Aldehyde-modified hyaluronic acid, method for preparing the same, and its uses

Abstract

The present invention relates to modified hyaluronic acid derivatives in which the -CH2-OH group of at least one N-acetyl-D-glucosamine unit is modified to an aldehyde group having the structure -CH2-O-CH2-CHO, a method for preparing the same, and uses thereof.

Description

[Technical Field]

[0001] The present invention relates to modified hyaluronic acid derivatives, methods for preparing them, and their use. Novel modified hyaluronic acid derivatives are characterized in that at least one -CH2-OH group of an N-acetyl-D-glucosamine unit is modified with an aldehyde group having the structure -CH2-O-CH2-CHO. [Background technology]

[0002] Injectable fillers are used today in many therapeutic and cosmetic applications to add volume to soft tissues. In cosmetic medicine, dermal fillers are increasingly used for rejuvenation of selected areas of the face and body. They allow for the enhancement of facial features (e.g., cheeks and lips), reduction of wrinkles (e.g., nasolabial folds) and muscle definition, and can restore some of the volume and elasticity lost in the skin and underlying tissues that occurs with aging. This makes the skin appear smoother and plumper, resulting in a more youthful appearance.

[0003] A wide variety of materials are known for use as soft tissue fillers. Most of these substances are absorbed into the body and therefore have a temporary effect (approximately 3 to 18 months) (e.g., collagen, hyaluronic acid (HA), poly-L-lactic acid (PLLA)). There are also some permanent (i.e., non-absorbable) fillers, such as FDA-approved fillers based on polymethyl methacrylate beads (PMMA microspheres). Some of these known soft tissue fillers contain lidocaine (a local anesthetic) which is added to reduce pain or discomfort associated with the injection.

[0004] Today, the most commonly used material for soft tissue fillers worldwide is hyaluronic acid (HA). This is due to its excellent ability to provide volume and a good safety profile. HA is a naturally occurring glycosaminoglycan found in the extracellular matrix, such as the dermis, and is composed of alternating residues of β-D-(1→3) glucuronic acid (GlcUA) and β-D-(1→4)-N-acetylglucosamine (GlcNAc). HA binds with water and swells into a gel, providing a smoothing / filling effect. In most cases, HA used in skin fillers is cross-linked for longer-lasting survival in the body (up to 18 months). [ka]

[0005] Various crosslinking approaches are known in the art for covalently joining the polymer chains of polysaccharide (e.g., HA) molecules to form a filler matrix having intermolecular and intramolecular crosslinks. A widely used approach is chemical crosslinking with chemical agents. These agents generally react with the hydroxyl and / or carboxyl functional groups of the polysaccharide. Commonly used crosslinking agents include divinyl sulfone (DVS), bifunctional or polyfunctional epoxides (e.g., 1,4-butanediol diglycidyl ether (BDDE)), 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE) and 1,2,7,8-diepoxyoctane (DEO)), PEG-based crosslinking agents (e.g., pentaerythritol tetraglycidyl ether (PETGE)), biscarbodiimide (BCDI) (e.g., phenylene bis-(ethyl)-carbodiimide and 1,6-hexamethylene bis-(ethylcarbodiimide)), diamine or multi-amine crosslinking agents (e.g., hexamethylenediamine (HMDA) and 3-[3-(3-aminopropoxy)-2,2-bis(3-aminopropoxymethyl)-propoxy]-propylamine (4AA)), bis(sulfosuccinimidyl)suberate (BS), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, epichlorohydrin, aldehydes (e.g., formaldehyde and glutaraldehyde), and hydrazides (bis-, tris- and polyvalent hydrazide compounds, e.g., adipic acid dihydrazide (ADH)), but are not limited thereto.

[0006] Other methods used for crosslinking injectable polysaccharide hydrogels include photochemical crosslinking of methacrylated polymers (Moller et al., Int.J.Artif.Organs 2011,34:93-102), Michael addition crosslinking (Shu et al., Biomacromolecules 2002,3:1304-1311), Schiff base reaction crosslinking (Tan et al., Biomaterials 2009,30:2499-2506), and "click" chemical approaches using reactions such as thiolene reactions or azide-alkyne cycloaddition (Hoyle et al., Chem.Soc.Rev.2010,39:1355-1387, van Dijk et al., Bioconjug.Chem.2009,20:2001-2016). Polysaccharide-based photocrosslinking fillers for soft tissue enhancement are also known in the art (see, for example, U.S. Patent Application Publication 2011 / 069475). Furthermore, the formation of “internal” intermolecular and / or intramolecular ester-based crosslinks (referred to as “self-crosslinking polymers” or “ACPs”) by esterifying the carboxyl functional groups of acidic polysaccharides with hydroxyl groups of the same or different polysaccharide molecules has been investigated in the art. In addition, U.S. Patent Application Publication 2006 / 0084759 describes tyramine-modified and crosslinked HA hydrogel materials, where crosslinking is achieved via peroxidase-mediated dityramine linking that can be performed in vivo.

[0007] The most commonly used approach for preparing crosslinked fillers is to perform crosslinking using BDDE. However, BDDE and its degradation products are low molecular weight compounds and are toxicologically harmful, so they need to be completely removed from the final product. The upper limit of BDDE content in dermal fillers (in most markets) is less than 2 ppm. Therefore, it is necessary to ensure that virtually all BDDE molecules (and their degradation products) are removed from the product. Thus, an important but very time-consuming part of the process for preparing BDDE crosslinked fillers is the purification after crosslinking. A widely used purification technique is dialysis. However, although dialysis is very effective in removing unwanted toxic impurities (i.e., BDDE and its degradation products), it is very time-consuming, i.e., often taking several days. Therefore, the production of BDDE crosslinked fillers consists of a very complex number of process steps, taking a long time, most often several days, and significantly increasing the cost of the final product. Also, since dialysis is mostly done manually, it can be a potential cause of gel contamination. Therefore, there is a great need for a method for preparing crosslinked fillers that overcomes the above disadvantages.

[0008] Also, conventional preformed hydrogels often have the drawback that they are very viscous, which hinders their injection through thin needles. One approach to address this problem is to use in situ gelling hydrogel compositions. These compositions are injected into tissues in liquid form rather than in the form of preformed gels and crosslink at the injection site to form an in situ crosslinked gel. Another approach to address the aforementioned problem is to add a lubricating phase (e.g., a non-crosslinked polysaccharide, such as HA) to the crosslinked hydrogel, which reduces the injection force. However, it has been shown that the lubrication step may not be necessary if the injected hydrogel has a fairly low viscosity.

[0009] Many in situ gels utilize aldehyde-modified polysaccharide (e.g., HA) derivatives that crosslink in situ with other polysaccharide (e.g., HA) derivatives to form crosslinked gels. Furthermore, using aldehyde-modified polysaccharide (e.g., HA) derivatives that form crosslinked gels upon reaction with other complementary polysaccharide (e.g., HA) derivatives eliminates the need for BDDE, thus overcoming the aforementioned problems related to BDDE crosslinking. Therefore, aldehyde-modified polysaccharide (e.g., HA) derivatives are promising candidates for preparing BDDE-free crosslinked fillers.

[0010] In this regard, refer to WO00 / 016818, which discloses the in situ formation of hydrogels by crosslinking aldehyde (or amine) functionalized derivatives of HA (e.g., adipic acid dihydrazide-HA) with homo- or heterobifunctional crosslinking agents (e.g., bifunctional N-hydroxysuccinimide ester crosslinking agents such as (SPA)1-PEG). Furthermore, WO2011 / 100469 discloses a crosslinked HA hydrogel for use as a glassy alternative biomaterial, prepared by reacting oxidized HA having an aldehyde functional group (oxi-HA) with a dihydrazide crosslinking agent, such as adipic acid dihydrazide (ADH). WO2009 / 108100 discloses an HA-based hydrogel prepared in situ by mixing aldehyde-modified HA and a hydrazide-modified polyvinyl alcohol (PVAH) crosslinking reagent to form a crosslinked structure exhibiting multiple hydroxyl groups.

[0011] Furthermore, WO2011 / 096475 discloses a method for preparing aldehyde-HA derivatives containing an aldehyde group by oxidizing the primary hydroxyl group of C6 of a glucosamine repeat unit using a TEMPO(2,2,6,6-tetramethyl-piperidinyloxyl) / co-oxidant system, and the use of said aldehyde-HA derivatives for preparing crosslinked HA hydrogels by reacting them with a diamine compound (e.g., hexanediamine) or amine-HA (e.g., hexanediamine-substituted HA). Furthermore, WO2017 / 063749 discloses the use of a first hyaluronic acid derivative and a second hyaluronic acid derivative for in situ formation of crosslinked hydrogels at a target site, the first hyaluronic acid derivative being functionalized at the hydrazide moiety and the second hyaluronic acid derivative being functionalized at the aldehyde moiety. A second aldehyde-functionalized hyaluronic acid derivative can be prepared by oxidizing a primary hydroxyl (-CH2OH) group to an aldehyde (-CHO) group.

[0012] Furthermore, non-patent literature also discloses gels formed in situ using aldehyde-modified polysaccharide derivatives. For example, Dahlmann et al. (Biomaterials 2013, 34:940-951) describe a fully defined in situ crosslinkable alginate and HA hydrogel for myocardial tissue engineering. The hydrogel is prepared by reacting aldehyde and hydrazide-functionalized alginate and HA in the presence of human type I collagen and neonatal rat cardiac cells (NRHC) to generate a hydrazone-crosslinked hydrogel-based bioartificial heart tissue. In addition, Ossipov et al. (Biomacromolecules 2010, 11:2247-2254) disclose the synthesis of hydrazide-functionalized HA using a specific symmetrical bifunctional reagent having a central divalent protecting group that can cause an amide-type reaction with the carboxylate residue of HA in aqueous solution. Hydrazide-functionalized HA can be used for in situ formation of hydrazone HA hydrogels by mixing it with aldehyde HA derivatives.

[0013] Varghese et al. (J.Am.Chem.Soc.2009,131:8781-8783) reported on HA derivatives doubly functionalized with a hydrazide group and an aminomethylenebisphosphonate group that can covalently bond with bisphosphonates (BP; anti-osteoclastic and antitumor small molecule drugs). When this doubly functionalized HA is mixed with aldehyde-functionalized HA, it results in the in situ formation of an injectable HA hydrogel for controlled release of BP drugs at the transplant site. Oommen et al. (Adv.Funct.Mater 2013,323:1273-1280) describe an HA hydrogel prepared by mixing an HA-aldehyde derivative with a carbodihydrazide (CDH)-functionalized HA derivative to obtain an HA hydrogel with hydrazone linkages. Furthermore, it has been reported that in situ HA hydrogel formation in the presence of therapeutic proteins (e.g., recombinant human growth factor BMP-2) has provided a hydrogel for in vivo applications that can deliver growth factors for bone tissue regeneration.

[0014] However, the methods commonly applied to add aldehyde functional groups to hyaluronic acid exhibit several drawbacks. For example, introducing aldehyde groups by periodic acid oxidation of unfunctionalized hyaluronic acid usually disrupts the cyclic sugar rings of the polysaccharide backbone, reducing the overall stability of hyaluronic acid and adding undesirable flexibility to the polysaccharide backbone. Specifically, reactions frequently used to introduce aldehyde functional groups into hyaluronic acid, such as oxidation with sodium periodate, require long reaction times and / or are difficult to control in terms of undesirable oxidation to carboxylic acid groups.

[0015] Furthermore, there is no universal filler suitable for all applications or all patients. For example, different applications require gels with varying degrees of stiffness / hardness (often expressed as modulus of elasticity). Considering the relatively high dynamic forces generated during facial muscle movement, higher stiffness gels are generally preferable for correcting areas such as nasolabial folds and marionette lines. In contrast, lower stiffness gels are suitable for areas where resistance to deformation is not important, or where anatomical structures do not require stiffness, but volume and flexibility are important, such as the lips (see, for example, Kablik et al., Dermatol Surg, 2009, 35, 302-312).

[0016] However, the properties, such as stiffness, of hydrogels prepared from aldehyde-modified polysaccharide (e.g., HA) derivatives and complementary polysaccharide (e.g., HA) derivatives (crosslinked in situ, i.e., after injection of the two complementary polysaccharide derivatives; or in vitro, i.e., crosslinked before injection) depend heavily on the properties of the specific modified polysaccharide (e.g., modified hyaluronic acid), particularly its molecular weight and degree of modification. For example, aldehyde-modified hyaluronic acid with a very low degree of aldehyde modification (i.e., a very small number of aldehyde groups) results in a relatively small amount of crosslinking, and therefore the resulting gel is quite flexible. Consequently, various aldehyde-modified hyaluronic acids are used in a variety of applications. Aldehydide-modified hyaluronic acid (e.g., aldehyde-modified hyaluronic acid with a low degree of modification) can be used in lip fillers to produce a "more flexible" hydrogel, while aldehyde-modified hyaluronic acid (e.g., aldehyde-modified hyaluronic acid with a high degree of modification) can be used in fillers for nasolabial folds or marionette lines to produce a "harder" hydrogel.

