equivalents of adenine, cytosine, or guanine dissolved in pure water were added dropwise. The reaction was carried out overnight at room temperature under light-shielded conditions. Then, this was dialyzed and freeze-dried to obtain HA-ABA-Adenine, HA-ABA-cytosine, and HA-ABA-guanine.

[0301] ` < 1 <1H NMR spectrum measurement and FT-IR spectrum measurement> For the obtained HA-ABA-Adenine, HA-ABA-cytosine, and HA-ABA-guanine, 1 1H NMR spectrum measurement and FT-IR spectrum measurement were performed. The results are shown in Figures 63 - 66. In the 1 1H NMR spectrum (Figure 63) of HA-ABA-Adenine, peaks (e) derived from HA and peaks (a - d) derived from adenine were observed. Of HA-ABA-cytosine 1 In the 1 H NMR spectrum (Figure 64), peaks (e) derived from HA and peaks (a-d) derived from cytosine were observed. Of HA-ABA-guanine 1 In the 1 H NMR spectrum (Figure 65), peaks (d) derived from HA and peaks (a-c) derived from guanine were observed. Also, in the FT-IR spectrum (Figure 66), absorptions derived from the bond between HA and ABA (stretching and ring vibration of C=O, C=C, C=N) were observed in all of HA-ABA-Adenine, HA-ABA-cytosine, HA-ABA-guanine, and HA-ABA. From these results, it was confirmed that conjugates of HA-ABA and DNA nucleotides (adenine, cytosine, or guanine) were formed. 1 The modification rate (drug introduction rate) of the DNA nucleotide as a drug was calculated from the H NMR spectrum. The conjugation rate of adenine to the carboxyl group (-COOH) of HA was 8%, indicating that 38% of ABA in HA-ABA reacted with adenine. The conjugation rate of cytosine to the carboxyl group (-COOH) of HA was 16%, indicating that 76.1% of ABA in HA-ABA reacted with cytosine. The conjugation rate of guanine to the carboxyl group (-COOH) of HA was 9%, indicating that 42.8% of ABA in HA-ABA reacted with guanine. The above results show that DNA nucleotides bind (conjugate) to HA-ABA simply by mixing HA-ABA and DNA nucleotides in an aqueous solvent. HA-ABA is expected to be a simple and versatile platform for developing hyaluronic acid-nucleic acid drug conjugates.

[0302] III. Carboxymethylcellulose derivative [Example III-1] Benzaldehyde-modified carboxymethylcellulose (CMC-ABA) [Synthesis of CMC-ABA [ka]

[0303] According to the above synthesis scheme, CMC-ABA was synthesized by a carbodiimide-mediated amidation reaction using the following procedure. (Synthetic Procedure) In a 100 mL eggplant-shaped flask, 0.3 g of CMC (Sigma-Aldrich, product number: 419338-100G, product name: Sodium carboxymethyl cellulose, weight-average molecular weight: ~700,000 (catalog value)) was dissolved in 100 mL of distilled water and stirred overnight to prepare a CMC solution. 2.16 g (0.016 mol) of HOBt (1-hydroxybenzotriazole monohydrate, Tokyo Chemical Industry Co., Ltd., product number: H0468, weight-average molecular weight: 135.13) was dissolved in 10 mL of DMSO and added dropwise to the CMC solution. 3.06 g (0.016 mol) of WSCD.HCl (Peptide Institute, product number: 1030, weight-average molecular weight: 191) was then dissolved in 15 mL of water and added dropwise to the CMC solution. The solution was then stirred for an additional 10 minutes. 1.38 g (0.011 mol) of 4-aminobenzaldehyde (ABA) was dissolved in 12 ml of tetrahydrofuran (THF, Fujifilm Wako Pure Chemical Industries, Ltd., product number: 206-08744, weight-average molecular weight: 72.11) and added dropwise to the CMC solution. 1 N NaOH (sodium hydroxide, Fujifilm Wako Pure Chemical Industries, Ltd., product number: 198-13765, weight-average molecular weight: 40) was added dropwise to the mixture while adjusting the pH to 7.5-8. The mixture was stirred at room temperature for 16-20 hours. The reaction mixture was centrifuged at 3000 rpm for 10 minutes. The supernatant was collected and dialyzed against purified water using a dialysis membrane (MWCO 6-8 kDa) (Spectra / Pro®) for 72 hours, followed by lyophilization to obtain CMC-ABA.

