Carbohydrate crosslinker

Abstract

The present invention relates to a hydrogel product comprising a glycosaminoglycan molecule as a swellable polymer wherein the glycosaminoglycan molecule is covalently cross-linked via a cross-linking portion comprising a spacer group selected from the group consisting of disaccharides, trisugars, tetrasugars, and oligosaccharides.

Description

[0001] This application is a divisional application of the application with application date of December 28, 2016, application number 201680076930.2, and invention name “Carbohydrate Cross-linking Agent”. Technical field of the invention

[0003] The present invention relates to the field of hydrogels comprising crosslinked polysaccharides and the use of such hydrogels in medical and / or cosmetic applications. More specifically, the present invention relates to hydrogels made of crosslinked glycosaminoglycans, in particular crosslinked hyaluronic acid, chondroitin or chondroitin sulfate. Background of the Invention

[0005] Water-absorbing gels or hydrogels are widely used in the biomedical field. They are usually prepared by chemical crosslinking of polymers into an infinite network. Although many polysaccharides absorb water until they are completely dissolved, crosslinked gels of the same polysaccharide can usually absorb a certain amount of water until they are saturated, that is, they have a limited liquid retention capacity, or swelling degree.

[0006] Hyaluronic acid, chondroitin and chondroitin sulfate are well-known biocompatible polymers. They are naturally occurring polysaccharides belonging to the group of glycosaminoglycans (GAGs). All GAGs are negatively charged heteropolysaccharide chains that have the ability to absorb large amounts of water.

[0007] Hyaluronic acid (HA) is one of the most widely used biocompatible polymers for medical and cosmetic purposes. HA is a naturally occurring polysaccharide belonging to the group of glycosaminoglycans (GAGs). Hyaluronic acid and products derived from hyaluronic acid are widely used in the biomedical and cosmetic fields, for example during viscosurgery and as dermal fillers.

[0008] Chondroitin sulfate (CS) is a highly abundant GAG found in mammalian connective tissue, where it binds to proteins as part of proteoglycans along with other sulfated GAGs. It has been previously shown that hydrogels containing CS can be successfully used in biomedical applications because they are similar to the natural extracellular matrix (Lauder, RM, Complement Ther Med 17:56-62, 2009). Chondroitin sulfate has also been used to treat osteoarthritis, for example as a dietary supplement.

[0009] The cross-linking of glycosaminoglycans prolongs the duration of the degradable polymers that form the network, which is useful in many applications. However, cross-linking also reduces the natural properties of glycosaminoglycans. Therefore, it is usually desirable to maintain a low degree of modification by effective cross-linking to preserve the natural properties and effects of the glycosaminoglycans themselves. SUMMARY OF THE INVENTION

[0011] It is an object of the present invention to provide hydrogels having glycosaminoglycans (GAGs) as swellable polymers.

[0012] It is a further object of the present invention to provide a method for cross-linking GAG molecules which method has a reduced impact on the natural properties of the GAG ​​molecules.

[0013] It is also an object of the present invention to provide a method for preparing hydrogels of GAG molecules by a mild and effective route.

[0014] For these and other purposes that will be apparent from the present disclosure, the present invention provides, according to a first aspect, a hydrogel product comprising glycosaminoglycan molecules as swellable polymers, wherein the glycosaminoglycan molecules are covalently cross-linked via cross-linking portions comprising spacer groups selected from the group consisting of: disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides.

[0015] With respect to the process of the present invention for preparing the hydrogel product described herein, the term "crosslinking agent" refers to a molecule having two or more functional groups, in particular a nucleophilic functional group attached to a non-reactive spacer group, in particular a disaccharide, trisaccharide, tetrasaccharide, or oligosaccharide. Each of the two or more functional groups is capable of reacting with a carboxylic acid group on a GAG molecule to form a stable covalent bond. Preferably, the crosslinking agent consists of two or more functional groups and a spacer.

[0016] With respect to the hydrogel products of the present invention described herein, the term "crosslinking moiety" refers to the portion or residue of a crosslinking agent through which the GAG ​​molecules are covalently attached after crosslinking. The crosslinking moiety is generally composed of: i) a spacer group and ii) a binding group formed after the functional group of the crosslinking agent reacts with the carboxylic acid group on the GAG. The spacer group may, for example, include a hyaluronic acid tetrasaccharide residue, a hyaluronic acid hexasaccharide residue, a trehalose residue, a lactose residue, a maltose residue, a sucrose residue, a cellobiose residue, or a raffinose residue.

[0017] The cross-linking of the cross-linking agent of the spacer group that comprises the group that is composed of disaccharides, trisaccharides, tetrasaccharides and oligosaccharides provides a hydrogel product based on carbohydrate type structure or its derivatives completely, which minimizes the interference of cross-linking to the natural property of glycosaminoglycan. Disaccharides, trisaccharides, tetrasaccharides or oligosaccharides are preferably well-defined in terms of structure and molecular weight. Preferably, the spacer group is selected from a kind of specific disaccharide structure, trisaccharide structure, tetrasaccharide structure or oligosaccharide structure. Preferably, disaccharides, trisaccharides, tetrasaccharides or oligosaccharides are monodisperse or have narrow molecular weight distribution. Use the cross-linking agent based on well-defined disaccharides, trisaccharides, tetrasaccharides or oligosaccharides together with efficient condensation reaction to allow product to be assembled in a controlled manner. Cross-linking agent itself can also contribute to the performance of maintenance or improvement of hydrogel, for example when adopting the structure (for example diamino hyaluronic acid tetrasaccharide) relevant to hyaluronic acid to be cross-linked, or when adopting the structure (for example trehalose) with high water retention to be cross-linked.

[0018] GAGs can be, for example, sulfated or non-sulfated glycosaminoglycans, such as hyaluronan, chondroitin sulfate, heparan sulfate, heparan precursor, heparin, dermatan sulfate, and keratan sulfate. In some embodiments, GAGs are hyaluronic acid, chondroitin, or chondroitin sulfate. In preferred embodiments, GAGs are hyaluronic acid.

[0019] In a preferred embodiment, the GAG ​​is a natural GAG. The GAG ​​used in conjunction with the present invention is preferably a naturally occurring GAG. The GAG ​​is preferably used in its natural state. That is, the chemical structure of the GAG ​​is preferably not altered or modified by the addition of functional groups or analogs. It is preferred to use the GAG ​​in its natural state because this will provide a cross-linked structure that more closely resembles the natural molecule, which preserves the natural properties and effects of the GAG ​​itself, and can minimize the immune response when the cross-linked GAG is introduced into the body.

[0020] The covalently cross-linked GAG molecule is preferably composed of a carbohydrate type structure or a derivative thereof or is substantially composed of a carbohydrate type structure or a derivative thereof. This means that the cross-linked GAG molecule is preferably free of or substantially free of synthetic non-carbohydrate structures or linking groups. This can be achieved by using a GAG in a natural state together with a cross-linking agent, which is composed of a carbohydrate type structure or a derivative thereof or is substantially composed of a carbohydrate type structure or a derivative thereof. The functional group of the cross-linking agent is then directly covalently bonded to the carboxyl group of the GAG. Therefore preferably, the cross-linked portion of the covalently cross-linked GAG is composed of or substantially composed of a disaccharide spacer, a trisaccharide spacer, a tetrasaccharide spacer, and an oligosaccharide spacer.

