High molecular weight beauty ingredients

Abstract

High molecular weight glycosaminoglycan (GAG) hydrogel compositions containing GAGs covalently crosslinked with a carbohydrate crosslinker and methods for making the high molecular weight GAG hydrogel compositions are described. Additionally, methods for using the high molecular weight glycosaminoglycan (GAG) hydrogel compositions for restorative or plastic surgery, cosmetic dermatology, facial contouring, body contouring, and gum augmentation are described.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Patent Application No. 62 / 942,624, filed December 2, 2019, which is incorporated herein by reference in its entirety.

[0002] Field The present disclosure relates to the field of high molecular weight cosmetic compositions, such as hydrogels containing cross-linked polysaccharides, and the use of such hydrogels in medical and / or cosmetic applications, such as implants for subcutaneous or intradermal injection that can be used in humans in reconstructive or plastic surgery and cosmetic dermatology. More specifically, the present disclosure relates to hydrogels containing cross-linked high molecular weight glycosaminoglycans (GAGs), particularly cross-linked hyaluronic acid, chondroitin, or chondroitin sulfate. [Background technology]

[0003] background Hydrogels are widely used in medicine and are prepared by chemically cross-linking polymers to form large-scale macromolecular networks. Both monomeric and minimally polymerized polysaccharides absorb water up to the saturation point, at which point the polysaccharides dissolve, while hydrogels containing the same polysaccharides, although cross-linked, can typically absorb water without dissolving, resulting in swelling of the hydrogel.

[0004] All glycosaminoglycans (GAGs) are long, linear, negatively charged heteropolysaccharides capable of absorbing large amounts of water. Hyaluronic acid, chondroitin, and chondroitin sulfate are well-known biocompatible GAGs used in medical and cosmetic applications. One of the most widely used biocompatible polymers for medical use is hyaluronic acid and its derivatives. To improve the durability of hyaluronic acid in vivo, it is necessary to modify the hyaluronic acid molecule by crosslinking and other means, such as crosslinking hyaluronic acid to form hyaluronic acid hydrogels.

[0005] Producing hydrogels from high-molecular-weight GAGs, such as hyaluronic acid, provides a suitable filler for multiple medical and cosmetic applications. However, hydrogels can degrade or hydrolyze during storage or under degradation conditions, such as heat sterilization or accelerated stability testing. One way to increase hydrogel stability is to increase the number of crosslinks in the hydrogel. However, increasing the number of crosslinks in high-molecular-weight GAGs can result in hydrogels that are stable under degradation conditions but that also result in phase separation of the hydrogel. This poses a challenge in diluting the hydrogel without causing phase separation.

[0006] An objective of the present disclosure is to overcome the problems associated with preparing hydrogels from high molecular weight GAGs that exhibit increased stability and are able to maintain the integrity of the hydrogel during degradation conditions such as heat sterilization, while maintaining the ability to dilute the hydrogel to a desired GAG concentration for applications such as filling the hydrogel into syringes. Summary of the Invention

[0007] Disclosure Overview The present disclosure generally relates to methods for producing hydrogels from cross-linked high molecular weight glycosaminoglycans (GAGs) that can maintain structural integrity under conditions that would hydrolyze or result in phase separation of the hydrogel. The disclosure further relates to hydrogel compositions produced by the methods.

[0008] In some embodiments, the present disclosure generally relates to a method for preparing a hydrogel comprising cross-linked glycosaminoglycan (GAG) molecules, the method comprising: (a) cross-linking a GAG having a molecular weight of at least 1.5 MDa with a cross-linking agent to obtain a glycosaminoglycan hydrogel cross-linked by amide bonds, wherein the concentration of the GAG ​​is 2% to 10% (w / w) and the molar ratio of cross-linking agent to GAG is 2% or less.

[0009] In some embodiments, when the GAG ​​concentration of (a) is 1% to 4.5% (w / w), the cross-linker concentration is 0.8 to 2 mol% per GAG disaccharide; when the GAG ​​concentration of (a) is 4.6% to 5.9% (w / w), the cross-linker concentration is 0.5 to 0.8 mol% per GAG disaccharide; and when the GAG ​​concentration of (a) is 6% to 12% (w / w), the cross-linker concentration is 0.3 to 0.5 mol% per GAG disaccharide.

[0010] In some embodiments, the crosslinker (a) is a dinucleophilic or polynucleophilic functional crosslinker. In some embodiments, the dinucleophilic or polynucleophilic functional crosslinker is an aliphatic or aromatic diamino derivative, a peptide, or a peptide sequence. In some embodiments, the dinucleophilic or polynucleophilic functional crosslinker comprises a spacer group selected from the group consisting of a disaccharide, a trisaccharide, a tetrasaccharide, and an oligosaccharide. In some embodiments, the dinucleophilic or polynucleophilic functional crosslinker is diaminotrehalose (DATH).

[0011] In some embodiments, the crosslinking step (a) comprises: (a1) providing or obtaining a solution of glycosaminoglycan (GAG) molecules; (a2) activating carboxyl groups on the glycosaminoglycan molecules with a coupling agent to form activated glycosaminoglycan molecules; and (a3) ​​crosslinking the activated glycosaminoglycan (GAG) molecules through the activated carboxyl groups using a binucleophilic or polynucleophilic functional crosslinker to obtain a glycosaminoglycan hydrogel crosslinked by amide bonds. In some embodiments, the coupling agent used in (a2) is a triazine-based coupling agent, such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM).

[0012] In some embodiments, the crosslinking in (a) is carried out at a pH of 5.0 to 9.0, preferably 6.0 to 8.0. In some embodiments, the method further comprises formulating the crosslinked hydrogel obtained from (a) to a final glycosaminoglycan (GAG) concentration of 10 to 30 mg / mL. In some embodiments, the method further comprises (b) sterilizing the crosslinked hydrogel obtained from (a).

[0013] In some embodiments, the glycosaminoglycan (GAG) is hyaluronic acid (HA). In some embodiments, the GAG ​​in (a) has a molecular weight of 2.0-10 MDa, preferably 2.5-3.5 MDa. In some embodiments, the concentration of the GAG ​​is 3-5% (w / w). In some embodiments, the molar ratio of the crosslinker to the GAG ​​is 0.9-1.1%. In some embodiments, the GAG ​​is hyaluronic acid (HA) having a molecular weight of 2.5-3.5 MDa, the crosslinker is diaminotrehalose (DATH), and the concentration of the HA is 3-5% (w / w), with the molar ratio of DATH to HA being 0.9-1.1%.

[0014] In some embodiments, the present disclosure generally relates to a hydrogel product obtained by any one of the methods described herein. In some embodiments, the present disclosure generally relates to a hydrogel product comprising glycosaminoglycan (GAG) molecules as a swellable polymer, the glycosaminoglycan molecules being cross-linked by amide bonds, the apparent molecular weight (Mwapp) of the cross-linked glycosaminoglycan molecules being greater than 1.0 MDa, and the thermal stability of the swellable polymer (NormGelC) being greater than 80% after 24 hours.

[0015] In some embodiments, the glycosaminoglycan molecules are covalently crosslinked via bridges comprising a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides. In some embodiments, the spacer group is trehalose. In some embodiments, the glycosaminoglycan (GAG) is hyaluronic acid (HA). In some embodiments, the hydrogel product is sterilized.

[0016] According to any one or more of the foregoing embodiments, the GAG ​​does not have a molecular weight less than 1.5 MDa, or optionally less than 1.4 MDa, 1.3 MDa, 1.2 MDa, 1.1 MDa, 1.0 MDa, 0.9 MDa, 0.8 MDa, or 0.7 MDa.

[0017] According to any one or more of the above embodiments, the hydrogel does not undergo degradation after cross-linking of glycosaminoglycans. According to any one or more of the above embodiments, the hydrogel undergoes degradation by the environment after cross-linking. However, the hydrogel may be final / 2C min C lower than the value min According to any one or more of the above embodiments, the hydrogel may have a C final C higher than / 2 min Indicates the value.

