Cross-linked material comprising polysaccharide moieties interconnected by ester bonds

Abstract

The present invention relates to a process for preparing a crosslinked material comprising reacting at least some of the carboxylic acid residues of a polysaccharide moiety with at least some of the hydroxyl residues of the polysaccharide moiety to form an ester bond that covalently crosslinks the polysaccharide moieties to each other wherein the reaction is initiated by one or more triazine-based activators. Furthermore, the invention relates to a cross-linked material obtainable by such a process, to a composition comprising the cross-linked material and to the use thereof.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480035175.8 (22) Application Date 2024.05.23 (30) Priority Data 23175399.7 2023.05.25 EP (85) PCT International Application Entering National Phase Date 2025.11.25 (86) PCT International Application Application Data PCT / EP2024 / 064195 2024.05.23 (87) PCT International Application Publication Data WO2024 / 240864 EN 2024.11.28 (71) Applicant: Merz Aesthetics GmbH Address: Frankfurt, Germany (72) Inventors: Clayton Kenzler, C. Dreby, R. Vukicevic, Radja El-Bana (74) Patent Agency: Beijing Guohao Tiancheng Intellectual Property Agency Co., Ltd. 11315 Patent Attorney: Nan Ting (51) Int.Cl. A61L 27 / 20 (2006.01) A61L 27 / 52 (2006.01) (54) Invention Title: Crosslinked Material Containing Polysaccharide Moments Interconnected by Ester Bonds (57) Abstract: This invention relates to a method for preparing a crosslinked material comprising reacting at least some carboxylic acid residues of a polysaccharide moiety with at least some hydroxyl residues of the polysaccharide moiety to form ester bonds covalently crosslinking the polysaccharide moiety with each other, wherein the reaction is initiated by one or more triazine activators. Furthermore, this invention relates to crosslinked materials obtainable by such method, compositions containing such crosslinked materials, and uses thereof. Claims 2 pages, Description 21 pages, Drawings 2 pages, CN 121285400 A 2026.01.06 CN 1 21 28 54 00 A 1. A method for preparing a crosslinked material, the method comprising: (i) contacting the following components with each other: (A) a polysaccharide moiety comprising a carboxylic acid residue or a salt thereof and a hydroxyl residue; (B) one or more triazine activators that react the carboxylic acid residue with the hydroxyl residue to form an ester bond; and (C) one or more solvents; and (ii) reacting at least some of the carboxylic acid residues of the polysaccharide moiety with at least some of the hydroxyl residues of the polysaccharide moiety to form ester bonds that covalently crosslink the polysaccharide moiety with each other; and (iii) optionally purifying the crosslinked material obtained from step (ii), wherein the ester bonds that covalently crosslink the polysaccharide moiety with each other are more numerous than the amide bonds that covalently crosslink the polysaccharide moiety with each other. Step (ii) is carried out in the absence of any activator other than triazine activators, and in particular, in the absence of carbodiimide activators, succinimide activators, glycidyl activators, p-nitrophenol esters and 2-chloromethylpyridine iodide.2. The method of claim 1, wherein the polysaccharide moiety comprises a molar ratio of hydroxyl residues to primary amino groups of at least 25:1, preferably at least 50:1, and particularly at least 100:1, and preferably, the polysaccharide moiety comprises hyaluronic acid, glycerol-grafted hyaluronic acid, heparin polysaccharide, chondroitin sulfate, and carboxymethyl cellulose, or is composed of hyaluronic acid, glycerol-grafted hyaluronic acid, heparin polysaccharide, chondroitin sulfate, and carboxymethyl cellulose. 3. The method of any one of claims 1 or 2, wherein the polysaccharide moiety comprises one or more hyaluronic acid moieties, or is composed of one or more hyaluronic acid moieties. 4. The method of any one of claims 1 to 3, wherein the weight-average molecular weight of the polysaccharide moiety is at least 50 kDa, preferably at least 200 kDa, and particularly in the range of 500 kDa to 4000 kDa. 5. The method of any one of claims 1 to 4, wherein one or more activators are selected from the group consisting of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinon or a salt thereof, and / or 2-chloro-4,6-dimethoxy-1,3,5-triazine or a salt thereof, and combinations thereof. 6. The method of any one of claims 1 to 5, wherein the activator is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinon or a salt thereof, particularly 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinon chloride. 7. The method of any one of claims 1 to 6, wherein the solvent is an aqueous solution, particularly an aqueous buffer solution, preferably an aqueous buffer solution with a pH in the range of 5 to 9, particularly in the range of 6 to 8. 8. The method of any one of claims 1 to 7, wherein the method is further characterized in that it does not include at least one of the following: (a) using an interconnecting linker portion between a carboxylic acid residue or its salt inserted into the polysaccharide moiety and a hydroxyl residue, particularly without a peptide linker portion or a heterogeneous linker portion; (b) using a carbodiimide activator; (c) using a reactive group selected from the group consisting of glycidyl ether, maleimide, acid anhydride, alkoxide, and combinations of two or more thereof; (d) a polysaccharide moiety having a primary amino group, a thiol group, an imide group, an imine group, or an epoxy group. 9. The method according to any one of claims 1 to 8, wherein the method is further characterized by: (a) comprising step (iii) purifying the crosslinked material by filtration, washing and / or dialysis, particularly cross-flow filtration, percolation and / or dead-end filtration; (b) steps (i) and (ii) are carried out in a single batch; (c) steps (i) and (ii) and optionally step (iii) are carried out at a temperature in the range of 5°C to 90°C, preferably 18°C ​​to 60°C, particularly 20°C to 50°C; and / or(d) Step (ii) is performed for at least 10 min, preferably 15 min to 48 h, particularly 30 min to 18 h. 10. The method of any one of claims 1 to 9, wherein the method comprises: (i) contacting the following components with each other: (A) a polysaccharide moiety having a weight average molecular weight of at least 50 kDa, comprising a carboxylic acid residue or a salt thereof and a hydroxyl residue; (B) one or more triazine activators that react the carboxylic acid residue with the hydroxyl residue to form an ester bond, particularly, wherein the activator is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium or a salt thereof; and (C) an aqueous solvent, preferably an aqueous solvent with a pH in the range of 5 to 9, particularly in the range of 6 to 8; and (ii) reacting at least some of the carboxylic acid residues with at least some of the hydroxyl residues to form ester bonds that covalently crosslink the polysaccharide moiety with each other; and (iii) optionally purifying the crosslinked material obtained from step (ii). 11. A crosslinked material that can be obtained by the method of any one of claims 1 to 10. 12. A crosslinked material comprising or consisting of one or more hyaluronic acid moieties with a weight average molecular weight of at least 50 kDa, wherein the hyaluronic acid moieties are covalently crosslinked to each other via ester bonds without interconnecting linker structures, the crosslinked material being characterized in that it does not contain diimide, epoxy, or xenobiotic linker moieties, preferably, wherein the crosslinked material comprises: (a) 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium or a salt thereof, (b) N-methylmorpholinium or a salt thereof; and / or (c) 4,6-dimethoxy-1,3,5-triazin-2-ol or a tautomer or salt thereof, and preferably, wherein the crosslinked material is obtained by the method of any one of claims 1 to 10. 13. A hydrogel comprising: (A) the crosslinked material of any one of claims 11 or 12; and (B) an aqueous solution; and (C) optionally one or more other pharmaceutically or cosmetically acceptable agents, particularly one or more local anesthetics. 14. An injectable composition comprising the crosslinking material of any one of claims 11 to 12 or the hydrogel of claim 13, wherein the crosslinking material is a supercompensator. 15. Use of the injectable composition of claim 14 for cosmetic applications, wherein the cosmetic applications include facial and body reshaping and rejuvenation, preferably including wrinkle filling, improvement of facial lines, breast reconstruction or enlargement, skin rejuvenation, buttock augmentation, cheekbone reshaping, soft tissue augmentation, filling of facial wrinkles, improvement of frown lines, improvement of nasolabial folds, improvement of marionette lines, improvement of angular lip lines, improvement of perioral wrinkles, improvement of crow's feet, improvement of eyebrows, subcutaneous support of cheek fat pads, improvement of tear troughs, improvement of…Nose augmentation, lip augmentation, cheek augmentation, perioral augmentation, infraorbital augmentation, correction of facial asymmetry, improvement of jawline contour, chin augmentation, or combinations of two or more thereof. Claims 2 / 2 pages 3 CN 121285400 A Crosslinked Material Containing Polysaccharide Moments Interconnected by Ester Bonds Technical Field

[0001] The present invention relates to a method for preparing a crosslinked material, the method comprising reacting at least some carboxylic acid residues of a polysaccharide moiety with at least some hydroxyl residues of the polysaccharide moiety to form ester bonds covalently crosslinking the polysaccharide moiety with each other, wherein the reaction is initiated by one or more triazine activators. Furthermore, the present invention relates to crosslinked materials obtainable by such method, compositions containing such crosslinked materials, and uses thereof. Background Art

[0002] Facial and body reshaping is receiving increasing attention. For example, filling wrinkles on the face and / or body, skin rejuvenation, breast reconstruction or enlargement, and other types of soft tissue augmentation are often of interest. To avoid the need for surgical intervention, some soft tissue fillers that can be injected subcutaneously or into the deep layers of the skin have been developed or are being developed.