[0021] According to a second aspect, the present invention provides a process for preparing a hydrogel product comprising cross-linked glycosaminoglycan molecules, the process comprising the following steps:

[0022] (a) providing a solution of glycosaminoglycan molecules;

[0023] (b) activating the carboxyl groups on the glycosaminoglycan molecules with a coupling agent to form activated glycosaminoglycan molecules;

[0024] (c) using a dinucleophile functional crosslinker or a polynucleophile functional crosslinker comprising a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides to crosslink the activated glycosaminoglycan molecules via the activated carboxyl groups of the activated glycosaminoglycan molecules to obtain crosslinked glycosaminoglycan molecules.

[0025] The present invention relates to the cross-linking of glycosaminoglycan molecules by covalent bonds, preferably amide bonds, which cross-linking generally uses an activator for the carboxyl groups on the backbone of the glycosaminoglycan molecules and a dinucleophilic cross-linking agent or a polynucleophilic cross-linking agent comprising a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides. The cross-linking according to the method of the present invention can be achieved by a mild and effective approach, resulting in high yields and minimal degradation of the GAG ​​molecules.

[0026] The cross-linking agent of dinucleophilic function or the cross-linking agent of polynucleophilic function comprises the spacer group selected from the group consisting of disaccharide, trisaccharide, tetrasaccharide and oligosaccharide, and the spacer group is retained in the cross-linking part between GAG molecules. The dinucleophilic function or polynucleophilic function disaccharide, trisaccharide, tetrasaccharide and oligosaccharide comprise at least two nucleophilic functional groups attached thereto. At least two nucleophilic functional groups are preferably separated by the spacer group selected from the group consisting of: disaccharide, trisaccharide, tetrasaccharide and oligosaccharide.

[0027] The cross-linking agent of dinucleophilic function or multinucleophilic function comprises two or more functional groups that can react with the functional carboxyl group of GAG, and the reaction results in the formation of covalent bond, preferably amide bond. The nucleophilic functional group is preferably capable of reacting with the carboxyl group on the glycosaminoglycan molecule to form amide bond. In some embodiments, the nucleophilic functional group of disaccharide, trisaccharide, tetrasaccharide and oligosaccharide is selected from the group consisting of: primary amine, hydrazine, hydrazide, carbazate, semi-carbazide, thiosemicarbazate, thiocarbazate and aminooxy group.

[0028] Dinucleophilic or polynucleophilic disaccharides, trisaccharides, tetrasaccharides and oligosaccharides can be derived from nucleophilic functional polysaccharides, such as chitobiose derived from chitin. Dinucleophilic or polynucleophilic disaccharides, trisaccharides, tetrasaccharides and oligosaccharides can also be disaccharides, trisaccharides, tetrasaccharides and oligosaccharides modified by introducing two or more nucleophilic functional groups.

[0029] A preferred group of di-nucleophilic functional cross-linking agents or poly-nucleophilic functional cross-linking agents includes homobifunctional or heterobifunctional primary amines, hydrazines, hydrazides, carbazates, semicarbazides, thiosemicarbazides, thiocarbazates and aminooxy groups.

[0030] In certain embodiments, the activation step (b) and the cross-linking step (c) occur simultaneously. In other embodiments, the activation step (b) occurs before the cross-linking step (c) and occurs separately from the cross-linking step (c).

[0031] In a preferred embodiment, step (c) further comprises providing particles of cross-linked GAG molecules, said particles having an average size in the range of 0.01 mm to 5 mm, preferably 0.1 mm to 0.8 mm.

[0032] In a preferred embodiment, the coupling agent of step (b) is a peptide coupling agent. The peptide coupling agent can be selected from the group consisting of: triazine-based coupling agents, carbodiimide coupling agents, imidazolium-derived coupling agents, Oxyma and COMU. Preferred peptide coupling agents are triazine-based coupling agents, including the group consisting of 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), preferably DMTMM. Another preferred peptide coupling agent is a carbodiimide coupling agent, preferably N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) combined with N-hydroxysuccinimide (NHS).

[0033] According to related aspects, the present invention also provides the use of the hydrogel product as a medicament, such as in the treatment of soft tissue disorders. Methods of treating a patient suffering from a soft tissue disorder by administering to the patient a therapeutically effective amount of the hydrogel product are provided. Methods of providing a corrective or aesthetic treatment to a patient by administering to the patient a therapeutically effective amount of the hydrogel product are also provided.

[0034] Other aspects and preferred embodiments of the invention will be apparent from the following detailed disclosure of the invention and the appended claims.

[0035] Itemized list of preferred embodiments

[0036] 1. A hydrogel product comprising glycosaminoglycan molecules as swellable polymers, wherein the glycosaminoglycan molecules are covalently cross-linked via cross-linking moieties comprising spacer groups selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides.

[0037] 2. The hydrogel product according to embodiment 1, wherein the glycosaminoglycan molecules are selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate, and mixtures thereof.

[0038] 3. The hydrogel product according to embodiment 2, wherein the glycosaminoglycan molecule is hyaluronic acid.

[0039] 4. The hydrogel product according to any one of the preceding embodiments, wherein at least 75% of the cross-links comprise a spacer group selected from the group consisting of a disaccharide, a trisaccharide, a tetrasaccharide, and an oligosaccharide.

[0040] 5. The hydrogel product according to embodiment 4, wherein at least 90% of the cross-links comprise a spacer group selected from the group consisting of a disaccharide, a trisaccharide, a tetrasaccharide, and an oligosaccharide.

[0041] 6. The hydrogel product according to embodiment 5, wherein at least 95% of the cross-links comprise a spacer group selected from the group consisting of a disaccharide, a trisaccharide, a tetrasaccharide, and an oligosaccharide.

[0042] 7. The hydrogel product according to any one of the preceding embodiments, wherein the spacer group is a hyaluronic acid tetrasaccharide residue, a hyaluronic acid hexasaccharide residue, a trehalose residue, a lactose residue, a maltose residue, a sucrose residue, a cellobiose residue or a raffinose residue.

[0043] 8. The hydrogel product according to embodiment 7, wherein the spacer group is a hyaluronic acid tetrasaccharide residue or a hyaluronic acid hexasaccharide residue.

[0044] 9. The hydrogel product according to embodiment 7, wherein the spacer group is a trehalose residue, a lactose residue, a maltose residue, a sucrose residue, a cellobiose residue or a raffinose residue.

[0045] 10. The hydrogel product according to any one of the preceding embodiments, wherein the spacer group is selected from the group consisting of: a disaccharide, a trisaccharide, and a tetrasaccharide.

[0046] 11. The hydrogel product according to any one of the preceding embodiments, wherein the cross-linking moieties are bound to the glycosaminoglycan molecules via amide bonds.