[0018] In some embodiments, (a) and (b) are performed stepwise from (a) to (b). In some embodiments, a1), a2), and a3) are performed stepwise from a1) to a2), a3). In some embodiments, (a) and (b) are not performed stepwise from (a) to (b). In some embodiments, a1), a2), and a3) are not performed stepwise from a1) to a2), a3).

[0019] In some embodiments, the present disclosure generally relates to methods of cosmetically treating skin, comprising applying to the skin a hydrogel product according to any one of the hydrogel compositions described herein.

[0020] [The present invention 1001] 1. A method for preparing a hydrogel comprising cross-linked glycosaminoglycan (GAG) molecules, comprising: Cross-linking a GAG having a molecular weight of at least 1.5 MDa with a cross-linking agent to obtain a glycosaminoglycan hydrogel cross-linked by amide bonds, wherein the concentration of the GAG ​​is 2% to 10% (w / w) and the molar ratio of the cross-linking agent to the GAG ​​is 2% or less. A preparation method comprising: [The present invention 1002] When the GAG ​​concentration is 1% to 4.5% (w / w), the concentration of the cross-linking agent is 0.8 to 2 mol% per GAG disaccharide; When the GAG ​​concentration is 4.6% to 5.9% (w / w), the concentration of the cross-linking agent is 0.5 to 0.8 mol% per GAG disaccharide; When the GAG ​​concentration is 6% to 12% (w / w), the concentration of the cross-linking agent is 0.3 to 0.5 mol% per GAG disaccharide. A method for preparing the hydrogel of the present invention. [The present invention 1003] 1001 or 1002, wherein the crosslinking agent is a dinucleophilic or polynucleophilic functional crosslinking agent. [The present invention 1004] 1003. The method of claim 1003, wherein said di- or poly-nucleophilic functional cross-linking agent is an aliphatic or aromatic diamino derivative, a peptide, or a peptide sequence. [The present invention 1005] 1005. The method of claim 1004, wherein said di- or poly-nucleophilic functional cross-linker comprises a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides. [The present invention 1006] 1005. The method of claim 1005, wherein said di- or poly-nucleophilic functional cross-linking agent is diaminotrehalose (DATH). [The present invention 1007] The crosslinking step (1) providing a solution of glycosaminoglycan (GAG) molecules; (2) activating a carboxyl group on the glycosaminoglycan molecule with a coupling agent to form an activated glycosaminoglycan molecule; (3) cross-linking the activated glycosaminoglycan (GAG) molecules through their activated carboxyl groups using a binucleophilic or polynucleophilic functional cross-linker to obtain glycosaminoglycan hydrogels cross-linked by amide bonds; Any of the methods 1003 to 1006 of the present invention, comprising: [The present invention 1008] 1007. The process of claim 1007, wherein said coupling agent is a triazine coupling agent, such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM). [The present invention 1009] The method according to any one of claims 1001 to 1008, wherein the crosslinking step is carried out at a pH of 5.0 to 9.0, preferably at a pH of 6.0 to 8.0. [The present invention 1010] 1009. The method of any of claims 1001 to 1009, further comprising the step of formulating said crosslinked hydrogel to a final glycosaminoglycan (GAG) concentration of 10 to 45 mg / mL. [The present invention 1011] The method of any one of claims 1001 to 1010, further comprising the step of sterilizing the crosslinked hydrogel. [The present invention 1012] The method of any one of claims 1001 to 1011, wherein the glycosaminoglycan (GAG) is hyaluronic acid (HA). [The present invention 1013] The method of any one of claims 1001 to 1012, wherein the GAG ​​has a molecular weight of 1.5 to 10 MDa, preferably 1.5 to 3.5 MDa. [The present invention 1014] The method of any one of claims 1001 to 1013, wherein the concentration of the GAG ​​is 3 to 5% (w / w). [The present invention 1015] The method of any one of claims 1001 to 1014, wherein the molar ratio of crosslinker to GAG is 0.9 to 1.1%. [The present invention 1016] Any of the methods of present inventions 1001 to 1015, wherein the GAG ​​is hyaluronic acid (HA) having a molecular weight of 1.5 to 3.5 MDa, the crosslinking agent is diaminotrehalose (DATH), and further, the concentration of HA is 3 to 5% (w / w), and the molar ratio of DATH to HA is 0.9 to 1.1%. [The present invention 1017] A hydrogel product obtained by any of the methods of the present inventions 1001 to 1016. [The present invention 1018] The swellable polymer comprises glycosaminoglycan (GAG) molecules, the glycosaminoglycan molecules being cross-linked by amide bonds, and the apparent molecular weight (Mw APP ) is greater than 1.0 MDa, and the thermal stability of said swellable polymer (NormGelC) is greater than 80% after 24 hours. [The present invention 1019] The hydrogel product of claim 1018, wherein the glycosaminoglycan molecules are covalently crosslinked via bridges comprising spacer groups selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides. [The present invention 1020] 1019. The hydrogel product of claim 10, wherein the spacer group is trehalose. [The present invention 1021] The hydrogel product of any of claims 1018 to 1020, wherein the glycosaminoglycan (GAG) is hyaluronic acid (HA). [The present invention 1022] A hydrogel product according to any one of claims 1018 to 1021, which is sterilized. [The present invention 1023] A method for cosmetically treating skin, comprising applying to the skin any of the hydrogel products of inventions 1018 to 1022. The following detailed description is exemplary and explanatory and is intended to provide further explanation of the present invention. [Brief explanation of the drawings]

[0021] [Figure 1] Shown are the normalized gel contents (NormGelC (%)) of four gel samples exhibiting varying HA (MW) and HA cross-linking at 90° C. over a period of approximately 70 hours. [Figure 2] The gel content (GelC%) corresponding to Samples 1 and 2 in Table 2 is shown after the gels were incubated at 90° C. for 24 or 48 hours. [Figure 3] The gel content (GelC%) corresponding to samples 3 and 4 in Table 2 is shown after the gels were incubated at 90° C. for 24 or 48 hours. [Figure 4] The gel content (GelC%) corresponding to samples 5 and 6 in Table 2 is shown after the gels were incubated at 90° C. for 24 or 48 hours. [Figure 5] The gel content (GelC%) corresponding to samples 7 and 8 in Table 2 is shown after the gels were incubated at 90°C for 24 or 48 hours. DETAILED DESCRIPTION OF THE INVENTION

[0022] Detailed Description of Disclosure I. Definition Although the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are provided to facilitate description of the subject matter of the present disclosure.

[0023] The term "a" or "an" can refer to one or more of its entities, i.e., it can refer to multiple referents. Thus, the terms "a" or "an," "one or more," and "at least one" are used interchangeably herein. In addition, reference to an "element" by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that only one of the element is present.

[0024] References throughout this specification to "one embodiment," "an embodiment," "one aspect," or "an aspect" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0025] As used herein, the terms "about" or "approximately," when preceding a numerical value, indicate a range of plus or minus 10% of that value.

[0026] As will be understood by those skilled in the art, for all purposes, particularly in terms of providing a written description, all ranges disclosed herein encompass all possible subranges and combinations of subranges. Any recited range can be readily recognized as fully descriptive and allowing for the same range to be divided into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily divided into a lower third, middle third, and upper third, etc. Also, as will be understood by those skilled in the art, all language such as "up to," "at least," "higher than," and "less than" refers to a range that is inclusive of the recited numbers and can be subsequently divided into the subranges described above. Finally, as will be understood by those skilled in the art, a range includes each individual element. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1 to 5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc.

[0027] As used herein, a "control" is an alternative sample used in an experiment for comparison purposes. A control can be "positive" or "negative." A "control sample" or "reference sample," as used herein, refers to a sample or reference material that serves as a control for comparison with an experimental sample. For example, an experimental sample may contain compounds A, B, and C in a vial, and a control may be the same type of sample that is treated identically to the experimental sample but lacks one or more of compounds A, B, or C.

[0028] As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired therapeutic and / or prophylactic effect, e.g., an amount that results in prevention of one or more outcomes or an increase in one or more outcomes.

[0029] As used herein, the terms "individual," "patient," or "subject" can be an individual organism, a vertebrate, a mammal, or a human. In preferred embodiments, the individual, patient, or subject is a human.