[0003] Soft tissue fillers are typically gels, such as hydrogels. Practitioners using such soft tissue fillers (especially dermal fillers) typically expect them to be free from toxic or immune side effects when applied under relevant conditions, exhibit good biocompatibility, be injectable without burden, and be based on natural materials. Simultaneously, the filler should remain within a spatially confined area and possess sufficient stability in biological systems, such as at the time of injection.

[0004] Materials suitable for soft tissue filling in this art are polysaccharides, such as hyaluronic acid (HA). Many polysaccharides, such as hyaluronic acid, have good bioacceptability. However, a significant drawback is that such polysaccharide-based materials (such as hyaluronic acid) biodegrade relatively quickly, and this filler material is not suitable for long-term solutions. The lifespan of this filler material in the subject of application is limited, often below the expected minimum range of several months. When such polysaccharides degrade rapidly in vivo, the viscosity decreases undesirably quickly, and the filling effect is not sufficiently durable. Furthermore, the storability and shelf life of hyaluronic acid-based fillers are often limited. Storage products containing unmodified polysaccharides are often prone to partial degradation. Then, the viscosity decreases, and subsequently applied products of this type that have been stored and thus partially degraded have an even shorter lifespan in the subjects to which they are applied.

[0005] Therefore, there is a need to provide other filler materials with suitable biostability.

[0006] Crosslinking polysaccharides (such as hyaluronic acid) with synthetic xenobiotic crosslinking agents, such as epoxide linkers butylene glycol diglycidyl ether (BDDE), such as (e.g.) US-A2012 / 0190644, WO 2015 / 149941 or WOAs described in 2020 / 030629, or crosslinked with a dinuphilic or polynuphilic functional crosslinker, such as that described in US-A 2020 / 0140626. Such materials have several disadvantages. For example, the residues of such unreacted or half-reacted divalent linkers can be harmful and limit the usability of such materials. Such reactive linkers have a maximum applicable content, and therefore this is also true for filler materials prepared using such substances for safety reasons. When administered to and degraded in a subject in need, xenobiotics and non-degradable or recalcitrant metabolites are generated, which is generally undesirable, especially in cosmetic and pharmaceutical uses. Therefore, it is necessary to avoid such xenobiotic linker portions.

[0007] To overcome such disadvantages, crosslinking with proteins, peptides, and amino acids has been considered. In this context, crosslinking polysaccharides, such as hyaluronic acid, with crosslinked proteins has been considered. For example, hyaluronic acid is crosslinked with elastin as described in WO 2011 / 119468. In addition, polysaccharides are also crosslinked with filamentous proteins, such as those described in WO 2022 / 268871. Application CN-A 105713211 describes hyaluronic acid linked to lysine, an amino acid containing two amino groups. In the absence of amino acids, it is unclear whether crosslinking can be achieved, and if so, the extent of crosslinking and which chemical bonds are involved. Specification 1 / 21 pages 4 CN 121285400 A

[0008] A disadvantage of such methods is that the presence of proteins, peptides, or amino acids is not always ideal. The process is quite complex because it involves several components and, depending on the protein used, the amide bonds within the protein sequence may break. In some cases, proteins and peptides may have undesirable immune responses. Therefore, it is desirable to crosslink polysaccharides, such as hyaluronic acid, without using such interconnected proteins, peptides, and amino acids.

[0009] In this context, it is thought that amide bonds can be formed by crosslinking polysaccharides having carboxylic acid residues and amino groups with each other. For this purpose, polysaccharides, such as glycosaminoglycans, are modified to include a free amino group conjugated with a free carboxyl group, as described in WO 2019 / 002369. However, such procedures require additional steps to provide non-natural intermediates, such as providing an amino group. Furthermore, the conjugated product thus obtained does not ideally contain an amide bond. It is desirable to directly conjugate natural polysaccharides via ester bonds.

[0010] There is a clear bias in the art that the formation of ester bonds between polysaccharides requires a strong activator, such as a carbodiimide activator (containing the structural motif -N=C=N-) or a 2-chloro-methylpyridine iodide. For example, EP-A0341745 teaches that esterification can be accomplished by using a 2-chloro-methylpyridine iodide. JP-A2019019201 teaches the use of a carbodiimide activator, such as N,N'-Dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). These reactants are undesirable for irritation, even toxicity, and may have other drawbacks.

[0011] On the other hand, when such activators are omitted, esterification does not occur, or is extremely slow and ineffective, even within harsh pH ranges, as illustrated in JP-A2003 / 252905.

[0012] In view of the above, there is an unmet need for an efficient method for preparing crosslinked materials that requires only low procedural workload, avoids the introduction of heterogeneous linker moieties into the hydrogel, and minimizes residual amounts of toxic reagents. Additionally, obtaining ester bonds between polysaccharide moieties is desirable. Particularly ideal is a method for providing injectable hydrogels for facial and body reshaping (e.g., that can be used as a super-volumizer).

[0013] Surprisingly, it has been found that crosslinked materials with beneficial properties can be obtained by reacting at least some carboxylic acid residues comprising the polysaccharide portion with at least some hydroxyl residues of the polysaccharide portion to form ester bonds that covalently crosslink the polysaccharide portions with each other, wherein the reaction is initiated by one or more triazine activators.

[0014] The first aspect relates to a method for preparing a crosslinked material, the method comprising:

[0015] (i) contacting the following components with each other:

[0016] (A) a polysaccharide moiety comprising a carboxylic acid residue or a salt thereof and a hydroxyl residue;

[0017] (B) one or more triazine activators, wherein the carboxylic acid residues react with the hydroxyl residues to form an ester bond; and

[0018] (C) one or more solvents; and

[0019] (ii) allowing at least some of the carboxylic acid residues of the polysaccharide moiety to react with at least some of the hydroxyl residues of the polysaccharide moiety to form an ester bond covalently crosslinking the polysaccharide moiety with each other; and

[0020] (iii) optionally purifying the crosslinked material obtained from step (ii);

[0021] wherein the ester bond covalently crosslinking the polysaccharide moiety with each other is greater than the amide bond covalently crosslinking the polysaccharide moiety with each other;

[0022] Step (ii) is carried out in the absence of any activator other than triazine activators, and in particular, in the absence of carbodiimide activators, succinimidyl activators, glycidyl activators, p-nitrophenol esters, and 2-chloromethylpyridine iodide.

[0023] Such crosslinked materials have been found to have unexpectedly beneficial properties. Viscosity can be maintained extensively. Similarly, enzymatic degradation can be ideally reduced. Surprisingly, the heterogeneous linker structure commonly used in the prior art can be avoided. The crosslinked materials of the present invention can be substantially composed of a polysaccharide moiety, which can also be found in nature. Also see page 2 / 21 of the specification of the present invention, CN 121285400 A.The cross-linked materials are referred to as “cross-linked polysaccharides,” “self-cross-linked materials,” or “self-cross-linked polysaccharides.” Cross-linked materials obtainable by the method of the present invention can have durable stability.

[0024] In an aqueous environment, the obtained cross-linked materials can form hydrogels.

[0025] Cross-linked materials can also be used to mimic the extracellular matrix, thereby inducing cell proliferation and / or cell migration and / or serving as a cellular skeleton. They can be successfully used as fillers that can be cell-filled (e.g., dermal fillers). The cross-linked materials of the present invention can have good shear-thinning properties. They preferably have thixotropic properties. Therefore, their viscosity may be low when subjected to stress. This allows for good injection while remaining relatively viscous in its target area (e.g., when applied to the subcutaneous region). Relatively low extrusion pressure is required. High-viscosity and low-extrusion-pressure gels can be obtained. They can optionally be used as supercompensators.

[0026] The method of the present invention can be carried out with relatively low input without excessive burden.