[0047] 12. The hydrogel product according to any one of the preceding embodiments, wherein at least 75% of the bonds between the glycosaminoglycan molecules and the cross-links are amide bonds.

[0048] 13. The hydrogel product according to embodiment 12, wherein at least 90% of the bonds between the glycosaminoglycan molecules and the cross-linking moieties are amide bonds.

[0049] 14. The hydrogel product according to embodiment 13, wherein at least 95% of the bonds between the glycosaminoglycan molecules and the cross-linking parts are amide bonds.

[0050] 15. The hydrogel product according to any one of the preceding embodiments, wherein less than 5% of the bonds between the glycosaminoglycan molecules and the cross-links are ester bonds.

[0051] 16. The hydrogel product according to embodiment 15, wherein less than 1% of the bonds between the glycosaminoglycan molecules and the cross-linking moieties are ester bonds.

[0052] 17. The hydrogel product according to any one of the preceding embodiments, wherein the cross-linked glycosaminoglycan molecules are in the form of gel particles having an average size in the range of 0.01 mm to 5 mm, preferably 0.1 mm to 0.8 mm.

[0053] 18. A hydrogel product according to any one of the preceding embodiments, in the form of an injectable formulation.

[0054] 19. A process for preparing a hydrogel product comprising cross-linked glycosaminoglycan molecules, the process comprising the following steps:

[0055] (a) providing a solution of glycosaminoglycan molecules;

[0056] (b) activating the carboxyl groups on the glycosaminoglycan molecules with a coupling agent to form activated glycosaminoglycan molecules;

[0057] (c) cross-linking the activated glycosaminoglycan molecules via the activated carboxyl groups of the activated glycosaminoglycan molecules using a dinucleophilic cross-linking agent or a polynucleophilic cross-linking agent comprising a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides to obtain cross-linked glycosaminoglycan molecules.

[0058] 20. The process according to embodiment 19, wherein the cross-linking of step (c) provides amide bonds between the glycosaminoglycan molecules and the cross-linking agent.

[0059] 21. The process according to any one of embodiments 19-20, wherein the activation step (b) and the cross-linking step (c) occur simultaneously.

[0060] 22. The process according to any one of embodiments 19-21, wherein the coupling agent and the cross-linking agent are added to the glycosaminoglycan simultaneously.

[0061] 23. The process according to any one of embodiments 19-20, wherein the activation step (b) occurs before and separately from the cross-linking step (c).

[0062] 24. The process according to any one of embodiments 19-23, wherein step (c) further comprises providing particles of cross-linked glycosaminoglycans, said particles having an average size in the range of 0.01 mm-5 mm, preferably 0.1 mm-0.8 mm.

[0063] 25. The process according to any one of embodiments 19-24, wherein the glycosaminoglycan molecules are selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate, and mixtures thereof.

[0064] 26. The process according to any one of embodiments 19-25, wherein the glycosaminoglycan molecule is hyaluronic acid.

[0065] 27. A process according to any one of embodiments 19-26, wherein the coupling reagent of step (b) is a peptide coupling reagent.

[0066] 28. The process according to embodiment 27, wherein the peptide coupling reagent is selected from the group consisting of triazine-based coupling reagents, carbodiimide coupling reagents, imidazolium-derived coupling reagents, Oxyma and COMU.

[0067] 29. A process according to embodiment 28, wherein the peptide coupling reagent is a triazine-based coupling reagent.

[0068] 30. The process according to embodiment 29, wherein the triazine-based coupling reagent is selected from the group consisting of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT).

[0069] 31. The process according to embodiment 30, wherein the triazine-based coupling reagent is DMTMM.

[0070] 32. A process according to embodiment 28, wherein the peptide coupling reagent is a carbodiimide coupling reagent.

[0071] 33. The process according to embodiment 32, wherein the carbodiimide coupling reagent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) combined with N-hydroxysuccinimide (NHS).

[0072] 34. A process according to any one of embodiments 19 to 33, wherein the spacer group is a hyaluronic acid tetrasaccharide residue, a hyaluronic acid hexasaccharide residue, a trehalose residue, a lactose residue, a maltose residue, a sucrose residue, a cellobiose residue or a raffinose residue.

[0073] 35. A process according to any one of embodiments 19-33, wherein the spacer group is a hyaluronic acid tetrasaccharide residue or a hyaluronic acid hexasaccharide residue.

[0074] 36. A process according to any one of embodiments 19-33, wherein the spacer group is a trehalose residue, a lactose residue, a maltose residue, a sucrose residue, a cellobiose residue or a raffinose residue.

[0075] 37. The process according to any one of embodiments 19-36, wherein the spacer group is selected from the group consisting of a disaccharide, a trisaccharide, and a tetrasaccharide.

[0076] 38. The process according to any one of embodiments 19-37, wherein the nucleophilic group of the cross-linking agent is selected from the group consisting of primary amine, hydrazine, hydrazide, carbazate, semicarbazide, thiosemicarbazide, thiocarbazate and aminooxy.

[0077] 39. The process according to embodiment 38, wherein the nucleophilic group of the disaccharide, trisaccharide, tetrasaccharide, and oligosaccharide is a primary amine.

[0078] 40. The process according to embodiment 39, wherein the cross-linking agent is a dinucleophilic cross-linking agent.

[0079] 41. The process according to embodiment 40, wherein the cross-linking agent is selected from the group consisting of diaminohyaluronan tetrasaccharide, diaminohyaluronan hexasaccharide, diaminotrehalose, diaminolactose, diaminomaltose, diaminosucrose, chitobiose or diaminoraffinose.

[0080] 42. The process according to any one of embodiments 19-41, further comprising the following steps:

[0081] (d) subjecting the cross-linked glycosaminoglycan molecules obtained in step (c) to an alkali treatment.

[0082] 43. A product obtainable by a process according to any one of embodiments 19-42.

[0083] 44. The hydrogel product according to any one of embodiments 1-18 and embodiment 43 for use as a medicament.

[0084] 45. The hydrogel product according to embodiment 44 is used for treating soft tissue disorders.

[0085] 46. ​​Use of the hydrogel product according to any one of embodiments 1-18 and embodiment 43 for the manufacture of a medicament for the treatment of soft tissue disorders.

[0086] 47. A method of treating a patient suffering from a soft tissue disorder by administering to the patient a therapeutically effective amount of a hydrogel product according to any one of embodiments 1-18 and embodiment 43.

[0087] 48. A method of providing a corrective or aesthetic treatment to a patient by administering to the patient a therapeutically effective amount of a hydrogel product according to any one of embodiments 1-18 and embodiment 43.

[0088] 49. A method of cosmetically treating the skin, the method comprising applying to the skin a hydrogel product according to any one of embodiments 1-18 and embodiment 43. DETAILED DESCRIPTION OF THE INVENTION

[0090] The present invention provides an advantageous process for preparing a hydrogel made of cross-linked glycosaminoglycan (GAG) molecules, the resulting hydrogel product and its use. GAGs are negatively charged heteropolysaccharide chains that have the ability to absorb large amounts of water. In the hydrogel product according to the present invention, the cross-linked GAG molecules are swellable polymers that provide gel properties. The preparation process described herein is gentle on the GAG ​​molecules, but provides effective cross-linking.

