Process for the deacetylation of biopolymers

Abstract

A process for at least partially deacetylating a biopolymer comprising acetyl groups, comprising: a1) providing a biopolymer comprising acetyl groups; a2) reacting the biopolymer comprising acetyl groups with hydroxylamine (NH2OH) or a salt thereof at a temperature of 100 °C or less for 2 to 200 hours to form an at least partially deacetylated biopolymer; and a3) recovering the at least partially deacetylated biopolymer.

Description

[0001] This application is a divisional application of the application that entered the Chinese national phase on August 27, 2018, with application number 201680082707.9.

[0002] Technical Field of the Invention

[0003] This invention relates to the field of hydrogels comprising cross-linked polysaccharides and the use of such hydrogels in medical and / or cosmetic applications. More specifically, this invention relates to hydrogels made from cross-linked glycosaminoglycans, particularly cross-linked hyaluronic acid, chondroitin, or chondroitin sulfate. Background of the Invention

[0004] Hydrogels, or water-absorbing gels, are widely used in the biomedical field. They are typically prepared by chemically crosslinking polymers into an infinite network. While many polysaccharides absorb water until they are completely dissolved, gels formed by crosslinking the same polysaccharides can usually absorb a certain amount of water until they are saturated; that is, they have limited liquid retention capacity or swelling degree.

[0005] Hyaluronic acid, chondroitin, and chondroitin sulfate are well-known biocompatible polymers. They are naturally occurring polysaccharides belonging to the glycosaminoglycan (GAG) group. All GAGs are negatively charged heteropolysaccharide chains that have the ability to absorb large amounts of water.

[0006] Hyaluronic acid (HA) is one of the most widely used biocompatible polymers for medical and cosmetic applications. HA is a naturally occurring polysaccharide belonging to the glycosaminoglycan (GAG) group. Hyaluronic acid and products derived from it are widely used in the biomedical and cosmetic fields, for example, during viscosurgery and as dermal fillers.

[0007] Chondroitin sulfate (CS) is a highly abundant GAG found in the connective tissues of mammals, where it, along with other sulfated GAGs, binds to proteins as a partial proteoglycan. It has been previously shown that CS-containing hydrogels can be successfully used for biomedical applications due to their similarity to the natural extracellular matrix (Lauder, RM, Complement Ther Med 17:56-62, 2009). Chondroitin sulfate is also used to treat osteoarthritis, for example, as a dietary supplement.

[0008] Crosslinking of glycosaminoglycans prolongs the lifespan of the biodegradable polymer network, which is useful in many applications. However, crosslinking can also reduce the natural properties of glycosaminoglycans. Therefore, it is generally desirable to maintain a low degree of modification through effective crosslinking to preserve the natural properties and functions of the glycosaminoglycans themselves. Invention Overview

[0009] The object of the present invention is to provide a hydrogel having glycosaminoglycans (GAGs) as a swellable polymer.

[0010] Another object of the present invention is to provide a method for crosslinking GAG molecules, which produces hydrogel products based entirely on carbohydrate-type structures.

[0011] Another object of the present invention is to provide a method for preparing hydrogels of GAG molecules in a mild and efficient manner.

[0012] Another object of the present invention is to provide a method for at least partially deacetylifying a biopolymer containing an N-acetyl group through a mild and efficient means.

[0013] According to the aspects shown herein, a method is provided for at least partially deacetylifying a biopolymer containing an acetyl group, the method comprising:

[0014] a 1) Provide biopolymers containing acetyl groups;

[0015] a2) Reacting a biopolymer containing an acetyl group with hydroxylamine (NH2OH) or a salt thereof at 100°C or lower for 2-200 hours to form a biopolymer that is at least partially deacetylated; and

[0016] a3) Recycle at least partially deacetylated biopolymers.

[0017] As used in this article, "biopolymer" refers to polymers produced by living organisms. Biopolymers are classified into three main categories: polynucleotides, polypeptides, and polysaccharides.

[0018] This invention is based on the understanding that hydroxylamine (NH2OH) and its salts can be advantageously used to deacetylate biopolymers containing N-acetyl groups under mild reaction conditions. Therefore, according to embodiments, the biopolymer contains N-acetyl groups, and the at least partially deacetylated biopolymer formed in step a2) is at least partially N-deacetylated.

[0019] By using the term "at least partially deacetylated" as used herein with respect to biopolymers, we mean that at least some of the N-acetyl groups in a biopolymer containing N-acetyl groups are cleaved, resulting in the formation of free amine groups in the biopolymer. By using the term "at least partially deacetylated" as used herein, we mean that a significant portion of the N-acetyl groups in the biopolymer, particularly at least 1%, preferably at least 2%, at least 3%, at least 4%, or at least 5%, of the N-acetyl groups in the biopolymer is converted into free amine groups.

[0020] By using the term "at least partially deacetylated" as used herein with respect to biopolymers, we mean a biopolymer containing N-acetyl groups, wherein at least some of the N-acetyl groups have been cleaved, resulting in the formation of free amine groups in the biopolymer. By using "at least partially deacetylated" as used herein, we mean that a significant portion of the N-acetyl groups in the biopolymer, particularly at least 1%, preferably at least 2%, at least 3%, at least 4%, or at least 5% of the N-acetyl groups in the biopolymer, has been converted into free amine groups.

[0021] Deacetylated biopolymers contain free amine groups and can be used in a variety of applications, such as crosslinking reactions, conjugation reactions, or grafting reactions that require the presence of free amine groups. In particular, deacetylated biopolymers, such as deacetylated glycosaminoglycans, can be used to prepare crosslinked biopolymers, such as crosslinked glycosaminoglycans, without the use of additional crosslinking agents that might reduce the natural properties of the glycosaminoglycans. In other words, deacetylated biopolymers can be used to prepare crosslinked biopolymers based entirely on the biopolymer structure, without the need for additional non-biopolymer crosslinking agents.

[0022] The deacetylation method of the present invention involves a hydroxylaminolysis reaction. It has been found that using hydroxylamine or its salts for deacetylation allows N-deacetylation under mild conditions, resulting in only slight degradation of the polymer backbone of sensitive polysaccharides such as HA. Therefore, using hydroxylamine or its salts for deacetylation allows the production of deacetylated HA with a retained high molecular weight. This contrasts with previously known methods, such as deacetylation using hydrazine or NaOH as deacetyling agents, where high degrees of deacetylation are inevitably accompanied by severe degradation of the polymer backbone.

[0023] The process of recovering at least partially deacetylated biopolymers may include simply retaining or using the deacetylated biopolymers as they were obtained. The process of recovering at least partially deacetylated biopolymers may also include any further treatment of the deacetylated biopolymers, including but not limited to washing and purification.

[0024] The biopolymer can be a modified biopolymer, such as a branched or cross-linked biopolymer. According to some embodiments, the biopolymer is a cross-linked biopolymer. According to a particular embodiment, the biopolymer is a biopolymer gel. The biopolymer can be, for example, a hyaluronic acid gel cross-linked with 1,4-butanediol diglycidyl ether (BDDE).

[0025] According to some embodiments, the acetyl-containing biopolymer used as a starting material in the deacetylation process is a polysaccharide. According to some embodiments, the acetyl-containing biopolymer is a glycosaminoglycan. According to some embodiments, the acetyl-containing biopolymer is selected from the group consisting of sulfated or non-sulfated glycosaminoglycans, such as hyaluronic acid, chondroitin, chondroitin sulfate, heparan sulfate, heparin precursor, heparin, dermatan sulfate, and keratin sulfate, preferably hyaluronic acid, chondroitin, and chondroitin sulfate, and mixtures thereof. According to some embodiments, the acetyl-containing biopolymer is hyaluronic acid.

[0026] Hyaluronic acid is one of the most widely used biocompatible polymers for medical applications. Hyaluronic acid and other GAGs are negatively charged heteropolysaccharide chains with the ability to absorb large amounts of water. Hyaluronic acid and products derived from it are widely used in the biomedical and cosmetic fields, for example, during elastic surgery and as dermal fillers.

[0027] Hydrogels, or water-absorbing gels, are widely used in the biomedical field. They are typically prepared by chemically crosslinking polymers into an infinite network. While natural hyaluronic acid and some crosslinked hyaluronic acid products absorb water until they are completely dissolved, crosslinked hyaluronic acid gels generally absorb a certain amount of water until they are saturated; that is, they have limited liquid retention capacity or swelling degree.

[0028] Because hyaluronic acid exists in most living organisms with the same chemical structure except for its molecular weight, it exhibits minimal foreign body reaction and allows for advanced medical applications. Cross-linking and / or other modifications of hyaluronic acid molecules are often necessary to improve its duration of presence in vivo. Furthermore, such modifications affect the liquid retention capacity of hyaluronic acid molecules. As a result, hyaluronic acid has been the subject of numerous modification attempts.

[0029] In a preferred embodiment, the glycosaminoglycan is a natural glycosaminoglycan. The glycosaminoglycan used in connection with this invention is preferably a naturally occurring glycosaminoglycan. The glycosaminoglycan is preferably used in its natural state. That is, the chemical structure of the glycosaminoglycan is preferably not altered or modified by the addition of functional groups or the like. Using glycosaminoglycans in their natural state is preferred because this provides a cross-linked structure more similar to the natural molecule, which preserves the natural properties and functions of the glycosaminoglycan itself, and minimizes the immune response when the cross-linked glycosaminoglycan is introduced into the body.

[0030] Polysaccharides, and especially glycosaminoglycans such as hyaluronic acid, chondroitin, and chondroitin sulfate, often tend to degrade their backbone under harsh reaction conditions (e.g., very high or low pH values ​​or high temperatures). Therefore, the method of the present invention is particularly useful for the deacetylation of such polysaccharides.

[0031] The deacetylation method of the present invention can be used to obtain at least partially deacetylated biopolymers, wherein a significant portion of the molecular weight of the starting material is retained.

[0032] According to some embodiments, the weight-average molecular weight of the recovered at least partially deacetylated biopolymer is at least 10%, preferably at least 20%, and more preferably at least 25% of the weight-average molecular weight of the biopolymer containing acetyl groups in step a1). The weight-average molecular weight of the recovered at least partially deacetylated biopolymer can also be higher, for example, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the weight-average molecular weight of the biopolymer containing acetyl groups in step a1).

[0033] According to some embodiments, the biopolymer containing acetyl groups has a weight-average molecular weight of at least 10 kDa. According to some embodiments, the biopolymer containing acetyl groups has a weight-average molecular weight of at least 100 kDa, at least 500 kDa, at least 750 kDa, or at least 1 MDa. According to some embodiments, the biopolymer containing acetyl groups has a weight-average molecular weight in the range of 1 MDa to 5 MDa, preferably in the range of 2 MDa to 4 MDa.

[0034] According to some embodiments, the recovered at least partially deacetylated biopolymer has a weight-average molecular weight of at least 10 kDa. According to some embodiments, the recovered at least partially deacetylated biopolymer has a weight-average molecular weight of at least 100 kDa, at least 500 kDa, at least 750 kDa, or at least 1 MDa. According to some embodiments, the recovered biopolymer containing acetyl groups has a weight-average molecular weight in the range of 0.1 MDa to 5 MDa, preferably in the range of 0.5 MDa to 5 MDa or 0.5 MDa to 3 MDa.