[0027] The claimed method is particularly advantageous compared to the procedures described in the prior art because it requires only two reactants (raw materials) besides the solvent: the polysaccharide moiety and one or more triazine activators. No other components, such as linkers, are required.

[0028] Omitting the xenobiotic linker structure (e.g., BDDE) allows for the administration of larger quantities of material to the subject, for example, via subcutaneous injection. Furthermore, it has a long lifespan in the subject after administration.

[0029] Due to the avoidance of reactive groups, the crosslinked materials obtained by the present invention can have a long shelf life and storage properties. They are also relatively thermally stable.

[0030] As used herein and as generally understood in the art, ester bonds can be understood in the broadest sense. Typically, ester bonds have a -O-CO- or -CO-O- structure, including their tautomerism.

[0031] As indicated above, the polysaccharide moiety is preferably linked to each other primarily via ester bonds. In other words, based on all bonds between the polysaccharide moieties (interconnecting groups between the polysaccharide moieties), more than 50 mol%, more than 75 mol%, or even more than 80 mol% are preferably ester bonds.

[0032] In a preferred embodiment, more ester bonds than amide bonds are formed in the crosslinked material. In a preferred embodiment, the crosslinked material contains an ester group: amide molar ratio of at least 25:1, preferably at least 50:1, and particularly at least 100:1. In a preferred embodiment, the crosslinked material is (substantially) free of amino groups.

[0033] As used herein, the terms “residue” and “group” are interchangeable.

[0034] As used herein, the terms “carboxylic acid residue” and “carboxyl group residue” are interchangeable, as generally understood in the art, i.e., residue -COOH or its salt -COO-.

[0035] In the polysaccharide moieties used as reactants in the method of the present invention, preferably hydroxyl residues (-OH) are more numerous than primary amino groups (-).NH2, including its salts, i.e., -NH3+ (and counterions).

[0036] In a preferred embodiment, the polysaccharide moiety contains a hydroxyl residue: primary amino group in a molar ratio of at least 25:1, preferably at least 50:1, and particularly at least 100:1. In a preferred embodiment, the polysaccharide moiety is (substantially) amino-free.

[0037] As used in the context of this invention, the term "polysaccharide moiety" may be understood in the broadest sense as any polysaccharide moiety known in the art that contains a carboxylic acid residue or a salt thereof and a hydroxyl residue. As used in the context of this invention, the term "polysaccharide" may be understood in the broadest sense as any polysaccharide in the art. According to the invention, at least one polysaccharide moiety contains at least one carboxylic acid residue or a salt thereof. The polysaccharide moiety also contains a hydroxyl residue. The polysaccharide may be a modified natural polysaccharide or a synthetic polysaccharide. In this context, the polysaccharide may be branched or unbranched. It should be understood that the term "polysaccharide moiety" may also include its salts and modified forms. In a preferred embodiment, the polysaccharide is unoxidized.

[0038] The polysaccharide fraction preferably has a weight-average molecular weight (Mw) of at least 1 kDa (1000 Da), more preferably at least 5 kDa, and even more preferably at least 10 kDa. In a preferred embodiment, the polysaccharide fraction has a weight-average molecular weight of at least 50 kDa, preferably at least 200 kDa, and particularly in the range of 500 to 4000 kDa. In a preferred embodiment, the polysaccharide fraction has a weight-average molecular weight (Mw) in the range of 10 to 10000 kDa, more preferably in the range of 25 to 7500 kDa, and particularly in the range of 50 to 4000 kDa.

[0039] The polysaccharide fraction may have any intrinsic viscosity (e.g., measured by a rheometer, as described in the Experimental Section below). In a preferred embodiment, the polysaccharide fraction may have an intrinsic viscosity of 1 to 4 m³ / kg (20°C, 1013 hPa, water). In a preferred embodiment, the polysaccharide fraction may have an intrinsic viscosity of 1.0 to 3.3 m³ / kg (20°C, 10¹³ hPa, water). In a preferred embodiment, the polysaccharide fraction may have an intrinsic viscosity of 1.1 to 3.2 m³ / kg, 2.0 to 3.1 m³ / kg, or 2.5 to 3.0 m³ / kg (at 20°C, 10¹³ hPa, and water, respectively).

[0040] In a preferred embodiment, the weight-average molecular weight of the polysaccharide fraction is 1500 to 3500 kDa (1.5 and 3.5 MDa). More preferably, its weight-average molecular weight may be in the range of 100 to 5000 kDa, in the range of 200 to 2000 kDa, in the range of 250 to 1500 kDa, in the range of 300 to 1000 kDa, in the range of 400 to 900 kDa, or in the range of 500 to 900 kDa.

[0041] In a preferred embodiment, the polysaccharide portion comprises one or more types of glycoacid portions or salts thereof.

[0042] In a preferred embodiment, the polysaccharide portion comprises one or more types of glycoacid portions or salts thereof, wherein the one or more types of glycoacid portions are selected from the group consisting of:

[0043] (B1) one or more uronic acid portions, particularly selected from the group consisting of glucuronic acid portions, galacturonic acid portions, iduronic acid portions, and combinations thereof;

[0044] (B2) one or more aldonic acid portions, particularly selected from the group consisting of glyceric acid portions, xylanic acid portions, gluconic acid portions, ascorbic acid portions, and combinations thereof;

[0045] (B3) one or more ketonic acid portions, particularly selected from the group consisting of neuraminic acid portions, ketodeoxyoctyl ketonic acid portions, and combinations thereof; and / or

[0046] (B4) one or more aldonic diacid portions, particularly selected from the group consisting of tartaric acid portions, mesogalactosic diacid portions, gluconic diacid portions, and combinations thereof.

[0047] In a preferred embodiment, the polysaccharide portion comprises a uronic acid portion. In a preferred embodiment, the polysaccharide portion comprises a glucuronic acid portion. In a preferred embodiment, the polysaccharide portion comprises a D-glucuronic acid portion.

[0048] In a preferred embodiment, the polysaccharide portion comprises or is composed of a D-glucose portion. In an alternative embodiment, the polysaccharide portion comprises or is composed of an L-glucose portion. In an alternative embodiment, the polysaccharide portion comprises or is composed of a combination of D-glucose and L-glucose portions. For example, in such a combination, a racemic mixture of sugar portions may be included, or a particular sugar portion may be a D-glucose portion, while others may be L-glucose portions. In a preferred embodiment, the polysaccharide portion comprises or is composed of one or more glycosaminoglycan portions.

[0049] In a preferred embodiment, the polysaccharide portion is selected from the group consisting of hyaluronic acid (HA) portions, heparin glycan portions, heparin, chondroitin sulfate, and mixtures of two or more thereof. In a preferred embodiment, the polysaccharide portion comprises or is composed of hyaluronic acid, glycerol-grafted hyaluronic acid, heparin glycan, chondroitin sulfate, and carboxymethyl cellulose. Such polysaccharides containing carboxylic acid groups are also commercially available.

[0050] In a preferred embodiment, the polysaccharide portion comprises or is composed of hyaluronic acid, glycerol-grafted hyaluronic acid, heparin polysaccharide, chondroitin sulfate, and carboxymethyl cellulose. It should be understood that such substances may also contain their salts.

[0051] In a preferred embodiment, the polysaccharide portion comprises or is composed of one or more hyaluronic acid portions. It should be understood that this may also contain their salts.

[0052] In a preferred embodiment, the crosslinking material of the present invention is a gel. In a preferred embodiment, the crosslinking material of the present invention is a polysaccharide-based gel. In a preferred embodiment, the crosslinking material of the present invention is a hyaluronic acid-based gel (HA gel). (See specification 4 / 21 pages 7 CN 121285400 A)This material may also be referred to as self-crosslinking hyaluronic acid (HA).

[0053] Hyaluronic acid (also known as HA, hyaluronic acid salt, or hyaluronic acid) can be understood in the broadest sense as any hyaluronic acid in this art. It may be a polysaccharide portion comprising a hyaluronic acid moiety (also known as a hyaluronic acid unit), preferably comprising at least 50 mol% of the hyaluronic acid moiety relative to the total content of the sugar moiety in the polysaccharide, more preferably at least 75 mol%, even more preferably at least 80 mol%, even more preferably at least 90 mol%. Hyaluronic acid may optionally comprise one or more sugar moieties other than hyaluronic acid.