[0091] Thus, the present invention provides a hydrogel of GAG molecules by crosslinking in aqueous media using a di-nucleophilic functional crosslinking agent or a poly-nucleophilic functional crosslinking agent capable of directly forming covalent bonds with the carboxylic acid groups of the GAG ​​molecules through a reaction involving the use of a coupling agent.

[0092] The GAG ​​according to the invention is preferably selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate. In a preferred embodiment, the GAG ​​molecule is hyaluronic acid. Hyaluronic acid (HA) is one of the most widely used biocompatible polymers for medical and cosmetic purposes. HA is a naturally occurring polysaccharide belonging to the group of glycosaminoglycans (GAGs). Hyaluronic acid and products derived from hyaluronic acid are widely used in the biomedical and cosmetic fields, for example during elastic surgery and as subcutaneous fillers.

[0093] Unless otherwise provided, the term "hyaluronic acid" encompasses all variants and combinations of variants of hyaluronic acid, hyaluronate or hyaluronan with various chain lengths and charge states and with various chemical modifications. That is, the term also encompasses a variety of hyaluronates of hyaluronic acid and a variety of counterions, such as sodium hyaluronate. Hyaluronic acid can be obtained from various sources of animal and non-animal origin. The source of non-animal origin includes yeast and preferably bacteria. The molecular weight of a single hyaluronic acid molecule is typically in the range of 0.1 MDa-10 MDa, but other molecular weights are possible.

[0094] The term "chondroitin" refers to a GAG having a disaccharide repeating unit consisting of alternating non-sulfated D-glucuronic acid and N-acetyl-D-galactosamine moieties. For the avoidance of doubt, the term "chondroitin" does not encompass any form of chondroitin sulfate.

[0095] The term "chondroitin sulfate" refers to a GAG having a disaccharide repeating unit consisting of alternating D-glucuronic acid and N-acetyl-D-galactosamine moieties. The sulfate moiety can be present in a variety of different positions. Preferred chondroitin sulfate molecules are 4-sulfate chondroitin (chondroitin-4-sulfate) and 6-sulfate chondroitin.

[0096] Chondroitin molecules can be obtained from a variety of sources, both animal and non-animal. Non-animal sources include yeast and preferably bacteria. The molecular weight of a single chondroitin molecule is typically in the range of 1 kDa-500 kDa, but other molecular weights are possible.

[0097] Cross-linked GAGs include cross-links between GAG molecular chains, which creates a continuous network of GAG molecules held together by the covalent cross-links.

[0098] The GAG ​​molecular chains are preferably cross-linked to each other via a cross-linking agent comprising a spacer group selected from the group consisting of a disaccharide, a trisaccharide, a tetrasaccharide, and an oligosaccharide.

[0099] Preferably, the cross-linking agent is bound to the glycosaminoglycan molecules via amide bonds.

[0100] The cross-linked GAG product is preferably biocompatible. This means that no or only very mild immune response occurs in the treated individual. That is, no or only very mild undesirable local effects or systemic effects occur in the treated individual.

[0101] The crosslinked product according to the invention is a gel or hydrogel. That is, it is a crosslinked system of GAG molecules which can be considered as water-insoluble, but greatly diluted, when subjected to a liquid, typically an aqueous liquid.

[0102] The gel mainly contains liquid by weight and may contain, for example, 90%-99.9% water, but due to the three-dimensional cross-linked network of GAG molecules within the liquid, it behaves like a solid. Due to its significant liquid content, the gel is structurally flexible and similar to natural tissue, which makes it very useful as a scaffold in tissue engineering and for tissue augmentation. It can also be used to treat soft tissue disorders and for corrective or aesthetic treatments. It is preferably used as an injectable preparation.

[0103] The cross-linking of GAG molecules can be achieved by activating with a coupling agent and then reacting with a cross-linking agent. The concentration of GAG molecules and the degree of cross-linking affect the mechanical properties of the gel, for example, the elastic modulus G' and the stability performance. The cross-linked GAG molecule gel can be characterized by a "modification degree". The modification degree range of GAG molecule gel is usually between 0.01 mol% and 15 mol%. The modification degree (mol%) describes the amount of the cross-linking agent bound to the GAG ​​molecule, that is, the molar amount of the cross-linking agent bound relative to the total molar amount of the repeated disaccharide units. The modification degree reflects the extent to which the GAG ​​molecule has been chemically modified by the cross-linking agent. The reaction conditions for activation and cross-linking and the suitable analytical techniques for determining the modification degree are well known to those skilled in the art, and they can easily adjust these and other related factors and thereby provide suitable conditions to obtain the desired modification degree, and verify the characteristics of the resulting product with respect to the modification degree.

[0104] The hydrogel product may also contain a fraction of GAG molecules that are not cross-linked, i.e. not bound to a three-dimensional cross-linked GAG molecule network. However, it is preferred that at least 50% by weight, preferably at least 60% by weight, more preferably at least 70% by weight, and most preferably at least 80% by weight of the GAG ​​molecules in the gel composition form part of a cross-linked GAG molecule network.

[0105] The cross-linked GAG molecules are preferably present in the form of gel particles.The gel particles preferably have an average size in the range of 0.01 mm to 5 mm, preferably 0.1 mm to 0.8 mm, for example 0.2 mm to 0.5 mm or 0.5 mm to 0.8 mm.

[0106] The hydrogel product may be present in an aqueous solution, but it may also be present in a dried form or in a precipitated form, for example in ethanol. The hydrogel product is preferably injectable.

[0107] The hydrogel product can be prepared by a process comprising the following steps:

[0108] (a) providing a solution of glycosaminoglycan molecules;

[0109] (b) activating the carboxyl groups on the glycosaminoglycan molecules with a coupling agent to form activated glycosaminoglycan molecules;

[0110] (c) cross-linking the activated glycosaminoglycan molecules via the activated carboxyl groups of the activated glycosaminoglycan molecules using a dinucleophilic cross-linking agent or a polynucleophilic cross-linking agent comprising a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides to obtain cross-linked glycosaminoglycan molecules.

[0111] The GAG ​​according to the present invention is preferably selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate. In a preferred embodiment, the GAG ​​molecule is hyaluronic acid.

[0112] In the activation step (b), the carboxyl groups on the GAG ​​molecules are activated with a coupling agent to form activated GAG molecules.

[0113] In a preferred embodiment, the peptide coupling reagent is selected from the group consisting of triazine-based coupling reagents, carbodiimide coupling reagents, imidazolium-derived coupling reagents, Oxyma and COMU.

[0114] The peptide coupling reagent is preferably a triazine-based coupling reagent, such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) and 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT). The preferred triazine-based peptide coupling reagent is DMTMM.