[0017] Therefore, there is a great desire for methods to produce modified hyaluronic acid, such as aldehyde-modified hyaluronic acid, for in vitro or in situ formation of cross-linked gels, which allows for modification (fine-tuning) of the properties of the resulting aldehyde-modified hyaluronic acid. In particular, there is a desire for methods that allow for easy modification of the properties of the resulting aldehyde-modified hyaluronic acid without significantly altering the overall synthesis method, i.e., methods that keep most parameters constant while changing only easily modifiable parameters such as the starting material concentration, the amount of oxidizing agent, and the reaction time. Such methods would allow for the production of various aldehyde-modified hyaluronic acid exhibiting various properties (e.g., different degrees of modification and molecular weight) using the same or similar experimental setup.

[0018] Problems that the invention aims to solve Considering the above, an object of the present invention is to provide a versatile and convenient method for producing aldehyde-modified hyaluronic acid suitable for in vitro or in situ formation of cross-linked hyaluronic acid-based hydrogels. Furthermore, an object of the present invention is to provide such aldehyde-modified hyaluronic acid particularly suitable for cosmetic applications. Another object of the present invention is to provide hydrogels prepared from aldehyde-modified hyaluronic acid particularly suitable for cosmetic applications. [Overview of the project]

[0019] The above objective is achieved by providing novel modified hyaluronic acid derivatives (also referred to herein as "aldehyde-modified hyaluronic acid derivatives" or "aldehyde-modified hyaluronic acid") and methods for preparing them. The novel modified hyaluronic acid derivatives are useful for in vitro and in situ formation of crosslinked hydrogels when reacted with a second polysaccharide derivative having at least one -CH2-OH group of an N-acetyl-D-glucosamine unit modified to an aldehyde group having the structure -CH2-O-CH2-CHO, and containing one or more nucleophilic functional groups capable of forming covalent bonds with one or more aldehyde groups of the novel modified hyaluronic acid derivative.

[0020] The two functionalized polysaccharide derivatives (i.e., the novel modified hyaluronic acid derivative and the second polysaccharide derivative) can be injected simultaneously in liquid form, thereby enabling simultaneous injection with low extrusion force even through a fine needle. Preferably, in situ gel formation does not produce harmful byproducts. The only byproduct is water, which is readily absorbed by the formed hydrogel and / or surrounding tissue. Furthermore, the hydrogel formed in situ possesses desirable properties with respect to tissue integration, skin improvement, tissue shaping ability, and volume-adding ability.

[0021] Furthermore, surprisingly, it was found that the novel aldehyde-modified hyaluronic acid derivatives are suitable for the in vitro preparation of crosslinked gels via reaction with a second complementary polysaccharide derivative, preferably also a hyaluronic acid derivative. In particular, it was found that the properties of such prepared gels can be controlled not only by the properties of a single derivative (e.g., degree of modification and molecular weight) but also by adjusting the concentration of each polysaccharide derivative in the crosslinking medium.

[0022] Advantageously, aldehyde groups with the structure -CH2-O-CH2-CHO are long enough to provide good sterically usable crosslinking, but short enough not to add excessive flexibility to the crosslinked gel. This makes novel modified hyaluronic acid derivatives particularly suitable for in situ formation of gels covering a wide range of stiffnesses. For example, using novel modified hyaluronic acid derivatives with a high degree of modification (i.e., a large number of aldehyde groups) results in the formation of numerous relatively short crosslinks (due to the large number of sterically usable aldehyde groups), thereby leading to the formation of a relatively stiff gel.

[0023] The modified hyaluronic acid derivative of the present invention can be prepared from glycerol-modified hyaluronic acid, which has at least one N-acetyl-D-glucosamine unit -CH2-OH group, and a -CH2-O-CH2-CHOH-CH2OH group in its structure -CH2-O-CH 2-This novel method for preparing new modified hyaluronic acid derivatives is characterized by modification of the -CH2-O-CH2-CHOH-CH2OH portion of the formula by oxidation to an aldehyde group containing CHO. This novel method avoids the drawbacks of methods commonly used to prepare aldehyde-modified hyaluronic acid derivatives, such as long reaction times, dramatic reduction in the molecular weight of hyaluronic acid, or undesirable oxidation to the carboxylic acid group.

[0024] This method demonstrates that the properties of new modified hyaluronic acid derivatives can be easily adjusted by simply changing basic parameters such as the starting material concentration, the amount of oxidizing agent, and the reaction time. For example, by changing the starting material concentration, the amount of oxidizing agent, and / or the reaction time, it is possible to fine-tune the degree of modification and / or molecular weight of the resulting new modified hyaluronic acid derivative.

[0025] In a first embodiment, the present invention relates to a modified hyaluronic acid derivative in which at least one -CH2-OH group of an N-acetyl-D-glucosamine unit is modified with an aldehyde group having the structure -CH2-O-CH2-CHO. Preferably, the modified hyaluronic acid derivative has the following structure [ka] (wherein the formula, Ac represents -C(O)CH3, and R is selected from hydrogen, alkali metal ions, preferably Na, or alkaline earth metal ions) comprising at least one disaccharide unit.

[0026] In a second aspect, the present invention relates to a method for preparing a modified HA derivative of the present invention. The method comprises the steps of: a) providing a glycerol-modified hyaluronic acid characterized in that the -CH2-OH group of at least one N-acetyl-D-glucosamine unit is modified to a portion of the following formula -CH2-O-CH2-CHOH-CH2OH; b) dissolving the glycerol-modified hyaluronic acid in an aqueous medium to obtain solubilized glycerol-modified hyaluronic acid; and c) reacting the solubilized glycerol-modified hyaluronic acid with an oxidizing agent, preferably periodic acid, more preferably sodium periodate, to convert at least a portion of the -CH2-O-CH2-CHOH-CH2OH group to an aldehyde group having the formula -CH2-O-CH2-CHO, thereby obtaining an aldehyde-modified hyaluronic acid derivative.

[0027] In a third embodiment, the present invention relates to a modified hyaluronic acid derivative obtained by the method of the present invention.

[0028] In a fourth aspect, the present invention relates to the use of the modified hyaluronic acid derivative of the present invention or a modified hyaluronic acid derivative obtained by the method of the present invention for in situ formation of crosslinked hydrogels in cosmetic applications.

[0029] In a fifth aspect, the present invention relates to the use of a modified hyaluronic acid derivative of the present invention or a modified hyaluronic acid derivative obtained by the method of the present invention for the formation of a pre-formed crosslinked hydrogel.

[0030] In a sixth aspect, the present invention relates to a modified hyaluronic acid derivative of the present invention, or a modified hyaluronic acid derivative obtained by the method of the present invention, for use in the in situ formation of cross-linked hydrogels for therapeutic applications.

[0031] Modified hyaluronic acid derivatives are generally used together with a second polysaccharide derivative comprising one or more nucleophilic functional groups capable of forming a covalent bond with one or more aldehyde groups of the modified hyaluronic acid derivative, wherein the second polysaccharide is preferably a hyaluronic acid derivative, the nucleophilic functional group is preferably a hydrazide functional group, and the second polysaccharide more preferably has the following structure: [ka] A hyaluronic acid derivative comprising at least one disaccharide unit having (wherein "Ac" is as defined above).

[0032] In the seventh embodiment, the present invention relates to the following structural units: [ka] This relates to a cross-linked hydrogel containing (wherein "Ac" and R are as defined above).

[0033] In an eighth aspect, the present invention relates to a crosslinked hydrogel obtained by contacting an aldehyde-modified HA derivative of the present invention or a modified hyaluronic acid derivative obtained by the method of the present invention with a second polysaccharide derivative comprising one or more nucleophilic functional groups capable of forming a covalent bond with one or more aldehyde groups of the modified HA derivative as defined herein.

[0034] In a ninth aspect, the present invention relates to a method, preferably a cosmetic method, for preparing a crosslinked hydrogel, the method comprising: a) providing a first precursor solution comprising a modified hyaluronic acid derivative of the present invention or a modified hyaluronic acid derivative obtained by the method of the present invention, and separately a second precursor solution comprising a second polysaccharide derivative as defined herein; b) mixing the first precursor solution and the second precursor solution into an in situ crosslinkable mixture; and c) injecting the in situ crosslinkable mixture into a target site in the body of a patient to form a crosslinked gel at the target site.

[0035] In a tenth embodiment, the present invention relates to a method for preparing a crosslinked hydrogel, preferably a pre-formed crosslinked hydrogel.

[0036] In an eleventh aspect, the present invention relates to a kit for in situ formation of a crosslinked hydrogel, comprising: (i) a first container containing a first precursor solution comprising a modified hyaluronic acid derivative as herein or a modified hyaluronic acid derivative obtained by the method of the present invention; (ii) a second container containing a second precursor solution comprising a second polysaccharide derivative as defined herein; and optionally (iii) instructions for use.

[0037] Specific embodiments of the present invention are described in the appended claims. [Brief explanation of the drawing]

[0038] For a more complete understanding of the present invention, please refer to the following description and accompanying drawings.

[0039] [Figure 1] This figure shows an exemplary chromatogram obtained to determine the molecular weight. [Figure 2] This figure shows an exemplary 1H NMR spectrum of a tyrosine-labeled aldehyde-modified hyaluronic acid derivative. [Figure 3] This figure shows the storage modulus (G') of gels 1 and 2 stored at 40°C. [Figure 4] This figure shows the loss coefficient (tanδ) of gels 1 and 2 stored at 40°C. [Modes for carrying out the invention]

[0040] This invention is based on the remarkable discovery that aldehyde-modified hyaluronic acid can be easily prepared from glycerol-modified hyaluronic acid. In particular, it has been found that the preparation of aldehyde-modified hyaluronic acid from glycerol-modified hyaluronic acid does not exhibit the drawbacks typically associated with the preparation of aldehyde-modified hyaluronic acid, such as a dramatic decrease in molecular weight, long reaction times, and undesirable side reactions (e.g., oxidation to carboxylic acid). Furthermore, it has been found that the properties of the resulting aldehyde-modified hyaluronic acid derivative can be easily modified (fine-tuned) by simply changing basic parameters such as the starting material concentration, the amount of oxidizing agent, and the reaction time.

[0041] In particular, it was found that the degree of modification and molecular weight of the resulting aldehyde-modified hyaluronic acid derivative can be fine-tuned by changing the starting material concentration (i.e., the concentration of glycerol-modified hyaluronic acid), the amount of oxidizing agent, and the reaction time. This is particularly advantageous because the degree of modification and molecular weight of the resulting aldehyde-modified hyaluronic acid derivative directly affect the gelling performance of the aldehyde-modified hyaluronic acid derivative. Therefore, aldehyde-modified hyaluronic acid derivatives having different properties, and thus yielding crosslinked gels with different properties, such as hardness, can be prepared by a single general synthetic method.

[0042] Furthermore, the modified hyaluronic acid derivatives of the present invention were found to be particularly suitable for both in situ and in vitro formation of crosslinked hydrogels, i.e., exhibiting good crosslinking properties. In particular, the modified hyaluronic acid derivatives of the present invention were found to exhibit good crosslinking properties with a second polysaccharide derivative containing one or more nucleophilic functional groups capable of forming covalent bonds with one or more aldehyde groups of the modified hyaluronic acid derivative. In this regard, the modified hyaluronic acid derivatives of the present invention and the second polysaccharide derivative were found to crosslink rapidly and efficiently to form a covalently crosslinked hydrogel, for example, at a target site in the body. No additives, catalysts, pH switches, UV irradiation, or other external stimuli (or "triggers") are required to induce the crosslinking reaction. In particular, no crosslinking agents are used and are not required. The only byproduct produced by the crosslinking reaction is typically water, which is readily absorbed by the hydrogel and / or surrounding tissue when crosslinking occurs in situ.