[0054] In a preferred embodiment, the hyaluronic acid is a natural glycosaminoglycan composed of repeating units of N-acetyl-D-glucosamine and D-glucuronic acid ([α-1,4-D-glucuronic acid-β-1,3-N-acetyl-D-glucosamine]n). Therefore, the repeating / monomer unit of hyaluronic acid may be the following examples or salts thereof:

[0055]

[0056] Therefore, the repeating structural motif of hyaluronic acid may be the following examples or salts thereof:

[0057]

[0058] Hyaluronic acid may also encompass glycerol-grafted hyaluronic acid.

[0059] Hyaluronic acid may be used in the manner described in WO 2022 / 268871. In the context of this invention, the weight average molecular weight (Mw) of hyaluronic acid is preferably at least 1 kDa (1000 Da), more preferably at least 5 kDa, even more preferably at least 10 kDa, even more preferably at least 50 kDa, even more preferably at least 100 kDa, even more preferably at least 200 kDa, even more preferably at least 300 kDa or higher.

[0060] In a preferred embodiment, the polysaccharide portion has the weight average molecular weight as described above.

[0061] Heparin glycan may be understood in the broadest sense as any heparin glycan. In a preferred embodiment, it may be as described in WO 2015 / 149941. Heparin polysaccharide (HEP) is a biopolymer belonging to the glycosaminoglycan (GAG) family of polysaccharides.

[0062] In the human body, it is an intermediate product in the biosynthesis of heparin and heparin sulfate. The structure of heparin polysaccharide is highly similar to that of hyaluronic acid (HA) because it has the same monosaccharide components as hyaluronic acid. The only differences from HA are that the β-(1,3) glycosidic bond between glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) in HA is replaced by the β-(1,4) glycosidic bond in HEP, and the β-(1,4) glycosidic bond between N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) in HA is replaced by the α-(1,4) glycosidic bond in HEP:

[0063] GlcUA-β-(1-4)-[GlcNAc-α-(1-4)-GlcUA-β-(1-4)]n-GlcNAc HEP

[0064] Typically, heparin glycans exhibit excellent biocompatibility. Heparin glycans carry a large number of negatively charged and hydroxyl residues, thus exhibiting high hydrophilicity and improved tissue compatibility. Furthermore, since heparin glycan polymers, even after modification, still contain fragments present in natural heparan sulfate and heparin polymers, they are typically non-immunogenic (e.g., do not induce antibodies). Additionally, due to the structural similarity between heparin glycans and hyaluronic acid, the same chemical modifications can be made to the functional groups, including oxidation to aldehydes, as is known in hyaluronic acid. The molecular weight (Mw) of the heparin glycan polymer used in the context of this invention can have any molecular weight.

[0065] Chondroitin sulfate can be understood in the broadest sense generally understood in this art. In an aqueous environment, it can be a structure comprising the following repeating / monomer units (the counterion is not depicted and can be any cation, such as sodium):

[0066]

[0067] The polysaccharide moiety can be one or more types of polysaccharide moieties. Such substances may have different molecular sizes, and / or different compositions of their monomer units, and / or different chemical structures. Optionally, the different polysaccharide moieties may be of different types.

[0068] In one embodiment, the polysaccharide moieties, particularly the hyaluronic acid moieties, have at least two different molecular weights, each containing a primary amino residue or a salt thereof. In other words, the polysaccharide moieties may also be a mixture of polysaccharide moieties of different molecular weights. In a preferred embodiment, the polysaccharide portion has at least two different molecular weights, and at least one polysaccharide portion has, preferably at least two polysaccharide portions, particularly all polysaccharide portions, each having a molecular weight in the range of 10 to 10000 kDa, in the range of 100 to 10000 kDa, or in the range of 100 to 5000 kDa, in the range of 100 to 3500 kDa, in the range of 200 to 2000 kDa, in the range of 250 to 1500 kDa, in the range of 300 to 1000 kDa, in the range of 400 to 900 kDa, or in the range of 500 to 900 kDa.

[0069] In a preferred embodiment, the polysaccharide portion has at least two different molecular weights, and at least one polysaccharide portion has, preferably at least two polysaccharide portions, particularly all polysaccharide portions, each having a molecular weight in the range of 1500 to 3500 kDa.

[0070] In a preferred embodiment, based on the total amount of the polysaccharide fraction, the total amount of the polysaccharide fraction does not contain more than 20% by weight, more than 10% by weight, more than 5% by weight, more than 1% by weight, more than 0.5% by weight, or more than 0.1% by weight of polysaccharide fractions with a molecular weight less than 200 kDa.

[0071] In a preferred embodiment, the polysaccharide portion comprises or is composed of at least two hyaluronic acid portions having at least two different molecular weights, and at least one hyaluronic acid portion is present, preferably at least two hyaluronic acid portions are present, particularly all hyaluronic acid portions each having a molecular weight in the range of 10 to 10000 kDa, in the range of 100 to 10000 kDa, or in the range of 100 to 5000 kDa, in the range of 100 to 3500 kDa, in the range of 200 to 2000 kDa, in the range of 250 to 1500 kDa, in the range of 300 to 1000 kDa, in the range of 400 to 900 kDa, or in the range of 500 to 900 kDa. In a preferred embodiment, the polysaccharide portion comprises or is composed of at least two hyaluronic acid portions having at least two different molecular weights, and at least one hyaluronic acid portion is present, preferably at least two hyaluronic acid portions are present, particularly all hyaluronic acid portions each having a molecular weight in the range of 1500 to 3500 kDa.

[0072] The amount of triazine activator used can be any suitable for crosslinking the carboxylic acid residues of the polysaccharide moiety with at least some hydroxyl residues of the polysaccharide moiety to form ester bonds that covalently crosslink the polysaccharide moiety with each other. Specification 6 / 21 pages 9 CN 121285400 A

[0073] Typically, the triazine activator activates the carboxylic acid residues of the polysaccharide moiety. Therefore, the amount of triazine activator can be reasonably defined as the molar equivalent (eq.) relative to the carboxylic acid residues of the polysaccharide moiety. In this context, it should be understood that the carboxylic acid residues also include their salts. In other words, the molar equivalent (eq.) of the triazine activator is defined as the molar ratio of [triazine activator]:[carboxylic acid residues of the polysaccharide moiety]. For this purpose, in the context of sodium hyaluronate (HA) salt, it can be approximately assumed that each HA monomer / repeating unit has one carboxylic acid residue when used as a sodium salt (molecular weight (MW) of about 402 g / mol). The degree of crosslinking can be adjusted with different molar equivalents (eq.) of triazine activator.

[0074] In a preferred embodiment, the molar ratio of [triazine activator] to [carboxylic acid residues of the polysaccharide moiety] is in the range of 1:100 to 100:1, preferably in the range of 1:10 to 10:1, more preferably in the range of 1:5 to 5:1, or in the range of 1:2 to 2:1.

[0075] As used in the context of this invention, the triazine activator can be any compound based on a triazine core structure that enables the carboxylic acid residues to react with hydroxyl residues to form an ester bond. It should be understood that it primarily refers to a compound in which the activator causes the carboxylic acid residues of the polysaccharide moiety to react with the hydroxyl residues of the polysaccharide moiety to form an ester bond.

[0076] In a preferred embodiment, the activator is typically not covalently included in the crosslinked material. Therefore, it can typically be optionally removed from the crosslinked material of this invention by any means, such as washing, filtration, etc.

[0077] Triazine activators may have any structure containing a triazine core suitable for crosslinking the carboxylic acid residues of the polysaccharide moiety with at least some hydroxyl residues of the polysaccharide moiety to form ester bonds that covalently crosslink the polysaccharide moiety with each other. For example, triazine activators may have the following structure:

[0078]

[0079] wherein R1, R2 and R3 are independently any residues, for example, each independently a residue selected from the group consisting of: hydrogen, deuterium, C6-C10-aryl, C2-C10-heteroaryl, linear or branched C1-C10-(cyclo)alkyl, linear or branched C1-C10-(cyclo)alkoxy, linear or branched C1-C10-hetero(cyclo)alkyl, linear or branched C2-C10-(cyclo)alkenyl, C2-C10-hetero(cyclo)alkenyl, linear or branched C2-C10-(cyclo)alkynyl and C2-C10-hetero(cyclo)alkenyl,

[0080] wherein each of the above residues is optionally substituted by one or more residues selected from the group consisting of: halogen, C6-C10-aryl, C2-C10-heteroaryl, linear or branched C1-C10-(cyclo)alkyl, linear or branched C1-C10-hetero(cyclo)alkyl, linear or branched C1-C10-(cyclo)alkoxy, linear or branched C2-C10-(cyclo)alkenyl, C2-C10-hetero(cyclo)alkenyl, linear or branched C2-C10-(cyclo)ynyl and C2-C10-hetero(cyclo)alkenyl,

[0081] or salts thereof.