[0115] Other preferred peptide coupling reagents are carbodiimide coupling reagents, preferably N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) coupled to N-hydroxysuccinimide (NHS).

[0116] In cross-linking step (c), the cross-linking of activated GAG molecules occurs via their carboxyl groups using a cross-linking agent. The cross-linking agent is a cross-linking agent of a dinucleophilic function or a cross-linking agent of a multinucleophilic function comprising a spacer group selected from the group consisting of: disaccharides, trisaccharides, tetrasaccharides and oligosaccharides. The cross-linking agent connects the GAG ​​chains to each other via the carboxyl groups on the GAG ​​main chain. The spacer group can be, for example, a hyaluronic acid tetrasaccharide residue, a hyaluronic acid hexasaccharide residue, a trehalose residue, a lactose residue, a maltose residue, a sucrose residue, a cellobiose residue or a raffinose residue. By the term "residue" it is meant that the structure of the compound is similar but not identical to the parent compound hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose or raffinose, respectively. The structure of the residue can be different from the structure of the parent compound because it has been provided with two or more nucleophilic functional groups, and is optionally covalently connected to the carboxyl group on the GAG ​​main chain via the nucleophilic functional group.

[0117] The dinucleophilic functional crosslinking agent or the polynucleophilic functional crosslinking agent comprises two or more functional groups capable of reacting with the functional carboxyl groups of the GAG, said reaction leading to the formation of a covalent bond, preferably an amide bond.

[0118] A preferred group of dinucleophilic crosslinking agents or polynucleophilic crosslinking agents includes homobifunctional or heterobifunctional primary amines, hydrazines, hydrazides, carbazates, semicarbazides, thiosemicarbazides, thiocarbazates and aminooxy groups. Non-limiting examples of such heterobifunctional crosslinking agents useful in the present invention include:

[0119]

[0120] Diaminotrehalose (6,6'-diamino-6,6'-dideoxytrehalose);

[0121]

[0122] Diaminosucrose (6,6'-diamino-6,6'-dideoxysucrose);

[0123]

[0124] Chitobiose (2,2'-diamino-2,2'-dideoxycellobiose);

[0125]

[0126] Diaminolactose (6,6'-diamino-6,6'-dideoxylactose);

[0127]

[0128] "Reduced N-deacetylated hyaluronan tetrasaccharide" or "reduced diaminohyaluronan tetrasaccharide"; and

[0129]

[0130] Diaminoraffinose (6,6"-diamino-6,6"-dideoxyraffinose).

[0131] Reaction schemes 1a-1h schematically illustrate examples of coupling via heterobifunctional primary amines (1a), aminooxy groups (1b), carbazate (1c), semicarbazide (1d), thiosemicarbazide (1e), thiocarbazate (1f), hydrazine (1g), and hydrazide (1h).

[0132]

[0133] Option 1a

[0134]

[0135] Option 1b

[0136]

[0137] Solution 1c

[0138]

[0139] Scheme 1d

[0140]

[0141] Option 1e

[0142]

[0143] Scheme 1f

[0144]

[0145] Solution 1g

[0146]

[0147] Program 1h

[0148] The dinucleophilic functional crosslinking agent or the polynucleophilic functional crosslinking agent comprises a spacer group selected from the group consisting of a disaccharide, a trisaccharide, a tetrasaccharide, and an oligosaccharide, which spacer group remains in the crosslinking part between the GAG ​​molecules.

[0149] The process can be carried out in an aqueous medium as a one-pot process and involves the covalent coupling of a di-nucleophilic functional cross-linking agent or a poly-nucleophilic functional cross-linking agent directly to the inherent carboxylic acid groups on the natural GAGs using a suitable coupling agent. In a preferred embodiment, the activation step (b) and the cross-linking step (c) occur simultaneously.

[0150] In another embodiment, the activation step (b) occurs prior to and separately from the cross-linking step (c).

[0151] The process for producing a cross-linked hydrogel generally comprises preparing a mixture of GAG molecules such as hyaluronic acid together with a cross-linking agent such as diaminotrehalose, DATH (0.001-10 molar equivalents of amine relative to the carboxylic acid groups, or preferably 0.001-1 molar equivalents) and a coupling agent such as DMTMM (0.01-10 molar equivalents relative to the carboxylic acid groups, or preferably 0.05-1 molar equivalents). The mixture is incubated at 5°C-50°C, preferably 10°C-40°C, or even more preferably 20°C-35°C during 2-120 hours, preferably 4-48 hours, followed by alkaline treatment, neutralization, precipitation, washing and vacuum drying to obtain a cross-linked polysaccharide as a solid. The precipitate is swollen in a phosphate buffer containing NaCl to form a hydrogel, which is preferably micronized into hydrogel particles having a size of 0.01 mm-5 mm, preferably 0.1 mm-1 mm.

[0152] Typical applications of the resulting hydrogel products include the preparation of injectable formulations for the treatment of soft tissue disorders including, but not limited to, orthodontic and aesthetic treatments.

[0153] In a more specific embodiment, cross-linking of chondroitin sulfate and DATH can be achieved as follows:

[0154] Diaminotrehalose (DATH) was synthesized as described in “Synthetic Carbohydrate Polymers Containing Trehalose Residues in the Main Chain: Preparation and Characteristic Properties”; Keisuke Kurita, *Naoko Masuda, Sadafumi Aibe, Kaori Murakami, Shigeru Ishii, and Shin-Ichiro Nishimurat; Macromolecules 1994, 27, 7544-7549.

[0155] Chondroitin sulfate (CS) (10kDa-200kDa) was weighed in a Falcon tube. A stock solution of diaminotrehalose (DATH) was prepared by dissolving DATH in a phosphate buffer at pH 7.4. DMTMM was weighed in a container, and the DATH solution was added to the DMTMM. The pH of the DMTMM-DATH solution was adjusted to about 7 by adding 1.2M HCl or 0.25M NaOH, and the mixture was then added to CS. The contents were fully homogenized, and then incubated at 15°C-55°C for 2-48 hours. The resulting material was pressed through a 1mm steel mesh twice and swollen in NaOH. The gel was neutralized to pH 7 with 1.2M HCl and precipitated with ethanol. The resulting precipitate was washed with 100mM NaCl in 70% ethanol, 70% ethanol, and ethanol. The resulting solid was dried in vacuo at 25°C. The precipitate was swollen in 0.7% NaCl phosphate buffer, pH 7.4 and pressed through a filter mesh three times.The cross-linked CS-gel was filled onto a syringe and sterilized.

[0156] In another more specific embodiment, cross-linking of HA with diaminosucrose can be achieved as follows:

[0157] Sucrose diamino was prepared as described in “Library of mild and economic protocols for the selective derivatization of sucrose under microwave irradiation”; M. Teresa Barros, Krasimira T. Petrova, Paula Correia-da-Silva and Taterao M. Potewar; Green Chem., 2011, 13, 1897-1906.