[0043] As used herein, the term “glycerol-modified hyaluronic acid” refers to hyaluronic acid characterized in that at least one N-acetyl-D-glucosamine unit is modified by a -CH2-OH group of the formula -CH2-O-CH2-CHOH-CH2OH moiety. Glycerol-modified hyaluronic acid is commercially available from “htl biotech” in France, or can be prepared by grafting glycidol onto hyaluronic acid according to the following reaction scheme. [ka]

[0044] Preferably, other groups besides the -OH group bonded to the C6 carbon of the GlcNac unit are not modified by reaction with glycidol. Preferably, the glycerol-modified hyaluronic acid contains 1 to 100, preferably 2 to 50, more preferably 5 to 20, and most preferably 10 to 20 -CH2-O-CH2-CHOH-CH2OH groups per 100 N-acetyl-D-glucosamine units present in the hyaluronic acid. The degree of modification has been shown to be particularly suitable for subsequently preparing the aldehyde-modified hyaluronic acid derivative of the present invention having a desired degree of (aldehyde) modification for preparing crosslinked hydrogels for cosmetic applications by reaction with complementary secondary polysaccharide derivatives, preferably hyaluronic acid derivatives. Within the scope of the present invention, it should be noted that other functional groups of hyaluronic acid, namely other -OH and -COOH groups, in particular other (secondary) -OH groups, can be glycerol-modified to a much lower degree. However, within the scope of the present invention, the glycerol-containing portion is preferably present only in the C6 carbon of the GlcNac unit, essentially only in it, or mainly in it.

[0045] As used herein, the term “in situ” means the site of administration, i.e., inside the patient’s body. Therefore, in order to form a hydrogel “in situ,” i.e., at the site of administration, the modified hyaluronic acid derivative of the present invention is generally administered co-administered with a second polysaccharide derivative containing one or more nucleophilic functional groups that can form a covalent bond with one or more aldehyde groups of the modified hyaluronic acid derivative of the present invention, or these compounds are separately applied together to a specific site (target site) inside the patient’s body, e.g., a site requiring tissue enhancement for cosmetic reasons, and covalently crosslinked at the site of co-injection. In the present invention, the terms “in situ” and “in vivo” may be used interchangeably. “Patient” in the sense of the present invention can be any individual or subject, e.g., a mammal, preferably a human, that requires “treatment” for a specific medical condition, state, or disease, such as for facial and cosmetic purposes. Therefore, the term “treatment,” as used herein, should be understood to include not only therapeutic / medical treatments but also, for example, facial and cosmetic treatments.

[0046] As used herein, the term "in vitro" means outside the body of a human or animal. Similarly, terms such as "pre-formed hydrogel" and "pre-formed gel" refer, as used herein, to hydrogels formed outside the body of a human or animal. Therefore, it should be understood that both the terms "pre-formed" and "in vitro" indicate that the hydrogel is formed outside the body of a human or animal before injection. Thus, a gel prepared "in vitro" is a "pre-formed" gel, i.e., a gel formed outside the body of a human or animal.

[0047] As used herein, the term “artificial container” refers to any container that is not a human or animal body or part thereof. Preferably, the artificial container is made of a non-biological material, preferably glass or plastic material, more preferably glass.

[0048] In relation to the present invention, the term “simultaneous injection” generally means that the modified hyaluronic acid derivative of the present invention and the second polysaccharide derivative are injected into a target site in the patient’s body as a single liquid composition, e.g., a solution. As used herein, the terms “injectable” or “inject” indicate that the in situ hydrogel-forming composition can be dispensed from a syringe or syringe system. In particular, the term “simultaneous injection” preferably means that the modified hyaluronic acid derivative of the present invention and the second polysaccharide derivative are mixed, especially uniformly, before exiting the tip of the needle and entering the target site in the patient’s body, and then injected as a mixture into the target site in the patient’s body. In the present invention, the terms “injection” or “simultaneous injection” may refer to intradermal, intercutaneous, or subcutaneous injection, or subcutaneous injection. Furthermore, the term “needle,” as used herein, is intended to include, or be synonymous with, a “cannula” or any other needle-like object suitable for injection.

[0049] The terms “hydrogel” or “gel,” as used herein, refer to a water-swollen three-dimensional network consisting of covalently crosslinked polymer chains. Preferably, the crosslinked (or “gelled”) hydrogel is cohesive. In the sense of the present invention, the terms “cohesive” or “cohesive” are defined as the ability of a material (e.g., a hydrogel) not to dissociate because its molecules are affinity for one another. Cohesiveness is an important property of gel implants (e.g., in situ gelled hydrogels as described herein) and is considered necessary for the integrity of the gel, so that the solid and liquid phases of the gel remain intact. In connection with the present invention, the cohesiveness of polysaccharide hydrogels, particularly HA-based hydrogels, may be determined using the Gavard-Sundaram cohesiveness scale (Sundaram et al., Plast. Reconstr. Surg. 136:678-686, 2015).

[0050] The terms “spontaneous” or “instinctively,” as used herein, are intended to mean that the aldehyde group of the modified hyaluronic acid derivative of the present invention and the nucleophilic group of the second polysaccharide derivative form a covalent bond without external stimuli (also called “triggers”) such as heat or UV light. In particular, it has been found that the hydrogel can be formed spontaneously under in vivo conditions, i.e., after co-injection into a target site in the patient’s body, without external stimuli (also called “triggers”) such as heat or UV light, and that a cross-linked polysaccharide hydrogel can be formed in situ at the target site.

[0051] Within the scope of the present invention, hydrogels formed in situ (or in vivo) are generally suitable as, used as, and / or function as soft tissue fillers. The term “soft tissue filler,” as used herein, generally refers to a material designed to fill cavities and / or add volume to an area of ​​soft tissue defect. This includes, for example, the enhancement, filling, or replacement of soft tissue. In this specification, the term “soft tissue” generally refers to tissues that connect, support, or surround other structures and organs of the body. Soft tissues include, for example, muscles, tendons (bands of fibers that connect muscles to bones), fibrous tissue, fat, blood vessels, nerves, and synovial tissue (tissue around joints). In relation to the present invention, soft tissue fillers are preferably skin fillers.

[0052] In a first embodiment, the present invention relates to a modified hyaluronic acid derivative in which at least one -CH2-OH group of an N-acetyl-D-glucosamine unit is modified with an aldehyde group having the structure -CH2-O-CH2-CHO.

[0053] As described above, this modified hyaluronic acid derivative was found to exhibit good crosslinking properties. Although we do not wish to be bound by any particular theory, the good crosslinking properties are attributed to the aldehyde group introduced by the method of the present invention, i.e., the aldehyde group having the structure -CH2-O-CH2-CHO, and the modification of at least one GlcNAc unit with -CH2 2This is thought to be due to the steric properties of the -OH group. Specifically, the aldehyde group is considered to be long enough to provide good steric usability for crosslinking, but short enough not to add excessive flexibility to the crosslinked gel.

[0054] Preferably, the modified hyaluronic acid derivative does not contain any chemical modifications other than an aldehyde group, preferably an aldehyde group present on the C6 carbon atom of a GlcNAc unit, having the structure -CH2-O-CH2-CHO.

[0055] Preferably, the modified hyaluronic acid derivatives of the present invention have a degree of modification of 1.0% to 20.0%, more preferably 1.0% to 15.0%, even more preferably 1.0% to 10.0%, even more preferably 1.5% to 10.0%, even more preferably 1.5% to 8.0%, even more preferably 1.8% to 7.0%, and even more preferably 2.0% to 6.9%. The degree of modification (MoD) is defined as the number of -CH2-O-CH2-CHO groups divided by the total number of N-acetyl-D-glucosamine units present in the modified hyaluronic acid derivative. For example, a MoD of 15.0% means that the modified hyaluronic acid derivative contains 15 -CH2-O-CH2-CHO groups per 100 N-acetyl-D-glucosamine units. For example, a MoD of 37.0% means that the modified hyaluronic acid derivative contains 37 -CH2-O-CH2-CHO groups per 100 N-acetyl-D-glucosamine units. This somewhat lower degree of modification has been found to be particularly suitable for preparing hydrogels for cosmetic applications where relatively low viscosity hydrogels are required. Furthermore, such low viscosity hydrogels can be injected through a fine needle, which allows for in vitro preparation of the hydrogel (i.e., formation of a pre-formed hydrogel) where its properties can be thoroughly determined and checked before injection, whereas this is not possible when the hydrogel is prepared in situ.

[0056] The hardness (or rigidity; e.g., indicated by the elastic modulus) of the hydrogel formed from the modified hyaluronic acid derivative of the present invention depends on the number and density of crosslinks formed in the hydrogel, and since the number and density of crosslinks formed in the hydrogel directly depend on the degree of modification of the modified hyaluronic acid derivative of the present invention, the degree of modification directly affects the hardness of the hydrogel formed from the modified hyaluronic acid derivative of the present invention. Therefore, the degree of modification is an important feature of the modified hyaluronic acid derivative of the present invention.

[0057] The degree of modification (MoD) can be determined, among other things, 1 by spectroscopic analysis and / or spectrometric analytical methods such as 1H NMR, UV / Vis and IR, titration, HPLC, SEC, viscosity, etc. Conveniently, the degree of modification is 1 determined by 1H NMR. An exemplary method for determining MoD is shown in the Examples section.

[0058] Molecular weight (also called molar mass) is another important property of the modified hyaluronic acid derivative that directly affects the properties of the gel formed therefrom. Preferably, the modified hyaluronic acid derivative has a weight average molecular weight of 0.1 to 2.5 MDa, more preferably 0.2 to 1.5 MDa, even more preferably 0.4 to 1.3 MDa, and even more preferably 0.6 to 1.1 MDa.

[0059] All numerical values in this specification representing the "molecular weight", "molar mass", "mean molecular weight", "mean molar mass", "average molecular weight", and "average molar mass" of a polysaccharide (e.g., HA) are understood to refer to the weight average molecular weight (or mass average molecular weight or weight average molar weight) in Daltons (Da) or M w (where w is by weight). The mass average molecular weight (M W ) is defined as follows: M w =Σ i N i Mi 2 / Σ i N i M i (In the formula, N i is the molar mass M i (This is the number of numerators.)

[0060] Various methods can be applied herein to determine the molecular weight of HA, including intrinsic viscosity measurement (e.g., European Pharmacopoeia 7.0-Hyaluronic Acid monograph No.1472,01 / 2011), capillary electrophoresis (CE) (e.g., Kinoshita et al., Biomed. Chromatogr., 2002,16:141-45), gel permeation chromatography (GPC) (e.g., Kim et al., Food Chem., 2008,109:63-770), and multi-angle laser light scattering (SEC-MALLS) combined with size exclusion chromatography (e.g., Hokputsa et al., Eur. Biophys. J. Biophys. Lett., 2003,32:450-456).

[0061] Within the framework of the present invention, the weight-average molecular weight (M) of the HA polymer. w The retention time is preferably determined by gel permeation chromatography (GPC) or viscosity measurement by the Mark-Houwink formula. In GPC techniques, the polymer solution is eluted through a matrix of packed polymer particles at a pressure of up to several megapascals (MPa). As is well known to those skilled in the art, the retention time can be correlated with the molar mass by using a low-dispersion standard.

[0062] In relation to the present invention, the mass-average molar mass (M w ) also means that this is generally called viscosity-average molar mass or M vDespite being called the viscosity average molecular weight, it can be determined by the Mark-Houwink formula. The Mark-Houwink formula shows the relationship between intrinsic viscosity (η) and molecular weight M, making it possible to determine the molecular weight of a polymer from intrinsic viscosity data and vice versa. In the context of the present invention, the intrinsic viscosity is preferably measured according to the procedure defined in European Pharmacopoeia 7.0 (Hyaluronic Acid Monograph No. 1472, 01 / 2011). Within the framework of the present invention, the average molecular weight is preferably the viscosity-average molecular weight (M) which can be calculated from the intrinsic viscosity using the Mark-Houwink formula. n ) [η]=KxM η a [η]=m 3 Intrinsic viscosity at / kg, M η = viscosity average molecular weight, K=2.26×10 -5 , and a = 0.796, where, as described above, the intrinsic viscosity is preferably measured according to the procedure defined in European Pharmacopoeia 7.0 (Hyaluronic Acid monograph No. 1472, 01 / 2011).

[0063] The determination of molecular weight is also illustrated in the Examples section.

[0064] Preferably, the modified hyaluronic acid derivative comprises at least one disaccharide unit having the following structure. [ka] In the formula, "Ac" represents -C(O)CH3, and R is selected from hydrogen, alkali metal ions, preferably Na, or alkaline earth metal ions. It should be understood that the carboxylic acid group can exist in a deprotonated form (i.e., R=negative charge) even without a specific counterion to balance the negative charge, instead of existing in a protonated form (i.e., R=H). This situation occurs, for example, in a solution where a counterion to balance the negative charge is solvated and randomly located near the deprotonated carboxylic acid.