[0082] In a preferred embodiment, at least one of residues R1, R2 and R3 is an optionally substituted morpholinium residue. In a preferred embodiment, at least one of residues R1, R2 and R3 is a C1-C4-alkyl-morpholinium residue. In a preferred embodiment, at least one of residues R1, R2 and R3 is a methylmorpholinium residue. In a preferred embodiment, at least one of residues R1, R2, and R3 is an optionally substituted linear or branched C1-C10-(cyclo)alkoxy residue. In a preferred embodiment, at least one of residues R1, R2, and R3 is an optionally substituted linear or branched C1-C4-alkoxy residue. In a preferred embodiment, at least one of residues R1, R2, and R3 is an optionally substituted linear or branched methoxy residue.

[0083] In a preferred embodiment, the molecular weight of the triazine activator is not more than 1500 g / mol, not more than 1000 g / mol, not more than 500 g / mol, and not more than 400 g / mol. Instruction manual, page 7 / 21, 10 CN 121285400 A

[0084] In a preferred embodiment, one or more activators are selected from the group consisting of: 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium (DMTMM) or a salt thereof and / or 2-chloro-4,6-dimethoxy-1,3,5-triazine orIts salts, and combinations thereof.

[0085] In a preferred embodiment, the activator is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholineonium (DMTMM) or a salt thereof.

[0086] The salt of DMTMM is preferably a salt of a cosmetically and / or pharmaceutically acceptable anion, such as chloride, acetate, bicarbonate (hydrocarbonate), or a mixture of two or more anions.

[0087] In a preferred embodiment, the activator is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride (CAS No 3945-69-5).

[0088] DMTMM is considered to have relatively low and substantially negligible toxicity at normal usage levels, is not carcinogenic, does not induce mutagenesis, and does not cause teratogenic / reproductive toxicity. Therefore, it is particularly suitable for the preparation of soft tissue fillers, such as dermal or connective tissue fillers.

[0089] When DMTMM or its salts are used as activators, 4-methylmorpholine (NMM) and / or 4,6-dimethoxy-1,3-5-triazine-2-ol (DMT) can be formed as degradation products. For example, the chloride salt of DMTMM can be used as an activator:

[0090]

[0091] In a preferred embodiment, the method is further characterized in that it does not include at least one of the following:

[0092] (a) using a linker portion between a carboxylic acid residue or its salt inserted into the polysaccharide moiety and a hydroxyl residue, particularly without a peptide linker portion or a heterogeneous linker portion;

[0093] (b) using a carbodiimide activator, particularly selected from the group consisting of: N,N'-dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and combinations of two or more thereof;

[0094] (c) using a reactive group selected from the group consisting of glycidyl ether, maleimide, acid anhydride, alkoxide, and combinations of two or more thereof;

[0095] (d) a polysaccharide moiety having a primary amino group, a thiol group, an imide group, an imine group, or an epoxy group.

[0096] In a preferred embodiment, the method is further characterized in that it does not include at least two of the following, more preferably at least three, and in particular all of them:

[0097] (a) using an interconnecting linker portion between a carboxylic acid residue or its salt inserted into the polysaccharide moiety and a hydroxyl residue, in particular without a peptide linker portion or a heterotrophic linker portion;

[0098] (b) using a carbodiimide activator, in particular selected from the group consisting of: N,N'-dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and combinations of two or more thereof;

[0099] (c) Using a reactive group selected from the group consisting of glycidyl ether, maleimide, acid anhydride, alkoxide, and combinations of two or more thereof;

[0100] (d) A polysaccharide moiety having a primary amino, thiol, imide, imine, or epoxy group. Specification 8 / 21 pages 11 CN 121285400 A

[0101] The solvent may be any solvent suitable for reacting the carboxylic acid residues or their salts with the hydroxyl residues of the polysaccharide moiety. In a preferred embodiment, the solvent is suitable for dissolving or suspending the polysaccharide moiety and for dissolving triazine activators. In a preferred embodiment, the solvent is suitable for dissolving both the polysaccharide and the triazine activator. In a preferred embodiment, the solvent is a polar solvent. In a preferred embodiment, the solvent is a proton solvent. In a preferred embodiment, the solvent is a proton polar solvent.

[0102] In a preferred embodiment, the solvent comprises, relative to the total mass of the solvent, more than 50% by weight, at least 60% by weight, at least 70% by weight, at least 80% by weight, at least 90% by weight, 95% by weight, or even 100% by weight, one or more of the following components selected from the group consisting of:

[0103] water;

[0104] one or more alcohols, preferably one or more C1-C5-alcohols, more preferably one or more C1-C5-alcohols selected from the group consisting of: methanol, ethanol, n-propanol, isopropanol, n-butanol (1-butanol), secondary butanol (2-butanol), isobutanol, (2-methylpropanol), tert-butanol (2-methylpropanol), pentyl-1-ol, 2-methylbutanol, 3-methylbutanol, 2,2-dimethylpropanol, pentyl-2-ol, 3-methylbutanol, pentyl-3-ol and / or 2-methylbutanol, and combinations of two or more thereof, particularly methanol and / or ethanol;

[0105] One or more primary amines, particularly one or more C1-C5-amines; one or more carbonic acids, preferably one or more C1-C5-carbonic acids selected from the group consisting of: formic acid, acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, particularly formic acid and / or acetic acid;

[0106] One or more primary or secondary amides, preferably one or more C1-C5-amides, particularly formamide;

[0107] One or more sulfoxides, preferably one or more C1-C5-amides, particularly dimethyl sulfoxide (DMSO);

[0108] and combinations of two or more thereof.

[0109] In a preferred embodiment, the solvent is an aqueous solution. An aqueous solvent can be understood in the broadest sense as a solvent containing water, wherein the water content, relative to the total mass of the solvent, is more than 50% by weight, at least 60% by weight, at least 70% by weight, at least 80% by weight, at least 90% by weight, at least 95% by weight, or even 100% by weight.In one embodiment, in addition to water, the aqueous buffer also comprises one or more components selected from the group consisting of: one or more alcohols (particularly one or more C1-C5-ols, such as methanol, ethanol, n-propanol, isopropanol, n-butanol (1-butanol), secondary butanol (2-butanol), isobutanol, (2-methylpropanol), tert-butanol (2-methylpropanol), pentyl-1-ol, 2-methylbutanol, 3-methylbutanol, 2,2-dimethylpropanol, pentyl-2-ol, 3-methylbutanol, pentyl-3-ol and / or 2-methylbutanol), one or more primary amines (particularly one or more C1-C5-amines), one or more carbonic acids (particularly one or more C1-C5-carbonic acids, such as formic acid, acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid), one or more primary amides or secondary amides (particularly one or more C1-C5-amides, such as formamide), one or more sulfoxides (particularly one or more C1-C5-... C5-amides, such as dimethyl sulfoxide (DMSO), one or more inorganic or organic cations (especially one or more inorganic or organic cations with a molecular weight less than 1000 Da, especially alkali metal cations or alkaline earth metal cations, other metal cations, protons, ammonium cations, etc.), one or more inorganic or organic anions (especially one or more inorganic or organic anions with a molecular weight less than 1000 Da, especially chlorides, sulfates, etc.), one or more silicates, and combinations of two or more thereof.

[0110] In a preferred embodiment, the solvent is an aqueous buffer (e.g., phosphate buffer (PBS), Tris buffer, borate buffer, acetate buffer, etc.). In a preferred embodiment, an alcoholic solvent is used, such as a mixture of water with ethanol, methanol, propanol, butanol and / or pentanol. In a preferred embodiment, water is used as the solvent. In a preferred embodiment, the solvent is an aqueous buffer with a pH in the range of 5 to 9, especially 6 to 8. For example, the solvent may be an aqueous buffer with a pH in the range of 6.1 to 7.9, 6.5 to 7.7, 6.7 to 7.5, or 7.0 to 7.5. Specification 9 / 21 pages 12 CN 121285400 A

[0111] Surprisingly, it was found that the reaction steps including pre-swelling do not require alkalization, and the reaction is carried out at a substantially neutral pH. This refutes the prejudice in the art that a strongly acidic or strongly alkaline pH range is required.

[0112] Water as used herein may be understood in the broadest sense. Water is preferably deionized water, distilled water, or tap water, especially deionized water or distilled water.