[0035] The deacetylation method disclosed herein is also applicable to shorter biopolymers or biooligomers, such as dimers, trimers, tetramers, etc.

[0036] According to some implementation schemes, the biopolymer containing acetyl groups is an oligomeric biopolymer having a weight-average molecular weight in the range of 0.3 kDa to 10 kDa.

[0037] According to some implementation schemes, the recovered at least partially deacetylated oligomeric biopolymers have a weight-average molecular weight in the range of 0.3 kDa to 10 kDa.

[0038] In deacetylation methods, biopolymers containing acetyl groups used as starting materials are typically fully or almost fully acetylated. By the term "fully acetylated" as used herein with respect to biopolymers, we mean a biopolymer in which all or substantially all of the free amine groups have been converted to N-acetyl groups. In other words, a "fully acetylated" biopolymer contains no or substantially no free amine groups. According to some embodiments, the acetyl-containing biopolymer used as a starting material in step a1) has a degree of acetylation in the range of 98%–100%.

[0039] According to some embodiments, the recovered at least partially deacetylated biopolymer has a degree of acetylation that is at least 1%, preferably at least 2%, preferably at least 3%, preferably at least 4%, and preferably at least 5% lower than that of the acetylated biopolymer containing acetyl groups in step a1). In other words, the recovered at least partially deacetylated biopolymer may have a degree of acetylation of less than 99%, preferably less than 98%, less than 97%, less than 97%, less than 96%, less than 95%, less than 94%, or less than 93%. The recovered at least partially deacetylated biopolymer may also have a degree of acetylation that is at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50% lower than that of the acetylated biopolymer containing acetyl groups in step a1). In a preferred embodiment, the at least partially deacetylated biopolymer has a degree of acetylation of less than 97%.

[0040] Deacetylation can be achieved using hydroxylamine or its salts. A hydroxylamine salt is a salt formed from hydroxylamine and an acid. A hydroxylamine salt can be, for example, a salt formed from hydroxylamine and an acid selected from the group consisting of mineral acids and organic acids, or mixtures thereof.

[0041] According to the embodiment, the hydroxylamine salt is a salt formed from hydroxylamine and a mineral acid. According to the embodiment, the acid is selected from the group consisting of sulfuric acid, hydrochloric acid, hydroiodic acid, hydrobromic acid, and phosphoric acid, and combinations thereof. Preferred mineral acids include hydrochloric acid, hydroiodic acid, and hydrobromic acid. Particularly preferred mineral acid is hydroiodic acid.

[0042] According to the implementation scheme, the hydroxylamine salt is a salt formed from hydroxylamine and an organic acid. According to the implementation scheme, the acid is selected from the group consisting of: acetic acid, propionic acid, neopentanoic acid, citric acid, oxalic acid, malonic acid, lactic acid, benzoic acid, and halogenated carboxylic acids such as trifluoroacetic acid (TFA) and trichloroacetic acid, and combinations thereof.

[0043] According to the implementation scheme, the acid is selected from the group consisting of acetic acid, propionic acid, neopentanoic acid, and halocarboxylic acids, preferably trifluoroacetic acid, and combinations thereof. According to the implementation scheme, the acid is a halocarboxylic acid, preferably trifluoroacetic acid.

[0044] According to the implementation scheme, the hydroxylamine salt is a salt formed by hydroxylamine and an acid selected from the group consisting of: hydrochloric acid, hydroiodic acid and hydrobromic acid, propionic acid, neopentanoic acid and trifluoroacetic acid, preferably hydroiodic acid or trifluoroacetic acid.

[0045] The reaction in step a2 is preferably carried out in a solvent capable of at least partially dissolving both the biopolymer containing an acetyl group and hydroxylamine or a salt thereof. The solvent can be, for example, water or an organic solvent or a mixture thereof. Non-limiting examples of preferred solvents include water or a mixture of water and a lower alcohol such as ethanol. However, many other solvents will be useful depending on the specific molecule containing the amide group to be cleaved and the choice of hydroxylamine or a salt thereof. One example of a useful organic solvent is tetrahydrofuran (THF).

[0046] According to the implementation scheme, the reaction in step a2) includes reacting a molecule containing an amide group with hydroxylamine in water.

[0047] The deacetylation process can preferably be carried out in water or an aqueous solution, which optionally also contains another solvent, such as ethanol. Therefore, according to some embodiments, step a1) includes contacting the biopolymer containing acetyl groups with hydroxylamine in water, such that an aqueous mixture or aqueous solution of the biopolymer and hydroxylamine is formed. In some embodiments, the concentration of hydroxylamine is at least 10%, preferably at least 20%, and preferably at least 30% by weight of the aqueous mixture or aqueous solution. Higher hydroxylamine concentrations can increase the reaction rate.

[0048] Hydroxylamine is typically provided in aqueous solution at a concentration of 50% by weight. In some embodiments, the biopolymer can be directly mixed and dissolved in an aqueous solution of hydroxylamine or a salt thereof, optionally diluted. Alternatively, a solid salt of hydroxylamine, such as hydroxylamine hydrochloride or hydroxylamine sulfate, can be dissolved in an aqueous solution of the biopolymer. Adding a salt of hydroxylamine and converting that salt to hydroxylamine can be performed as an alternative or supplementary method for dissolving a biopolymer containing an acetyl group in an aqueous solution of hydroxylamine.

[0049] The molar concentration of hydroxylamine in the reaction mixture is preferably in the range of 5M-20M. For example, a concentration of 50% hydroxylamine by weight corresponds approximately to a molar concentration of 16M.

[0050] The inventors have surprisingly discovered that when hydroxylamine salt is used instead of hydroxylamine itself, the same reaction rate can be obtained at a significantly lower molar concentration. Therefore, the molar concentration of hydroxylamine salt in the reaction mixture is preferably in the range of 0.01M-10M, and more preferably in the range of 0.1M-5M.

[0051] According to some embodiments, in step a1), the biopolymer containing acetyl groups is dissolved in an aqueous solution of hydroxylamine or a salt thereof. According to some embodiments, in step a1), a salt of hydroxylamine is dissolved in an aqueous solution of the biopolymer containing acetyl groups. According to some embodiments, the biopolymer containing acetyl groups is dissolved in an aqueous solution of hydroxylamine, and a salt of hydroxylamine is dissolved in an aqueous solution of the biopolymer containing acetyl groups in hydroxylamine.

[0052] According to the embodiment, the reaction temperature in step a2) is 100°C or lower. Preferably, the reaction temperature in step a2) is selected to avoid excessive degradation of the biopolymer. According to some embodiments, the temperature in step a2) is in the range of 10°C-90°C, preferably 20°C-80°C, preferably 30°C-70°C, and preferably 30°C-50°C. According to the embodiment, the reaction in step a2) involves reacting a molecule containing an amide group with hydroxylamine or a salt thereof at a temperature in the range of 10°C-100°C, preferably 20°C-90°C, preferably 30°C-70°C, and preferably 30°C-50°C. This temperature can be, for example, in the range of 70°C-90°C, such as about 80°C, or in the range of 30°C-50°C, such as about 40°C.

[0053] The reaction time in step a2) depends on the desired degree of deacetylation. The reaction time is preferably selected to avoid excessive degradation of the biopolymer and also depends on temperature and pH. The reaction time can typically be any time from 5 minutes to 200 hours or more. According to some embodiments, the reaction in step a2) involves reacting the amide-containing molecule with hydroxylamine or a salt thereof for 2-200 hours. According to some embodiments, the reaction in step a2) involves reacting the amide-containing molecule with hydroxylamine or a salt thereof for 2-150 hours, preferably 5-150 hours, preferably 5-100 hours. In other embodiments, such as when using higher temperatures or pH, the reaction time can be much shorter, for example, in the range of 5 minutes to 2 hours, in the range of 30 minutes to 2 hours, or in the range of 1-2 hours.

[0054] Preferably, the pH in step a2) is selected to avoid excessive degradation of the biopolymer. According to some embodiments, the reaction in step a2) is carried out at a pH in the range of 4-12. According to some embodiments, the reaction in step a2) is carried out at a pH in the range of 9-11. According to some embodiments, the reaction in step a2) is carried out at a pH in the range of 4-9, preferably in the range of 6-9, preferably in the range of 6-8 or 7-8. Generally, a lower pH is preferred to avoid degradation of the biopolymer.

[0055] The inventors have discovered through extensive experimentation that the addition of a pH-lowering agent can significantly increase the reaction rate in step a2), particularly when hydroxylamine is used. This effect is both surprising and highly advantageous. Notably, the corresponding addition of a pH-lowering agent to the hydrazine deacetylation reaction does not result in any increase in the reaction rate. A lower pH value during the reaction is also preferred to avoid excessive degradation of the biopolymer. Therefore, according to some embodiments, by adding a pH-lowering agent, the pH of the reaction is reduced to a value in the range of 4-9, preferably in the range of 6-9, and more preferably in the range of 6-8 or 7-8. The pH-lowering agent can be selected, for example, from the group consisting of mineral acids, organic acids, and pH-lowering salts, and combinations thereof. In a preferred embodiment, the pH-lowering agent comprises hydroxylamine hydrochloride or hydroxylamine sulfate, preferably hydroxylamine hydrochloride.

[0056] According to some implementation schemes, the reaction in step a2) is carried out in an inert atmosphere and / or in the dark.

[0057] The products obtained by the above-described deacetylation method can have properties significantly different from those obtained by other known deacetylation methods. According to other aspects shown herein, at least partially deacetylated glycosaminoglycans obtained by the above method are provided. The partially deacetylated glycosaminoglycans obtained by the above method have a combination of reduced degree of acetylation and retained high weight-average molecular weight. This combination of significant degree of deacetylation and retained high weight-average molecular weight cannot be obtained by existing techniques for chemical deacetylation.

[0058] According to other aspects shown herein, deacetylated glycosaminoglycans are provided having a degree of acetylation of 99% or less, preferably 98% or less, preferably 97% or less, preferably 96% or less, and a weight-average molecular weight of 0.1 MDa or more, preferably 0.3 MDa or more, preferably 0.5 MDa or more.

[0059] This invention is based on the understanding that hydroxylamine (NH2OH) or its salts can be advantageously used to deacetylate biopolymers containing N-acetyl groups under mild reaction conditions. Therefore, according to other aspects shown herein, use of hydroxylamine or its salts for at least partial deacetylation of biopolymers containing acetyl groups is provided. The use may also be characterized as described above with respect to deacetylation methods.

[0060] According to other aspects shown herein, a method for preparing a hydrogel product comprising cross-linked glycosaminoglycan molecules is provided, the method comprising the following steps:

[0061] a) Provide a solution comprising at least partially deacetylated glycosaminoglycans and optionally a second glycosaminoglycan;

[0062] b) Activate the carboxyl groups on at least partially deacetylated glycosaminoglycans and / or optional second glycosaminoglycans with a coupling agent to form activated glycosaminoglycans;

[0063] c) Using at least partially deacetylated amino groups of a glycosaminoglycan, and by means of activated carboxyl groups of an activated glycosaminoglycan, to crosslink an activated glycosaminoglycan to provide a glycosaminoglycan crosslinked via amide bonds.