[0065] The modified hyaluronic acid derivative may further contain at least one disaccharide unit having the following structure. [ka] In the formula, "Ac" is as defined above, and R 1 , R 2 , R 3 and R 4 It is selected independently from H and -CH2-CHO, and R 5 R is selected from hydrogen, alkali metal ions, preferably Na, alkaline earth metal ions, and -CH2-CHO, however, 1 , R 2 , R 3 , R 4 and R 5 At least one of them is -CH2-CHO, R 1 If R is -CH2-CHO, 2 , R 3 , R 4 and R 5 At least one of them is -CH2-CHO.

[0066] Preferably, the number of aldehyde groups present on the C6 carbon atom of the GlcNAc unit (i.e., R 1 The ratio obtained by dividing (=-CH2-CHO) by the total number of aldehyde groups present in the modified hyaluronic acid derivative (including the aldehyde group present on the C6 carbon atom of the GlcNAc unit) is 0.60 to 1, preferably 0.65 to 1, more preferably 0.70 to 1, even more preferably 0.75 to 1, even more preferably 0.80 to 1, even more preferably 0.85 to 1, even more preferably 0.90 to 1, even more preferably 0.95 to 1, even more preferably 0.97 to 1, even more preferably 0.98 to 1, even more preferably 0.99 to 1, and most preferably 1.

[0067] In a second aspect, the present invention relates to a method for preparing a modified hyaluronic acid derivative of the present invention. The method comprises the steps of: a) providing a glycerol-modified hyaluronic acid characterized in that the -CH2-OH group of at least one N-acetyl-D-glucosamine unit is modified to a portion of the following formula -CH2-O-CH2-CHOH-CH2OH; b) dissolving the glycerol-modified hyaluronic acid in an aqueous medium to obtain a solubilized glycerol-modified hyaluronic acid; and c) reacting the solubilized glycerol-modified hyaluronic acid with an oxidizing agent to convert at least a portion of the -CH2-O-CH2-CHOH-CH2OH group to an aldehyde group having the formula -CH2-O-CH2-CHO, thereby obtaining an aldehyde-modified hyaluronic acid derivative.

[0068] Preferably, the oxidizing agent is periodate or lead(IV) acetate, preferably periodate. More preferably, the oxidizing agent is sodium periodate.

[0069] As described above, the method of the present invention was found not to exhibit the drawbacks typically associated with the preparation of aldehyde-modified hyaluronic acid, such as a dramatic decrease in molecular weight, long reaction times, and undesirable side reactions (e.g., oxidation to carboxylic acid). For example, even when sodium periodate, which is known to oxidize the polymer backbone and thereby lead to the breakdown of the polymer backbone and a decrease in the molecular weight of hyaluronic acid, was used as the oxidizing agent, it was found that the method of the present invention resulted in little to no oxidation of the polymer backbone.

[0070] The reason for this surprising behavior is as follows: periodates are more favorable to cis-diols than trans-diols, and oxidizing trans-diols with periodates requires relatively harsh conditions and / or long reaction times. Although the hydroxyl groups of the polymer backbone are trans-oriented to one another, the -OH groups of the glycerol moiety (e.g., -CH2-O-CH2-CHOH-CH2OH) are freely rotatable. Therefore, the vicinal diol of the glycerol moiety is sufficiently accessible for periodate oxidation, and the oxidation reaction (i.e., step c)) can be carried out under relatively short and mild conditions so that oxidation does not occur in the polymer backbone. For example, it has been found that the oxidation reaction in step c) can be carried out in less than an hour, and even in 10 minutes.

[0071] Furthermore, it was found that the properties of the modified hyaluronic acid derivative obtained in the first embodiment can be easily altered (fine-tuned) simply by changing basic parameters such as the starting material concentration, the amount of oxidizing agent, and the reaction time.

[0072] Preferably, the method of the present invention is d) Preferably by adding ethylene glycol to stop the reaction in step c); e) A step of purifying the modified hyaluronic acid derivative by precipitating the modified hyaluronic acid derivative in an organic solvent, preferably ethanol, isopropanol, or a mixture thereof, redissolving the precipitate in physiological saline, and then precipitating the modified hyaluronic acid derivative again in the organic solvent; f) further comprises one or more steps of drying the modified hyaluronic acid derivative obtained in step e).

[0073] Preferably, glycerol-modified hyaluronic acid has a weight-average molecular weight of 0.1 to 5.0 MDa, more preferably 1.0 to 3.0 MDa, more preferably 1.0 to 2.0 MDa, even more preferably 1.1 to 1.9, even more preferably 1.2 to 1.8 MDa, more preferably 1.3 to 1.7 MDa, and even more preferably 1.4 to 1.6 MDa. Glycerol-modified hyaluronic acid may also have a weight-average molecular weight of 2.0 to 5.0 MDa, or 2.5 to 5.0 MDa, or 3.0 to 5.0 MDa, or 3.0 to 4.5 MDa, or 3.0 to 4.0 MDa.

[0074] Furthermore, glycerol-modified hyaluronic acid preferably has a degree of modification of 5-25%, more preferably 10-20%. In this case, the degree of modification is defined as the number of -CH2-O-CH2-CHOH-CH2OH groups divided by the total number of N-acetyl-D-glucosamine units present in the glycerol-modified hyaluronic acid. For example, a MoD of 25.0% means that the glycerol-modified hyaluronic acid contains 25 -CH2-O-CH2-CHOH-CH2OH groups per 100 N-acetyl-D-glucosamine units. A MoD of 50.0% means that the glycerol-modified hyaluronic acid contains 50 -CH2-O-CH2-CHOH-CH2OH groups per 100 N-acetyl-D-glucosamine units. The degree of modification has been shown to be particularly suitable for preparing aldehyde-modified hyaluronic acid derivatives with a desired (somewhat low) degree of aldehyde modification for preparing cross-linked hydrogels for cosmetic applications.

[0075] Preferably, step c) is carried out at a temperature of 4 to 35°C, more preferably 15 to 35°C, even more preferably 20 to 30°C, even more preferably 20 to 25°C, even more preferably 21 to 23°C, and even more preferably about 22°C. These somewhat lower reaction temperatures have been shown to ensure that the aldehyde-modified hyaluronic acid derivatives obtained according to the present invention have a suitable degree of modification for preparing hydrogels for cosmetic applications.

[0076] Step c) is preferably carried out for a period of 5 to 120 minutes, preferably 5 to 65 minutes, more preferably 10 to 60 minutes, even more preferably 10 to 50 minutes, even more preferably 10 to 40 minutes, even more preferably 10 to 30 minutes, and even more preferably 10 to 20 minutes. These somewhat shorter reaction times have also been shown to ensure that the aldehyde-modified hyaluronic acid derivatives obtained according to the present invention have the "correct" degree of modification for preparing hydrogels for cosmetic applications.

[0077] The degree of (aldehyde) modification increased with increasing reaction time, and the molecular weight of the prepared modified hyaluronic acid derivative was found to decrease slightly with increasing reaction time.

[0078] Furthermore, it is preferable that an oxidizing agent, preferably sodium periodate, is present in an amount of 0.01 to 0.5 molar equivalents, preferably 0.01 to 0.3 molar equivalents, more preferably 0.04 to 0.3 molar equivalents, and even more preferably 0.04 to 0.1 molar equivalents, based on the molar amount of the disaccharide repeating units of glycerol-modified hyaluronic acid.

[0079] It was found that the degree of (aldehyde) modification increased with increasing amounts of oxidizing agent, while the molecular weight decreased with increasing amounts of oxidizing agent.

[0080] Furthermore, glycerol-modified hyaluronic acid is preferably present in an amount of 2 to 50 g / L, more preferably 2 to 40 g / L, more preferably 3 to 38 g / L, and more preferably 4 to 36 g / L, based on the total reaction volume.

[0081] It was found that the degree of (aldehyde) modification increased with increasing amounts of glycerol-modified hyaluronic acid, while the molecular weight decreased with increasing amounts of glycerol-modified hyaluronic acid.

[0082] In a third embodiment, the present invention relates to a modified hyaluronic acid derivative obtained by the method described in the second embodiment.

[0083] Furthermore, in this embodiment, it is preferable that the modified hyaluronic acid derivative does not contain any chemical modifications other than an aldehyde group present on the C6 carbon atom of an aldehyde group, preferably a GlcNAc unit, having the structure -CH2-O-CH2-CHO.

[0084] Furthermore, the modified hyaluronic acid derivative obtained by the method described in the second embodiment preferably has a degree of modification of 1.0% to 20.0%, more preferably 1.0% to 15.0%, even more preferably 1.0% to 10.0%, even more preferably 1.5% to 10.0%, even more preferably 1.5% to 8.0%, even more preferably 1.8% to 7.0%, and even more preferably 2.0% to 6.9%.

[0085] Furthermore, the modified hyaluronic acid derivative obtained by the method described in the second embodiment preferably has a weight-average molecular weight of 0.1 to 2.5 MDa, preferably 0.2 to 1.5 MDa, more preferably 0.4 to 1.3 MDa, and even more preferably 0.6 to 1.1 MDa.

[0086] Furthermore, the modified hyaluronic acid derivative obtained by the method described in the second embodiment preferably contains at least one disaccharide unit having the following structure. [ka] In the formula, "Ac" is as defined above, and R is selected from hydrogen, alkali metal ions, preferably Na, or alkaline earth metal ions. In this case as well, it should be understood that the carboxylic acid group can exist in a deprotonated form (i.e., R=H) even without the presence of a specific counterion to balance the negative charge, instead of in a protonated form (i.e., R=H).

[0087] Furthermore, the modified hyaluronic acid derivative obtained by the method described in the second embodiment may further comprise at least one disaccharide unit having the following structure. [ka] In the formula, "Ac" is as defined above, and R 1 , R 2 , R 3 and R 4 It is selected independently from H and -CH2-CHO, and R 5 R is selected from hydrogen, alkali metal ions, preferably Na, alkaline earth metal ions, and -CH2-CHO, however, 1 , R 2 , R 3 , R 4 and R 5 At least one of them is -CH2-CHO, R 1 If R is -CH2-CHO, 2 , R 3 , R 4 and R 5 At least one of them is -CH2-CHO.

[0088] Furthermore, in this embodiment as well, the number of aldehyde groups present on the C6 carbon atom of the GlcNAc unit (i.e., R 1 The ratio obtained by dividing (=-CH2-CHO) by the total number of aldehyde groups present in the modified hyaluronic acid derivative (including the aldehyde group present on the C6 carbon atom of the GlcNAc unit) is 0.60 to 1, preferably 0.65 to 1, more preferably 0.70 to 1, even more preferably 0.75 to 1, even more preferably 0.80 to 1, even more preferably 0.85 to 1, even more preferably 0.90 to 1, even more preferably 0.95 to 1, even more preferably 0.97 to 1, even more preferably 0.98 to 1, even more preferably 0.99 to 1, and most preferably 1.

[0089] In a fourth aspect, the present invention relates to the use of the modified hyaluronic acid derivative of the present invention for in situ formation of crosslinked hydrogels in cosmetic applications.

[0090] The cosmetic applications of the present invention are non-therapeutic. Preferably, the cosmetic applications of the present invention are non-surgical.

[0091] Preferably, the modified hyaluronic acid derivatives described in the first and third embodiments are used in situ formation of cross-linked hydrogels to treat wrinkles and skin lines, including frown lines, nasolabial folds, double chin, marionette lines, jawline, cheek commissures, perioral and crow's feet, skin depressions, scars, temples, subcutaneous support of the eyebrows, cheek and cheek fat pads, under-eye grooves, nose, lips, cheeks, chin, perioral region, infraorbital region, and facial asymmetry.

[0092] In a fifth aspect, the present invention relates to the use of the (aldehyde)-modified hyaluronic acid derivative of the present invention or a modified hyaluronic acid derivative obtained by the method of the present invention for the formation of a pre-formed cross-linked hydrogel.

[0093] The hydrogel is preferably formed by reacting the (aldehyde)-modified hyaluronic acid derivative of the present invention with a second polysaccharide derivative containing one or more nucleophilic functional groups capable of forming covalent bonds with one or more aldehyde groups. Using the (aldehyde)-modified hyaluronic acid derivative of the present invention to prepare crosslinked hydrogels offers several advantages over commonly used BDDE-based production methods. Firstly, since no toxic chemicals are used and preferably only water is produced as a byproduct, less time is required to purify the resulting hydrogel, and therefore the overall production is much simpler and faster. Furthermore, since purification is a potential source of contamination, the risk of contamination can be greatly reduced.