[0113] In a preferred embodiment, the method does not include a purification step (iii). In another embodiment, the method includes at least one purification step (iii). In one embodiment, the method includes at least one step of filtration, washing, and / or...Alternatively, dialysis, particularly cross-flow filtration, percolation, and / or dead-end filtration, may be used to purify the cross-linked material (iii);

[0114] Dialysis can be performed in any manner. In a preferred embodiment, dialysis is performed using a dialysis membrane with a molecular weight cutoff (MWCO) of 1 to 25 kDa, 5 to 20 kDa, or 10 to 15 kDa. The purification time can be adjusted according to the molecular structure and can range from at least 1 hour, at least 6 hours, 12 to 78 hours, 1 to 7 days, or 1 to 3 days. Optionally, the swelling ratio can be controlled by the weight of the membrane, and dialysis can be stopped once the product shows the desired concentration of the cross-linked material.

[0115] It should be understood that filtration can be cross-flow filtration, dead-end filtration, or a combination of both. Filtration can be performed in any manner. In the context of filtration, the filter can have any pore size suitable for purifying the cross-linked material, i.e., preferably retaining the cross-linked material and allowing reactants and optionally unreacted polysaccharides to pass through. Optionally, the pore size can be in the range of 5 nm to 2 μm, more particularly 30 nm to 600 nm, more particularly 80 nm to 300 nm, and particularly 5 nm to 60 nm. The filter can be any material, such as ceramic, metal, polymer material, or a combination thereof.

[0116] Optionally, the filtration can be dynamic filtration, such as that described in WO 2020 / 030629. Therefore, step (iii) may optionally include dynamic filtration of the crosslinked material, which optionally includes the following steps:

[0117] a) transferring the crosslinked material to a dynamic filtration device equipped with semi-permeable filter discs and permeating the gel, which includes the following steps:

[0118] i) concentrating the crosslinked material to a predetermined concentration by applying a rotational speed in the range of 20 rpm to 500 rpm and an overpressure in the range of 0.5 to 6 bar; or pumping the crosslinked material directly into the processing chamber of the dynamic filtration device;

[0119] ii) permeating by applying a rotational speed in the range of 20 rpm to 500 rpm and an overpressure in the range of 0.5 to 6 bar to reduce unwanted molecules;

[0120] b) optionally adding a mixture containing non-crosslinked material and water to the crosslinked material.

[0121] In one embodiment, the dynamic filtration device is equipped with 1 to 10 semi-permeable filter discs. In DCF, any rotation speed and pressure can be used, for example, a rotation speed in the range of 20 rpm to 500 rpm and a pressure in the range of 0.5 to 3 bar. In DCF, any concentration can be used, for example, 10 to 70 mg / g.

[0122] In an embodiment of the invention, crossflow filtration is dynamic crossflow filtration (DCF). Therefore, in an embodiment, the method is further characterized by including...Step (iii) involves purifying the crosslinked material using DCF. This is illustrated below. Optionally, the DCF may be as described in WO 2020 / 030629.

[0123] Optionally, one or more additional components (e.g., one or more local anesthetics (e.g., lidocaine, as described below), one or more cell proliferation factors, one or more dyes, and combinations of two or more thereof) may be added before, during, or after step (iii) of purifying the crosslinked material by filtration, washing, and / or dialysis.

[0124] In one embodiment, steps (i) and (ii) are performed in a single batch. In another embodiment, steps (i) and (ii) are performed in multiple batches.

[0125] Steps (i) and (ii), and optionally step (iii), may each be performed within any temperature range. In a preferred embodiment, step (i) is performed in the range of 5°C to 90°C, preferably in the range of 18°C ​​to 60°C, particularly more preferably in the range of 18°C ​​to 25°C or in the range of 20°C to 50°C (page 10 / 21 of the specification, CN 121285400 A). In a preferred embodiment, step (ii) is performed in the range of 5°C to 90°C, preferably in the range of 18°C ​​to 60°C, particularly in the range of 20°C to 50°C. In a preferred embodiment, step (iii) is performed in the range of 5°C to 90°C, or in the range of 18°C ​​to 60°C, or in the range of 20°C to 50°C. In a preferred embodiment, steps (i) and (ii) and optional step (iii) are performed in the range of 5°C to 90°C, preferably in the range of 18°C ​​to 60°C, particularly in the range of 20°C to 50°C. In a preferred embodiment, steps (i) and (ii) are performed at temperatures with a difference of no more than 10°C, no more than 5°C, or no more than 2°C. For example, steps (i) and / or (ii) and / or (iii) may be performed at temperatures of about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, or about 30°C. Steps (i) and (ii), and optionally step (iii), may be performed at any pressure. For example, the pressure may be ambient pressure (e.g., typically an external pressure of about 970 to 1100 hPa).

[0126] Step (i), which brings the components into contact with each other, may be performed in any manner. In a preferred embodiment, the method is further characterized in that step (i) includes mixing the components, namely the polysaccharide and one or more activators and one or more solvents, and optionally one or more other components. This mixing may be performed in any manner, for example by stirring and / or shaking.

[0127] In a preferred embodiment, the polysaccharide portion is first reacted with a solvent or solvent before being contacted with one or more activators.Solvent mixing. This step may include incubation (i.e., pre-swelling) of the polysaccharide portion with a solvent or a mixture of solvents. For example, such optional incubation steps may be carried out (e.g., at a temperature of 4°C to 40°C) for at least 10 min, 30 min to 7 days, 1 hour to 2 days, 6 to 36 hours, or 12 to 24 hours. One or more activators may optionally be premixed with a portion of the solvent and then added to the mixture of the polysaccharide portion with a solvent or a mixture of solvents.

[0128] Reaction step (ii) may be carried out as long as it is suitable for completing the desired reaction of the carboxylic acid residue or its salt with the hydroxyl residue. Step (ii) may be carried out for any time suitable for this purpose. Optionally, step (ii) may be carried out for 1 min to 1 week or longer, 2 min to 5 days, 3 min to 4 days, 5 min to 72 hours, 5 min to 24 hours, 10 min to 12 hours, 30 min to 6 hours, 1 hour to 5 hours, or 2 to 4 hours. Step (ii) can be performed at any temperature suitable for this purpose, for example, at 0°C to 100°C, at 4°C to 95°C, at 10°C to 70°C, at 15°C to 30°C, at 18°C ​​to 25°C, at 20°C to 70°C, at 20°C to 40°C, or at 60°C to 70°C. In a preferred embodiment, step (ii) is performed for no more than 72 hours, no more than 48 hours, or no more than 24 hours. In a preferred embodiment, step (ii) is performed for at least 10 minutes, preferably 15 minutes to 48 hours, particularly 30 minutes to 18 hours, at a temperature in the range of 5°C to 90°C, preferably 18°C ​​to 60°C, particularly 20°C to 50°C. In a preferred embodiment, the method is further characterized in that step (ii) is performed for at least 10 minutes at a temperature in the range of 5°C to 90°C. In a preferred embodiment, the method is further characterized in that step (ii) is carried out at a temperature in the range of 18°C ​​to 60°C for 30 min to 18 h. Step (ii) can be carried out at any pH. For example, it can be carried out at a pH in the range of 5 to 9, particularly 6 to 8, 6.1 to 7.9, 6.5 to 7.7, 6.7 to 7.5, or 7.0 to 7.5.

[0129] During reaction step (ii), any concentration range of the polysaccharide fraction can be used. In a preferred embodiment, a polysaccharide fraction of 0.01 to 800 mg / mL, 0.1 to 500 mg / mL, or 0.5 to 200 mg / mL is used. In a preferred embodiment, a polysaccharide fraction of 1 to 100 mg / mL, 2 to 75 mg / mL, 5 to 50 mg / mL, 20-25 mg / mL, or 25 to 35 mg / mL is used.

[0130] Optional purification step (iii), if performed, can be carried out for any time suitable for this purpose. Optionally, step (iii) can be carried out for 1 minute to 1 week or longer, 2 minutes to 5 days, 3 minutes to 4 days, 5 minutes to 72 hours, 5 minutes to 24 hours, 10 minutes to 12 hours, 30 minutes to 6 hours, 1 hour to 5 hours, or 2 to 4 hours. Step (iii) can be carried out at any temperature suitable for this purpose, for example at 0°C to 100°C, at 4°C to 95°C, at 10°C to 70°C, at 15°C to 30°C, at 18°C ​​to 25°C, at 20°C to 70°C, at 20°C to 40°C, or at 60°C to 70°C. Instruction manual 11 / 21 pages 14 CN 121285400 A

[0131] In a preferred embodiment, the method is further characterized by:

[0132] (a) it includes step (iii) of purifying the crosslinked material by filtration, washing and / or dialysis, particularly cross-flow filtration, percolation and / or dead-end filtration;

[0133] (b) steps (i) and (ii) are carried out in a single batch;

[0134] (c) steps (i) and (ii) and optional step (iii) are carried out at a temperature in the range of 5°C to 90°C, preferably 18°C ​​to 60°C, particularly 20°C to 50°C; and / or

[0135] (d) step (ii) is carried out for at least 10 min, preferably 15 min to 48 h, particularly 30 min to 18 h.