[0030] As used herein, the phrase "soft tissue" refers to tissue that connects, supports, or surrounds other structures and organs of the body. Soft tissue includes muscle, fibrous tissue, and fat.

[0031] As used herein, the phrase "soft tissue augmentation" refers to any type of volumetric augmentation of soft tissue, including, but not limited to, facial contouring (e.g., more prominent cheeks, chin, or lips), correction of depression deformities (e.g., post-traumatic or HIV-associated lipoatrophy), and correction of deep facial wrinkles associated with aging. Thus, soft tissue augmentation can be used for cosmetic or medical purposes, such as after trauma or degenerative disease. Soft tissue augmentation also refers to skin fillers, body contouring, and gum fillers.

[0032] As used herein, the phrase "non-animal origin" refers to sources that exclude animals, but include sources such as yeast, bacterial, or synthetic.

[0033] As used herein, the term "bioresorbable" refers to a degradation event or events. Bioresorbable materials may dissolve, be phagocytosed, or simply degrade over a period of time, so that the material is removed from the body, organ, tissue, location, or cell over a period of time. The material or its degradation products may be metabolized, incorporated into other molecules or compounds, or excreted.

[0034] As used herein, the term "sterile" refers to free from or removed from pathogenic microorganisms.

[0035] As used herein, the term "sterile" refers to free from living organisms, generally free from living microorganisms.

[0036] As used herein, the term "injectable" refers to the ability to inject a composition of the present disclosure through a needle.

[0037] As used herein, the terms "MW" or "Mw" refer to the mass average molecular mass.

[0038] As used herein, the term "MW app " refers to apparent MW, which is a simulated value of the molecular weight of the GAG ​​in the hydrogel.

[0039] As used herein, the term "SwF" refers to swelling factor analysis in saline, which is the volume of saline per gram of gel swollen to its maximum, usually expressed in mL / g.

[0040] As used herein, "gel content" or "GelC" refers to the percentage of total HA bound in gel form and is further described as the amount of HA in a sample that does not pass through a 0.22 micrometer filter. GelC is calculated from the amount of HA collected in the filtrate and is reported as a percentage of the total amount of HA in the gel sample.

[0041] As used herein, "SwD" refers to the swelling index, which is the reciprocal of the concentration of gel-formed GAG in a fully swollen gel in 0.9% saline, i.e., the volume or mass of fully swollen gel that can be formed per gram of dry cross-linked GAG. SwD generally describes the maximum liquid absorption (0.9% saline) capacity of a product. SwD is preferably expressed as g / g, mL / g, or a dimensionless number. TIFF0007730323000001.tif10128

[0042] SwD can also be expressed as: TIFF0007730323000002.tif9128

[0043] As used herein, "CrR DATH " refers to the effective cross-linking ratio analyzed by LC-SEC-MS, more specifically: TIFF0007730323000003.tif9128

[0044] A CrR of 1.0 indicates that all of the crosslinker has crosslinked.

[0045] As used herein, "C min " is the theoretical minimum GAG concentration. It is the concentration of gel-form GAG in a fully swollen gel in 0.9% saline, typically expressed in mg / g or mg / mL. C min -1 =SwD

[0046] As used herein, "C final " is the intended concentration of GAG in the final hydrogel product. In some embodiments, C final is 2 × C min is greater than.

[0047] The present technology is not limited with respect to the specific aspects described in this application, which are intended as single illustrations of individual aspects of the technology. As will be apparent to those skilled in the art, many modifications and variations of the present technology can be made without departing from its spirit and scope. In addition to those enumerated herein, functionally equivalent methods and apparatuses within the scope of the present technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to fall within the scope of the present technology. It is understood that the present technology is not limited to particular methods, reagents, compound compositions, or biological systems, which can, of course, vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0048] II. Hydrogels and Methods for Making Hydrogels Fillers, such as dermal fillers, have been used to repair, reconstruct, or enhance defects in the body's hard or soft tissue contours due to aging, injury, or acquired or congenital deformities of the face, body, and internal organs. Fillers can be natural or synthetic substances used to reduce wrinkles and / or fine lines, restore lost volume, hydrate the skin, reduce the appearance of nasolabial folds, augment and contour the lips, improve scarring (dimples, hypertrophic, and keloid scars), strengthen weakened vocal cords, and provide other soft tissue improvements. Utilized materials include fat, paraffin, human collagen, bovine collagen, silicone, hyaluronic acid, lactic acid, and glycolic acid. The FDA approval of bovine collagen in 1981 ushered in a new era of soft tissue fillers. Since then, many soft tissue fillers have emerged. The dramatic increase in the number of fillers currently available and under investigation has been driven by many factors, including improvements in biotechnology and society's emphasis on aesthetic appearance. With the introduction of newer fillers, there is a continuing need to evaluate their risk / benefit profile and define their limitations to maximize cosmetic results and safety for patients. Common filler / hydrogel compositions include GAGs such as hyaluronic acid.

[0049] Methods for producing GAG hydrogels are disclosed in PCT Publication Nos. WO2017 / 114867, WO2017 / 114861, WO2017 / 114864, and WO2017 / 114865, U.S. Pregrant Publication Nos. 2019 / 0023812A1, 2019 / 0016830A1, 2019 / 0023855A1, and 2007 / 0066816A1, and U.S. Patent Application Nos. 8,858,999, 6,831,172, 8,887,243, and 6,703,444.

[0050] A common route to crosslinking hyaluronic acid is the use of diglycidyl ethers, such as 1,4-butanediol diglycidyl ether (BDDE). Alternatively, amide coupling, using diamine- or multiamine-functional crosslinkers in conjunction with a coupling agent, is an attractive route to preparing crosslinked hyaluronic acid molecules useful for hydrogel production. For example, the use of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) for activation of carboxylates, followed by condensation with diamino structures, such as diaminotrehalose (DATH), has been shown to be an efficient method for producing hydrogels composed of crosslinked hyaluronic acid with minimal degradation of the biopolymer.

[0051] In some embodiments, the cross-linking is performed via bridges comprising spacer groups selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides, which provides a hydrogel product based entirely on carbohydrate structures or derivatives thereof and minimizes the cross-linking's interference with the native properties of the GAGs utilized to generate the hydrogel.

[0052] In some embodiments, the crosslinker itself contributes to maintaining or increasing the properties of the hydrogel, for example, when crosslinking with structures related to hyaluronic acid (e.g., diaminohyaluronic acid tetrasaccharide) or with structures with high water retention properties (e.g., trehalose).

[0053] In some embodiments, the GAG ​​is sulfated or non-sulfated GAG, such as hyaluronan, chondroitin sulfate, heparan sulfate, heparosan, heparin, dermatan sulfate, and keratan sulfate. In some embodiments, the GAG ​​is hyaluronic acid, chondroitin, or chondroitin sulfate. In one embodiment, the GAG ​​is hyaluronic acid. In some embodiments, the GAG ​​is a natural GAG. In some embodiments, the GAG ​​is a GAG of natural origin. In some embodiments, the GAG ​​is used in its natural state (i.e., the chemical structure of the GAG ​​is not changed or modified, such as by adding a functional group). It is preferable to use the GAG ​​in its natural state. This is because it preserves the natural properties and effects of the GAG ​​itself and provides a crosslinked structure that more closely resembles a natural molecule, which can minimize immune responses when the crosslinked GAG is introduced into the body.

[0054] In some embodiments, the GAGs are covalently crosslinked. In some embodiments, the covalently crosslinked GAG molecules consist essentially of or consist of carbohydrate-type structures or derivatives thereof. In some embodiments, the crosslinked GAGs or hydrogels are free of or essentially free of synthetic non-carbohydrate structures or linkers. This can be achieved by using GAGs in their native state with a crosslinker that comprises, consists essentially of, or consists essentially of carbohydrate-type structures or derivatives thereof. In some embodiments, the functional group of the crosslinker is covalently bonded directly to the carboxyl group of the GAG. In some embodiments, the crosslinks of the covalently crosslinked GAGs comprise, consist essentially of, or consist essentially of disaccharide, trisaccharide, tetrasaccharide, and oligosaccharide spacer groups.