[0064] According to some embodiments, the at least partially deacetylated glycosaminoglycan used in step a) of the method for preparing the hydrogel product is a deacetylated glycosaminoglycan having a degree of acetylation of 99% or less, preferably 98% or less, preferably 97% or less, preferably 96% or less, and a weight-average molecular weight of 0.1 MDa or higher, preferably 0.3 MDa or higher, preferably 0.5 MDa or higher. According to some embodiments, the at least partially deacetylated glycosaminoglycan used in step a) of the method for preparing the hydrogel product is obtained by the above-described deacetylation method.

[0065] According to some embodiments, the at least partially deacetylated glycosaminoglycan used in step a) of the method for preparing a hydrogel product is a deacetylated glycosaminoglycan selected from the group consisting of: deacetylated hyaluronic acid, deacetylated chondroitin, and deacetylated chondroitin sulfate, and mixtures thereof. Preferably, the at least partially deacetylated glycosaminoglycan used in step a) of the method for preparing a hydrogel product is deacetylated hyaluronic acid.

[0066] According to some embodiments, the optional second glycosaminoglycan used in step a) of the method for preparing the hydrogel product is a glycosaminoglycan selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate, and mixtures thereof. Preferably, the optional second glycosaminoglycan used in step a) of the method for preparing the hydrogel product is hyaluronic acid.

[0067] Methods for preparing hydrogel products involve crosslinking glycosaminoglycan molecules via covalent bonds, preferably amide bonds, typically using activators for the carboxyl groups on the glycosaminoglycan backbone and at least partially deacetylated amino groups of the glycosaminoglycan. The crosslinking according to the method of the invention can be achieved through a mild and efficient approach, resulting in high yields and minimal degradation of the glycosaminoglycan molecules.

[0068] By directly crosslinking glycosaminoglycans through the formation of amide bonds between the amino and carboxyl groups present on the glycosaminoglycans, hydrogel products with a completely carbohydrate-type structure are provided. This minimizes the interference of crosslinking with the natural properties of glycosaminoglycans.

[0069] In some embodiments, activation step b) and crosslinking step c) occur simultaneously. In other embodiments, activation step b) occurs before and separately from crosslinking step c).

[0070] In a preferred embodiment, the method further includes providing cross-linked glycosaminoglycan particles having an average size in the range of 0.01 mm to 5 mm, preferably 0.1 mm to 0.8 mm.

[0071] In a preferred embodiment, the coupling agent in 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 comprising 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), preferably DMTMM. Another preferred peptide coupling agent is a carbodiimide coupling agent, preferably N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS). Another preferred peptide coupling agent is 2-chloro-1-methylpyridinium iodide (CMPI).

[0072] Hydrogel products obtained by the method of the present invention are provided, according to other aspects shown herein.

[0073] According to relevant aspects, this disclosure also provides the use of hydrogel products as medicines, such as in the treatment of soft tissue disorders. Methods for treating patients with soft tissue disorders by applying a therapeutically effective amount of the hydrogel product to the patient are provided. Methods for providing corrective or aesthetic treatment to patients by applying a therapeutically effective amount of the hydrogel product are also provided.

[0074] According to other aspects shown herein, hydrogel products obtained by the method of the present invention are provided for use as pharmaceuticals.

[0075] According to other aspects shown herein, a hydrogel product obtained by the method of the present invention is provided for the treatment of soft tissue disorders.

[0076] According to other aspects shown herein, the use of hydrogel products obtained by the method of the present invention in the manufacture of medicaments for the treatment of soft tissue disorders is provided.

[0077] According to other aspects shown herein, a method is provided for treating patients with soft tissue disorders by administering a therapeutically effective amount of a hydrogel product obtained by the method of the present invention to the patient.

[0078] According to other aspects shown herein, a method is provided to provide corrective or aesthetic treatment to a patient by administering a therapeutically effective amount of a hydrogel product obtained by the method of the present invention to the patient.

[0079] According to other aspects shown herein, a method for treating skin through cosmetics is provided, the method comprising applying a hydrogel product obtained by the method of the present invention to the skin.

[0080] Other aspects and preferred embodiments of the invention will become apparent from the following detailed disclosure and appended claims. Brief description of the attached diagram

[0081] Figure 1 This describes a reaction scheme for the formation of cross-linked hyaluronic acid, which includes 1) deacetylation of hyaluronic acid to form partially deacetylated hyaluronic acid, 2) cross-linking of the partially deacetylated hyaluronic acid by amide formation, and 3) reacetylation of the free amine groups and alkaline hydrolysis of the ester bonds formed during cross-linking and reacetylation.

[0082] Figure 2 This describes a reaction scheme for the formation of cross-linked hyaluronic acid, which includes 1) deacetylation of hyaluronic acid to form partially deacetylated hyaluronic acid, 2) cross-linking of partially deacetylated hyaluronic acid with undeacetylated hyaluronic acid by amide formation, and 3) reacetylation of free amine groups and alkaline hydrolysis of ester bonds formed during cross-linking and reacetylation. Invention Details

[0083] This disclosure provides an advantageous process for preparing hydrogels made from cross-linked glycosaminoglycan (GAG) molecules, the resulting hydrogel products, and their uses. GAGs are negatively charged heteropolysaccharide chains with the ability to absorb large amounts of water. In the hydrogel products according to this disclosure, the cross-linked GAG molecules are swellable polymers that provide gel properties. The preparation process described herein is mild on the GAG ​​molecules but provides efficient cross-linking.

[0084] The method of the present invention for preparing hydrogel products containing cross-linked glycosaminoglycan molecules includes the following steps:

[0085] a) Provide a solution comprising at least partially deacetylated glycosaminoglycans and optionally a second glycosaminoglycan;

[0086] b) Activate the carboxyl groups on at least partially deacetylated glycosaminoglycans and / or optional second glycosaminoglycans with a coupling agent to form activated glycosaminoglycans;

[0087] c) Crosslinking an activated glycosaminoglycan using at least partially deacetylated amino groups and activated carboxyl groups of the activated glycosaminoglycan to provide a glycosaminoglycan crosslinked via amide bonds; and optionally the following steps:

[0088] d) Acylate the residual amine groups of the cross-linked glycosaminoglycan provided in step c) to form an acylated cross-linked glycosaminoglycan;

[0089] and / or

[0090] e) subject the cross-linked glycosaminoglycan provided in step c) or d) to alkali treatment to hydrolyze the ester crosslinks formed as byproducts during the amide crosslinking in step c).

[0091] The hydrogel products discussed in this article are obtained through amide coupling of glycosaminoglycan molecules. Amide coupling using diamine or polyamine-functionalized crosslinking agents in conjunction with coupling agents is an attractive approach for preparing crosslinked glycosaminoglycan molecules suitable for use in hydrogel products. Crosslinking can be achieved using non-carbohydrate-based di- or multinucleofile crosslinkers such as hexamethylenediamine (HMDA), or carbohydrate-based di- or multinucleofile crosslinkers such as diaminotrehalose (DATH) in conjunction with the glycosaminoglycan. Crosslinking can also be achieved using at least partially deacetylated glycosaminoglycans, alone or in combination with a second glycosaminoglycan, whereby the deacetylated glycosaminoglycan itself acts as a di- or multinucleofile crosslinking agent.

[0092] Therefore, this disclosure provides a GAG molecular hydrogel by crosslinking in an aqueous medium using a crosslinking agent containing at least two nucleophilic functional groups, such as amine groups, which are capable of forming covalent bonds directly with the carboxylic acid groups of the GAG ​​molecule through a reaction involving the use of a coupling agent.

[0093] Crosslinking agents containing at least two nucleophilic functional groups can be, for example, non-carbohydrate-based binucleophilic or polynucleophilic crosslinking agents or carbohydrate-based binucleophilic or polynucleophilic crosslinking agents.

[0094] Carbohydrate-based amphiphilic or polyphilic crosslinking agents are preferred because they provide hydrogel products based entirely on carbohydrate-type structures or their derivatives, minimizing interference with the natural properties of glycosaminoglycans from crosslinking. The crosslinking agent itself can also help maintain or enhance the properties of the hydrogel, for example, when crosslinked with structures associated with hyaluronic acid or when crosslinked with structures possessing high water-retention capacity.

[0095] Carbohydrate-based amphiphilic or polyphilic crosslinking agents can be selected, for example, from the group consisting of disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, and polysaccharides with amphiphilic or polyphilic functions.

[0096] In a preferred embodiment, the amphiphilic or polyphilic crosslinking agent is a polysaccharide that is at least partially deacetylated, i.e., an acetylated polysaccharide that has been at least partially deacetylated to provide a polysaccharide with free amine groups. The at least partially deacetylated glycosaminoglycan can be crosslinked alone or in combination with a second glycosaminoglycan, whereby the deacetylated glycosaminoglycan itself acts as an amphiphilic or polyphilic crosslinking agent.

[0097] In a preferred embodiment, the cross-linked GAG is obtained through the following:

[0098] 1) Using free amine and carboxylic acid groups present in at least partially deacetylated GAG to crosslink at least partially deacetylated GAG with partially deacetylated GAG, such as Figure 1 As shown; or

[0099] 2) Crosslinking at least partially deacetylated GAG with non-deacetylated GAG using free amine groups present in at least partially deacetylated GAG and carboxylic acid groups present in GAG, such as... Figure 2 As shown in the image.

[0100] According to some embodiments, glycosaminoglycans are selected from the group consisting of sulfated or non-sulfated glycosaminoglycans, such as hyaluronic acid, chondroitin, chondroitin sulfate, heparin sulfate, bacterial heparin precursors, heparin, dermatan sulfate, and keratin sulfate. According to some embodiments, glycosaminoglycans are selected from the group consisting of hyaluronic acid, chondroitin, and chondroitin sulfate, and mixtures thereof. According to some embodiments, the glycosaminoglycan is hyaluronic acid.

[0101] Hyaluronic acid (HA) is one of the most widely used biocompatible polymers for medical and cosmetic applications. HA is a naturally occurring polysaccharide belonging to the glycosaminoglycan (GAG) group. Hyaluronic acid consists of two alternating monosaccharide units, DN-acetylglucosamine (GlcNAc) and D-glucuronic acid (GlcA), assembled via β(1→3) and β(11→) glycosidic bonds, respectively. Hyaluronic acid and products derived from it are widely used in the biomedical and cosmetic fields, for example, during elastic surgery and as a dermal filler.

[0102] Unless otherwise stated, the term "hyaluronic acid" encompasses all variants and combinations of variants of hyaluronic acid, hyaluronic acid salts / esters, or hyaluronic acid with various chain lengths and charge states, and with various chemical modifications. In other words, the term also covers various hyaluronic acid salts, such as sodium hyaluronate, with various counterions. Hyaluronic acid can be obtained from a variety of animal and non-animal sources. Non-animal sources include yeast and, preferably, bacteria. The molecular weight of a single hyaluronic acid molecule is typically in the range of 0.1 MDa to 10 MDa, but other molecular weights are possible.