[0158] Hyaluronic acid (HA) (10kDa-1000kDa) is weighed in a container. A stock solution of diaminosucrose is prepared by dissolving diaminosucrose in a phosphate buffer at pH 7.4. DMTMM is weighed in a container, and the diaminosucrose solution is added to DMTMM. The pH of the DMTMM-diaminosucrose solution is adjusted to about 7 by adding 1.2M HCl or 0.25M NaOH, and the mixture is then added to HA. The contents are fully homogenized, and then incubated at 15°C-55°C for 2-48 hours. The resulting material is pressed through a 1mm steel mesh twice and swollen in NaOH. The gel is neutralized to pH 7 with 1.2M HCl and precipitated with ethanol. The resulting precipitate is washed with 100mM NaCl in 70% ethanol, washed with 70% ethanol and washed with ethanol. The resulting solid is vacuum dried at 25°C. The precipitate is swollen in a 0.7% NaCl phosphate buffer at pH 7.4 and pressed through a filter mesh three times. The cross-linked HA-gel was filled onto a syringe and sterilized.

[0159] In another more specific embodiment, cross-linking of HA and chitobiose can be achieved as follows:

[0160] Hyaluronic acid (HA) (10kDa-1000kDa) is weighed in a container. A stock solution of chitobiose is prepared by dissolving chitobiose (purchased from Carbosynth Ltd. UK) in a phosphate buffer of pH 7.4. DMTMM is weighed in a container, and the chitobiose solution is added to DMTMM. The pH of the DMTMM-chitobiose solution is adjusted to about 7 by adding 1.2M HCl or 0.25M NaOH, and then the mixture is added to HA. The contents are fully homogenized, and then incubated at 15°C-55°C for 2-48 hours. The resulting material is pressed through a 1mm steel mesh twice and swelled in NaOH. The gel is neutralized to pH 7 with 1.2m HCl, and then precipitated with ethanol. The resulting precipitate is washed with 100mM NaCl in 70% ethanol, washed with 70% ethanol and washed with ethanol. The resulting solid is dried in vacuo at 25°C. The precipitate was swollen in 0.7% NaCl phosphate buffer, pH 7.4 and pressed through a filter three times.The cross-linked HA-gel was filled onto a syringe and sterilized.

[0161] In another more specific embodiment, cross-linking of HA to reduced diamino HA-tetrasaccharide can be achieved as follows:

[0162] Hyaluronic acid (HA) (10kDa-1000kDa) is weighed in a container. A stock solution of reduced diamino HA-tetrasaccharide is prepared by dissolving the reduced diamino HA-tetrasaccharide in a phosphate buffer of pH 7.4. DMTMM is weighed in a container, and the reduced diamino HA-tetrasaccharide solution is added to DMTMM. The pH of DMTMM and the reduced diamino HA-tetrasaccharide solution is adjusted to about 7 by adding 1.2M HCl or 0.25M NaOH, and then the mixture is added to HA. The contents are fully homogenized and incubated at 15°C-55°C for 2-48 hours. The resulting material is pressed through a 1mm steel mesh twice and swollen in NaOH. The gel is neutralized to pH 7 with 1.2M HCl and precipitated with ethanol. The resulting precipitate is washed with 100mM NaCl in 70% ethanol, 70% ethanol, and ethanol. The resulting solid is dried in vacuo at 25°C. The precipitate was swollen in 0.7% NaCl phosphate buffer, pH 7.4 and pressed through a filter three times.The cross-linked HA-gel was filled onto a syringe and sterilized.

[0163] In another more specific embodiment, cross-linking of HA with dihydrazino trehalose can be achieved as follows:

[0164] α,α-D-trehalose (1 equivalent) (anhydrous) (Carbosynth Ltd, UK) is dissolved in dry dimethylformamide (DMF), and triethylamine (2-6 equivalents) is subsequently added. The flask is cooled to 0°C (ice / water) and under N2 atmosphere. 4-nitrophenyl chloroformate (2-6 equivalents) is added dropwise to the flask. The resulting mixture is allowed to stir at room temperature for 2-48 hours, then concentrated, purified by FC, and dried in vacuo. The product is dissolved in DMF and hydrazine monohydrate (2-20 equivalents) is added to the solution, and stirred at 0°C-50°C for 4-48 hours. The reaction is then concentrated, purified by FC, and dried in vacuo to obtain α,α-D-6,6'-dideoxy-6,6'-dihydrazine formate trehalose (dihydrazine formate trehalose, DCT).

[0165] Hyaluronic acid (HA) (10kDa-1000kDa) was weighed in a container. A stock solution of DCT was prepared by dissolving dihydrazinocarboxylate trehalose (DCT) in a phosphate buffer at pH 7.4. DMTMM was weighed in a container, and the DCT solution was added to the DMTMM. The pH of the DMTMM-DCT solution was adjusted to about 7 by adding 1.2M HCl or 0.25M NaOH, and the mixture was then added to HA. The contents were fully homogenized and incubated at 15°C-55°C for 2-48 hours. The resulting material was pressed through a 1mm steel mesh twice and swollen in NaOH. The gel was neutralized to pH 7 with 1.2M HCl and precipitated with ethanol. The resulting precipitate was washed with 100mM NaCl in 70% ethanol, 70% ethanol, and ethanol. The resulting solid was dried in vacuo at 25°C. The precipitate was swollen in 0.7% NaCl phosphate buffer at pH 7.4 and then pressed through a filter mesh three times.The cross-linked HA-gel was filled onto a syringe and sterilized.

[0166] In another more specific embodiment, cross-linking of HA with diaminooxytrehalose can be achieved as follows:

[0167] N-hydroxyphthalimide (2-10 equivalents) and triphenylphosphine (2-10 equivalents) are added to a stirred suspension of α,α-D-trehalose (1 equivalent) (anhydrous) (Carbosynth Ltd, UK) in anhydrous THF, and the mixture is stirred for 5-60 minutes. Diisopropyl azodicarboxylate (DIAD, 2-10 equivalents) is then added dropwise at 0°C-40°C, and the mixture is stirred for 2-48 hours at 0°C-40°C. The solvent is removed in vacuo, and the crude product is purified by FC and dried in vacuo. A suspension of the product in a mixture of MeOH and CH2Cl2 is treated with hydrazine monohydrate (2-20 equivalents), and the mixture is stirred for 2-24 hours at 0°C-40°C, then concentrated, purified by FC, and dried in vacuo to obtain diaminooxytrehalose.

[0168] Hyaluronic acid (HA) (10kDa-1000kDa) is weighed in a container. A stock solution of DAOT is prepared by dissolving diaminooxytrehalose (DAOT) in a phosphate buffer at pH 7.4. DMTMM is weighed in a container, and the DAOT solution is added to the DMTMM. The pH of the DMTMM-DAOT solution is adjusted to about 7 by adding 1.2MHCl or 0.25M NaOH, and then the mixture is added to HA. The contents are fully homogenized and incubated at 15°C-55°C for 2-48 hours. The resulting material is pressed through a 1mm steel mesh twice and swollen in NaOH. The gel is neutralized to pH 7 with 1.2M HCl and precipitated with ethanol. The resulting precipitate is washed with 100mM NaCl in 70% ethanol, 70% ethanol, and ethanol. The resulting solid is vacuum dried at 25°C. The precipitate is swollen in a 0.7% NaCl phosphate buffer at pH 7.4 and then pressed through a filter mesh three times. The cross-linked HA-gel was filled onto a syringe and sterilized. Example

[0169] Without wishing to be limited thereto, the present invention will be explained hereinafter by way of examples.