[0055] In some embodiments, the cross-linked GAGs comprise cross-links between GAG molecular chains, which form a continuous network of GAG molecules held together by covalent cross-links.

[0056] In some embodiments, the crosslinked GAGs are water-insoluble, but upon exposure to a liquid, typically an aqueous liquid, form a gel or hydrogel, which is a substantially diluted crosslinked system of the GAG.

[0057] In some embodiments, a process for preparing a hydrogel product comprising crosslinked glycosaminoglycan molecules comprises, consists of, or consists essentially of: (a) providing a solution of glycosaminoglycan molecules; (b) activating carboxyl groups on the glycosaminoglycan molecules with a coupling agent to form activated glycosaminoglycan molecules; and (c) crosslinking the activated glycosaminoglycan molecules via their activated carboxyl groups using a di- or poly-nucleophilic functional crosslinker comprising a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides to obtain crosslinked glycosaminoglycan molecules.

[0058] In some embodiments, GAGs are typically cross-linked by covalent bonds, such as amide bonds, using a di- or poly-nucleophilic functional cross-linker that comprises an activator of the carboxyl group on the GAG ​​molecule backbone and a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides. In some embodiments, cross-linking of GAGs can be achieved by a gentle and efficient route, resulting in high yields with minimal degradation of the GAG ​​molecule.

[0059] In some embodiments, the di- or poly-nucleophilic functional cross-linker contains a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides that remains in the cross-link between GAG molecules. In some embodiments, the di- or poly-nucleophilic functional disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides contain at least two nucleophilic functional groups attached thereto. In some embodiments, the at least two nucleophilic functional groups are separated by a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides.

[0060] In some embodiments, the dinucleophilic or polynucleophilic functional crosslinker contains two or more functional groups capable of reacting with the functional carboxyl groups of GAGs, resulting in the formation of a covalent bond, such as an amide bond. In some embodiments, the nucleophilic functional group can react with a carboxyl group on a glycosaminoglycan molecule to form an amide bond. In some embodiments, the nucleophilic functional group of the disaccharide, trisaccharide, tetrasaccharide, and oligosaccharide is selected from the group consisting of primary amine, hydrazine, hydrazide, carbazate, semicarbazide, thiosemicarbazide, thiocarbazate, and aminoxy. In some embodiments, the dinucleophilic or polynucleophilic functional disaccharide, trisaccharide, tetrasaccharide, and oligosaccharide can be derived from a nucleophilic functional polysaccharide, such as chitobiose derived from chitin. In some embodiments, the dinucleophilic or polynucleophilic functional disaccharide, trisaccharide, tetrasaccharide, and oligosaccharide can be a disaccharide, trisaccharide, tetrasaccharide, and oligosaccharide modified by the introduction of two or more nucleophilic functional groups.

[0061] In some embodiments, di- or poly-nucleophilic functional crosslinkers include homo- or hetero-bifunctional primary amines, hydrazines, hydrazides, carbazates, semicarbazates, thiosemicarbazates, thiocarbazates, and aminoxy.

[0062] In some embodiments, the cross-linking agent is selected from the group consisting of diaminohyaluronic acid tetrasaccharide, diaminohyaluronic acid hexasaccharide, diaminotrehalose (DATH), diaminolactose, diaminomaltose, diaminosucrose, diaminochitobiose, chitobiose, or diaminoraffinose.

[0063] In some embodiments, the activation step and the cross-linking step are performed simultaneously. In some embodiments, the activation step is performed before and separately from the cross-linking step.

[0064] In some embodiments, a post-crosslinking step involves providing particles of crosslinked GAG molecules having an average size ranging from 0.01 to 5 mm, preferably 0.1 to 0.8 mm.

[0065] In some embodiments, the particles are 20 to 800 μm in size. In some embodiments, the particles are about 100 to about 500 μm in size. In some embodiments, the size can be length, diameter, or width. Generally, this refers to the diameter. In some embodiments, the particles are 20 to 800 μm, 20 to 700 μm, 20 to 600 μm, 20 to 500 μm, 20 to 400 μm, 20 to 300 μm, 20 to 200 μm, 100 to 800 μm, 100 to 700 μm, or 100 to 300 μm in size.

[0066] In some embodiments, the coupling agent is a peptide coupling reagent. In some embodiments, the coupling reagent is selected from 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). A preferred triazine-based peptide coupling reagent is DMTMM. Another preferred peptide coupling reagent is a carbodiimide coupling reagent, preferably a combination of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0067] In some embodiments, crosslinking of activated GAG molecules is carried out via their carboxyl groups using a crosslinker. In some embodiments, the crosslinker is a di- or poly-nucleophilic functional crosslinker containing a spacer group selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides. In some embodiments, the crosslinker connects GAG chains to each other via carboxyl groups on the GAG ​​backbone. In some embodiments, the spacer group may be a hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose, or raffinose residue. The term "residue" means that the structure of the compound is similar but not identical to the proprietary compounds hyaluronic acid tetrasaccharide, hyaluronic acid hexasaccharide, trehalose, lactose, maltose, sucrose, cellobiose, or raffinose, respectively. The structure of the residue may differ from that of the parent compound in that two or more nucleophilic functional groups are provided, optionally covalently bonded via the carboxyl groups on the GAG ​​backbone.

[0068] According to related aspects, the present invention also provides for the use of the hydrogel product as a medicament, such as in the treatment of soft tissue disorders. Methods are provided for treating a patient suffering from a soft tissue disorder by administering a therapeutically effective amount of the hydrogel product to the patient. Methods are also provided for providing corrective or cosmetic treatment to a patient by administering a therapeutically effective amount of the hydrogel product to the patient.

[0069] In some embodiments, hydrogels are mostly liquid by weight, and can contain 90-99.9% water, but behave like solids due to a three-dimensional cross-linked GAG molecular network within the liquid. Their significant liquid content makes them structurally flexible and similar to natural tissue, making them highly useful in tissue engineering and as scaffolds for tissue augmentation. They are also useful for the treatment of soft tissue disorders and for corrective or cosmetic treatments. In some embodiments, hydrogels are used as injectable formulations.

[0070] Disclosed herein are methods of using injectable compositions for restorative or plastic surgery, aesthetic dermatology, facial contouring, body contouring, and gum augmentation. In some embodiments, the compositions are freeze-dried or lyophilized. In some embodiments, the compositions comprise hydrogels containing aqueous solutions.

[0071] In some embodiments, cross-linked glycosaminoglycan products, such as hyaluronic acid products, are produced from high molecular weight (HMW) glycosaminoglycans using amide cross-links.

[0072] In some embodiments, crosslinked hyaluronic acid products from HMW hyaluronic acid using DATH / DMTMM chemistry are formulated to provide a suitable GAG ​​concentration for use (C of 10-45 mg / mL). final In some embodiments, the GAG ​​is hyaluronic acid. In some embodiments, the suitable GAG ​​concentration is for dermatological, dental, medical, or reconstructive surgical uses.

[0073] In some embodiments, suitable GAG ​​concentrations are 10 to 50 mg / mL, 10 to 45 mg / mL, 10 to 40 mg / mL, 10 to 35 mg / mL, 10 to 30 mg / mL, 10 to 25 mg / mL, 10 to 20 mg / mL, 10 to 15 mg / mL, 15 to 40 mg / mL, 15 to 40 mg / mL, 15 to 35 mg / mL, 15 to 30 mg / mL, 15 to 25 mg / mL, 15 to 20 mg / mL, 20 to 50 mg / mL, 20 to 45 mg / mL, 20 to 40mg / mL, 20~35mg / mL, 20~30mg / mL, 20~25mg / mL, 25~50mg / mL, 25~45mg / mL, 25~40mg / mL, 25~35mg / mL, 25~30mg / mL, 30~5 0mg / mL, 30~45mg / mL, 30~40mg / mL, 30~35mg / mL, 35~50mg / mL, 35~45mg / mL, 35~40mg / mL, 40~50mg / mL, or 40~45mg / mL.