[0103] 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 ambiguity, the term "chondroitin" does not cover any form of chondroitin sulfate.

[0104] 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 moieties can be present in various different positions. Preferred chondroitin sulfate molecules are 4-chondroitin sulfate and 6-chondroitin sulfate.

[0105] Chondroitin molecules can be obtained from a variety of animal and non-animal sources. Non-animal sources include yeast and, preferably, bacteria. The molecular weight of a single chondroitin molecule is typically in the range of 1 kDa to 500 kDa, but other molecular weights are possible.

[0106] According to some embodiments, the at least partially deacetylated glycosaminoglycan used in step a) of the method for preparing the hydrogel product is a deacetylated glycosaminoglycan having a degree of acetylation of 99% or less, preferably 98% or less, preferably 97% or less, preferably 96% or less, and a weight-average molecular weight of 0.1 MDa or higher, preferably 0.5 MDa or higher. According to some embodiments, the at least partially deacetylated glycosaminoglycan used in step a) of the method for preparing the hydrogel product is obtained by the above-described deacetylation method.

[0107] According to some embodiments, the at least partially deacetylated glycosaminoglycan used in step a) of the method for preparing a hydrogel product is a deacetylated glycosaminoglycan selected from the group consisting of: deacetylated sulfated or non-sulfated glycosaminoglycans, such as deacetylated hyaluronic acid, deacetylated chondroitin, deacetylated chondroitin sulfate, deacetylated heparan sulfate, deacetylated bacterial heparin precursor, deacetylated heparin, deacetylated dermatin sulfate, and deacetylated keratin sulfate. Preferably, the at least partially deacetylated glycosaminoglycan used in step a) of the method for preparing a hydrogel product is selected from the group consisting of: deacetylated hyaluronic acid, deacetylated chondroitin, and deacetylated chondroitin sulfate, and mixtures thereof. Preferably, the at least partially deacetylated glycosaminoglycan used in step a) of the method for preparing a hydrogel product is deacetylated hyaluronic acid.

[0108] According to some embodiments, the optional second glycosaminoglycan used in step a) of the method for preparing the hydrogel product is a glycosaminoglycan selected from the group consisting of sulfated or non-sulfated glycosaminoglycans, such as hyaluronic acid, chondroitin, chondroitin sulfate, heparin sulfate, bacterial heparin precursor, heparin, dermatan sulfate, and keratin sulfate. Preferably, the optional second glycosaminoglycan used in step a) of the method for preparing the hydrogel product is selected from the group consisting of hyaluronic acid, chondroitin and chondroitin sulfate, and mixtures thereof. Preferably, the optional second glycosaminoglycan used in step a) of the method for preparing the hydrogel product is hyaluronic acid.

[0109] By directly crosslinking glycosaminoglycans through the formation of amide bonds between the amino and carboxyl groups present on the glycosaminoglycans, hydrogel products with a completely carbohydrate-type structure are provided. This minimizes the interference of crosslinking with the natural properties of glycosaminoglycans.

[0110] Methods for preparing hydrogel products involve crosslinking glycosaminoglycan molecules via covalent bonds, preferably amide bonds, typically using activators for the carboxyl groups on the glycosaminoglycan backbone and at least partially deacetylated amino groups of the glycosaminoglycan. The crosslinking according to the method of the invention can be achieved through a mild and efficient approach, resulting in high yields and minimal degradation of the glycosaminoglycan molecules.

[0111] According to some implementation schemes, activation step b) and crosslinking step c) occur simultaneously.

[0112] According to some implementation schemes, the coupling agent in step b) is a peptide coupling agent. Crosslinking using peptide coupling agents is advantageous compared to many other common crosslinking methods (such as BDDE crosslinking) because it allows crosslinking to occur at neutral pH and minimizes the degradation of glycosaminoglycan molecules.

[0113] According to some implementation schemes, the peptide coupling agent is selected from the group consisting of: triazine-based coupling agents, carbodiimide coupling agents, imidazolium-derived coupling agents, Oxyma, and COMU.

[0114] According to some embodiments, the peptide coupling agent is a triazine-based coupling agent. According to some embodiments, the triazine-based coupling agent 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). According to some embodiments, the triazine-based coupling agent is DMTMM.

[0115] According to some embodiments, the peptide coupling agent is a carbodiimide coupling agent. According to some embodiments, the carbodiimide coupling agent is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) combined with N-hydroxysuccinimide (NHS).

[0116] The terms “crosslinked glycosaminoglycan” or “crosslinked glycosaminoglycan molecule” herein refer to glycosaminoglycans containing generally covalently crosslinked portions between glycosaminoglycan molecular chains, which create a continuous network of glycosaminoglycan molecules held together by the crosslinked portions.

[0117] Cross-linked GAG products are preferably biocompatible. This means that no or only very mild immune responses occur in the treated individuals. That is, no or only very mild, undesirable local or systemic effects occur in the treated individuals.

[0118] The cross-linked product according to this disclosure is a gel or hydrogel. That is, it can be considered as a cross-linked system of GAG molecules that are water-insoluble but substantially diluted when subjected to liquids, typically aqueous liquids.

[0119] The cross-linked GAG molecules preferably exist 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.

[0120] According to some implementations, step c) further includes providing cross-linked glycosaminoglycan particles having 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.

[0121] The gel, by weight, is primarily liquid and can contain, for example, 90%–99.9% water, but it behaves like a solid due to the three-dimensional cross-linked GAG molecular network within the liquid. Because of its significant liquid content, the gel is structurally flexible and resembles natural tissue, making it highly 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 formulation.

[0122] The hydrogel product may also contain portions of uncrosslinked GAG molecules, i.e., GAG molecules not bound to a three-dimensional crosslinked GAG molecular 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 crosslinked GAG molecular network.

[0123] Hydrogel products can exist in aqueous solutions, but they can also exist in a dry or precipitated form, for example, in ethanol. Hydrogel products are preferably injectable.

[0124] According to an embodiment, at least partially deacetylated glycosaminoglycans are obtained by a novel method for at least partially deacetylation of biopolymers, wherein the biopolymer is a glycosaminoglycan, the method comprising:

[0125] a1) Provides biopolymers containing acetyl groups;

[0126] a2) Reacting a biopolymer containing an acetyl group with hydroxylamine (NH2OH) or a salt thereof at 100°C or lower for 2-200 hours to form a biopolymer that is at least partially deacetylated; and

[0127] a3) Recycle at least partially deacetylated biopolymers.

[0128] Hydroxylamine (NH2OH) and its salts have been found to be advantageous for deacetylation of biopolymers containing acetyl groups under mild reaction conditions. Deacetylated biopolymers can be used in a variety of applications, such as crosslinking, conjugation, or grafting reactions that require the presence of free amine groups.

[0129] The deacetylation method of the present invention involves a hydroxylamine hydrolysis reaction. It has been found that using hydroxylamine or a salt thereof for deacetylation allows N-deacetylation under mild conditions, resulting in only slight degradation of the polymer backbone of sensitive polysaccharides such as HA. Therefore, using hydroxylamine or a salt thereof for deacetylation allows the production of deacetylated HA with a retained high molecular weight. This contrasts with previously known methods, such as deacetylation using hydrazine or NaOH as deacetyling agents, in which high degrees of deacetylation are inevitably accompanied by severe degradation of the polymer backbone.

[0130] According to embodiments, the biopolymer containing acetyl groups is a glycosaminoglycan, preferably selected from the group consisting of hyaluronic acid, chondroitin sulfate, and mixtures thereof. According to some embodiments, the biopolymer containing acetyl groups is hyaluronic acid.

[0131] Hyaluronic acid can be obtained from a variety of animal and non-animal sources. Non-animal sources include yeast and, preferably, bacteria. The molecular weight of a single hyaluronic acid molecule is typically in the range of 0.1 MDa to 10 MDa, but other molecular weights are possible.

[0132] In some embodiments, the concentration of the hyaluronic acid is in the range of 1 mg / ml to 100 mg / ml. In some embodiments, the concentration of the hyaluronic acid is in the range of 2 mg / ml to 50 mg / ml. In specific embodiments, the concentration of the hyaluronic acid is in the range of 5 mg / ml to 30 mg / ml or in the range of 10 mg / ml to 30 mg / ml. In some embodiments, the hyaluronic acid is cross-linked. The cross-linked hyaluronic acid contains cross-linking portions between hyaluronic acid chains, which create a continuous network of hyaluronic acid molecules that are held together by covalent cross-linking portions, physical entanglement of hyaluronic acid chains, and various interactions such as electrostatic interactions, hydrogen bonding, and van der Waals forces.

[0133] Crosslinking of hyaluronic acid can be achieved by modification with a chemical crosslinking agent. The chemical crosslinking agent can be, for example, selected from the group consisting of divinyl sulfone, multiepoxide, and diepoxide. According to an embodiment, hyaluronic acid is crosslinked using a bifunctional or multifunctional crosslinking agent containing two or more glycidyl ether functional groups. According to an embodiment, the chemical crosslinking agent is selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), 1,2-ethylenediol diglycidyl ether (EDDE), and diepoxyoctane. According to a preferred embodiment, the chemical crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE).

[0134] Polysaccharides, and especially glycosaminoglycans such as hyaluronic acid, chondroitin, and chondroitin sulfate, often tend to degrade their backbone under harsh reaction conditions (e.g., very high or low pH values ​​or high temperatures). Therefore, the method of the present invention is particularly useful for the deacetylation of such polysaccharides.

[0135] The deacetylation method of the present invention can be used to obtain at least partially deacetylated biopolymers, wherein a significant portion of the molecular weight of the starting material is retained.

[0136] According to some embodiments, the weight-average molecular weight of the recovered at least partially deacetylated biopolymer is at least 10%, preferably at least 20%, and more preferably at least 25% of the weight-average molecular weight of the biopolymer containing acetyl groups in step a1). The weight-average molecular weight of the recovered at least partially deacetylated biopolymer can also be higher, for example, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the weight-average molecular weight of the biopolymer containing acetyl groups in step a1).

[0137] According to some embodiments, the biopolymer containing acetyl groups has a weight-average molecular weight of at least 10 kDa. According to some embodiments, the biopolymer containing acetyl groups has a weight-average molecular weight of at least 100 kDa, at least 500 kDa, at least 750 kDa, or at least 1 MDa. According to some embodiments, the biopolymer containing acetyl groups has a weight-average molecular weight in the range of 1 MDa to 5 MDa, preferably in the range of 2 MDa to 4 MDa.

[0138] According to some embodiments, the recovered at least partially deacetylated biopolymer has a weight-average molecular weight of at least 10 kDa. According to some embodiments, the recovered at least partially deacetylated biopolymer has a weight-average molecular weight of at least 100 kDa, at least 500 kDa, at least 750 kDa, or at least 1 MDa. According to some embodiments, the recovered at least partially deacetylated biopolymer has a weight-average molecular weight in the range of 0.1 MDa to 5 MDa, preferably in the range of 0.5 MDa to 5 MDa or 0.5 MDa to 3 MDa.