[0304] < 1 H NMR and FT-IR Spectroscopy About CMC-ABA 11H NMR spectrum measurement and FT-IR spectrum measurement were performed. The results are shown in Figs. 67 to 68. 1 In the 1H NMR spectrum (Fig. 67), peaks (e) derived from CMC and peaks (a - d) derived from ABA were observed. Also, in the FT-IR spectrum (Fig. 68), absorptions derived from the bonds between CMC and ABA (stretching of C=O of amide bond, stretching of C-H of aldehyde, and stretching of N-H) in CMC-ABA were observed. Furthermore, 1 From the 1H NMR spectrum, the modification rate of ABA to the carboxyl group (-COOH) of CMC was calculated to be 0.4866 (86.6 mol%).

[0305] IV. Carboxymethyl Dextran Derivative [Example IV-1] Benzaldehyde-Modified Carboxymethyl Dextran (CMDX-ABA) [Synthesis of CMDX-ABA] [Chemical formula]

[0306] According to the above synthesis scheme, CMDX-ABA was synthesized from the following procedure by amidation reaction via carbodiimide. [Synthesis procedure] In a 100 mL eggplant-shaped flask, 0.5 g (0.0030 mol) of CMDX (Meito Sangyo Co., Ltd., product name: carboxymethyl dextran, product number: CMD-500-0613, weight-average molecular weight: 580,000 (catalog value)) was dissolved in 50 mL of purified water and stirred overnight to prepare a CMDX solution. 2.83 g (0.021 mol) of HOBt was dissolved in 10 mL of DMSO and added dropwise to the CMDX solution. 4.01 g (0.021 mol) of WSCD.HCl was then dissolved in 20 mL of water and added dropwise to the CMDX solution. The solution was then stirred for an additional 10 min. 1.81 g (0.015 mol) of 4-aminobenzaldehyde (ABA) was dissolved in 12 mL of THF and added dropwise to the CMDX solution. The pH of the mixture was adjusted to a range of 7.5 to 8 by adding 1 N NaOH dropwise. The mixture was then stirred at room temperature for 16 to 20 hours. The reaction mixture was centrifuged at 3000 rpm for 10 minutes. The supernatant was dialyzed against pure water for 72 hours using a dialysis membrane (MWCO 6-8 kDa) (Spectra / Pro®) and then lyophilized to obtain CMDX-ABA.

[0307] < 1 H NMR and FT-IR Spectroscopy About CMDX-ABA 1 H NMR spectroscopy and FT-IR spectroscopy were performed, and the results are shown in Figures 69 and 70. 1 In the H NMR spectrum (Figure 69), peaks (e, f) derived from CMDX and peaks (ad) derived from ABA were observed. In the FT-IR spectrum (Figure 70), absorptions derived from the bond between CMDX and ABA (stretching of C=O of the amide bond, stretching of CH and NH of the aldehyde) were observed in CMDX-ABA. 1 From the 1 H NMR spectrum, the ABA modification ratio of the carboxyl group (-COOH) of CMDX was calculated to be 0.43 (43 mol%).

[0308] [Example IV-2] Benzaldehyde-Modified Carboxymethyl Dextran-DNA Nucleotide Conjugates (CMD-ABA-Adenine, CMD-ABA-Cytosine, CMD-ABA-Guanine) <Synthesis of CMD-ABA-Adenine, CMD-ABA-Cytosine, and CMD-ABA-Guanine> Conjugates (CMD-ABA-Adenine, CMD-ABA-Cytosine, CMD-ABA-Guanine) of the DNA nucleotides adenine, cytosine, and guanine with benzaldehyde-modified carboxymethyl dextran were synthesized. Specifically, the CMDX-ABA obtained in Example IV-1 was dissolved in pure water at a concentration of 1 mg / ml, and 2.5 equivalents of adenine, cytosine, or guanine dissolved in pure water were added dropwise. The reaction was carried out overnight at room temperature under light-shielded conditions. Then, this was dialyzed and freeze-dried to obtain CMD-ABA-Adenine, CMD-ABA-Cytosine, and CMD-ABA-Guanine.