[0074] In some embodiments, suitable GAG ​​concentrations are about 10 to about 50 mg / mL, about 10 to about 45 mg / mL, about 10 to about 40 mg / mL, about 10 to about 35 mg / mL, about 10 to about 30 mg / mL, about 10 to about 25 mg / mL, about 10 to about 20 mg / mL, about 10 to about 15 mg / mL, about 15 to about 40 mg / mL, about 15 to about 40 mg / mL, about 15 to about 35 mg / mL, about 15 to about 30 mg / mL, about 15 to about 25 mg / mL, about 15 to about 20 mg / mL, about 20 to about 50 mg / mL, about 20 to about 45 mg / mL, about 20 to about 40 mg / mL, about 20 to about 35 mg / mL, about 20 to about 30 mg / mL, about 20 to about 25 mg / mL, about 25 to about 50 mg / mL, about 25 to about 45 mg / mL, about 25 to about 40 mg / mL, about 25 to about 35 mg / mL, about 25 to about 30 mg / mL, about 30 to about 50 mg / mL, about 30 to about 45 mg / mL, about 30 to about 40 mg / mL, about 30 to about 35 mg / mL, about 35 to about 50 mg / mL, about 35 to about 45 mg / mL, about 35 to about 40 mg / mL, about 40 to about 50 mg / mL, or about 40 to about 45 mg / mL.

[0075] In some embodiments, hyaluronic acid encompasses all variants and combinations of variants of hyaluronic acid, hyaluronate, or hyaluronan of various chain lengths and charge states, as well as various chemical modifications, including crosslinking.

[0076] In some embodiments, hyaluronic acid includes various hyaluronate salts of hyaluronic acid with various counterions, such as sodium hyaluronate.In some embodiments, the list of hyaluronic acid also includes various modifications of hyaluronic acid, such as oxidation, for example, the oxidation of -CHOH group to -CHO and / or -COOH; the periodate oxidation of adjacent hydroxyl groups, followed by possible reduction, for example, the reduction of -CHO to -CHOH, or the formation of imine by coupling with amine, followed by reduction to secondary amine; sulfation; deamidation, followed by possible deamination or amide formation with new acid; esterification; crosslinking; substitution with various compounds, for example, using crosslinking agent or carbodiimide-assisted coupling (including the coupling of different molecules, such as protein, peptide and active pharmaceutical ingredient, to hyaluronic acid); and deacetylation.In some embodiments, hyaluronic acid can be further modified by the coupling of isourea, hydrazide, cyanogen bromide, monoepoxide and monosulfone.

[0077] In some embodiments, hyaluronic acid can be obtained from a variety of animal and non-animal sources. In some embodiments, non-animal sources include yeast or bacteria. In some embodiments, the molecular weight of a single hyaluronic acid molecule is typically in the range of 0.1 to 10 mDa, although other molecular weights are possible.

[0078] In some embodiments, the present disclosure relates to at least partially deacetylating a biopolymer / hydrogel comprising acetyl groups, comprising: a) providing a biopolymer comprising acetyl groups; b) reacting the biopolymer comprising acetyl groups with hydroxylamine 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 c) recovering the at least partially deacetylated biopolymer. In some embodiments, the hydrogel is prepared from HMW GAGs by the methods described herein.

[0079] In some embodiments, the present disclosure relates to a method for preparing a hydrogel product comprising crosslinked GAGs, the method comprising: a) providing a GAG crosslinked by an amide bond, wherein the crosslinked GAG comprises residual amine groups; and b) acylating the residual amine groups of the crosslinked GAG provided in a) to form an acylated crosslinked GAG. In some embodiments, the hydrogel is prepared from an HMW GAG by the methods described herein.

[0080] In some embodiments, the present disclosure relates to a method for preparing a hydrogel product comprising crosslinked GAGs, the method comprising: a) providing GAGs crosslinked by amide bonds, wherein the crosslinked GAGs comprise ester crosslinks formed as by-products during the amide crosslinking; and b) subjecting the crosslinked GAGs to alkaline treatment to hydrolyze the ester crosslinks formed as by-products during the amide crosslinking. In some embodiments, the hydrogel is prepared from HMW GAGs by the methods described herein.

[0081] When low DATH loadings are used in the DATH / DMTMM system to produce hydrogels from high molecular weight (HMW) glycosaminoglycans such as hyaluronic acid, a gel suitable for filler compositions is initially formed, but the hydrogel may hydrolyze during storage or exposure to degradation conditions (e.g., heat sterilization, accelerated stability testing). This indicates that more crosslinks are required to keep the gel intact and protect it from hydrolysis. However, increasing the amount of DATH / DMTMM to increase the number of crosslinks in the gel results in a high C phase that phase separates at suitable GAG ​​concentrations. minIn other words, when making a gel from HMW GAGs with sufficient crosslinking to stabilize the gel during autoclaving, it is not possible to dilute the gel to 20 mg / ml (10-45 mg / ml) without obtaining phase separation. This then adds complexity to the process for filling the gel into a syringe, for example. In some embodiments, the hydrogel is homogeneous. A homogeneous product is not phase separated. In some embodiments, the hydrogel is formulated to a concentration suitable for dermatological use (e.g., 10-45 mg / mL) yet retains the ability to swell in the presence of excess saline.

[0082] In some embodiments, the methods of producing the hydrogel do not result in phase separation of the hydrogel. In some embodiments, the hydrogels produced or derived from the methods disclosed herein do not phase separate. In some embodiments, the method of diluting the hydrogel after heat sterilization does not result in phase separation of the hydrogel.

[0083] In some embodiments, the hydrogel is diluted in PBS buffer. In some embodiments, the hydrogel is diluted in 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM phosphate buffer. In some embodiments, the hydrogel is diluted in about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM phosphate buffer. In some embodiments, the hydrogel is diluted into 1 mM to 20 mM, 1 mM to 15 mM, 1 mM to 10 mM, 1 mM to 5 mM, 5 mM to 20 mM, 5 mM to 15 mM, 5 mM to 10 mM, 10 mM to 20 mM, 10 mM to 15 mM, or 15 mM to 20 mM phosphate buffer. In some embodiments, the hydrogel is diluted into about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, about 1 mM to about 5 mM, about 5 mM to about 20 mM, about 5 mM to about 15 mM, about 5 mM to about 10 mM, about 10 mM to about 20 mM, about 10 mM to about 15 mM, or about 15 mM to about 20 mM phosphate buffer.

[0084] In some embodiments, the hydrogel is diluted to a solution having a pH of about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4, about 7.6, about 7.8, or about 8.0. In some embodiments, the hydrogel is diluted to a solution having a pH of 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, or 8.0. In some embodiments, the hydrogel is diluted to a solution having a pH of 6.0-8.0, 6.0-7.0, 7.0-8.0, 6-7.5, 7.0-7.5, or 6.5-7.5.

[0085] In some embodiments, the HA concentration in the cross-linking reaction is about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%. In some embodiments, the HA concentration in the cross-linking reaction is 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, the HA concentration for the cross-linking reaction is 1%-3%, 2%-5%, 3%-5%, 4%-5%, 2%-3%, 2%-4%, 3%-5%, 3%-4%, 1%-20%, 1%-15%, 1%-10%, 1%-5%, 1%-2%, 2%-5%, 2%-10%, 2%-15%, 2%-20%, 5%-20%, 5%-15%, 5%-10%, 10%-20%, 10%-15%, or 15%-20%.

[0086] In some embodiments, the HA concentration in the cross-linking reaction is 10% or less. In some embodiments, the HA concentration in the cross-linking reaction is 9% or less. In some embodiments, the HA concentration in the cross-linking reaction is 8% or less. In some embodiments, the HA concentration in the cross-linking reaction is 7% or less. In some embodiments, the HA concentration in the cross-linking reaction is 6% or less. In some embodiments, the HA concentration in the cross-linking reaction is 5% or less. In some embodiments, the HA concentration in the cross-linking reaction is 4% or less. In some embodiments, the HA concentration in the cross-linking reaction is 3% or less.