[0170] Definition and Analysis

[0171] SwF - Swelling Factor analysis in saline.

[0172] [PS] - Polysaccharide concentration, e.g. HA concentration. PS concentration is determined by LC-SEC-UV or NIR.

[0173] GelP-Gel fraction (sometimes also called gel content or GelC) is a description of the percentage of polysaccharides that are part of the gel network. A number of 90% means that only 10% of the polysaccharides are not part of the network. The amount of free polysaccharides in the gel is determined by LC-SEC-UV.

[0174] SwC - Swelling coefficient is the total liquid uptake of one gram of polysaccharide, uncorrected for the gel fraction.

[0175]

[0176] The SwCC-corrected swelling capacity (sometimes also referred to as SwDC) is the total liquid uptake of one gram of polysaccharide, corrected for the gel fraction.

[0177]

[0178] The CrR-effective cross-linking ratio was analyzed by LC-SEC-MS and was defined as:

[0179]

[0180] A CrR of 1.0 means that all crosslinking agents have been crosslinked.

[0181] Alkaline or thermal hydrolysis

[0182] In some of the examples below, the product was subjected to alkaline or thermal hydrolysis in order to hydrolyze the ester bonds formed during the crosslinking process. Alkaline / thermal hydrolysis produces only amide crosslinks in the final product. Alkaline / thermal hydrolysis was performed as follows:

[0183] Alkaline hydrolysis

[0184] The material was swollen in 0.25 M NaOH (1 g material: 9 g 0.25 M NaOH, resulting in pH 13) for at least 1 hour at room temperature. The gel was neutralized to pH 7 with 1.2 M HCl and then precipitated with ethanol. The resulting precipitate was washed with 100 mM NaCl in 70% ethanol to remove excess reagent, and then with 70% ethanol to remove salts, and finally with ethanol to remove water. The ethanol was removed in a vacuum desiccator overnight.

[0185] The precipitate was swollen in 0.7% NaCl phosphate buffer at pH 7.4 and then pressed through a fine filter mesh three times. The gel was filled on syringes and sterilized. In some cases, several syringes were not sterilized to observe the effect of sterilization.

[0186] Thermal hydrolysis

[0187] The material was swollen at room temperature in 0.7% NaCl phosphate buffer at pH 7.4. If necessary, the pH was adjusted to 7.2-7.5. The gel was left at 70°C for 20-24 hours and then passed through a fine filter three times to reduce the particle size. The gel was filled on a syringe and sterilized. In some cases, several syringes were not sterilized to observe the effect of sterilization.

[0188] Synthesis of hyaluronic diaminotrehalose

[0189] Diaminotrehalose (DATH) was synthesized as described in “Synthetic Carbohydrate Polymers Containing Trehalose Residues in the Main Chain: Preparation and Characteristic Properties”; Keisuke Kurita, *Naoko Masuda, Sadafumi Aibe, Kaori Murakami, Shigeru Ishii, and Shin-Ichiro Nishimurat; Macromolecules 1994, 27, 7544-7549.

[0190] Example 1 - Cross-linking of hyaluronic acid and diaminotrehalose (DATH)

[0191] A series of experiments (Examples 1-1 to 1-5) were conducted involving crosslinking hyaluronic acid (HA) of different molecular weights with DATH of different molar ratios using 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM) as a coupling agent. The ratios between HA, DATH and DMTMM are listed in Table 1 below.

[0192] Hyaluronan (molecular weight (M w ) from about 100kDa to about 1000kDa) was weighed in a Falcon tube. A stock solution of DATH was prepared by dissolving diaminotrehalose (DATH) (0.001-0.005 equivalent) in a phosphate buffer at pH 7.4. DMTMM (0.05 equivalent) was weighed in a PTFE container, and the DATH solution was added to the DMTMM to dissolve it. The pH of the DMTMM-DATH solution was adjusted to 6-7 with 1.2M HCl or 0.25M NaOH, and then added to HA. The contents were fully homogenized, and then incubated at 35°C for 24 hours.

[0193] The resulting material was pressed through a 1 mm steel mesh twice and then treated with a NaOH solution. The gel was neutralized to pH 7 with 1.2 M HCl and then precipitated with ethanol. The resulting precipitate was washed with 100 mM NaCl in 70% ethanol to remove excess reagent, and then washed with 70% ethanol to remove salts, and finally washed with ethanol to remove water. The ethanol was removed in a vacuum desiccator overnight.

[0194] The precipitate was swollen in 0.7% NaCl phosphate buffer at pH 7.4 and then pressed through a filter mesh three times.The gel was filled onto a syringe and sterilized.

[0195] Table 1.

[0196]

[0197] NA - Not available, because the analysis was not completed

[0198] Example 2 - Cross-linking of hyaluronic acid and diaminotrehalose (DATH)

[0199] A series of experiments (Examples 2-1 to 2-11) were conducted involving crosslinking hyaluronic acid (HA) of different molecular weights with DATH of different molar ratios using 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium chloride (DMTMM) as a coupling agent. The ratios between HA, DATH and DMTMM are listed in Table 2 below.

[0200] Hyaluronic acid is weighed in a reaction vessel. A stock solution of a crosslinking agent (DATH) is prepared by dissolving the crosslinking agent (DATH) in a phosphate buffer of pH 7.4. DMTMM is weighed in a PTFE container, and the crosslinking agent solution is added to the DMTMM to dissolve it. The pH of the DMTMM-crosslinking agent solution is adjusted to 6-7 with 1.2M HCl or 0.25M NaOH, and then added to HA. The contents are fully homogenized, and then incubated at 35°C for 24 hours. The resulting material is pressed through a 1mm steel mesh twice, and then treated with heat or alkali. The results are shown in Table 2.

[0201] Table 2. Overview of cross-linking of hyaluronic acid with (DATH)

[0202]

[0203] Empty cells - analysis not completed

[0204] Example 3 - Cross-linking of hyaluronic acid and chitobiose (CB)

[0205] Hyaluronic acid is weighed in a reaction vessel. A stock solution of a crosslinking agent (chitobiose) is prepared by dissolving the crosslinking agent (chitobiose) in a phosphate buffer of pH 7.4. DMTMM is weighed in a PTFE container, and the crosslinking agent solution is added to DMTMM to dissolve it. The pH of the DMTMM-crosslinking agent solution is adjusted to 6-7 with 1.2M HCl, and then added to HA. The contents are fully homogenized, and then incubated at 35°C for 24 hours. The resulting material is pressed through a 1mm steel mesh twice, and then treated with heat or alkali according to a general procedure. The ratios between HA, chitobiose and DMTMM are listed in Table 3 (Examples 3-1 to 3-2) below.