[0094] Furthermore, since cosmetic applications require hydrogels with somewhat lower viscosity, the prepared pre-formed hydrogels were found to exhibit good injectability through fine needles. However, if it is necessary to reduce the injection force, a non-crosslinked polysaccharide, preferably hyaluronic acid, can be added as a lubricating phase.

[0095] The hydrogel is formed preferably in a buffer medium, preferably a physiological buffer, more preferably a phosphate buffer, citrate buffer, or acetate buffer, and even more preferably a phosphate buffer. Preferably, the buffer is present in an artificial container.

[0096] In a sixth aspect, the present invention relates to a modified hyaluronic acid derivative for use in the in situ formation of cross-linked hydrogels for therapeutic applications, preferably for the treatment of stress urinary incontinence, vaginal dryness, vesicoureteral reflux, vocal cord dysfunction, and vocal cord internalization.

[0097] Preferably, and this applies to all embodiments using hydrogels prepared from aldehyde-modified hyaluronic acid derivatives according to the present invention, the modified hyaluronic acid derivative is used together with a second polysaccharide derivative comprising one or more nucleophilic functional groups capable of forming covalent bonds with one or more aldehyde groups of the modified HA derivative.

[0098] Preferably, and this applies to all embodiments using the second polysaccharide derivative, the second polysaccharide derivative is derived from a natural polysaccharide or a semi-synthetic polysaccharide. Specific examples of suitable polysaccharides include cellulose, dextran, starch, alginate, hyaluronic acid, pectin, chitin, chondroitin sulfate, dermatan sulfate, heparin, heparin sulfate, heparosan, and the like.

[0099] It is particularly preferable that the second polysaccharide derivative is a hyaluronic acid derivative. In this case, the nucleophilic functional group is preferably bonded to the hyaluronic acid skeleton via the carboxylic acid group of the D-glucuronic acid moiety.

[0100] Preferably, the nucleophilic functional group that can form a covalent bond with one or more aldehyde groups of the modified HA derivative is an amino, aminooxy, carbazate, or hydrazide moiety, and is preferably a hydrazide moiety.

[0101] As used herein, the term “hydrazide moiety” includes a hydrazide functional group and a hydrazide terminal group or residue, and typically has a total number of carbon atoms of 15, 10, 5, 4, 3, or 2 or less. The hydrazide moiety is preferably a hydrazide (i.e., [polysaccharide]-C(O)-NH-NH2) or a dihydrazide moiety, particularly a general formula [Polysaccharide]-C(=O)-NH-NH-R 1 This is the dihydrazide portion of -C(=O)-NH-NH2, R in the formula 1 =covalent bond, C(=O), C(=O)-OR 2 , (C=O)-R 2 And R 2 =A linear or branched C1, C2, C3, C4, C5, or C6 alkyl or alkenyl group. Particularly preferred for use herein is carbodhidrazide (CDH). When CDH is used as the hydrazide moiety and bonded to a carboxyl group of a polysaccharide, the resulting modified polysaccharide has the following pendant hydrazide terminal moiety: polysaccharide-C(=O)-R, where R is -NH-NH-C(=O)-NH-NH2.

[0102] Preferably, the nucleophilic functional group of the second polysaccharide derivative is bonded to the polysaccharide backbone via a free carboxylic acid group of the polysaccharide from which the second polysaccharide derivative is derived. Modification of the carboxylic acid group can be carried out by any method known in the art using a water-soluble coupling reagent. For example, a suitable method involves the use of standard carbodiimide chemistry, such as the use of EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) as a coupling reagent for coupling the hydrazide terminal portion with a carboxyl group to form the corresponding polysaccharide acylhydrazide (see, e.g., WO95 / 15168). Other usable coupling reagents include triazine compounds such as DMTMM (4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride; see, e.g., WO2016 / 097211), active esters such as N,N-disuccinimidyl carbonate, and tetramethylaminium salts (e.g., HATU).

[0103] Preferably, the second polysaccharide is a hyaluronic acid derivative comprising at least one disaccharide unit having the following structure. [ka] In the formula, "Ac" is as defined above.

[0104] Upon contact with each other, the aldehyde group of the modified hyaluronic acid derivative and the nucleophilic functional group of the second polysaccharide derivative spontaneously form covalent bonds after the modified hyaluronic acid derivative and the second polysaccharide derivative are simultaneously injected into a target site in the body, thereby forming a cross-linked hydrogel at the target site. Alternatively, the aldehyde group of the modified hyaluronic acid derivative and the nucleophilic functional group of the second polysaccharide derivative spontaneously form covalent bonds after they come into contact in vitro. In this case, the modified hyaluronic acid derivative and the second polysaccharide are preferably present in the buffer defined above, and the buffer is preferably present in an artificial container.

[0105] In a seventh embodiment, the present invention relates to a crosslinked hydrogel comprising the following structural units. [ka] In the formula, "Ac" represents -C(O)CH3, and R is selected from hydrogen, alkali metal ions, preferably Na, and alkaline earth metal ions.

[0106] In a preferred embodiment, the cross-linked hydrogel is a pre-formed cross-linked hydrogel. Preferably, the pre-formed hydrogel is contained within a ready-to-use delivery system, such as a pre-filled syringe. Preferably, the pre-formed cross-linked hydrogel is sterile and can be immediately injected into a patient's body, preferably for cosmetic purposes. The hydrogel is preferably sterilized by wet heating (e.g., autoclaving). Preferably, the hydrogel is first filled into a delivery system, such as a syringe, and the resulting ready-to-use delivery system, such as a pre-filled syringe, is then subjected to sterilization. Preferably, the pre-formed cross-linked hydrogel further contains a non-cross-linked polysaccharide, preferably hyaluronic acid, as a lubricating phase. The lubrication step reduces the injection force through a fine needle, as is commonly used in cosmetic applications, and ensures that the hydrogel can be injected through a fine needle even when it is in a cross-linked form. Preferably, the non-crosslinked polysaccharide is present in an amount of 1% to 30% by weight, more preferably 5% to 20% by weight, even more preferably 7% to 15% by weight, even more preferably 8% to 12% by weight, even more preferably 9% to 11% by weight, and even more preferably about 10% by weight, based on the total weight of the polysaccharide. Also preferably, the pre-formed crosslinked hydrogel further comprises a local anesthetic, preferably lidocaine, a polyhydric alcohol (also called a polyol), vitamins, alkali metal and alkaline earth metal salts, metals, antioxidants, amino acids, and ceramic particle anesthetic, preferably lidocaine. Preferably, the local anesthetic is present in an amount of 0.05% to 2% by weight, more preferably 0.1% to 1% by weight, even more preferably 0.1% to 0.8% by weight, even more preferably 0.1% to 0.6% by weight, even more preferably 0.1% to 0.4% by weight, even more preferably 0.2% to 0.4% by weight, and even more preferably about 0.3% by weight, based on the total weight of the polysaccharide. Preferably, the polyol is present in an amount of 0.5% to 5.0% by weight, more preferably 1.0% to 3.0% by weight, even more preferably 1.5% to 2.5% by weight, even more preferably 1.8% to 2.2% by weight, and even more preferably about 2.0% by weight.The polyol is preferably mannitol.

[0107] In an eighth aspect, the present invention relates to a crosslinked hydrogel obtained by contacting a modified hyaluronic acid derivative of the present invention with a second polysaccharide derivative defined above, wherein the crosslinked hydrogel preferably comprises the following structural units. [ka] In the formula, "Ac" represents -C(O)CH3, and R is selected from hydrogen, alkali metal ions, preferably Na, and alkaline earth metal ions.

[0108] Preferably, the crosslinked hydrogels described in the seventh and eighth embodiments have a storage modulus of 50 to 1000 Pa, more preferably 50 to 500 Pa, even more preferably 50 to 300 Pa, and even more preferably 50 to 150 Pa or 150 to 300 Pa at 1 Hz. The storage modulus can be determined by rheological measurements performed at 25°C with a vibration stress of 1 Pa using frequency scanning of 0.1 to 10 Hz.

[0109] According to one embodiment, contact is made in situ, i.e., within the patient's body. According to another embodiment, contact is made in vitro, i.e., outside the body of a human or animal, preferably in the buffering medium defined above. Preferably, the buffering medium is placed in an artificial container.

[0110] In a ninth aspect, the present invention relates to a method, preferably a cosmetic method, for preparing a crosslinked hydrogel. This method comprises the steps of: a) providing a first precursor solution containing the modified hyaluronic acid derivative of the present invention and separately a second precursor solution containing the second polysaccharide derivative described above; b) mixing the first and second precursor solutions into an in situ crosslinkable mixture; and c) injecting the in situ crosslinkable mixture into a target site in the patient's body to form a crosslinked gel at the target site.

[0111] Preferably, the first and second precursor solutions provided in step a) are sterile.

[0112] When used herein, the terms “sterilization” or “aseptic” are intended to refer to heat sterilization, particularly wet heat sterilization (e.g., steam sterilization), and preferably to autoclaving. Autoclaving can be performed at 120°C to 132°C for 0.3 to 20 minutes, or at 121°C to 130°C for 0.5 to 10 minutes, for example, at 121°C for 0.5 to 2 minutes.

[0113] The in situ crosslinkable mixture obtained in step b) can be easily injected through a fine needle with low injection force, providing, for example, skin improvement, skin shaping, or good volume-adding effects. This also advantageously allows for the use of fine needles, which on the one hand enhances patient comfort (reduced pain and back pressure during injection) and further allows the practitioner to accurately and safely (without vascular blockage) inject the hydrogel into desired target sites such as various layers of skin.

[0114] Mixing and injection can be achieved using a double-barrel syringe as described below, or any other suitable syringe system in which the first and second precursor solutions are physically separated before injecting the crosslinkable mixture in situ through a needle (or cannula) inside the patient's body, as well as simultaneous extrusion and accompanying mixing. Therefore, simultaneous injection must be fast enough to avoid preliminary crosslinking before depositing the crosslinkable mixture in situ at the target site in the body. On the other hand, the gelation time must be reasonably short to avoid the in situ crosslinkable mixture spreading into the surrounding tissue.

[0115] Therefore, preferably, in step a), the first and second precursor solutions are present in different barrels of a multi-barrel syringe, preferably a double-barrel syringe, and the mixing in step b) occurs during extrusion from the multi-barrel syringe.

[0116] The term "multi-barrel syringe," as used herein, is intended to mean a syringe having at least two separate barrels and potentially having two or more plungers. The term "double-barrel syringe system," as used herein, is intended to mean any system or device, typically a syringe, having two separate barrels and potentially having one or two plungers. Furthermore, a multi-barrel, for example, a double-barrel syringe system, generally includes a tip cap, or a needle or cannula with or without a needle shield, to seal the end of the syringe system. The barrels generally have storage capacity to accommodate sufficient first and second precursor solutions. The barrels may be made of glass, plastic, or other suitable material and may have different shapes, inner diameters, material compositions, transparency, etc. Furthermore, a multi-barrel syringe system may be a double-barrel syringe system in the form of two integrally connected syringes, i.e., two integrally connected barrels, and a syringe having a mono or double plunger assembly for dispensing contents from the barrels. The syringe system may also include two detachably connected barrels and two or one detachably connected plungers.

[0117] Furthermore, the first precursor solution and / or the second precursor solution may contain cells including stem cells, as well as additional substances such as adipocytes, fats, lipids, growth factors, cytokines, drugs, and bioactive factors. More specifically, the first precursor solution and / or the second precursor solution may contain local anesthetics, polyhydric alcohols (also called polyols), vitamins, alkali metal and alkaline earth metal salts, metals, antioxidants, amino acids, and ceramic particles.

[0118] In the context of the present invention, the addition of a local anesthetic is particularly desirable in terms of its ability to reduce pain during injection. Exemplary local anesthetics include ambucaine, amoranon, amylocaine, benoxynate, benzocaine, vetoxycaine, bifenamine, bupivacaine, butacaine, butamben, butanilicaine, butetamine, butoxycaine, calticaine, chloroprocaine, cocaethylene, cocaine, cyclomethicaine, dibucaine, dimethisoquine, dimethocaine, diperodon, dicyclomine, ecgonidine, ecgonin, ethyl chloride, etidocaine, betaeucaine, euprosin, phenalcomine, formocaine, hexylcaine, hydroxytetracaine, isobutyl p-aminobenzoate, mesiopropylmethylamine. This includes, but is not limited to, leucinocaine acid, leboxadrol, lidocaine, mepivacaine, meprilcaine, metabutoxycaine, methyl chloride, myrtecaine, nepain, octocaine, orthocaine, oxetazine, paretoxycaine, phenacaine, phenol, pipelocaine, pyridocaine, polidocanol, pramoxin, prilocaine, procaine, propanocaine, proparacaine, propipokaine, propoxycaine, pseudococaine, pyrocaine, ropivacaine, salicylic alcohol, tetracaine, tricaine, trimecaine, zolamine, and their salts.