[0136] In a preferred embodiment, the method comprises:

[0137] (i) contacting the following components with each other:

[0138] (A) a polysaccharide moiety having a weight average molecular weight of at least 50 kDa, comprising a carboxylic acid residue or a salt thereof and a hydroxyl residue;

[0139] (B) one or more triazine activators that cause the carboxylic acid residues to react with the hydroxyl residues to form an ester bond, particularly wherein the activator is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium or a salt thereof; and

[0140] (C) an aqueous solvent; preferably an aqueous solvent with a pH in the range of 5 to 9, particularly 6 to 8; and

[0141] (ii) reacting at least some of the carboxylic acid residues with at least some of the hydroxyl residues to form an ester bond that covalently crosslinks the polysaccharide moiety with each other; and

[0142] (iii) optionally purifying the crosslinked material obtained from step (ii).

[0143] In optional additional steps, the obtained crosslinked material may be further processed. For example, it may be optionally homogenized and / or sieved (screened). In this context, the terms sieve, filter, and screen are used interchangeably. This may result in a particularly homogeneous material (e.g., a hydrogel). In optional additional steps, the crosslinked material may be sterilized.

[0144] In a preferred embodiment, the method includes the following steps:

[0145] (i) Contacting the following components with each other:

[0146] (A) a polysaccharide moiety comprising a carboxylic acid residue or a salt thereof and a hydroxyl residue,

[0147] (B) one or more triazine activators that cause the carboxylic acid residue to react with the hydroxyl residue to form an ester bond, and

[0148] (C) an aqueous solvent; preferably an aqueous solvent,

[0149] wherein the polysaccharide moiety is premixed with a portion of the aqueous solvent and incubated (e.g., 30 min to 7 days);

[0150] (ii) Reacting at least some of the carboxylic acid residues with at least some of the hydroxyl residues at a temperature of 20°C to 60°C for 10 min to 2 days to form an ester bond that covalently crosslinks the polysaccharide moiety moiety with each other; and

[0151] (iii) optionally purifying the crosslinked material obtained from step (ii) by dialysis; and

[0152] (iv) optionally homogenizing and / or sieving and / or mixing with an anesthetic (e.g., lidocaine) and / or optionally sterilizing.

[0153] In a preferred embodiment, the method comprises:

[0154] i) contacting the following components with each other:

[0155] (A) a polysaccharide moiety having a weight average molecular weight of at least 50 kDa, comprising a carboxylic acid residue or a salt thereof and a hydroxyl residue,

[0156] wherein the polysaccharide moiety is premixed with a portion of an aqueous solvent and incubated (e.g., for 30 min to 7 days),

[0157] (B) one or more triazine activators that cause the carboxylic acid residue to react with the hydroxyl residue to form an ester bond, particularly wherein the activator is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium or a salt thereof; and specification 12 / 21 page 15 CN 121285400 A

[0158] (C) an aqueous solvent; preferably an aqueous solvent with a pH in the range of 5 to 9, particularly 6 to 8; and

[0159] (ii) reacting at least some carboxylic acid residues with at least some hydroxyl residues at a temperature of 20 to 60°C for 10 min to 2 days to form ester bonds that covalently crosslink the polysaccharide moieties to each other; and

[0160] (iii) optionally purifying the crosslinked material obtained from step (ii) by dialysis; and

[0161] (iv) optionally homogenizing and / or sieving and / or mixing with an anesthetic (e.g., lidocaine) and / or optionally sterilizing.

[0162] It should be understood that the crosslinked materials obtainable by the method of the present invention have special technical characteristics, such as structural features and the absence of irritating (bi)functional linkers and activators.

[0163] Therefore, another aspect of the present invention relates to crosslinked materials obtainable by the method of the present invention.

[0164] It should be understood that the definitions and preferred embodiments shown in the context of the method of the present invention are applicable to the crosslinked materials of the present invention after necessary modifications.

[0165] In embodiments of the present invention, the crosslinked material has been prepared by using DMTMM as an activator. In embodiments of the present invention, the crosslinked material (particularly prior to final purification) comprises:

[0166] (a) 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium or a salt thereof,

[0167] (b) N-methylmorpholinium or a salt thereof; and / or

[0168] (c) 4,6-dimethoxy-1,3,5-triazin-2-ol or a tautomer or salt thereof.

[0169] In embodiments of the invention, the crosslinking material (particularly prior to final purification) comprises NMM and / or DMT. In embodiments of the invention, the crosslinking material (particularly prior to final purification) comprises at most 0.1% by weight, preferably 0.01 to 1000 ppm, 0.1 to 100 ppm, or 1 to 50 ppm of NMM and / or DMT relative to the total weight of the crosslinking material (gel).

[0170] Another aspect of the invention relates to a crosslinked material comprising or consisting of one or more hyaluronic acid moieties with a weight average molecular weight of at least 50 kDa, wherein the hyaluronic acid moieties are covalently crosslinked with each other via ester bonds without interconnecting linker structures, characterized in that the crosslinked material does not contain diimide, epoxy, or heterogeneous linker moieties.

[0171] As used herein, the term “without interconnecting linker structures” can be understood in the broadest sense as the absence of any other chemical moieties not derived from (and also not present in) the polysaccharide moieties (e.g., the hyaluronic acid moieties) introduced into the chemical structure conjugating the polysaccharide moieties with the ester bonds. In other words, the ester bonds are preferably formed by introducing oxygen atoms derived from the polysaccharide moieties (e.g., the hyaluronic acid moieties) and carbon atoms derived from the polysaccharide moieties (e.g., the hyaluronic acid moieties).

[0172] In a preferred embodiment, the crosslinked material is further characterized in that it does not contain imide or diimide groups. In a preferred embodiment, the crosslinked material is further characterized in that it does not contain imine groups. In a preferred embodiment, the crosslinked material is further characterized in that it does not contain epoxy groups. In a preferred embodiment, the crosslinked material is further characterized in that it does not contain heterogeneous linker portion groups.

[0173] In a preferred embodiment, the crosslinked material is further characterized in that it does not contain the following groups that interconnect the polysaccharide portions:

[0174] (a) diimide group

[0175] (b) imide group;

[0176] (c) imine group;

[0177] (d) epoxy group; and / or

[0178] (e) heterogeneous linker portion.

[0179] In a preferred embodiment, the crosslinked material is further characterized in that it does not contain the following groups that interconnect the polysaccharide portions:

[0180] (a) diimide group

[0181] (b) imide group;

[0182] (c) imine group;

[0183] (d) epoxy group; and

[0184] (e) heterogeneous linker portion.

[0185] In a preferred embodiment, the crosslinking material is obtained by the method of the present invention.

[0186] Depending on the intended use of the crosslinked material, it may be ready for immediate use or storable. For example, it may be stored under any conditions suitable for this purpose, such as at ambient temperature (e.g., 18°C ​​to 30°C, preferably 18°C ​​to 25°C), in a refrigerator (e.g., at 0°C to 15°C, preferably 3°C to 10°C), in a freezer (e.g., -30°C to 0°C, preferably -25°C to -10°C), in an ultra-low temperature freezer (e.g., -100°C to -200°C, preferably -90°C to -55°C), on liquid nitrogen, on dry ice, or even in one or more liquid rare gases. It may be stored in a dry state as a hydrogel, a gel and / or suspension containing other non-aqueous solvents, an emulsion, a colloid, or a solution.

[0187] Optionally, the solvents used in steps (i) and (ii) and / or optional step (iii) may be partially or completely removed. Because the components used, including the activator, may have relatively low toxicity, the solvents used in steps (i) and (ii) and / or optional step (iii) may also optionally remain in the crosslinked material. If removed, the solvents used in steps (i) and (ii) and / or optional step (iii) can be removed by any means, such as by substitution with another solvent (e.g., another aqueous buffer) and / or by evaporation.