[0309] <FT-IR Spectrum Measurement> FT-IR spectrum measurements were performed on the obtained CMD-ABA-Adenine, CMD-ABA-Cytosine, and CMD-ABA-Guanine. The results are shown in Figure 71. In the FT-IR spectrum (Figure 71), for CMD-ABA-Adenine, CMD-ABA-Cytosine, CMD-ABA-Guanine, at 1740 cm -1 absorption due to the stretching of the C=N of the imino bond, at 1640 - 1690 cm -1 absorption due to the stretching of the C=O of the amide bond, and at 1550 - 1640 cm -1 absorption due to the stretching of N-H were observed. From these results, it was confirmed that conjugates of CMD-ABA and the DNA nucleotides (adenine, cytosine, or guanine) were formed.

[0310] V. Chitosan Derivatives [Example V-1] Benzaldehyde-modified chitosan (Chitosan-CBA) <Synthesis of Chitosan-CBA

Chemical formula

[0311] According to the above synthesis scheme, Chitosan-CBA was synthesized from the following procedure by amidation reaction via carbodiimide. 1 g (0.0062 mol) of chitosan (Sigma Aldrich, product number: 448877, CAS number: 9012-76-4, degree of deacetylation 75-85%, weight average molecular weight MW: 190,000 - 310,000 Da) was dissolved in 150 ml of 1.5% acetic acid solution to obtain a chitosan solution. Then, 0.93 g (0.0062 mol) of 4-carboxybenzaldehyde (CBA) was dissolved in 20 ml of water. 3.7 g (0.027 mol) of HOBt was dissolved in 10 ml of DMSO and added dropwise to the CBA solution. Subsequently, 5.32 g (0.027 mol) of WSCD / HCl was dissolved in 10 ml of distilled water and added dropwise to the above CBA solution, and stirred for 15 - 20 minutes. Then, the CBA solution was added dropwise to the chitosan solution (for 5 - 7 minutes), the pH of the reaction mixture was maintained at about 5.5, and stirred at room temperature for 20 - 24 hours. Next, using a dialysis tube (MWCO: 6 - 8 kDa) (Spectra / Pro (registered trademark)), the solution was dialyzed against deionized water for 72 hours and then freeze-dried to obtain Chitosan-CBA.

[0312] < 1 1H NMR spectrum measurement, FT-IR spectrum measurement For Chitosan-CBA, 1 1H NMR spectrum measurement and FT-IR spectrum measurement were performed. The results are shown in Figures 72 to 73. From these results, the synthesis of CBA-modified chitosan (Chitosan-CBA) in which the carboxyl group of CBA and the amino group of chitosan were bonded was confirmed. Also, 1From the 1 H NMR spectrum, the CBA modification ratio for the amino groups (-NH2) of chitosan was calculated to be 0.20.

Claims

1. A polysaccharide derivative in which a group represented by the following formula (A) is introduced into an acidic, basic or amphoteric polysaccharide: 【Hua 61】 (In the formula, R １ is a hydrogen atom or C １-４ represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or the pyridine ring is a halogen atom, —CF ３ , -NO ２ , a carboxyl group and —SO ３ and optionally substituted with one or more substituents independently selected from H; Y is -NH-, -C(=O)-, -S-, -O-, C １-6 Alkylene, -(CH ２ CH ２ O) n -, ... * indicates the linkage to the polysaccharide.)

2. The polysaccharide derivative according to claim 1, wherein the polysaccharide is selected from alginic acid, a derivative thereof or a salt thereof, hyaluronic acid, a derivative thereof or a salt thereof, carboxymethylcellulose, a derivative thereof or a salt thereof, carboxymethyldextran, a derivative thereof or a salt thereof, carboxymethylstarch, a derivative thereof or a salt thereof, heparin, a derivative thereof or a salt thereof, heparan sulfate, a derivative thereof or a salt thereof, chondroitin sulfate, a derivative thereof or a salt thereof, dermatan sulfate, a derivative thereof or a salt thereof, chitosan, a derivative thereof or a salt thereof, regenerated oxidized cellulose, a derivative thereof or a salt thereof, and pectinic acid, a derivative thereof or a salt thereof.