[0119] Preferably, the anesthetic is lidocaine, such as in the form of lidocaine HCl. The first and / or second precursor solution may have lidocaine concentrations of, for example, 0.05% to 8.0% by weight, 0.1% to 4.0% by weight, 0.2% to 3.0% by weight, 0.3% to 2.0% by weight, or 0.4% to 1.0% by weight.

[0120] Suitable polyols for use in this specification include, but are not limited to, glycerol, mannitol, sorbitol, propylene glycol, erythritol, xylitol, maltitol, and lactitol. Mannitol and glycerol are particularly suitable for use in this specification. Furthermore, the polyol is preferably a glycol and optionally combined with one or more of the aforementioned polyol compounds, particularly mannitol. Suitable vitamins are vitamin C, vitamin E, and B vitamins, namely B2, B3, B5, B6, B7, B9, and B 12 It contains one or more vitamins. The vitamins may be present to stimulate and maintain cellular metabolism and therefore to promote collagen production. Vitamins C, E, and B6 are particularly preferred for use here. A preferred salt for use in soft tissue filler compositions is zinc salt. The ceramic particles are preferably hydroxyapatite particles, such as calcium hydroxyl apatite (CaHA) particles.

[0121] Alternatively, the first precursor solution consists of a modified hyaluronic acid derivative, and / or the second precursor solution consists of a second polysaccharide derivative.

[0122] Furthermore, the first precursor solution may consist of a modified hyaluronic acid derivative and an aqueous buffer, and / or the second precursor solution may consist of a second polysaccharide derivative and an aqueous buffer.

[0123] The amount of modified hyaluronic acid derivative present in the first precursor solution may be 0.1% to 5.0% by weight, preferably 0.5% to 4.0% by weight, more preferably 1.0% to 3.0% by weight, and most preferably 1.5% to 2.5% by weight. The amount of the second polysaccharide derivative present in the second precursor solution may be 0.1% to 5.0% by weight, preferably 0.5% to 4.0% by weight, more preferably 1.0% to 3.0% by weight, and most preferably 1.5% to 2.5% by weight. Furthermore, the weight ratio of the simultaneously injected modified hyaluronic acid derivative to the second polysaccharide derivative is preferably 15:85 to 85:15, more preferably 30:70 to 70:30, and most preferably 40:60 to 60:40 or 50:50 (modified hyaluronic acid derivative to second polysaccharide derivative).

[0124] The first and second precursor solutions typically have a low complex viscosity of 0.001 Pa·s to 5.0 Pa·s, particularly 0.005 Pa·s to 3.0 Pa·s, preferably 0.01 Pa·s to 2.0 Pa·s, and more preferably 0.1 Pa·s to 1.8 Pa·s, as determined by vibrational rheological measurements at 1 Hz and 25°C. Furthermore, both the first and second precursor solutions can be characterized by a low extrusion force of 0.01 N to 15 N, preferably 0.1 N to 10 N, more preferably 0.5 N to 7.5 N, and most preferably 0.01 N to 50 N or 1.0 to 5.0 N, as measured through a 30 G needle (TSK Laboratory) at an extrusion rate of approximately 0.21 mm / sec using a standard 1.0 ml glass syringe (BD Hypak SCF, 1 ml long RF-PRTC, ISO11040, inner diameter 6.35 mm).

[0125] The crosslinkable mixture in situ, when measured as described above, preferably has a complex viscosity of 0.1 Pa·s to 100 Pa·s, or 0.1 Pa·s to 75 Pa·s, or 1.0 Pa·s to 75 Pa·s, more preferably 1 Pa·s to 50 Pa·s, or 5 Pa·s to 50 Pa·s. Furthermore, the injection force of the composition, when measured as described above, preferably has a complex viscosity of 0.01 N to 20 N or 0.01 to 10 N, more preferably 0.1 N to 10 N, and most preferably 1.0 N to 5.0 N.

[0126] The in situ crosslinkable mixture that enters the patient's body, i.e., the mixture of the two precursor solutions, preferably contains a total amount of 0.1% to 5.0% by weight of the modified hyaluronic acid derivative and the second polysaccharide derivative.

[0127] According to the present invention, the total amount of hydrazide and aldehyde-functionalized HA derivatives present in the liquid composition is preferably 0.1% to 5.0% by weight, particularly 0.5% to 4.0% by weight, more preferably 1.0% to 3.0% by weight, and most preferably 1.5% to 2.5% by weight. Furthermore, the modification ratio of hydrazide-functionalized HA derivative to aldehyde-functionalized HA derivative is preferably 15:85 to 75:25, more preferably 25:75 to 60:40, particularly preferably 40:60 to 60:40, and most preferably 50:50.

[0128] The injected in situ crosslinkable mixture rapidly and efficiently crosslinks in situ to form a covalently crosslinked hydrogel at the target site in the body. No additives, catalysts, pH switches, UV irradiation, or other external stimuli (or "triggers") are required to induce the crosslinking reaction. In particular, no crosslinking agents are used and are not required. The only byproduct generated by the crosslinking reaction is typically water, which is readily absorbed by the hydrogel and / or surrounding tissue.

[0129] Furthermore, the in situ cross-linked hydrogels exhibit desirable mechanical, chemical, and rheological properties for use as soft tissue fillers. In particular, they have a high capacity to generate volume. In addition, the in situ cross-linked hydrogels of the present invention are biodegradable while having an extended in vivo residence time. Moreover, they may preferably contain anesthetics (e.g., lidocaine) and various other components (e.g., cells including stem cells and adipocytes, fats, lipids, growth factors, and vitamins). Therefore, the in situ cross-linkable compositions of the present invention are particularly suitable for use as skin fillers for cosmetic purposes.

[0130] In a tenth aspect, the present invention relates to a method for preparing a crosslinked hydrogel. It should be understood that this method is performed outside the body of a human or animal. In other words, the crosslinked hydrogel prepared by this method is a pre-formed crosslinked hydrogel. This method comprises the following steps: Step a): A step of providing the buffer defined above. Step b): Adding a modified hyaluronic acid derivative according to the present invention (or prepared according to the method of the present invention) and a second polysaccharide derivative as defined above to the buffer. The modified hyaluronic acid derivative and the second polysaccharide derivative may be added to the buffer simultaneously or sequentially in any possible order. Optionally, a non-crosslinked polysaccharide, preferably hyaluronic acid, may be added to the buffer in step b). The non-crosslinked polysaccharide may also be added simultaneously with or after either the modified hyaluronic acid derivative or the second polysaccharide derivative. Step c): A step of crosslinking a modified hyaluronic acid derivative and a second polysaccharide derivative in a buffer, thereby preparing a crosslinked gel. Step d): Optionally, add the non-crosslinked polysaccharide defined above. Step e): Optionally, sift and degas the hydrogel obtained in step c) or d). Step f): Optionally, fill a container, preferably a syringe, more preferably a ready-to-use syringe, with the hydrogel obtained in step c), d), or e). Step g): Optionally, a step to sterilize the container containing the gel obtained in step f).

[0131] Preferably, step d) is part of the method. Also preferably, steps e), f), and g) are part of the method. More preferably, steps d), e), f), and g) are part of the method.

[0132] The present invention's method for preparing crosslinked hydrogels is significantly more advantageous than BDDE-based methods for preparing crosslinked hydrogels. In particular, the present invention's method is remarkably simpler and less prone to error. This is because the BDDE-based method for preparing hydrogels generally involves the following steps: 1.HA dissolution 2. Adding BDDE 3. Crosslinking reaction at high temperatures 4. Neutralization 5. Filling the dialysis tubes 6. Dialysis with buffer solution (PBS, etc.) 7. Adjustment of gel concentration and addition of additives (e.g., lubricating phase, lidocaine, etc.) 8. Sieving and degassing 9. Filling the syringe 10.Sterilization While the present invention includes the following general steps: 1. Dissolve both components in the buffer solution. 2. Addition of a lubricating phase (if any) 3. Waiting period (crosslinking reaction at room temperature only) 4. Sieving and degassing 5. Filling the syringe 6. Because it only includes sterilization. Therefore, the method of the present invention involves significantly fewer process steps, which makes the steps of the present invention much simpler, faster, and therefore less expensive. Furthermore, since the method of the present invention avoids the relatively error-prone purification by dialysis, the method of the present invention demonstrates a significantly lower risk of contamination. Moreover, since the method of the present invention does not require harmful BDDE, the method of the present invention poses fewer health and environmental risks.

[0133] It has been found that the properties of the gel, particularly its rheological properties, can be fine-tuned by the degree of modification of the modified hyaluronic acid derivative (and the second polysaccharide derivative). Furthermore, the properties of the gel, particularly its rheological properties, can be fine-tuned by adjusting the polymer concentration in the buffer. Generally, higher polymer concentrations result in a higher storage modulus (G') of the prepared crosslinked hydrogel. Preferably, the concentration of the modified hyaluronic acid derivative of the present invention in the buffer is 1 g / L to 40 g / L, more preferably 2 g / L to 30 g / L, even more preferably 5 g / L to 25 g / L, even more preferably 10 g / L to 25 g / L, even more preferably 12 g / L to 25 g / L, and even more preferably 15 g / L to 25 g / L. Preferably, the concentration of the second polysaccharide derivative in the buffer is 1 g / L to 40 g / L, more preferably 2 g / L to 30 g / L, even more preferably 5 g / L to 25 g / L, even more preferably 10 g / L to 25 g / L, even more preferably 12 g / L to 25 g / L, and even more preferably 15 g / L to 25 g / L.

[0134] Preferably, the crosslinking in step c) is carried out for 1 to 24 hours, more preferably 2 to 20 hours, even more preferably 2 to 18 hours, even more preferably 2 to 16 hours, even more preferably 3 to 16 hours, even more preferably 4 to 15 hours, even more preferably 4 to 14 hours, even more preferably 5 to 12 hours, even more preferably 6 to 10 hours, even more preferably 7 to 9 hours, and even more preferably about 8 hours.

[0135] Preferably, the crosslinking in step c) is carried out at a temperature of 20°C to 35°C, more preferably 20°C to 30°C, and even more preferably 20°C to 25°C.

[0136] According to a preferred embodiment, the non-crosslinked polysaccharide is added in step b). According to another preferred embodiment, the non-crosslinked polysaccharide is added in step d). According to another preferred embodiment, the non-crosslinked polysaccharide is added in steps b) and d). Preferably, the non-crosslinked polysaccharide is added in steps b) and / or d) so that it is present at a final concentration of 1% to 30% by weight, more preferably 5% to 20% by weight, even more preferably 7% to 15% by weight, even more preferably 8% to 12% by weight, even more preferably 9% to 11% by weight, and even more preferably about 10% by weight, based on the total amount of polysaccharide.

[0137] In an eleventh aspect, the present invention relates to a kit for in situ formation of a crosslinked hydrogel, comprising (i) a first container containing a first precursor solution as described above, and (ii) a second container containing a second precursor solution as described above.

[0138] It is understood that various embodiments of the first and second precursor solutions described above also apply to this embodiment. This includes, for example, the first and second precursor solutions being present in different barrels of a multi-barrel syringe, preferably a double-barrel syringe. A system of multi-barrel syringes containing the first and second precursor solutions in different barrels may also be referred to as a “delivery system”.

[0139] The kit may also include an instruction manual.

[0140] The term "container" is not limited to, for example, glass or plastic bottles, vials, carpools, or any other sealed container.

[0141] The “Instructions for Use” are preferably instructions for use in cosmetic or therapeutic applications, particularly in the replacement or filling of biological tissue or the increase in the volume of biological tissue for cosmetic or therapeutic purposes as defined herein, and especially preferably instructions for use as a skin filler in cosmetic applications.

[0142] Herein, the present invention is further illustrated by the following non-limiting examples. [Examples]

[0143] chemicals The chemical substances used in connection with this invention were obtained from the following suppliers and used without further purification. [Table 1]

[0144] device The following experimental equipment was used in connection with the present invention. [Table 2]

[0145] General procedure for synthesizing the aldehyde-modified hyaluronic acid derivative of the present invention Glycerol-modified HA (in powder or fiber form) is weighed out, and the required amount of solvent (purified water or buffer solution) is added. The mixture is stirred to obtain a homogeneous polymer solution. Then, an oxidizing agent (sodium periodate) is added, and the solution is vigorously stirred for the desired time. To stop the reaction, a deactivating agent (e.g., vicinal diol (1,2-diol), preferably ethylene glycol) is added, followed by the addition of NaCl. Purification is carried out by pouring the reaction mixture into an organic solvent (e.g., ethanol or isopropanol). The precipitate is collected, dissolved again in physiological saline, and then precipitated a second time with an organic solvent (e.g., ethanol). The product is collected, rinsed with an organic solvent (e.g., ethanol), and dried under vacuum for a certain period of time (e.g., overnight).