[0087] In some embodiments, the mole % of DATH in the crosslinking reaction is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2%. In some embodiments, the mole % of DATH in the crosslinking reaction is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2%. In some embodiments, the mole % of DATH in the crosslinking reaction is 0.1% to 2%, 0.1% to 1.5%, 0.1% to 1%, 0.1% to 0.5%, 0.2% to 1%, 0.2% to 0.5%, 0.2% to 0.3%, 0.5% to 1%, 0.5% to 1.5%, 0.5% to 2%, 1% to 2%, or 1% to 1.5%.

[0088] In some embodiments, the GAG ​​is selected from the group consisting of 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 1100 kDa, 1200 kDa, 1300 kDa, 1400 kDa, 1500 kDa, 1600 kDa, 1700 kDa, 1800 kDa, 1900 kDa, 2000 kDa, 2500 kDa, having a molecular weight of greater than 3000 kDa, 3500 kDa, 4000 kDa, 4500 kDa, 5000 kDa, 5500 kDa, 6000 kDa, 6500 kDa, 7000 kDa, 7500 kDa, 8000 kDa, 8500 kDa, 9000 kDa, 9500 kDa, or 10000 kDa.

[0089] In some embodiments, the GAG ​​is about 700 kDa, about 800 kDa, about 900 kDa, about 1000 kDa, about 1100 kDa, about 1200 kDa, about 1300 kDa, about 1400 kDa, about 1500 kDa, about 1600 kDa, about 1700 kDa, about 1800 kDa, about 1900 kDa, about 2000 kDa, about 2500 kDa, It has a molecular weight of greater than about 3000 kDa, about 3500 kDa, about 4000 kDa, about 4500 kDa, about 5000 kDa, about 5500 kDa, about 6000 kDa, about 6500 kDa, about 7000 kDa, about 7500 kDa, about 8000 kDa, about 8500 kDa, about 9000 kDa, about 9500 kDa, or about 10000 kDa.

[0090] In some embodiments, the thermal stability of the hydrogel (NormGelC or GelC) is at least 80% after 24 hours. In some embodiments, the thermal stability of the hydrogel (NormGelC or GelC) is at least 80% after 48 hours. In some embodiments, the thermal stability of the hydrogel (NormGelC or GelC) is at least 80% after 24 hours at about 90°C. In some embodiments, the thermal stability of the hydrogel (NormGelC or GelC) is at least 80% after 24 hours or 48 hours at a temperature of at least 70°C. In some embodiments, the thermal stability of the hydrogel (NormGelC or GelC) is at least 80% after 24 hours or 48 hours at a temperature of at least 90°C.

[0091] In some embodiments, the thermal stability of the hydrogel (NormGelC or GelC) is at least 70%, 75%, 80%, 85%, 90%, or 95% after 24 or 48 hours. In some embodiments, the thermal stability of the hydrogel (NormGelC or GelC) is at least 70%, 75%, 80%, 85%, 90%, or 95% after 24 or 48 hours at a temperature of at least 70° C. or at least 90° C.

[0092] In some embodiments, the thermal stability of the hydrogel (NormGelC or GelC) is at least about 70%, 75%, 80%, 85%, 90%, or 95% after 24 or 48 hours. In some embodiments, the thermal stability of the hydrogel (NormGelC or GelC) is at least 70%, 75%, 80%, 85%, 90%, or 95% after 24 or 48 hours at a temperature of at least 70° C. or at least 90° C.

[0093] In some embodiments, the thermal stability of the hydrogel (NormGelC or GelC) decreases by less than 5%, 10%, 15%, 20%, 25%, or 30% after 24 or 48 hours at a temperature of at least 70° C. or at least 90° C. In some embodiments, the thermal stability of the hydrogel (NormGelC or GelC) decreases by less than about 5%, about 10%, about 15%, about 20%, about 25%, or 30% after 24 or 48 hours at a temperature of at least 70° C. or at least 90° C.

[0094] In some embodiments, the temperature at which thermal stability is determined is at least 70°C, 72°C, 74°C, 76°C, 78°C, 80°C, 82°C, 84°C, 86°C, 88°C, 90°C, 92°C, 94°C, 96°C, 98°C, 100°C, 102°C, 104°C, 106°C, 108°C, or 110°C. In some embodiments, the temperature at which thermal stability is determined is at least about 70°C, about 72°C, about 74°C, about 76°C, about 78°C, about 80°C, about 82°C, about 84°C, about 86°C, about 88°C, about 90°C, about 92°C, about 94°C, about 96°C, about 98°C, about 100°C, about 102°C, about 104°C, about 106°C, about 108°C, or about 110°C.

[0095] In some embodiments, the period of time after manufacture at which thermal stability is determined is at least 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 ​​hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, 74 hours, 76 hours, 78 hours, or 80 hours. In some embodiments, the period of time after manufacture during which thermal stability is determined is at least about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours, about 48 hours, about 50 hours, about 52 hours, about 54 hours, about 56 hours, about 58 hours, about 60 hours, about 62 hours, about 64 hours, about 66 hours, about 68 hours, about 70 hours, about 72 hours, about 74 hours, about 76 hours, about 78 hours, or about 80 hours.

[0096] In some embodiments, the composition is bioabsorbable. In some embodiments, the hydrogel is bioabsorbable. In some embodiments, the composition is bioabsorbed within a period of about 1 year to about 3 years. In some embodiments, the composition is bioabsorbed within a period of 1 year to 3 years. In some embodiments, the hydrogel is bioabsorbed within a period of about 1 year to about 3 years. In some embodiments, the hydrogel is bioabsorbed within a period of 1 year to 3 years.

[0097] In some embodiments, the composition further comprises a local anesthetic. In some embodiments, the composition comprises at least one local anesthetic. In some embodiments, the local anesthetic is an amide-type local anesthetic. In some embodiments, the local anesthetic is an ester-type local anesthetic.

[0098] In some embodiments, the local anesthetic is bupivacaine, butanilicaine, carticaine, sinticocaine (dibucaine), clibucaine, ethyl parapiperidinoacetylaminobenzoate, etidocaine, lignocaine (lidocaine), mepivacaine, oxetazaine, prilocaine, ropivacaine, tolycaine, trimecaine, vadocaine, articaine, levobupivacaine, amylocaine, cocaine, procaine, The antihistamine is selected from the group consisting of panocaine, chlormecaine, cyclomethycaine, proxymetacaine, amethocaine (tetracaine), benzocaine, butacaine, butoxycaine, butyl aminobenzoate, chloroprocaine, dimethocaine (larocaine), oxybuprocaine, piperocaine, parethoxycaine, procaine (novocaine), propoxycaine, and tricaine, or a combination thereof.

[0099] In some embodiments, the concentration of the local anesthetic in the composition is 1 to 5 mg / mL. In some embodiments, the concentration of the local anesthetic in the composition is about 1 to about 5 mg / mL. In some embodiments, the concentration of the local anesthetic in the composition is 2 to 4 mg / mL. In some embodiments, the concentration of the local anesthetic in the composition is about 2 to about 4 mg / mL. In some embodiments, the concentration of the local anesthetic in the composition is 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, or 5 mg / mL. In some embodiments, the concentration of the local anesthetic in the composition is about 0.5 mg / mL, about 1 mg / mL, about 1.5 mg / mL, about 2 mg / mL, about 2.5 mg / mL, about 3 mg / mL, about 3.5 mg / mL, about 4 mg / mL, about 4.5 mg / mL, or about 5 mg / mL.

[0100] In some embodiments, the composition is injectable. In some embodiments, the injectable composition is an injectable implant. In some embodiments, the present disclosure relates to an injectable implant comprising any one of the compositions disclosed herein. In some embodiments, the injectable implant is for subdermal, intradermal, subcutaneous, intramuscular, submuscular, or intragingival injection.

[0101] In some embodiments, the present disclosure relates to a pre-filled syringe comprising any one of the compositions disclosed herein. In some embodiments, the present disclosure relates to a pre-filled vial comprising any one of the compositions disclosed herein.

[0102] In some embodiments, the kit includes a pre-filled syringe containing any one of the compositions disclosed herein. In some embodiments, the kit includes a pre-filled vial containing any one of the compositions disclosed herein, a syringe, and one or more hypodermic needles. In some cases, the kit includes an antimicrobial composition for administration to an injection site.