3. The polysaccharide derivative according to claim 1 or 2, wherein the group represented by formula (A) is a group selected from the following formula (A-1) or (A-2): 【Hua 62】 (In formulas (A-1) and (A-2), R １ is a hydrogen atom or C １-４ represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or the pyridine ring is a halogen atom, —CF ３ , -NO ２ , a carboxyl group and —SO ３ and optionally substituted with one or more substituents independently selected from H; L １ is a single bond or —C(═O)—, —S—, —O—, C １―６ Alkylene, -(CH ２ CH ２ O) ｎ represents a divalent group selected from the group consisting of -, ... L ２ is a single bond, or —NH—, —S—, —O—, C １―６ Alkylene, -(CH ２ CH ２ O) n represents a divalent group selected from the group consisting of -, ... n is an integer from 1 to 9 * indicates the linkage to the polysaccharide.)

4. the polysaccharide is a polysaccharide containing a carboxyl group and / or an amino group, The polysaccharide derivative according to any one of claims 1 to 3, wherein the group represented by formula (A) is introduced into the polysaccharide by forming an amide bond with a carboxyl group or an amino group of the polysaccharide.

5. the polysaccharide is a polysaccharide containing a carboxyl group, The group represented by formula (A) is a group represented by formula (A-1), The polysaccharide derivative according to any one of claims 1 to 4, wherein the group represented by formula (A-1) is introduced into the polysaccharide by substituting -OH of a carboxyl group of the polysaccharide to form an amide bond.

6. The polysaccharide derivative according to any one of claims 1 to 5, comprising at least one structural unit selected from the following formulae (c11), (c12), (c13), (c14) and (c15): 【Chemistry 63】 (In the formula, R １１ , R １２ , R １３ , and R １４ are each independently a hydrogen atom, C １－６ Alkyl, and —C(═O)—C １－６ alkyl, R ２１ , R ２２ , R ２３ , and R ２４ each independently represents a hydrogen atom, R ３１ , R ３２ , and R ３３ One to three of them are 【Hua 64】 represents R ３１ , R ３２ , and R ３３ The remainder of each independently represents a hydrogen atom, C １－６ Alkyl, —C(═O)—C １－６ Alkyl and —CH ２ COOH, R ４１ , R ４２ , and R ４３ One to three of them are 【Chemistry 65】 represents R ４１ , R ４２ , and R ４３ The remainder of each independently represents a hydrogen atom, C １－６ Alkyl, and —C(═O)—C １－６ Alkyl and —CH ２ COOH, R １ is a hydrogen atom or C １－４ represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or the pyridine ring is a halogen atom, —CF ３ , -NO ２ , a carboxyl group and —SO ３ and optionally substituted with one or more substituents independently selected from H; Y is -L １ represents —NH—, where L １ is bonded to ring P, L １ is a single bond, C １－６ Alkylene, and -(CH ２ CH ２ O) n -, and n is an integer from 1 to 9.

7. the polysaccharide is a polysaccharide containing an amino group, The group represented by formula (A) is a group represented by formula (A-2), The polysaccharide derivative according to any one of claims 1 to 4, wherein the group represented by formula (A-2) is introduced into the polysaccharide by substituting a hydrogen atom of an amino group of the polysaccharide to form an amide bond.

8. The polysaccharide derivative according to any one of claims 1 to 4 and 7, comprising a constitutional unit represented by the following formula (c16): 【Hua 66】 (In the formula, R ８１ and R ８２ are each independently a hydrogen atom, C １－６ Alkyl, and —C(═O)—C １－６ alkyl, R １ is a hydrogen atom or C １－４ represents alkyl, Ring P is a phenyl ring or a pyridine ring, and the phenyl ring or the pyridine ring is a halogen atom, —CF ３ , -NO ２ , a carboxyl group and —SO ３ and optionally substituted with one or more substituents independently selected from H; Y is -L ２ represents —C(═O)—, where L ２ is bonded to ring P, L ２ is a single bond, C １―６ Alkylene, -(CH ２ CH ２ O) n -, -(CH ２ ) ｍ１ - (CH ２ CH ２ O) n - (CH ２ ) ｍ２ -, and -(CH ２ ) ｍ１ -O-(CH ２ CH ２ O) n - (CH ２ ) ｍ２ - is selected from, n is an integer from 1 to 9, m1 and m2 each independently represent an integer of 1 to 9.