[0146] Characteristic evaluation 1.Molecular weight The molecular weight of the material was determined using size exclusion chromatography combined with a multi-angle light scattering detector, refractive index detector, and viscometer. A sample was prepared by dissolving 2 mg of dried modified HA in 10 mL of PBS buffer at 25°C for 4 hours to a concentration of 0.2 mg / mL. The sample solution was filtered through a 0.45 μm filter and placed in a 1.8 mL vial for chromatography. Then, 50 μL of this solution was injected into the system (eluent: PBS buffer, flow rate: 0.7 mL / min, column temperature: 35°C). An example chromatogram is shown in Figure 1.

[0147] 2. Degree of qualification The degree of modification of the aldehyde-modified hyaluronic acid derivative of the present invention is 1 Measurements were performed using 1H NMR. Specifically, the prepared aldehyde-modified hyaluronic acid derivative was first labeled with tyrosine hydrazide: 66 mg of the aldehyde-modified hyaluronic acid derivative was dissolved in 15 mL of water, followed by the addition of 88 mg of tyrosine hydrazide. The reaction proceeded for 20 hours, after which 150 mg of NaCl was added. The reaction mixture was precipitated in 75 mL of ethanol. The precipitate was collected and dissolved in physiological saline (150 mg of NaCl in 15 mL of water). Ethanol precipitation was repeated. The solid was collected and dried under vacuum. Subsequently, the labeled aldehyde-modified hyaluronic acid derivative was treated for 1H NMR measurement by digesting 20 mg of the material with 300 units of hyaluronidase overnight at 40°C. The MoD was then determined by comparing the peak area of ​​approximately 6.9 ppm (aromatic protons from tyrosine hydrazide) with the peak area of ​​approximately 2.0 ppm (-CH3 protons from the polymer backbone). Figure 2 shows an exemplary 1H NMR spectrum of a tyrosine-labeled aldehyde-modified hyaluronic acid derivative.

[0148] Example 1 (Effect of starting material (glycerol-modified HA) concentration on molecular weight and MoD of aldehyde-modified hyaluronic acid derivative) The reaction was carried out according to the general procedure described above. The reaction time was kept constant at 0.1 equivalents for 10 minutes. NaO4 was applied and the reaction was carried out at 22°C. The concentration of glycerol-modified HA was varied. The following table shows the effect of glycerol-modified HA on the properties of the prepared material. [Table 3]

[0149] Example 1.1 1.49 g of glycerol-modified HA (with a drying loss (LoD) of 6.2%) was weighed out, followed by the addition of 315 g of water. The mixture was stirred at 22°C for 16 hours to obtain a homogeneous solution. Then, 0.074 g (0.35 mmol) of sodium periodate dissolved in 35 g of water was added, and the mixture was vigorously stirred at 22°C for 10 minutes. To stop oxidation, 3.9 mL (69 mmol) of ethylene glycol was quickly added. Then, 1.75 g of NaCl was added with stirring until a homogeneous mixture was obtained, and the solution was precipitated in 1.75 L of ethanol. The polymer was collected, placed in a new dish, and 1.75 g of NaCl was added, dissolving them in 350 mL of water. The homogeneous mixture was poured into 1.75 L of fresh ethanol, the solid was collected, and the product was dried under vacuum to obtain white fibers. Material properties: MoD of 2.1% (mol / mol) and Mw of 1.1 MDa

[0150] Example 1.4 13.4 g of glycerol-modified HA (LoD 6.2%) was weighed out, followed by 315 g of water. The mixture was stirred at 22°C for 16 hours to obtain a homogeneous solution. Then, 0.663 g (3.1 mmol) of sodium periodate dissolved in 35 g of water was added, and the mixture was vigorously stirred at 22°C for 10 minutes. To stop oxidation, 34.7 mL (620 mmol) of ethylene glycol was quickly added. Then, 2800 mL of water and 16 g of NaCl were added, and the mixture was stirred until a homogeneous mixture was obtained. The solution was precipitated in 16 L of ethanol. The polymer was collected, placed in a new dish, 16 g of NaCl was added, and they were dissolved in 3000 mL of water. The homogeneous mixture was poured into 16 L of fresh ethanol, the solid was collected, and the product was dried under vacuum to obtain white fibers. Material properties: MoD 6.1% (mol / mol) and Mw 0.6 MDa

[0151] Example 2 (Effect of the amount of oxidizing agent (sodium periodate) on the molecular weight and MoD of aldehyde-modified hyaluronic acid derivatives) The reaction was carried out according to the general procedure described above. The concentration of glycerol-modified HA was kept constant at 12 g / L during the reaction, the reaction time was 10 minutes, and the reaction was carried out at room temperature. The only parameter that was varied was the amount of sodium periodate. The properties of the prepared material are shown in the following table (the equivalent amount of NaO4 is shown based on the molar amount of disaccharide repeating units of glycerol-modified hyaluronic acid). [Table 4]

[0152] Example 2.1 4.48 g of glycerol-modified HA (LoD 6.2%) was weighed out, followed by 315 g of water. The mixture was stirred at 22°C for 16 hours to obtain a homogeneous solution. Then, 0.11 g (0.51 mmol) of sodium periodate dissolved in 35 g of water was added, and the mixture was vigorously stirred at 22°C for 10 minutes. To stop oxidation, 11.6 mL (207 mmol) of ethylene glycol was added. Then, 700 mL of water and 5.25 g of NaCl were added, and the mixture was stirred until a homogeneous mixture was obtained. The solution was precipitated in 5 L of ethanol. The polymer was collected, placed in a new dish, 5.25 g of NaCl was added, and they were dissolved in 1000 mL of water. The homogeneous mixture was poured into 5 L of fresh ethanol, the solid was collected, and the product was dried under vacuum to obtain white fibers. Material properties: MoD 2.9% (mol / mol) and Mw 1.0 MDa

[0153] Example 2.4 4.48 g of glycerol-modified HA (LoD 6.2%) was weighed out, followed by 315 g of water. The mixture was stirred at 22°C for 16 hours to obtain a homogeneous solution. Then, 0.44 g (2.1 mmol) of sodium periodate dissolved in 35 g of water was added, and the mixture was vigorously stirred at 22°C for 10 minutes. To stop oxidation, 11.6 mL (207 mmol) of ethylene glycol was added. Then, 700 mL of water and 5.25 g of NaCl were added, and the mixture was stirred until a homogeneous mixture was obtained. The solution was precipitated in 5 L of ethanol. The polymer was collected, placed in a new dish, 5.25 g of NaCl was added, and they were dissolved in 1000 mL of water. The homogeneous mixture was poured into 5 L of fresh ethanol, the solid was collected, and the product was dried under vacuum to obtain white fibers. Material properties: MoD 6.8% (mol / mol) and Mw 0.6 MDa

[0154] Example 3 (Effect of reaction time on molecular weight and MoD of aldehyde-modified hyaluronic acid derivative) The reaction was carried out according to the general procedure described above. The concentration of glycerol-modified HA was kept constant at 12 g / L, 0.1 equivalents during the reaction. NaLO4 was used, and the reaction was carried out at 22°C. The only parameter that was varied was the reaction time. The properties of the prepared material are shown in the following table. [Table 5]

[0155] Example 3.4 4.93 g of glycerol-modified HA (LoD 6.2%) was weighed out, followed by 345 g of water. The mixture was stirred at 22°C for 16 hours to obtain a homogeneous solution. Then, 0.22 g (1.0 mmol) of sodium periodate dissolved in 39 g of water was added, and the mixture was vigorously stirred at 22°C for 60 minutes. To stop oxidation, 12.7 mL (227 mmol) of ethylene glycol was added. Then, 770 mL of water and 6 g of NaCl were added, and the mixture was stirred until a homogeneous mixture was obtained. The solution was precipitated in 6 L of ethanol. The polymer was collected, placed in a new dish, 6 g of NaCl was added, and they were dissolved in 1100 mL of water. The homogeneous mixture was poured into 6 L of fresh ethanol, the solid was collected, and the product was dried under vacuum to obtain white fibers. Material properties: MoD 6.1% (mol / mol) and Mw 0.7 MDa.

[0156] Example 4: Application of aldehyde-modified hyaluronic acid derivative to the preparation of an in situ hydrogel Hyaluronic acid having an aldehyde group can be used to prepare hydrogels that can be crosslinked in situ. Specifically, sodium hyaluronate salts having an aldehyde group (electrophile) can covalently react with polysaccharides having a nucleophilic group (e.g., a hydrazide group) to form in situ crosslinked gels. Depending on the degree of modification and molecular weight of the aldehyde-modified hyaluronic acid derivative, gels with different rheological properties can be obtained. To confirm the gelation properties of the prepared aldehyde-modified hyaluronic acid derivatives, the materials were dissolved in water or buffer, filled into glass syringes, and sterilized. Similarly, hydrazide-modified sodium hyaluronate salts were dissolved in water or buffer, filled into syringes, and sterilized. The contents of the two solutions were then mixed to crosslink (chemically react) the polymers with each other. That is, the hydrazide groups of one polymer reacted with the aldehyde groups of the other material, forming a gel by the formation of hydrazone bonds (see scheme below). The prepared gels were characterized by measuring their rheological properties 24 hours after mixing. The storage modulus (G') was measured at 25°C using a rheometer with a cone-plate configuration (diameter 50 mm, angle 0.1°, CP50-1, gap size 0.1 mm). The samples were vibrated with a stress of 1 Pa using frequency scans from 0.1 to 10 Hz, but the values ​​shown are at 1 Hz. [ka] Example 4.1: An aldehyde-modified hyaluronic acid derivative (material from Example 1.1: MoD=2.1%, Mw=1.1MDa) was dissolved in 3 mM phosphate buffer (pH 7), filled into a glass syringe, and steam-sterilized (127°C, 6.5 min). A hydrazide-modified sodium hyaluronate (MoD=2.1%, Mw=1.3MDa) was dissolved in 3 mM phosphate buffer (the buffer contained 0.6 wt% lidocaine and was pH 7), filled into a glass syringe, and steam-sterilized (127°C, 6.5 min). Then, 0.6 g of the aldehyde-modified hyaluronic acid derivative solution was weighed into an empty glass syringe, followed by the addition of 0.6 g of the hydrazide-modified sodium hyaluronate solution. The syringes were closed with plunger stoppers and connected to the empty syringe via a Luer lock connector, and the contents were pushed from one syringe to the other 40 times. The syringe containing the contents was then closed, and crosslinking was performed at 22°C for 24 hours. After that, the rheological properties of the gel were measured. The gel reached a storage modulus of 68 Pa. Example 4.2: An aldehyde-modified hyaluronic acid derivative (material from Example 2.1: MoD=2.9%, Mw=1.0MDa) was dissolved in 3 mM phosphate buffer (pH 7), filled into a glass syringe, and steam-sterilized (127°C, 6.5 min). A hydrazide-modified sodium hyaluronate (MoD=2.1%, Mw=1.3MDa) was dissolved in 3 mM phosphate buffer (the buffer contained 0.6 wt% lidocaine and was pH 7), filled into a glass syringe, and steam-sterilized (127°C, 6.5 min). Then, 0.6 g of the aldehyde-modified hyaluronic acid derivative solution was weighed into an empty glass syringe, followed by the addition of 0.6 g of the hydrazide-modified sodium hyaluronate solution. The syringes were closed with plunger stoppers and connected to the empty syringe via a Luer lock connector, and the contents were squeezed from one syringe to the other 40 times. The syringe containing the contents was then closed, and crosslinking was performed at 22°C for 24 hours. After that, the rheological properties of the gel were measured. The gel reached a storage modulus of 164 Pa. Example 4.3: An aldehyde-modified hyaluronic acid derivative (material from Example 2.4: MoD=6.8%, Mw=0.6MDa) was dissolved in 3 mM phosphate buffer (pH 7), filled into a glass syringe, and steam-sterilized (127°C, 6.5 min). A hydrazide-modified sodium hyaluronate (MoD=2.1%, Mw=1.3MDa) was dissolved in 3 mM phosphate buffer (the buffer contained 0.6 wt% lidocaine and was pH 7), filled into a glass syringe, and steam-sterilized (127°C, 6.5 min). Then, 0.6 g of the aldehyde-modified hyaluronic acid derivative solution was weighed into an empty glass syringe, followed by the addition of 0.6 g of the hydrazide-modified sodium hyaluronate solution. The syringes were closed with plunger stoppers and connected to the empty syringe via a Luer lock connector, and the contents were squeezed from one syringe to the other 40 times. The syringe containing the contents was then closed, and crosslinking was performed at 22°C for 24 hours. After that, the rheological properties of the gel were measured. The gel reached a storage modulus of 277 Pa.