[0103] In some embodiments, kits are contemplated for use in practicing the methods described herein. In some embodiments, the kits contain all solutions, buffers, compounds, containers, and / or instructions sufficient to carry out the methods described herein.

[0104] In some embodiments, the composition further comprises sodium chloride. In some embodiments, the composition has a sodium chloride concentration of 0.9% w / v. In some embodiments, the composition further comprises a phosphate buffer. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises sodium chloride, a phosphate buffer, and a pharmaceutically acceptable carrier.

[0105] In some embodiments, the composition comprises one or more density-increasing agents. In some embodiments, the density-increasing agents may be selected from sorbitol, mannitol, and fructose.

[0106] In some embodiments, the composition includes a buffer. A buffer is a chemical or compound added to a solution that allows the solution to resist changes in pH, either as a result of dilution or the addition of small amounts of acid or base. An effective buffer system uses a solution containing a conjugate acid-base pair (or buffer) in large amounts and approximately equal concentrations. The buffer used herein can be any such chemical that is pharmaceutically acceptable, including, but not limited to, phosphate and citrate salts (conjugate acids and / or conjugate bases). In some embodiments, the buffer includes phosphate-buffered saline (PBS) or an alternative phosphate buffer.

[0107] In some embodiments, the composition is aseptic.In some embodiments, the composition is sterilized.In some embodiments, the composition is sterilized by filtration sterilization, heat sterilization, or irradiation sterilization.In some embodiments, the components of the composition are sterilized before mixing or forming the whole composition, thus resulting in a composition that includes two or more components that are sterilized before forming the composition.

[0108] In some embodiments, the GAGs do not have a molecular weight less than 1.5 MDa. In some embodiments, the GAGs do not have a molecular weight less than 1.4 MDa. In some embodiments, the GAGs do not have a molecular weight less than 1.3 MDa. In some embodiments, the GAGs do not have a molecular weight less than 1.2 MDa. In some embodiments, the GAGs do not have a molecular weight less than 1.1 MDa. In some embodiments, the GAGs do not have a molecular weight less than 1.0 MDa. In some embodiments, the GAGs do not have a molecular weight less than 0.9 MDa. In some embodiments, the GAGs do not have a molecular weight less than 0.8 MDa. In some embodiments, the GAGs do not have a molecular weight less than 0.7 MDa.

[0109] In some embodiments, the hydrogel does not undergo degradation after cross-linking of glycosaminoglycans. In some embodiments, the hydrogel undergoes degradation by the environment after cross-linking. However, the hydrogel does not undergo degradation by the environment after cross-linking. final / 2C min C lower than the value min In some embodiments, the hydrogel has a C final C higher than / 2 min Indicates the value.

[0110] Other aspects and preferred embodiments of the present invention will become apparent from the following detailed disclosure of the invention and the appended claims.

[0111] II. Methods of Using Hydrogels In some embodiments, the present disclosure includes a method of performing a restorative or cosmetic dermatological treatment. In some embodiments, the restorative or cosmetic dermatological treatment comprises injecting a composition disclosed herein into a subject. In some embodiments, the injection is subdermal, intradermal, subcutaneous, intramuscular, submuscular, or intragingival.

[0112] In some embodiments, the methods of the present disclosure relate to intragingival injections to fill the gums as a result of gum recession. In some embodiments, the methods relate to injection of a composition into one or more tissues of the oral cavity.

[0113] In some embodiments, the injections are for skin filling, body contouring, facial contouring, and gum filling.

[0114] In some embodiments, the injection of the compositions disclosed herein is for dermal filling. In some embodiments, the method of dermal filling comprises injecting the composition to fill skin cracks. In some embodiments, the method of dermal filling comprises injecting the composition to fill fine lines on the face, neck, hands, feet, knees, and elbows. In some embodiments, the method of dermal filling comprises injecting the composition to fill fine lines on the face, neck, hands, feet, knees, and elbows. In some embodiments, the method of dermal filling comprises injecting the composition to fill fine lines on the face, neck, hands, feet, knees, and elbows.

[0115] In some embodiments, the method of dermal filler comprises injecting the composition to fill a scar. In some embodiments, the method of dermal filler comprises injecting the composition to fill a depressed scar. In some embodiments, the method of dermal filler comprises injecting the composition to fill a hypertrophic scar. In some embodiments, the method of dermal filler comprises injecting the composition to fill a keloid scar.

[0116] In some embodiments, the method of dermal filler involves injecting a composition to restore and / or correct signs of facial fat atrophy (lipoatrophy) in people with human immunodeficiency virus (HIV).

[0117] In some embodiments, the method of skin filling comprises injecting the composition into the back of the hand or the top of the foot.

[0118] In some embodiments, the method of dermal filling comprises injecting a composition to strengthen weakened vocal cords.

[0119] In some embodiments, the method of dermal filler involves injecting a composition to restore volume lost to a body part as a result of aging, disease, or injury.

[0120] In some embodiments, the method of facial contouring comprises injecting a composition into the face to modify the facial contour, hi some embodiments, the method of facial contouring comprises injecting a composition into the lips to enhance the size and / or shape of the lips.

[0121] In some embodiments, the facial contouring method comprises injecting a composition into the face to increase facial symmetry. In some embodiments, the facial contouring method comprises injecting a composition to change the shape of the face to an oval, round, square, triangle, inverted triangle, rectangle, or oblong. In some embodiments, the facial contouring method comprises injecting a composition to increase the overall width of the face. In some embodiments, the facial contouring method comprises injecting a composition to increase the overall length of the face.

[0122] In some embodiments, the method of facial contouring comprises injecting a composition into the face to increase the width of the forehead and / or cheekbones, hi some embodiments, the method of facial contouring comprises injecting a composition into the face to increase the length of the jawline.

[0123] In some embodiments, the method of facial contouring comprises injecting a composition into the face to change the size and / or shape of the chin. In some embodiments, the method of facial contouring comprises injecting a composition into the face to change the size and / or shape of the forehead. In some embodiments, the method of facial contouring comprises injecting a composition into the face to change the size and / or shape of the cheeks. In some embodiments, the method of facial contouring comprises injecting a composition into the face to change the size and / or shape of the eyebrows.

[0124] In some embodiments, the method of facial contouring comprises injecting a composition into the face to correct the appearance associated with retrognathia. In some embodiments, the method of facial contouring comprises injecting a composition into the face to correct the appearance associated with prognathia.

[0125] In some embodiments, methods of body shaping include injecting a composition into the body to modify various external size and shape of the body, hi some embodiments, methods of body shaping include injecting a composition into the body to modify various external size and shape of the body to increase symmetry.

[0126] In some embodiments, the method of body contouring comprises injecting the composition into the body to modify the size and shape of the breasts, buttocks, sacrum, groin, lower back, abdomen, chest, feet, legs, knees, popliteus muscles, thighs, arms, hands, elbows, and / or ankles.

[0127] In some embodiments, the body contouring method comprises injecting the composition into the body to fill depressions. In some embodiments, the depressions are the result of aging, disease, injury, or predisposition. In some embodiments, the body contouring method comprises injecting the composition into the body to reduce the appearance of cellulite. [Example]

[0128] Example 1 Formation of high molecular weight hydrogels Example 1 provides a general process for making the high molecular weight GAG hydrogels described herein.

[0129] A solution of hyaluronic acid (HA), diaminotrehalose (DATH), and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) is mixed and crosslinked. After the crosslinking process, the gel is divided and diluted to a set concentration. The gel is then heated to approximately 70°C for approximately 24 hours. The gel then undergoes particle size reduction (PSR) and precipitation, followed by washing and drying to form a powder. The gel powder is then mixed with a suitable buffer solution, causing the gel to swell. The gel is then autoclaved.

[0130] Table 1: Hydrogel compositions corresponding to Figure 1 TIFF0007730323000004.tif54145ND=Not determined

[0131] Gel content was analyzed under pyrolysis conditions at 90°C and evaluated over a period of just over 70 hours (Figure 1). Gel content values ​​were normalized for ease of comparison and are shown as "NormGelC (%)" on the y-axis of Figure 1. The data indicate that high molecular weight HA provides gels that maintain a higher degree of gel content under pyrolysis conditions compared to gels made from lower molecular weight HA.