[0139] The deacetylation method disclosed herein is also applicable to shorter biopolymers or biooligomers, such as dimers, trimers, tetramers, etc.

[0140] According to some implementation schemes, the biopolymer containing acetyl groups is an oligomeric biopolymer having a weight-average molecular weight in the range of 0.3 kDa to 10 kDa.

[0141] According to some implementation schemes, the recovered at least partially deacetylated oligomeric biopolymers have a weight-average molecular weight in the range of 0.3 kDa to 10 kDa.

[0142] In deacetylation methods, biopolymers containing acetyl groups used as starting materials are typically fully or almost fully acetylated. By the term "fully acetylated" as used herein with respect to biopolymers, we mean a biopolymer in which all or substantially all of the free amine groups have been converted to N-acetyl groups. In other words, a "fully acetylated" biopolymer contains no or substantially no free amine groups. According to some embodiments, the acetyl-containing biopolymer used as a starting material in step a1) has a degree of acetylation in the range of 98%–100%.

[0143] According to some embodiments, the recovered at least partially deacetylated biopolymer has a degree of acetylation that is at least 1%, preferably at least 2%, preferably at least 3%, preferably at least 4%, and preferably at least 5% lower than that of the acetylated biopolymer containing acetyl groups in step a1). In other words, the recovered at least partially deacetylated biopolymer may have a degree of acetylation of less than 99%, preferably less than 98%, less than 97%, less than 97%, less than 96%, less than 95%, less than 94%, or less than 93%. The recovered at least partially deacetylated biopolymer may also have a degree of acetylation that is at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50% lower than that of the acetylated biopolymer containing acetyl groups in step a1).

[0144] Deacetylation can be achieved using hydroxylamine or its salts. A hydroxylamine salt is a salt formed from hydroxylamine and an acid. A hydroxylamine salt can be, for example, a salt formed from hydroxylamine and an acid selected from the group consisting of mineral acids and organic acids, or mixtures thereof.

[0145] According to the embodiment, the hydroxylamine salt is a salt formed from hydroxylamine and a mineral acid. According to the embodiment, the acid is selected from the group consisting of sulfuric acid, hydrochloric acid, hydroiodic acid, hydrobromic acid, and phosphoric acid, and combinations thereof. Preferred mineral acids include hydrochloric acid, hydroiodic acid, and hydrobromic acid. Particularly preferred mineral acid is hydroiodic acid.

[0146] According to the implementation scheme, the hydroxylamine salt is a salt formed from hydroxylamine and an organic acid. According to the implementation scheme, the acid is selected from the group consisting of: acetic acid, propionic acid, neopentanoic acid, citric acid, oxalic acid, malonic acid, lactic acid, benzoic acid, and halogenated carboxylic acids such as trifluoroacetic acid (TFA) and trichloroacetic acid, and combinations thereof.

[0147] According to the implementation scheme, the acid is selected from the group consisting of acetic acid, propionic acid, neopentanoic acid, and halocarboxylic acids, preferably trifluoroacetic acid, and combinations thereof. According to the implementation scheme, the acid is a halocarboxylic acid, preferably trifluoroacetic acid.

[0148] According to the implementation scheme, the hydroxylamine salt is a salt formed by hydroxylamine and an acid selected from the group consisting of: hydrochloric acid, hydroiodic acid and hydrobromic acid, propionic acid, neopentanoic acid and trifluoroacetic acid.

[0149] The reaction in step a2 is preferably carried out in a solvent capable of at least partially dissolving both the biopolymer containing an acetyl group and hydroxylamine or a salt thereof. The solvent can be, for example, water or an organic solvent or a mixture thereof. Non-limiting examples of preferred solvents include water or a mixture of water and a lower alcohol such as ethanol. However, many other solvents will be useful depending on the specific biopolymer and the choice of hydroxylamine or a salt thereof. One example of a useful organic solvent is tetrahydrofuran (THF).

[0150] According to the implementation scheme, the reaction in step a2) includes reacting a molecule containing an amide group with hydroxylamine in water.

[0151] The deacetylation process can preferably be carried out in water or an aqueous solution, which optionally also contains another solvent, such as ethanol. Therefore, according to some embodiments, step a1) includes contacting the biopolymer containing acetyl groups with hydroxylamine in water, such that an aqueous mixture or aqueous solution of the biopolymer and hydroxylamine is formed. In some embodiments, the concentration of hydroxylamine is at least 10%, preferably at least 20%, and preferably at least 30% by weight of the aqueous mixture or aqueous solution. Higher hydroxylamine concentrations can increase the reaction rate.

[0152] Hydroxylamine is typically provided in the form of an aqueous solution, usually at a concentration of 50% by weight. In some embodiments, the biopolymer can be directly mixed and dissolved in an aqueous solution of hydroxylamine, optionally diluted. Alternatively, a solid salt of hydroxylamine, such as hydroxylamine hydrochloride or hydroxylamine sulfate, can be dissolved in an aqueous solution of the biopolymer. Adding a salt of hydroxylamine and converting that salt to hydroxylamine can be performed as an alternative or supplementary method for dissolving a biopolymer containing an acetyl group in an aqueous solution of hydroxylamine.

[0153] The molar concentration of hydroxylamine in the reaction mixture is preferably in the range of 5M-20M. For example, a concentration of 50% hydroxylamine by weight corresponds approximately to a molar concentration of 16M.

[0154] The inventors have surprisingly discovered that when hydroxylamine salt is used instead of hydroxylamine itself, the same reaction rate can be obtained at a significantly lower molar concentration. Therefore, the molar concentration of hydroxylamine salt in the reaction mixture is preferably in the range of 0.01M-10M, and more preferably in the range of 0.1M-5M.

[0155] According to some embodiments, in step a1), the biopolymer containing acetyl groups is dissolved in an aqueous solution of hydroxylamine or a salt thereof. According to some embodiments, in step a1), a salt of hydroxylamine is dissolved in an aqueous solution of the biopolymer containing acetyl groups. According to some embodiments, the biopolymer containing acetyl groups is dissolved in an aqueous solution of hydroxylamine, and a salt of hydroxylamine is dissolved in an aqueous solution of the biopolymer containing acetyl groups in hydroxylamine.

[0156] The inventors have surprisingly discovered that when hydroxylamine salt is used instead of hydroxylamine itself, the same reaction rate can be obtained at a significantly lower molar concentration. Therefore, the molar concentration of hydroxylamine salt in the reaction mixture is preferably in the range of 0.01M-10M, and more preferably in the range of 0.1M-5M.

[0157] According to some embodiments, in step a1), the biopolymer containing acetyl groups is dissolved in an aqueous solution of hydroxylamine or a salt thereof. According to some embodiments, in step a1), a salt of hydroxylamine is dissolved in an aqueous solution of the biopolymer containing acetyl groups. According to some embodiments, the biopolymer containing acetyl groups is dissolved in an aqueous solution of hydroxylamine, and a salt of hydroxylamine is dissolved in an aqueous solution of the biopolymer containing acetyl groups in hydroxylamine.

[0158] The reaction temperature in step a2) is preferably 100°C or lower. The reaction temperature in step a2) is selected to avoid excessive degradation of the biopolymer. According to some embodiments, the temperature in step a2) is in the range of 10°C-90°C, preferably 20°C-80°C, preferably 30°C-70°C, and preferably 30°C-50°C. According to embodiments, the reaction in step a2) comprises reacting a molecule containing an amide group with hydroxylamine or a salt thereof at a temperature in the range of 10°C-100°C, preferably 20°C-90°C, preferably 30°C-70°C, and preferably 30°C-50°C. This temperature can be, for example, in the range of 70°C-90°C, such as about 80°C, or in the range of 30°C-50°C, such as about 40°C.

[0159] The reaction time in step a2) depends on the desired degree of deacetylation. The reaction time is preferably selected to avoid excessive degradation of the biopolymer and also depends on temperature and pH. The reaction time can typically be any time from 5 minutes to 200 hours or more. According to some embodiments, the reaction in step a2) involves reacting the amide-containing molecule with hydroxylamine or a salt thereof for 2-200 hours. According to some embodiments, the reaction in step a2) involves reacting the amide-containing molecule with hydroxylamine or a salt thereof for 2-150 hours, preferably 5-150 hours, preferably 5-100 hours. In other embodiments, such as when using higher temperatures or pH, the reaction time can be much shorter, for example, in the range of 5 minutes to 2 hours, in the range of 30 minutes to 2 hours, or in the range of 1-2 hours.

[0160] Preferably, the pH in step a2) is selected to avoid excessive degradation of the biopolymer. According to some embodiments, the reaction in step a2) is carried out at a pH in the range of 4-12. According to some embodiments, the reaction in step a2) is carried out at a pH in the range of 9-11. According to some embodiments, the reaction in step a2) is carried out at a pH in the range of 4-9, preferably in the range of 6-9, and preferably in the range of 6-8 or 7-8. Generally, a lower pH (e.g., approximately neutral pH), such as in the range of 6-8 or 7-8, is preferred to avoid degradation of the biopolymer.

[0161] The inventors have discovered through extensive experimentation that the addition of a pH-lowering agent can significantly increase the reaction rate in step a2), particularly when hydroxylamine is used. This effect is both surprising and highly advantageous. Notably, the corresponding addition of a pH-lowering agent to the hydrazine deacetylation reaction does not result in any increase in the reaction rate. A lower pH value during the reaction is also preferred to avoid excessive degradation of the biopolymer. Thus, according to some embodiments, by adding a pH-lowering agent, the pH of the reaction is reduced to a value in the range of 4-9, preferably in the range of 6-9, and more preferably in the range of 6-8 or 7-8. The pH-lowering agent can be selected, for example, from the group consisting of mineral acids, organic acids, and pH-lowering salts, and mixtures or combinations thereof. Examples of useful mineral acids include, but are not limited to, sulfuric acid, hydrochloric acid, hydroiodic acid, hydrobromic acid, and phosphoric acid. Examples of useful organic acids include, but are not limited to, acetic acid, propionic acid, pentanoic acid, citric acid, oxalic acid, malonic acid, lactic acid, benzoic acid, and halogenated carboxylic acids such as trifluoroacetic acid and trichloroacetic acid. Examples of useful pH-lowering salts include, but are not limited to, ammonium chloride, ammonium bromide, ammonium iodide, hydroxylamine hydrochloride, and hydroxylamine sulfate. In preferred embodiments, the pH-lowering agent comprises hydroxylamine hydrochloride or hydroxylamine sulfate, most preferably hydroxylamine hydrochloride. In some embodiments, the pH-lowering agent is hydroiodic acid (HI). In some embodiments, the pH-lowering agent is trifluoroacetic acid (TFA).

[0162] According to some implementation schemes, the reaction in step a2) is carried out in an inert atmosphere and / or in the dark.

[0163] Products obtained by the above deacetylation method can have properties that are significantly different from those obtained by other known deacetylation methods.

[0164] The hydrogel product obtained by the method of the present invention may optionally undergo step d): acylation of the residual amine groups of the cross-linked glycosaminoglycan provided in step c) to form an acylated cross-linked glycosaminoglycan. This process is also referred to herein as reacylation or reacetylation.