[0206] Example 4 - Cross-linking of hyaluronic acid with diaminotetra-HA (DA-4HA)

[0207] Diaminotetrakis-HA (DA-4HA) was synthesized according to the following scheme:

[0208]

[0209] Step 1

[0210] A solution of HA-4 (500 mg, 0.61 mmol) in water (5 ml) was treated with sodium borohydride (23.05 mg, 0.61 mmol) at room temperature and the resulting solution was stirred for 3 hours and concentrated to dryness to give the reduced product 1 (532 mg, assumed to be 100%) as a white foam.

[0211] LCMS(t r =0.28 minutes, ES+=779.4 (M-2Na+2H)

[0212] Step 2

[0213] The reduction product 1 (532mg) was dissolved in NH2OH aqueous solution (5ml, 50% v / v / ), and solid NH4I (100mg) was added. The resulting suspension was heated at 70°C for 48 hours, cooled to room temperature and concentrated to dryness to obtain a residue. The residue was precipitated in pure EtOH, and the resulting precipitate was collected by filtration and dried to constant weight to obtain a 1:1 mixture of diamine 2 and monoamine 3 in quantitative yield. The crude reaction product was used without additional purification.

[0214] 2: LCMS (t r =0.16 min, ES+=695.36 (M-2Na+2H)

[0215] 3:LCMS(t r =0.19 minutes, ES+=737.47 (M-2Na+2H)

[0216] Hyaluronic acid is weighed in a reaction vessel. A stock solution of a cross-linking agent (diamino tetra-HA) and a stock solution of DMTMM are prepared by dissolving the cross-linking agent (diamino tetra-HA) and DMTMM synthesized as described above in a phosphate buffer of pH 7.4, respectively. The pH of the solution is adjusted to 7, and then added to HA. The contents are fully homogenized, and then incubated at 23 ° C for 24 hours. The resulting material is pressed through a 1 mm steel mesh twice, and then heat-treated according to a general procedure. The ratios between HA, diamino tetra-HA and DMTMM are listed in Table 3 (Example 4) below.

[0217] Example 5 - Cross-linking of bacterial heparin precursor (HEP) and diaminotrehalose (DATH)

[0218] Coupling agent DMTMM and cross-linking agent DATH are weighed in a separate reaction vessel and dissolved in phosphate buffer (pH 7.4). The solution pH is adjusted to pH 7-7.5 with 1.2M HCl or 0.25M NaOH. Thereafter, DMTMM solution and DATH solution are successively added to the bacterial heparin precursor weighed in the reaction vessel. The contents are fully homogenized, and then incubated at 35°C for 24 hours. The resulting material is pressed through a 1mm steel mesh twice, and then heat-treated according to a general procedure. The ratio between bacterial heparin precursor, DATH and DMTMM is listed in Table 3 (Examples 5-1 to 5-2) below.

[0219] Example 6 - Cross-linking of chondroitin sulfate (CS) and diaminotrehalose (DATH)

[0220] Coupling agent DMTMM and cross-linking agent DATH are weighed in a separate reaction vessel and dissolved in phosphate buffer (pH 7.4). The solution pH is adjusted to pH 7-7.5 with 1.2M HCl or 0.25M NaOH. Thereafter, DMTMM solution and DATH solution are successively added to the chondroitin sulfate weighed in the reaction vessel. The contents are fully homogenized, and then incubated at 35°C for 24 hours. The resulting material is pressed through a 1mm steel mesh twice, and then heat-treated according to a general procedure. The ratio between chondroitin sulfate, DATH and DMTMM is listed in Table 3 (Examples 6-1 to 6-2) below.

[0221] Table 3. Summary of crosslinking of Examples 3-6

[0222]

[0223] PS = polysaccharide, HA = hyaluronan, HEP = bacterial heparin precursor, CS = chondroitin sulfate, DA-4HA = diaminotetra-HA, CB = chitobiose

[0224] Empty cells - analysis not completed.

Claims

1. A hydrogel product comprising hyaluronic acid (HA) molecules as swellable polymers, wherein: The HA molecules are covalently cross-linked via amide bonds formed between activated carboxyl groups on the HA molecules and amino groups on diaminotrehalose (DATH), The cross-linked HA molecules contain no or substantially no synthetic non-carbohydrate structures or linkers. The hydrogel product has less than 50% by weight of the hydrogel product of non-cross-linked HA molecules, The hydrogel product has an effective crosslink ratio (CrR) greater than or equal to 0.63, and The chemical structure of the HA molecules was not modified by adding functional groups prior to cross-linking.

2. The hydrogel product of claim 1, wherein at least 90% of the bonds between the HA molecules and the diaminotrehalose are amide bonds.

3. The hydrogel product of claim 1 or claim 2, wherein the hydrogel product has an effective cross-linking ratio (CrR) greater than or equal to 0.

99.

4. The hydrogel product according to any one of claims 1 to 3, in the form of an injectable preparation.

5. A process for preparing a hydrogel product comprising cross-linked hyaluronic acid (HA) molecules, the process comprising the following steps: (a) activating the carboxyl groups on the HA molecules with a coupling agent to form activated HA molecules; (b) cross-linking the activated HA molecules via activated carboxyl groups of the activated HA molecules using diaminotrehalose (DATH) to obtain cross-linked HA molecules; and (c) hydrolyzing the ester bonds in the cross-linked HA molecules by alkaline hydrolysis to obtain the hydrogel product, wherein the hydrogel product has less than 50% by weight of the hydrogel product of non-cross-linked HA molecules; wherein the cross-linked HA molecules contain no or substantially no synthetic non-carbohydrate structures or linkers, wherein the hydrogel product has an effective crosslink ratio (CrR) greater than or equal to 0.63, and The chemical structure of the HA molecules is not modified by adding functional groups before cross-linking.

6. The process according to claim 5, wherein the cross-linking of step (b) provides an amide bond between the HA molecule and DATH.

7. A process according to claim 5 or 6, wherein the coupling agent is a triazine based coupling agent, preferably DMTMM.

8. The process according to any one of claims 5-7, wherein hydrolyzing the ester bonds comprises swelling the cross-linked HA molecules in an alkaline solution for at least 1 hour.

9. The process of claim 8, wherein the alkaline solution comprises a hydroxide.

10. The process of any one of claims 5-9, wherein the hydrogel product has an effective cross-linking ratio (CrR) greater than or equal to 0.

99.

11. The process according to any one of claims 5 to 10, wherein during the activation (a), the coupling agent is present in a ratio of 2.4 mol% or more relative to the disaccharide repeating units of the HA.

12. The process according to any one of claims 5 to 11, wherein during the activation (a), the coupling agent is present in a ratio of 5.0 mol% or less relative to the disaccharide repeating units of the HA.

13. A hydrogel product obtained according to the process of any one of claims 5-12.

14. Use of a hydrogel product according to any one of claims 1 to 4 or 13 for the preparation of a medicament for the cosmetic treatment of the skin.

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