9. The polysaccharide derivative according to any one of claims 1 to 8, wherein the modification rate of the polysaccharide derivative with the group represented by the following formula (A) is 0.01 to 1:

10. A polysaccharide derivative-drug conjugate comprising a drug containing a primary amino group and the polysaccharide derivative according to any one of claims 1 to 9, A polysaccharide derivative-drug conjugate, in which a structure represented by the following formula (D) is formed between a primary amino group contained in the drug and a group represented by formula (A) contained in the polysaccharide: 【Chemical Formula 67】 (wherein Drug represents the drug moiety excluding the primary amino group, Ring P, R １ , and * are as defined in claim 1.)

11. The polysaccharide derivative-drug conjugate of claim 10, wherein the drug is released from the polysaccharide derivative-drug conjugate under low pH conditions.

12. The polysaccharide derivative-drug conjugate according to claim 10 or 11, wherein the drug is at least one selected from the group consisting of low molecular weight compounds, medium molecular weight compounds, peptides, nucleic acids, nucleic acid derivatives, aptamers, vitamins, monoamines, amino acids, polyamines, antibodies, fluorescent dyes, and contrast agents.

13. A crosslinked structure comprising the polysaccharide derivative according to any one of claims 1 to 9, wherein the polysaccharide derivative is crosslinked via a crosslinking group.

14. A crosslinked structure comprising the polysaccharide derivative according to any one of claims 1 to 9 and at least one of an amino group-containing polymer and an amino group-containing low molecular weight compound, each of which contains two or more primary amino groups, hydrazide groups, or aminooxy groups, wherein the primary amino groups, hydrazide groups, or aminooxy groups contained in the amino group-containing polymer and the amino group-containing low molecular weight compound are covalently bonded to a group represented by formula (A) contained in the polysaccharide derivative via a Schiff base.

15. The crosslinked structure according to claim 14, wherein the amino group-containing polymer is at least one selected from the group consisting of linear, branched, or dendritic polyamines; polyalkylene glycols substituted with amino groups, hydrazide groups, or aminooxy groups; polyallylamine; polyvinylamine; polyacrylamine; amino group-containing polysaccharides; amino group-containing proteins; and polyamino acids.

16. A crosslinked structure-drug conjugate comprising a drug containing a primary amino group and the crosslinked structure according to any one of claims 13 to 15, wherein the primary amino group contained in the drug and the group represented by formula (A) contained in the crosslinked structure are covalently bonded via a Schiff base.

17. A composition comprising the polysaccharide derivative according to any one of claims 1 to 9, the polysaccharide derivative-drug conjugate according to any one of claims 10 to 12, the crosslinked structure according to any one of claims 13 to 15, or the crosslinked structure-drug conjugate according to claim 16.

18. A gel, sponge, film, or capsule comprising the polysaccharide derivative according to any one of claims 1 to 9, the polysaccharide derivative-drug conjugate according to any one of claims 10 to 12, the crosslinked structure according to any one of claims 13 to 15, the crosslinked structure-drug conjugate according to claim 16, or the composition according to claim 17.

19. A tissue-adhesive material comprising the polysaccharide derivative according to any one of claims 1 to 9, the polysaccharide derivative-drug conjugate according to any one of claims 10 to 12, the crosslinked structure according to any one of claims 13 to 15, or the crosslinked structure-drug conjugate according to claim 16.

20. A drug transport carrier or separation material comprising the polysaccharide derivative according to any one of claims 1 to 9 or the crosslinked structure according to any one of claims 13 to 15.

21. A method for producing a polysaccharide derivative represented by the following formula (C1): A method comprising: subjecting a polysaccharide containing a carboxyl group and a compound (a1) represented by the following formula (a1) to a condensation reaction in a solvent: 【Chemistry 68】 (In the formula, L １ is a single bond, C １－６ Alkylene, and -(CH ２ CH ２ O) n -, where n is an integer from 1 to 9; Rings P and R １ is as defined in claim 1.

22. The method according to claim 21, wherein the reaction between the compound (a1) and the polysaccharide is carried out under conditions of pH 5 to 10.

23. A method for producing the polysaccharide derivative-drug conjugate according to any one of claims 10 to 12, comprising mixing the polysaccharide derivative according to any one of claims 1 to 9 with a drug containing a primary amino group in a solvent.

Images