[0165] It has been found that the acylation, such as acetylation, of residual free amine groups in hydrogel products containing amide-crosslinked glycosaminoglycan molecules can be used to alter the mechanical properties of the hydrogel products. Without being limited to any specific scientific explanation, it is anticipated that the acylation of free amine groups could reduce the formation of zwitterionic complexes that act as additional crosslinking sites in the hydrogel product, thereby leading to the formation of a softer gel.

[0166] According to some embodiments, step d) includes acetylation of the residual amine groups of the cross-linked glycosaminoglycan provided in step c) to form an acetylated cross-linked glycosaminoglycan. Glycosaminoglycans in their natural form are N-acetylated. Therefore, acetylation of free amine groups in a hydrogel product can be expected to produce a hydrogel product more similar to natural glycosaminoglycans.

[0167] According to some implementations, step d) includes reacting the cross-linked glycosaminoglycan provided in step c) with an acetylation agent under reaction conditions suitable for forming an acetylated cross-linked glycosaminoglycan.

[0168] According to some implementation schemes, the acetylation agent is selected from the group consisting of acetic anhydride, isopropyl acetate, and pre-activated esters of acetic acid.

[0169] Reacetylation can be carried out according to standard protocols using, for example, a pre-activated ester of acetic anhydride, isopropyl acetate, or acetic acid, typically in an aqueous or alcoholic solution or a mixture thereof, or under pure conditions. Preferably, the reacetylation process can be carried out in a solid-state reaction using an alcohol, preferably methanol or ethanol, an acetylation agent, and, if desired, an organic or inorganic base.

[0170] Potential problems such as over-acetylation, O-acetylation, ester formation, and / or anhydride formation can be addressed by including a post-crosslinking alkali treatment step. If desired, the reacetylation step can be excluded from the process to supply zwitterionic hydrogels.

[0171] The hydrogel product obtained by the method of the present invention optionally undergoes step e): subjecting the cross-linked glycosaminoglycan provided in step c) or d) to alkali treatment to hydrolyze the ester cross-linked portion formed as a byproduct during amide cross-linking in step c).

[0172] Amide coupling using diamine or polyamine-functionalized crosslinking agents in conjunction with coupling agents is an attractive approach for preparing crosslinked glycosaminoglycan molecules suitable for hydrogel products. Crosslinking can be achieved using non-carbohydrate-based amphiphilic or polyphilic crosslinking agents such as hexamethylenediamine (HMDA), or carbohydrate-based amphiphilic or polyphilic crosslinking agents such as diaminotrehalose (DATH) in conjunction with the glycosaminoglycan. Crosslinking can also be achieved using at least partially deacetylated glycosaminoglycans, alone or in combination with a second glycosaminoglycan, whereby the deacetylated glycosaminoglycan itself acts as an amphiphilic or polyphilic crosslinking agent.

[0173] It has been found that the coupling or crosslinking of glycosaminoglycans using coupling agents to form amide bonds is often associated with the simultaneous formation of a portion of ester bonds. The size of the ester bond moiety can vary depending on the reaction conditions, concentration, and coupling agent used. Compared to amide bonds, ester bonds are more prone to degradation during the handling and storage of hydrogel products, such as high-temperature sterilization (autoclaving). This means that the properties of hydrogel products containing ester bonds, or a combination of ester and amide bonds, will tend to change over time as the ester bonds degrade. To obtain hydrogels that retain their original properties over a longer period of time, it is preferable that the glycosaminoglycans are crosslinked via amide bonds.

[0174] The inventors have now discovered that glycosaminoglycans with both amide and ester crosslinks can be hydrolyzed by alkali treatment, where the ester crosslinks formed as byproducts during amide crosslinking are not simultaneously degraded. It has also been found that, with appropriate reaction conditions, the hydrolysis of the ester bonds can be achieved without excessive degradation of the glycosaminoglycan backbone.

[0175] The method for preparing a hydrogel product optionally includes step d): acylation of the residual amine groups of the cross-linked glycosaminoglycan provided in step c) to form an acylated cross-linked glycosaminoglycan.

[0176] Acylation, such as acetylation, of residual free amine groups in hydrogel products containing amide-crosslinked glycosaminoglycan molecules can be used to alter the mechanical properties of the hydrogel product. Without being limited to any specific scientific explanation, it is anticipated that acylation of free amine groups can reduce the formation of zwitterionic complexes that act as additional crosslinking sites in the hydrogel product, thereby leading to the formation of a softer gel.

[0177] According to some embodiments, step d) includes acetylation of the residual amine groups of the cross-linked glycosaminoglycan provided in step c) to form an acetylated cross-linked glycosaminoglycan. Glycosaminoglycans in their natural form are N-acetylated. Therefore, acetylation of free amine groups in hydrogel products can be expected to produce hydrogel products that are more similar to natural glycosaminoglycans.

[0178] The acylation of glycosaminoglycans with acylating agents to form amide bonds is generally associated with the simultaneous formation of a portion of ester bonds. The size of the ester bond moiety can vary depending on the reaction conditions, concentration, and acylating agent used. Compared to amide bonds, ester bonds are more prone to degradation during the handling and storage of hydrogel products, such as high-temperature sterilization (autoclaving). This means that the properties of hydrogel products containing ester bonds, or a combination of ester and amide bonds, will tend to change over time as the ester bonds degrade. To obtain hydrogels that retain their original properties over a longer period of time, it is preferable that the glycosaminoglycans are acylated via amide bonds.

[0179] The inventors have now discovered that cross-linked glycosaminoglycans having both amide and ester cross-linking portions can be hydrolyzed by alkali treatment without simultaneously degrading the amide bonds formed during acylation. It has also been found that, with appropriate reaction conditions, the hydrolysis of the ester bonds can be achieved without excessive degradation of the glycosaminoglycan backbone.

[0180] Alkali treatment selectively hydrolyzes the less stable ester bonds formed by O-acetylation and anhydrides from crosslinking processes or reacetylation processes, resulting in an increased amide / ester bond ratio in the material.

[0181] 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 corrective and aesthetic treatments.

[0182] As used in this article for various polymers such as polysaccharides, the term "molecular weight" refers to the polymer's weight-average molecular weight M. w This is well defined in the scientific literature. Weight-average molecular weight can be determined by, for example, static light scattering, small-angle neutron scattering, X-ray scattering, and sedimentation velocity. The unit of molecular weight is Da or g / mol.

[0183] Those skilled in the art will recognize that the present invention is by no means limited to the preferred embodiments described above. Rather, many modifications and variations are possible within the scope of the appended claims. Furthermore, based on a study of the drawings, the disclosure, and the appended claims, those skilled in the art can understand and implement variations of the disclosed embodiments in practicing the claimed invention. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite articles "a" or "an" do not exclude multiple. The mere fact that certain measures are stated in mutually different dependent claims does not imply that combinations of these measures cannot be advantageously used. Example

[0184] Not wishing to be limited thereto, the present invention will be illustrated below by way of examples.

[0185] Definition and Analysis

[0186] Mw - weight average molecular mass

[0187] Swelling factor analysis in SwF-salt, the volume (mL / g) of 1g gel swollen to its maximum value.

[0188] SwC - Swelling capacity in brine, total fluid uptake per gram of PS (mL / g).

[0189] SwCC Ps- Corrected swelling, for GelP, total fluid uptake (mL / g) of 1 gram of PS.

[0190]

[0191] [PS] - Polysaccharide concentration (mg / g).

[0192] GelP – The gel portion describes the percentage of PS that is part of the gel network. A 90% figure means that 10% of the polysaccharides are not part of the gel network.

[0193] CrD 酰胺 - The degree of crosslinking of amides (%) was analyzed using SEC-MS and defined as:

[0194]

[0195]

[0196] DoA – Degree of acetylation. Degree of acetylation (DoA) is the molar ratio of acetyl groups to hyaluronic acid disaccharide. DoA can be calculated from NMR spectra by comparing the integral of the acetyl signal of the hyaluronic acid disaccharide residue with the integral of the C2-H signal of the deacetylated glucosamine residue, according to the following equation.

[0197]

[0198] NMR - 1 ¹H NMR spectra were recorded on a BRUKER Biospin AVANCE 400 spectrometer. Chemical shifts are reported as δ values ​​from the internal standard TMS in a suitable organic solution to a low magnetic field. The purity and structure of the product were confirmed by LCMS (254 nm) on a Waters 2690 photodiode array detector system under the following conditions: column, symmetric C-18; solvent A, water 0.1% formic acid; solvent B, CH₃CN; flow rate, 2.5 mL / min; run time, 4.5 min; gradient, from 0% solvent B to 100% solvent B; mass detector, micro mass ZMD. Purification was performed directly by mass-triggered preparative LCMS on a Waters X-Terra reversed-phase column (C-18, 5 μm silica, 19 mm diameter, 100 mm length, 40 mL / min flow rate) and a gradually decreasing polarity mixture of water (containing 0.1% formic acid) and acetonitrile as eluent. The portion containing the desired compound is evaporated to dryness to obtain the final compound, which is usually a solid.

[0199] Example 1 - Deacetylation of hyaluronic acid via hydroxylamineolysis

[0200] 0.2 g or 20 g of HA (Mw 2500 kDa, DoA 100%) was dissolved in hydroxylamine (Sigma-Aldrich 50 vol% solution) or a mixture of hydroxylamine and water, as shown in Table 1. The solutions were incubated at 30°C–70°C in the dark under argon for 5–353 hours. After incubation, the mixture was precipitated by ethanol. The resulting precipitate was filtered, washed with ethanol, and then redissolved in water. The solution was purified by ultrafiltration and subsequently lyophilized to give deacetylated HA (de-Ac HA) as a white solid. Examples 1-1 to 1-14 were carried out using about 0.2 g of HA, and Examples 1-15 to 1-16 were carried out using 20 g of HA.

[0201] Compared with hydrazine hydrolysis (Example 2) and alkaline methods (Examples 3 and 4), deacetylation via hydroxylamine hydrolysis is more efficient and better preserves the Mw of the HA backbone.

[0202] Table 1.

[0203]

[0204]

[0205] a: SEC-UV b: SEC-MALS

[0206] Example 2 - Deacetylation of hyaluronic acid via hydrazine hydrolysis - Comparative Example

[0207] As shown in Table 2, 0.2 g of HA (Mw 2500 kDa, DoA 100%) was dissolved in 10 mL of a 1% solution of hydrazine sulfate monohydrate. The reaction was carried out in the dark at 30-55°C under argon for 24-120 hours. The mixture was precipitated with ethanol. The precipitate was filtered, washed with ethanol, and then redissolved in water. The final deacetylated HA product was obtained after ultrafiltration and freeze-dried. Compared with hydroxylamine hydrolysis (Example 1), deacetylation via hydrazine hydrolysis yielded greater degradation of the HA backbone, i.e., a lower Mw in the deacetylated product.

[0208] Table 2.