[0157] Example 5 (Importance of using glycerol-modified HA in oxidation reactions) It is known that aldehyde groups can be introduced into the structure of carbohydrates by oxidation with sodium periodate. However, the reaction time required for this synthesis is long, and because the oxidation occurs in the polymer's main chain, the molecular weight of the polymer decreases significantly as a result of the reaction. To avoid this, glycerol-modified HA can be used for oxidation because the suspended glycerol units are more susceptible to oxidation than the hydroxyl groups in the HA main chain. To confirm this, natural HA and glycerol-modified HA were oxidized under the same conditions and their properties were tested. The reaction conditions were as follows: polymer concentration: 12 g / L; reaction temperature: 22°C; sodium periodate 0.1 e (relative to the number of hyaluronic acid repeating units). The gelation performance of oxidized natural HA and oxidized glycerol-modified HA is shown in the following table. [Table 6] The results indicate that, under the mild conditions used for the oxidation of glycerol-modified HA, natural HA cannot produce a material that can form a gel when mixed with a hydrazide-modified hyaluronic acid solution.

[0158] Example 6 (Preparation of pre-formed hydrogel) Two gels with different concentrations were prepared [10 mg / mL (Example 6.1); 20 mg / mL (Example 6.2)]. Example 6.1 2.5 g of hydrazide-modified HA (average molecular weight 1.5 MDa and degree of modification 2.1%) and 2.5 g of aldehyde-modified HA (prepared according to the present invention; average molecular weight 1.1 MDa and degree of modification 4.3%) were weighed into a Kenwood bowl, followed by the addition of 450 ml of 10 mM phosphate buffer (pH 7, containing 2 wt% mannitol and 0.3 wt% lidocaine). The mixture was stirred for 8 hours, then 50 mL of a solution containing uncrosslinked HA at a concentration of 10 mg / mL in 10 mM phosphate buffer was added. The resulting product was then filled into a syringe and sterilized at 127°C for 6.5 minutes. The gel was characterized by measuring rheological properties (before and after sterilization), extrusion force (after sterilization), pH, and osmotic pressure. The obtained data are shown in the following table. [Table 7] Example 6.2 5.0 g of hydrazide-modified HA (average molecular weight 1.5 MDa and degree of modification 2.1%) and 5.0 g of aldehyde-modified HA (prepared according to the present invention; average molecular weight 1.1 MDa and degree of modification 4.3%) were weighed into a Kenwood bowl, followed by the addition of 450 mL of 10 mM phosphate buffer (pH 7, containing 2 wt% mannitol and 0.3 wt% lidocaine). The mixture was stirred for 8 hours, then 50 mL of a solution containing uncrosslinked HA at a concentration of 20 mg / mL in 10 mM phosphate buffer was added. The resulting product was then filled into a syringe and sterilized at 127°C for 6.5 minutes. The gel was characterized by measuring rheological properties (before and after sterilization), extrusion force (after sterilization), pH, and osmotic pressure. The obtained data are shown in the following table. [Table 8] As seen in Examples 6.1 and 6.2, the properties of the resulting hydrogels can be fine-tuned simply by changing the polymer concentration.

[0159] Example 7 (Thermal stability of gel prepared according to the method of the present invention) To test the thermal stability of preformed gels prepared according to the method of the present invention, two formulations were prepared and placed in a furnace at 40°C. The rheological properties of the gels were investigated at various time points (weeks 0, 3, 6, 9, and 12). Gel 1: 0.45 g of hydrazide-modified HA (molecular weight 1.8 MDa, degree of modification 3.7%) was dissolved in physiological saline containing 0.6 wt% lidocaine in a syringe. In a second syringe, 0.45 g of aldehyde-modified HA (prepared according to the present invention; molecular weight 1.2 MDa and degree of modification 3.7%) was dissolved in 30 mL of 25 mM PBS buffer (containing 1.25 g of mannitol). Syringes containing HA-hydrazide solution and HA-aldehyde solution were connected via Luer lock connectors, and the contents were vigorously mixed. The homogeneous mixture was then filled into a glass BD syringe and sterilized at 127°C for 6.5 minutes. The syringe containing the gel was placed in a furnace at 40°C. Gel 2: 0.45 g of hydrazide-modified HA (molecular weight 1.8 MDa, degree of modification 3.7%) was dissolved in physiological saline containing 0.6 wt% lidocaine in a syringe. In a second syringe, 0.45 g of aldehyde-modified HA (prepared according to the present invention; molecular weight 1.2 MDa and degree of modification 3.7%) was dissolved in 30 mL of 3 mM PBS buffer (without mannitol). Syringes containing HA-hydrazide solution and HA-aldehyde solution were connected via Luer lock connectors, and the contents were vigorously mixed. The mixture was then filled into a glass syringe and sterilized at 127°C for 6.5 minutes. The syringes containing the gel were placed in a furnace at 40°C. The results are shown in Figures 3 and 4. As can be seen, both gels exhibit similarly good thermal stability.

Claims

1. At least one N-acetyl-D-glucosamine unit -CH ２ -OH group, structure -CH ２ -O-CH ２ It is modified with an aldehyde group having -CHO, with a degree of modification ranging from 1.0% to 20.0%, where the degree of modification is -CH ２ -O-CH ２ A modified hyaluronic acid derivative defined as the number of -CHO groups divided by the total number of N-acetyl-D-glucosamine units present in the modified hyaluronic acid derivative.

2. The modified hyaluronic acid derivative according to claim 1, wherein the degree of modification is 1.0% to 15.0%, and / or the modified hyaluronic acid derivative has a weight-average molecular weight of 0.1 to 2.5 MDa.

3. The following structure 【Chemistry 1】 (wherein R is selected from hydrogen, alkali metal ions, and alkaline earth metal ions) comprises at least one disaccharide unit, Optionally, the following structure 【Chemistry 2】 (wherein, R １ , R ２ , R ３ and R ４ are independently selected from H and -CH ２ -CHO, R ５ is hydrogen, an alkali metal ion, an alkaline earth metal ion, and -CH ２ -CHO, provided that R １ , R ２ , R ３ , R ４ and R ５ at least one of which is -CH ２ -CHO, and when R １ is -CH ２ -CHO, at least one of R ２ , R ３ , R ４ and R ５ is -CH ２ -CHO), and further comprising at least one disaccharide unit of the modified hyaluronic acid derivative according to claim 1 or 2.

4. A method for preparing a modified hyaluronic acid derivative according to any one of claims 1 to 3, a) At least one N-acetyl-D-glucosamine unit -CH ２ -OH group is in the following formula -CH ２ -O-CH ２ -CHOH-CH ２ A step of providing glycerol-modified hyaluronic acid characterized by modification of the OH portion; b) The step of dissolving the glycerol-modified hyaluronic acid in an aqueous medium to obtain solubilized glycerol-modified hyaluronic acid; c) The solubilized glycerol-modified hyaluronic acid is reacted with an oxidizing agent to form the -CH ２ -O-CH ２ -CHOH-CH ２ At least a portion of the OH group, formula -CH ２ -O-CH ２ A method comprising the step of converting to an aldehyde group having -CHO, thereby obtaining an aldehyde-modified hyaluronic acid derivative, wherein step c) is performed for a period of 5 to 65 minutes.

5. d) Optionally, the reaction in step c) is stopped by adding vicinal diol (1,2-diol); e) Optionally, purify the modified hyaluronic acid derivative by precipitating it in an organic solvent, redissolving the precipitate in physiological saline, and then precipitating the modified hyaluronic acid derivative again in the organic solvent; The method according to claim 4, further comprising, optionally, one or more steps of drying the modified hyaluronic acid derivative obtained in step e).

6. The glycerol-modified hyaluronic acid has a weight-average molecular weight of 0.1 to 5.0 MDa and / or a degree of modification of 5 to 25%, wherein the degree of modification is -CH ２ -O-CH ２ -CHOH-CH ２ The number of OH groups is defined as the number obtained by dividing the number of N-acetyl-D-glucosamine units present in the glycerol-modified hyaluronic acid by the total number of N-acetyl-D-glucosamine units present in the glycerol-modified hyaluronic acid. Furthermore / or, (i) Step c) is performed at a temperature of 4 to 35°C; and / or (ii) Step c) is performed for a period of 10 to 60 minutes; and / or (iii) The oxidizing agent is present in an amount of 0.01 to 0.5 molar equivalents based on the molar amount of the disaccharide repeating units of the glycerol-modified hyaluronic acid; and / or (iv) The method according to claim 4 or 5, wherein the glycerol-modified hyaluronic acid is present in an amount of 2 to 50 g / L.

7. A modified hyaluronic acid derivative according to any one of claims 1 to 3, for use in in-situ formation of cross-linked hydrogels for therapeutic applications treating stress urinary incontinence, vaginal dryness, vesicoureteral reflux, vocal cord dysfunction, and vocal cord internalization.

8. The modified hyaluronic acid derivative for use according to claim 7, wherein the modified hyaluronic acid derivative is used together with a second polysaccharide derivative comprising one or more nucleophilic functional groups capable of forming a covalent bond with one or more aldehyde groups of the modified hyaluronic acid derivative, the second polysaccharide being a hyaluronic acid derivative, the nucleophilic functional group being a hydrazide functional group, or the second polysaccharide being a hyaluronic acid derivative comprising at least one disaccharide unit having the following structure. 【Transformation 5】

9. (i) When the modified hyaluronic acid derivative and the second polysaccharide derivative are simultaneously injected into a target site in the body, the aldehyde group of the modified HA derivative and the nucleophilic functional group of the second polysaccharide derivative spontaneously form covalent bonds, thereby forming a crosslinked hydrogel at the target site, or (ii) The modified hyaluronic acid derivative for use according to claim 8, wherein the aldehyde group of the modified HA derivative and the nucleophilic functional group of the second polysaccharide derivative spontaneously form a covalent bond when they come into contact with the modified hyaluronic acid derivative and the second polysaccharide derivative, and the modified HA derivative and the second polysaccharide derivative are present in a buffer medium.

10. The next structural unit: 【Transformation 6】 (In the formula, "Ac" is -C(O)CH ３ A cross-linked hydrogel containing (where R is selected from hydrogen, alkali metal ions, and alkaline earth metal ions).

11. The crosslinked hydrogel according to claim 10, which is a pre-formed crosslinked hydrogel.

12. The crosslinked hydrogel according to claim 11, further comprising a non-crosslinked polysaccharide and / or an anesthetic.

13. A crosslinked hydrogel obtained by contacting a modified hyaluronic acid derivative according to any one of claims 1 to 3 with a second polysaccharide derivative according to claim 8, comprising the following structural units: 【Transformation 7】 (In the formula, "Ac" is -C(O)CH ３ A cross-linked hydrogel containing (where R is selected from hydrogen, alkali metal ions, and alkaline earth metal ions).

14. The crosslinked hydrogel according to claim 13, wherein the contact is performed in situ.

15. The crosslinked hydrogel according to claim 13, wherein the contact is performed in vitro and in a buffer solution.

16. A method for preparing a crosslinked hydrogel, a) the step of providing a buffer; b) Adding a modified hyaluronic acid derivative according to any one of claims 1 to 3, adding a second polysaccharide derivative according to claim 8 to the buffer, and optionally adding a non-crosslinked polysaccharide to the buffer; c) Crosslinking the modified hyaluronic acid derivative and the second polysaccharide derivative in the buffer solution to prepare a crosslinked hydrogel; d) Optionally, the step of adding a non-crosslinked polysaccharide to the crosslinked hydrogel obtained in step c); e) optionally, the step of sieving and degassing the hydrogel obtained in step c) or d); f) optionally, the step of filling a container with the hydrogel obtained in step c), d), or e); g) A method comprising the optional step of sterilizing the container containing the gel obtained in step f).

17. A kit for in-situ formation of a crosslinked hydrogel, comprising: (i) a first container containing a first precursor solution comprising a modified hyaluronic acid derivative according to any one of claims 1 to 3; (ii) a second container containing a second precursor solution comprising a second polysaccharide derivative according to claim 8; and optionally (iii) instructions for use.

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