[0132] Example 2 Preparation and evaluation of high molecular weight (HMW) and low molecular weight (LMW) hyaluronic acid (HA) hydrogels General procedure for crosslinking HMW and LMW HA Freshly prepared stock solutions of DMTMM and DATH crosslinkers were prepared in water. The reaction solutions were prepared by adding the desired volumes of DMTMM (mol% DATH x 8.5) and DATH (see Table 1) stock solutions to water. The reaction solutions were mixed and added directly to pre-weighed HA (0.4 MDa or 2.1 MDa) in a reaction vessel. The mixture was thoroughly mixed and incubated for 3 minutes. After 24 ± 2 hours, the resulting material was passed through a 1 mm steel mesh. After homogenizing the material at 70 °C for 24 ± 2 hours, the gel was subjected to particle size reduction (PSR) using 3 × 315 μm filters and then precipitated by adding EtOH. The resulting powder was dried under vacuum overnight and reconstituted in 7 mM phosphate, 0.7% NaCl, and 3 mg / g Lid-HCl at neutral pH. The gel was loaded into syringes and then autoclaved.

[0133] Table 2: Reaction conditions and properties of the resulting gel TIFF0007730323000005.tif84150

[0134] The gels were incubated in sealed glass vials in a water bath at 90°C for 24 or 48 hours. At the given times, the samples were cooled to room temperature and the gels were analyzed. See Figures 2-4.

[0135] Figure 2 shows the gel content (GelC%) corresponding to Samples 2-1 and 2-2 in Table 2. Figure 3 shows the gel content (GelC%) corresponding to Samples 2-3 and 2-4 in Table 2. Figure 4 shows the gel content (GelC%) corresponding to Samples 2-5 and 2-6 in Table 2. Figure 5 shows the gel content (GelC%) corresponding to Samples 2-7 and 2-8 in Table 2.

[0136] MW in HMW gel app General procedure for determining A pre-weighed amount of HA (2.1 MDa) was mixed with water in a reaction vessel. The mixture was mixed thoroughly for 3 minutes and incubated at ambient temperature. After 24 hours, the solution was diluted with water, NaCl was added (final concentration 0.9%), and the resulting material was incubated at 70°C. After 24 hours of incubation, the solution was loaded into syringes. Subsequently, the syringes for sample 2-10 were autoclaved, but the syringes for sample 2-9 were not. The Mw of the solutions was determined by SEC-MALLS.

[0137] Table 3: Reaction conditions and properties of the resulting gel TIFF0007730323000006.tif53136

[0138] The methods illustratively described herein may suitably be practiced in the absence of any element(s), limitation(s) not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," and the like, should be interpreted broadly and without limitation. Furthermore, the terms and expressions used herein are used as terms of description and not of limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the shown and described features or portions thereof. It will be recognized that various modifications are possible within the scope of the present disclosure as claimed. Thus, while the present disclosure has been specifically disclosed by preferred embodiments and optional features, it will be understood that modifications and variations of the present disclosure embodied herein may occur to those skilled in the art, and that such modifications and variations are considered to be within the scope of the present disclosure.

[0139] The present disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings belonging to the generic disclosure also forms part of the present method. This includes the general description of the present method with a conditional or negative limitation that removes any subject matter from the genus, regardless of whether the omitted material is specifically recited herein. The present technology is not limited to the specific embodiments described in this application, which are intended as single illustrations of individual aspects of the technology. As will be apparent to those skilled in the art, many modifications and variations of the present technology can be made without departing from its spirit and scope. In addition to those recited herein, functionally equivalent methods and apparatuses within the scope of the present technology will be apparent to those skilled in the art from the foregoing description. Such modifications and variations are intended to be within the scope of the present technology. It is understood that the present technology is not limited to particular methods, reagents, compound compositions, or biological systems, which may, of course, vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0140] Those skilled in the art will readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Modifications and other uses of the present disclosure will occur to those skilled in the art. These modifications are encompassed within the spirit of the disclosure and are defined by the scope of the claims which describe non-limiting embodiments of the disclosure.

[0141] Additionally, when features or aspects of the disclosure are described in terms of a Markush group, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual element or subgroup of elements of the Markush group.

[0142] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes.

[0143] However, mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the general knowledge in any country in the world.

Claims

1. 1. A method for preparing a hydrogel comprising cross-linked glycosaminoglycan (GAG) molecules, the method comprising the step of cross-linking a GAG having a molecular weight of at least 1.5 MDa with a cross-linking agent to obtain a glycosaminoglycan hydrogel having GAG molecules cross-linked by amide bonds, wherein: the cross-linking agent comprises diaminotrehalose (DATH); The concentration of GAG is 2% to 10% (w / w), and A method for preparing said cross-linking agent to GAG molar ratio is at least 0.1 mol% per GAG disaccharide and not more than 2 mol% per GAG disaccharide.

2. when the GAG ​​concentration is 2% to 4.5% (w / w), the concentration of the cross-linker is 0.8 to 2 mol% per GAG disaccharide; when the GAG ​​concentration is 4.6% to 5.9% (w / w), the cross-linker concentration is 0.5 to 0.8 mol% per GAG disaccharide; When the GAG ​​concentration is 6% to 10% (w / w), the concentration of the cross-linker is 0.3 to 0.5 mol% per GAG disaccharide. A method for preparing the hydrogel of claim 1.

3. The crosslinking step (1) providing a solution of glycosaminoglycan (GAG) molecules; (2) activating a carboxyl group on the glycosaminoglycan molecule with a coupling agent to form an activated glycosaminoglycan molecule; (3) using the crosslinking agent to crosslink the activated glycosaminoglycan (GAG) molecules through their activated carboxyl groups to obtain a glycosaminoglycan hydrogel crosslinked by amide bonds; 3. The method of claim 1 or 2, comprising:

4. 4. The method of claim 3, wherein the coupling agent is a triazine-based coupling agent, such as 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM).

5. The method according to any one of claims 1 to 4, wherein the cross-linking step is carried out at a pH of 5.0 to 9.0, or at a pH of 6.0 to 8.

0.

6. 6. The method of any one of claims 1 to 5, further comprising formulating the crosslinked hydrogel to a final glycosaminoglycan (GAG) concentration of 10 to 45 mg / mL.

7. The method of any one of claims 1 to 6, further comprising the step of sterilizing the crosslinked hydrogel.

8. The method according to any one of claims 1 to 7, wherein the glycosaminoglycan (GAG) is hyaluronic acid (HA).

9. The method of any one of claims 1 to 8, wherein the GAG ​​has a molecular weight of 1.5 to 10 MDa, or 2.1 to 3.5 MDa.

10. The method according to any one of claims 1 to 9, wherein the concentration of the GAG ​​is 3 to 5% (w / w).

11. 11. The method according to any one of claims 1 to 10, wherein the molar ratio of cross-linker to GAG is 0.9 to 1.1 mol% per GAG disaccharide.

12. 12. The method of any one of claims 1 to 11, wherein the GAG ​​is hyaluronic acid (HA) having a molecular weight of 1.5 to 3.5 MDa, the cross-linking agent is diaminotrehalose (DATH), and the concentration of HA is 3 to 5% (w / w), and the molar ratio of DATH to HA is 0.9 to 1.1 mol% per GAG disaccharide.

13. 1. A hydrogel product comprising glycosaminoglycan (GAG) molecules as swellable polymers, the glycosaminoglycan molecules are cross-linked by amide bonds formed between the GAG ​​and diaminotrehalose (DATH); the GAG ​​molecule has a molecular weight of at least 1.5 MDa; and A hydrogel product, wherein the thermal stability (measured as NormGelC) of said swellable polymer is greater than 80% after 24 hours.

14. 14. The hydrogel product of claim 13, wherein the glycosaminoglycan (GAG) is hyaluronic acid (HA).

15. 15. The hydrogel product of claim 13 or 14, which is sterilized.

16. A pharmaceutical composition for cosmetically treating the skin, comprising a hydrogel product according to any one of claims 13 to 15.

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