[0209]

[0210] Example 3 - Deacetylation of hyaluronic acid via homogeneous alkaline hydrolysis - Comparative Example

[0211] HA (1000 kDa) was weighed into a reaction vessel, NaOH solution was added, and the mixture was stirred until a homogeneous solution was obtained. The mixture was incubated without stirring as shown in Table 3, and then diluted with water and EtOH. The mixture was neutralized by adding 1.2 M HCl and precipitated by adding EtOH. The precipitate was washed with ethanol (70 w / w%), then washed with ethanol again, and vacuum dried overnight to obtain a solid. Deacetylation via homogeneous alkaline hydrolysis yielded more degradation of the HA backbone compared to hydroxylamine hydrolysis (Example 1), i.e., a lower Mw of the deacetylated product.

[0212] Table 3.

[0213]

[0214] Example 4 - Deacetylation of hyaluronic acid via heterogeneous alkaline hydrolysis - Comparative Example

[0215] HA (1000 kDa) was weighed into the reaction vessel, and NaOH (70% w / w%) from EtOH was added, as shown in Table 4. The heterogeneous mixture was incubated and then neutralized by adding 1.2 M HCl. The precipitate was washed with ethanol (75 w / w%), then washed with ethanol again, and dried under vacuum overnight to obtain a solid.

[0216] Compared to hydroxylamine hydrolysis (Example 1), deacetylation via heterogeneous alkaline hydrolysis results in greater degradation of the HA backbone, i.e., a lower Mw for the deacetylated product.

[0217] Table 4.

[0218]

[0219] Example 5 - Crosslinked deacetylated HA

[0220] Dissolve the coupling agent DMTMM in Na-phosphate buffer (pH 7.4). Adjust the pH of the DMTMM mixture if necessary, and then add the solution to the deacetylated HA. Homogenize the reaction mixture by shaking for 3.5 minutes and mixing with a spatula or by pressing the mixture through a filter. Place the reaction mixture in a water bath at 35°C for 24 hours. Stop the reaction by removing it from the water bath and cutting the gel into small pieces with a spatula or pressing it through a filter. Adjust the pH of the reaction mixture to >13 with 0.25M NaOH, stirring for approximately 60 minutes, and then neutralize with 1.2M HCl. After neutralization, precipitate the gel in ethanol and wash with ethanol (70 w / w%), and vacuum dry overnight. Swell the dried gel in phosphate buffer in 0.7% NaCl for at least two hours. Control the pH and adjust to 7.4 if necessary. Reduce the size of the gel particles using a fine filter. Fill syringes with the gel and sterilize the syringes by autoclaving. The results provided in Table 5 show the formation of hydrogels by crosslinking deacetylated HAs with different Mw and DoA using DMTMM.

[0221] Table 5.

[0222]

[0223] Example 6 - Crosslinking a mixture of deacetylated HA and HA

[0224] Dissolve HA and deacetylated HA in 40 mL of water (Milli-Q) in a 50 mL Falcon tube with end-over-end stirring for 24 hours. After complete dissolution, freeze-dry the sample. Dissolve the coupling agent DMTMM in Na-phosphate buffer (pH 7.4), measure the pH of the DMTMM mixture, and then add it to the freeze-dried mixture. Homogenize the reaction mixture and place it in a water bath at 35°C for 24 hours. Stop the reaction by removing it from the water bath and cut the gel into small pieces with a spatula. Adjust the reaction mixture to pH > 13 with 0.25 M NaOH for approximately 60 minutes. Neutralize the gel with 1.2 M HCl. After neutralization, precipitate the gel with ethanol and wash with ethanol (70%), then vacuum dry overnight. Swell the dried gel in phosphate buffer in 0.7% NaCl for at least two hours. Control the pH and adjust to 7.4 if necessary. Reduce the size of the gel particles using a fine filter. The gel was filled into a syringe, which was then sterilized by autoclaving. The results provided in Table 5 illustrate the formation of a hydrogel by crosslinking deacetylated HA with HA using DMTMM.

[0225] Table 6.

[0226]

[0227] Example 7 - Crosslinking a mixture of HMW-deacetylated HA and LMW-deacetylated HA

[0228] Two different Mws of deacetylated HA were mixed together. The coupling agent DMTMM was dissolved in Na-phosphate buffer (pH 7.4), and the pH of the DMTMM mixture was adjusted if necessary. The solution was then added to the deacetylated HA. The reaction mixture was homogenized by mixing with a spatula or by pressing the mixture through a filter. The reaction mixture was incubated at 23°C for 24 hours. The reaction was stopped by removing it from the incubator and cutting the gel into small pieces with a spatula or pressing it through a filter. The reaction mixture was adjusted to pH > 13 with 0.25M NaOH, stirred for about 60 minutes, and then neutralized to pH 7.4 with 1.2M HCl.

[0229] Table 7.

[0230]

[0231] Example 8 - Heterogeneous Reacetylation of Hydrogels

[0232] The coupling agent DMTMM was dissolved in Na-phosphate buffer (pH 7.4). If necessary, the pH of the DMTMM mixture was adjusted, and the solution was then added to the deacetylated HA. The reaction mixture was homogenized by shaking for 3.5 minutes and mixing with a spatula or by pressing the mixture through a filter. The reaction mixture was placed in a water bath at 35°C for 24 hours. The reaction was stopped by removing the mixture from the water bath and cutting the gel into small pieces with a spatula or pressing it through a filter. The reaction mixture was adjusted to pH > 13 with 0.25M NaOH, stirred for 60 minutes, and then neutralized with 1.2M HCl. After neutralization, the gel was precipitated in ethanol and washed with ethanol (70 w / w%), and then vacuum dried overnight.

[0233] The precipitated gel was suspended in MeOH and Ac₂O (20 equivalences / HA disaccharide) was added. The suspension was incubated at 40°C for 24 hours, then filtered. The resulting solid was washed with 70 w / w EtOH, followed by washing with EtOH again, and then vacuum dried overnight. The acetylated gel was dissolved in 0.25 M NaOH, stirred for 60 minutes, and then neutralized with 1.2 M HCl. After neutralization, the gel was precipitated in ethanol and washed with ethanol (70 w / w%), and then vacuum dried overnight. The dried gel was swollen in phosphate buffer in 0.7% NaCl for at least two hours.

[0234] As a control experiment (Examples 8-3), HA (310 kDa) was suspended in MeOH, and Ac₂O (20 equivalents / HA disaccharide) was added. The suspension was incubated at 40°C for 24 hours, then filtered. The resulting solid was washed with 70 w / w% EtOH and then vacuum dried overnight. The product was dissolved in 0.25 M NaOH, stirred for 60 minutes, and then neutralized with 1.2 M HCl. After neutralization, the gel was precipitated in ethanol and washed with ethanol (70 w / w%), and then vacuum dried overnight. The Mw of the resulting product was analyzed. The results are summarized in Table 8.

[0235] Table 8.

[0236]

[0237] Example 9 - Homogeneous reacetylation of hydrogels

[0238] The coupling agent DMTMM was dissolved in Na-phosphate buffer (pH 7.4), and the pH was controlled and adjusted as necessary. The DMTMM solution was then added to the deacetylated HA. The suspension was homogenized by shaking for 3.5 minutes and mixing with a spatula or by pressing the mixture through a filter. The reaction mixture was incubated at 23°C for 24 hours. The reaction was stopped by removing the mixture from the incubator and mixing the gel twice with a spatula or pressing it through a 1 mm wire mesh. 0.25 M NaOH (pH > 13) was then added to the resulting material, and the mixture was stirred for 60 minutes, followed by neutralization with 1.2 M HCl. After neutralization, the gel particle size was reduced by passing it through a fine filter. The gel was then precipitated in EtOH and washed with 70 w / w% EtOH and EtOH. The resulting material was vacuum dried overnight.

[0239] The precipitated gel powder was added to deionized water and allowed to stand for 60 minutes. Triethanolamine (1.5 equimolar / HA disaccharide) and Ac₂O (1 equimolar / HA disaccharide) were added to the gel suspension. The reaction mixture was mixed at 23°C for 60 minutes. Then, 0.25M NaOH (pH>13) was added to the acetylated gel, and the mixture was mixed for 45 minutes, followed by neutralization with 1.2M HCl. After neutralization, the gel was precipitated in EtOH and washed with 70 w / w% EtOH + 100 mM NaCl, 70 w / w% EtOH, and then EtOH, and vacuum dried overnight. The dried gel was swollen in Na-phosphate buffer at room temperature for at least two hours and then passed through a fine filter to reduce particle size.

[0240] As a control experiment (Examples 9-3), deacetylated HA (1700 kDa) was added to deionized water and allowed to stand for 60 minutes. Triethanolamine (1.2 equimolars / HA disaccharide) and Ac₂O (1 equimolar / HA disaccharide) were added to the HA mixture. The reaction mixture was mixed at 23°C for 60 minutes, then 0.25 M NaOH (pH > 13) was added, and the mixture was mixed for 40 minutes, followed by neutralization with 1.2 M HCl. After neutralization, the mixture was precipitated in EtOH, washed with 70 w / w% EtOH + 100 mM NaCl, 70 w / w% EtOH, and then EtOH, and vacuum dried overnight. The Mw and DoA of the resulting product were analyzed. The results are summarized in Table 9.

[0241] Table 9.

[0242]

[0243] Example 10 - Alkaline hydrolysis of cross-linked HA gel

[0244] Dissolve the coupling agent DMTMM in Na-phosphate buffer (pH 7.4). Adjust the pH of the DMTMM mixture if necessary, and then add the solution to the deacetylated HA. Homogenize the reaction mixture by shaking for 3.5 minutes and mixing with a spatula or by pressing the mixture through a filter. Place the reaction mixture in a water bath at 35°C for 24 hours. Stop the reaction by removing it from the water bath and cutting the gel into small pieces with a spatula or pressing it through a filter.

[0245] The gel was separated into two fractions. For one fraction, the pH was adjusted to >13 with 0.25M NaOH and stirred for approximately 60 minutes, followed by neutralization with 1.2M HCl. After neutralization, the gel was precipitated in ethanol and washed with ethanol (70 w / w%), then washed again with ethanol and vacuum dried overnight. If desired, the dried gel was swollen in phosphate buffer in 0.7% NaCl at room temperature for at least two hours and then passed through a fine filter to reduce particle size. If necessary, the pH of the gel was controlled and adjusted to 7.2–7.5.

[0246] Dilute the second portion of the gel with water and adjust the pH to 6.5–7.5. After neutralization, precipitate the gel with ethanol, wash with ethanol (70 w / w%), then wash again with ethanol, and vacuum dry overnight. If desired, swell the dried gel in phosphate buffer in 0.7% NaCl at room temperature for at least two hours, and then reduce the particle size through a fine filter. If necessary, control the pH of the gel and adjust it to 7.2–7.5.

[0247] Alkali treatment is performed to hydrolyze intermolecular and intramolecular ester bonds formed between HA chains during the crosslinking step, and potential O-acetic acid esters and anhydrides formed during the reacetylation step, as well as residual active esters formed by the coupling agent. Alkali hydrolysis exclusively leads to amide bonds in the material.

[0248] As a control experiment (Examples 10-13 to 10-15, Table 10.3), HA was added to Na-phosphate buffer (pH 7.4). The reaction mixture was homogenized by shaking for 3.5 minutes and pressing the mixture through a filter. The reaction mixture was placed in a water bath at 5°C, 35°C, or 50°C for 24 hours. The reaction was stopped by removing the mixture from the water bath and pressing it through a filter. The mixture was adjusted to pH > 13 with 0.25M NaOH for 60-100 minutes. The mixture was neutralized with 1.2M HCl. After neutralization, the HA was precipitated with ethanol, washed with 70% ethanol, washed again with ethanol, and vacuum dried overnight. The Mw of the resulting product was analyzed. The results summarized in Tables 10.1-10.3 show that the alkaline treatment after crosslinking provides the gel with increased swelling properties and lower CrD.

[0249] Table 10.1

[0250]

[0251] Table 10.2

[0252] Example Starting Mw(kDa) Initial DoA (%) DMTMM(mol%) Time (min) CrD* 10-9 1700 95 2.4 0 0.34 10-10 1700 95 2.4 60 0.30 10-11 950 89 4.0 0 0.88 10-12 950 89 4.0 60 0.73

[0253] For the non-alkali-treated gels in Table 10.2, *CrD also includes the ester crosslinking portion.

[0254] Table 10.3

[0255] Example Starting Mw(kDa) Temperature (°C) Time (min) Ultimately, Mw(kDa) 10-13 1360 5 100 <![CDATA[1340 b ]]> 10-14 920 35 60 <![CDATA[860 a <!-- 25 -->]]> 10-15 1360 50 70 <![CDATA[1230 b ]]>

[0256] a: SEC-UV b: SEC-MALS

[0257] Example 11 -N-((2R,3R,4S)-1,3,4,5-tetrahydroxy-6-(triphenylmethyloxy)hex-2-yl)acetamide Preparation

[0258]

[0259] A solution of N-((2R,3S,5S)-2,4,5-trihydroxy-6-triphenylmethyloxymethyl-tetrahydro-pyran-3-yl)-acetamide (556 mg, 1.20 mol, 1.00 equivalent) in a mixture of THF and H₂O (20 mL, 4:1) was treated with solid sodium borohydride (49.92 mg, 1.32 mol, 1.10 equivalent) [gas escape]. The reaction mixture was stirred at room temperature for 2 h and concentrated to dryness to give N-((2R,3R,4S)-1,3,4,5-tetrahydroxy-6-(triphenylmethyloxy)hex-2-yl)acetamide (500 mg, 89.54%) as a white solid, which was used without further purification.

[0260] LCMS: t R =1.01 minutes, purity =100%; ES+, 464.26(MH)-.

[0261] Example 12-N-((2R,3R,4S)-1,3,4,5-tetrahydroxy-6-(triphenylmethyloxy)hex-2-yl)acetamide Deacetylation

[0262] A suspension of N-((2R,3R,4S)-1,3,4,5-tetrahydroxy-6-(triphenylmethyloxy)hex-2-yl)acetamide (1 equivalent) in hydroxylamine (10 times its volume) was treated with an acid additive to lower the pH to 7 or left untreated, as shown in Table 11, Examples 12-1 to 12-9. The mixture was heated at 80°C until complete deacetylation was achieved. Deacetylation of N-((2R,3R,4S)-1,3,4,5-tetrahydroxy-6-(triphenylmethyloxy)hex-2-yl)acetamide with hydrazine (pH 13) under the same conditions as in Example 2 was also included as Examples 13-10.

[0263] The results are presented in Table 11. The results show that the deacetylation procedure with hydroxylamine proceeds much faster than the deacetylation procedure with hydrazine, and is significantly faster by the addition of a pH-lowering agent.

[0264] Table 11.

[0265]

[0266]

[0267] The reaction mixture was directly purified by preparative LCMS to give (2R,3R,4S)-2-amino-6-(triphenylmethyloxy)hexane-1,3,4,5-tetraol as a white solid.

[0268] LCMS: t R =0.88 minutes, purity = 99%; ES+, 422.11 (MH)- .

[0269] 1 H NMR (DMSO-d6) δ: 7.47-7.37 (m, 6H), 7.30 (dd, J=8.3, 6.7Hz, 6H), 7.26-7.15 (m, 3H), 3.92 (m, 1H), 3.83-3.74 (m, 1H) , 3.62-3.53(m, 1H), 3.52-3.41(m, 1H), 3.34-3.27(m, 1H), 3.22-3.16(m, 1H), 3.13-3.04(m, 1H), 3.01-2.91(m, 1H)

[0270] Example 13: Preparation of N-(4-aminophenylethyl)acetamide

[0271]

[0272] Pure p-cresol acetate (1.65 g, 11.0 mmol, 1.00 equivalent) was added to 4-(2-aminoethyl)aniline (1.50 g; 11.01 mmol; 1.00 equivalent), and the reaction mixture was stirred at room temperature for 30 h. The resulting orange solution was directly adsorbed onto silica gel and purified by rapid chromatography (silica gel, DCM / MeOH 0%–5%) to give N-(4-aminophenylethyl)acetamide (1.76 g, 89.7% yield).

[0273] LCMS: t R =0.58 minutes, purity =99.5%; ES+, 179.5(M+H)+.

[0274] 1 H-NMR (400MHz, DMSO-d6) δ 1.78 (s, 3H), 2.50 (m, 2H hidden by DMSO signal) 3.14 (m, 2H), 4.83 (s, 2H), 6.49 (d, J = 7.5Hz, 2H), 6.84 (d, J = 7.5Hz, 2H), 7.82 (s, 1H).

[0275] Example 14: Preparation of tert-butyl (4-(2-acetaminoethyl)phenyl)carbamate

[0276]

[0277] Triethylamine (0.51 ml, 3.65 mmol, 1.30 equivalent) was added to a stirred solution of N-[2-(4-amino-phenyl)-ethyl]-acetamide (500 mg, 2.81 mmol, 1.00 equivalent) in DCM (20 ml) at room temperature, followed by the addition of di-tert-butyl dicarbonate (673.48 mg, 3.09 mmol, 1.10 equivalent). The reaction mixture was stirred at room temperature for 1 h, washed with water (5 ml), a saturated solution of NaHSO4 (containing water) (5 ml), and water (3 × 5 ml), dried over MgSO4, and concentrated to dryness to give tert-butyl (4-(2-acetaminoethyl)phenyl)carbamate (496 mg, 63% yield) as a light orange solid.

[0278] LCMS: t R =1.11 minutes, purity =100%; ES+, 279.5 (M+H).

[0279] 1 H-NMR (DMSO-d6) δ 1 H NMR (400MHz, DMSO-d6) δ1.57 (s, 9H), 1.87 (s, 3H), 2.75-2.64 (m, 2H), 3.36-3.20 (m , 2H), 7.27-7.07 (m, 2H), 7.45 (d, J=8.3Hz, 2H), 7.94 (t, J=5.6Hz, 1H), 9.31 (s, 1H).

[0280] Example 15 - Preparation of NH2OH.HI

[0281] At 0°C, 57% HI (aqueous) was carefully added dropwise over a 5-minute period to a stirred solution of 50% NH₂OH (9.28 ml, 0.15 mol, 1.00 equivalent) until a pH of 7 was achieved. A dense white crystalline solid formed, which was collected by filtration and carefully washed with ice-cold water to give hydroxylamine hydroiodide (6.80 g, 28%).

[0282] Example 16: Preparation of NT-NH2OH.TFA

[0283] TFA was carefully added dropwise over a 5-minute period at 0°C to a stirred solution of 50% NH₂OH (containing water) (9.28 ml, 0.15 mol, 1.00 equivalent) until a pH of 7 was achieved. The reaction mixture was concentrated under nitrogen bubbling to give hydroxylamine trifluoroacetate (11.0 g, 98%) as a clear, colorless oil.

[0284] Example 17 - NH2OH and its salts relative to commonly used transamidation agents, for example Comparative Study of NH2NH2.H2O and NaOH

[0285]

[0286] The salt (5 equivalents) was added to a stirred solution / suspension of (4-(2v-acetamidoethyl)phenyl)-tert-butyl carbamate (50 mg, 0.18 mmol) in a selected solvent (5 times its volume), and the resulting mixture was heated at 80 °C for the time required to complete the reaction. The results are summarized in Table 12.

[0287] LCMS: t R =0.81 minutes, purity =100%; ES+, 237.51(M+H)+.

[0288] 1 ¹H NMR (DMSO-d6) δ¹H NMR (400MHz, DMSO-d6) δ 9.26 (s, 1H), 8.40 (s, 1H), 7.38 (d, J = 8.0Hz, 2H), 7.11 (d, J = 8.0Hz, 2H), 2.89 (m, 2H), 2.80–2.63 (m, 2H), 1.47 (s, 9H) (separated as formate).

[0289] Table 12.

[0290]

[0291] *Volume = 1g = 1ml = 1 volume.

Claims

1. A method for preparing a hydrogel product comprising cross-linked glycosaminoglycan molecules, the method comprising: (a) Obtaining a solution comprising at least partially deacetylated glycosaminoglycans and a second glycosaminoglycan, wherein the at least partially deacetylated glycosaminoglycan has a degree of acetylation between 50% and 99%; (b) Activating the carboxyl groups on at least partially deacetylated glycosaminoglycans and / or the second glycosaminoglycan with a coupling agent to form activated glycosaminoglycans; (c) Using a carbohydrate-based amphiphilic or polyphilic crosslinking agent to crosslink the activated glycosaminoglycan only by amide bonds to provide a crosslinked glycosaminoglycan; as well as (d) Acylation of the residual amine groups of the cross-linked glycosaminoglycan obtained in step (c) to form an acylated cross-linked glycosaminoglycan; and (e) subject the cross-linked glycosaminoglycan provided in step (c) or (d) to alkali treatment to hydrolyze the ester cross-linked portion formed as a byproduct during the amide cross-linking in step (c).

2. The method according to claim 1, wherein the crosslinking agent is a diamine or polyamine functional crosslinking agent.

3. The method according to claim 1, wherein the glycosaminoglycan is selected from hyaluronic acid, chondroitin, chondroitin sulfate, heparan sulfate, bacterial heparin precursor, heparin, dermatan sulfate, and keratin sulfate.

4. The method according to claim 1, wherein the at least partially deacetylated glycosaminoglycan and / or the second glycosaminoglycan is deacetylated hyaluronic acid.

5. The method of claim 1, wherein the at least partially deacetylated glycosaminoglycan and the second glycosaminoglycan are deacetylated hyaluronic acid.

6. The method according to claim 1, wherein steps (b) and (c) occur simultaneously.

7. The method of claim 1, wherein step (b) occurs prior to step (c).

8. The method of claim 1, wherein the hydrogel product comprises uncrosslinked glycosaminoglycans.

9. The method of claim 7, wherein the cross-linked glycosaminoglycan molecules comprise at least 80% by weight of the hydrogel product.

10. The method of claim 1, wherein the at least partially deacetylated glycosaminoglycan has a degree of acetylation between 93% and 99%.

11. The method of claim 1, wherein the second glycosaminoglycan obtained in step (a) is not altered or modified by the addition of functional groups.

12. The method according to claim 1, wherein at least one of the crosslinking agents is trehalose.

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