[0188] The crosslinked material can be used for any purpose and can be in any form. For example, it can be a gel when its pores contain a liquid or viscous medium. Generally, such a liquid or viscous medium can be any liquid or viscous medium that will not disintegrate the crosslinked material. The liquid or viscous medium is preferably an aqueous liquid. As described above, the crosslinked material of the present invention can be used to prepare hydrogels.

[0189] Thus, another aspect of the present invention relates to hydrogels comprising:

[0190] (A) the crosslinked material of the present invention; and

[0191] (B) an aqueous solution; and

[0192] (C) optionally one or more other pharmaceutically or cosmetically acceptable agents, particularly one or more local anesthetics.

[0193] It should be understood that the definitions and preferred embodiments shown in the context of the methods and crosslinking materials of the present invention are applicable to the hydrogels of the present invention after necessary modifications.

[0194] Therefore, the present invention also relates to a method for preparing a hydrogel. Optionally, such a method may include the step of adding an aqueous buffer to a purified crosslinking material.

[0195] The crosslinking materials and / or hydrogels of the present invention can be used for any purpose. For example, such materials can be used as injectable compositions.

[0196] Therefore, another aspect of the present invention relates to injectable compositions comprising the crosslinking materials or hydrogels of the present invention, wherein the crosslinking material is preferably a supercompensator.

[0197] It should be understood that the definitions and preferred embodiments shown in the context of the methods, crosslinking materials and hydrogels of the present invention are applicable to the injectable compositions of the present invention after necessary modifications.

[0198] In a preferred embodiment, the crosslinked materials, hydrogels, and / or injectable compositions of the present invention can be used as soft tissue fillers, particularly dermal fillers or connective tissue fillers.

[0199] The liquid or viscous carrier according to the invention included in the injectable composition can be any injectable carrier. Typically, the liquid or viscous carrier is a pharmaceutically and / or cosmetically acceptable carrier, and therefore, when applied to mammals in the sense of the present invention, the carrier is non-toxic to mammals, particularly humans. The liquid or viscous carrier preferably comprises or is composed of one or more solvents, such as water, aqueous buffers (e.g., saline or phosphate-buffered saline), dimethyl sulfoxide (DMSO), ethanol, vegetable oils, paraffin oils, or combinations thereof. The liquid or viscous carrier more preferably comprises or isotonic buffers, more particularly physiological saline solutions or buffered physiological saline solutions.

[0200] Other components optionally present can be any components. For example, such other components may be selected from the group consisting of one or more local anesthetics, one or more cell proliferation factors, one or more dyes, and combinations of two or more thereof.

[0201] Such other components may be added at any point in time, such as before, during or after the purification of the crosslinking material. For example, one or more other components may be added during purification step (iii). In another embodiment of the invention, one or more other components may be added to the prepared and optionally purified crosslinking material.

[0202] Local anesthetics may make the individual more comfortable to inject. Cell proliferation factors may improve cell invasion into the crosslinking material of the invention. Dyes may improve the localization of the injection (e.g., pharmaceutically acceptable fluorescent dyes such as fluorescein or rose red), or may improve the invisibility of the originally whitish crosslinking material (e.g., by making it flesh-colored). Any other pharmaceutically active compound may also be added. The crosslinking material of the invention may then be administered in a sustained-release form.

[0203] The local anesthetics suitable for use in this article include (but are not limited to) ambucaine, amolanone, amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine, butacaine, butamben, butanilicaine, butethamine, butoxycaine, carticaine, chloroprocaine, and hexylCocaethylene, cocaine, cyclomethycaine, dibucaine, dimethysoquin, dimethocaine, diperodon, dicyclomine, ecgonidine, ecgonine, ethyl chloride, eticaine, beta-eucaine, euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate mesylate, levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, myrtecaine, naepaine, octacaine, orthocaine, oxethazaine, parethoxycaine, phenacaine, phenol, piperocaine, piridocaine, polidocanol, pramoxine, prilocaine e) Procaine, propanocaine, proparacaine, propipocaine, propoxycaine, psudococaine, pyrrocaine, ropivacaine, succinate, tetracaine, tolycaine, trimecaine, zolamine, and their salts. Combinations of two or more of the described anesthetics may also be used herein, such as lidocaine combined with other "caine" anesthetics, such as prilocaine.

[0204] According to the intended use of the crosslinked materials, hydrogels, and / or injectable compositions of the present invention, they can be provided in various packages. They can be stored under any conditions suitable for this purpose, such as at ambient temperature (e.g., 18°C ​​to 30°C, preferably 18°C ​​to 25°C), in a refrigerator (e.g., 0°C to 15°C, preferably 3°C to 10°C), in a freezer (e.g., -30°C to 0°C, preferably -25°C to -10°C), in an ultra-low temperature freezer (e.g., -100°C to -200°C, preferably -90°C to -55°C), on liquid nitrogen, on dry ice, or even in one or more liquid rare gases. For example, they can be provided in small glass vials, injection devices, etc. They can be administered to the subject by injection (e.g., via syringe or infusion). It can be stored in a dry state, as a hydrogel, a gel and / or suspension containing other non-aqueous solvents, an emulsion, a colloid, or a solution.

[0205] The present invention also relates to the use of the injectable composition according to the invention for cosmetic applications. More preferably, the present invention also relates to the use of the injectable composition according to the invention for cosmetic applications including facial and body reshaping and rejuvenation.

[0206] As used herein, cosmetic applications and cosmetic uses can be understood in the broadest sense as any non-therapeutic application intended to improve appearance. They may also be referred to as aesthetic applications and aesthetic uses, respectively. In the context of the present invention, it should be understood that cosmetic applications and cosmetic uses may include any route of administration, which may optionally include injection and / or topical application of individual materials or another suitable route of administration.

[0207] Another aspect of the invention relates to the use of the injectable compositions of the invention in cosmetic applications, including facial and body reshaping and rejuvenation, preferably including wrinkle filling, improvement of facial lines, breast reconstruction or enlargement, skin rejuvenation, buttock augmentation, cheekbone reshaping, soft tissue augmentation, filling of facial wrinkles, improvement of frown lines, improvement of nasolabial folds, improvement of marionette lines, improvement of corners of the mouth, improvement of perilipine wrinkles, improvement of crow's feet, improvement of eyebrows, subcutaneous support of cheek fat pads, improvement of tear troughs, improvement of nose appearance, lip augmentation, cheek augmentation, perioral augmentation, infraorbital augmentation, correction of facial asymmetry, improvement of jawline, chin augmentation, or a combination of two or more thereof.

[0208] It should be understood that the definitions and preferred embodiments shown in the context of the methods, crosslinking materials, hydrogels and injectable compositions of the invention are applicable to the uses of the invention after necessary modifications.

[0209] This use may be for therapeutic and / or cosmetic purposes. In a preferred embodiment, the invention relates to the use of the injectable compositions of the invention for reducing facial wrinkles. In embodiments, the use of the invention may be for cosmetic purposes, preferably non-therapeutic purposes. The invention can be used by beauty professionals, cosmetic specialists, or health care experts.

[0210] In a preferred embodiment, the injectable composition of the present invention is used to improve skin quality, treat fine lines, treat deep wrinkles, or restore volume, or as a super-volumizing filler for breasts or buttocks. The present invention also relates to methods of facial and body reshaping and rejuvenation (preferably including the specific uses described above), the method comprising applying the injectable composition according to the present invention.

[0211] When therapeutic use is involved, the present invention relates to the use of the injectable composition according to the present invention in methods for: facial and body reshaping and rejuvenation, preferably including filling wrinkles, improving facial lines, breast reconstruction or enlargement, skin rejuvenation, buttock augmentation, cheekbone reshaping, soft tissue augmentation, filling facial wrinkles, improving frown lines, improving nasolabial folds, improving marionette lines, improving corners of the mouth, improving perioral wrinkles, improving crow's feet, improving eyebrows, subcutaneous support of the cheek fat pads, improving tear troughs, improving nose appearance, lip augmentation, cheek augmentation, perioral augmentation, infraorbital augmentation, correcting facial asymmetry, improving jawline, chin augmentation, or a combination of two or more of these.

[0212] In other words, the present invention also relates to the method of use for facial and body reshaping and rejuvenation, preferably including filling wrinkles, improving facial lines, breast reconstruction or enlargement, skin rejuvenation, buttock augmentation, cheekbone reshaping, soft tissue augmentation, filling facial wrinkles, improving frown lines, improving nasolabial folds, improving marionette lines, improving corners of the mouth, improving perioral wrinkles, improving crow's feet, improving eyebrows, subcutaneous support of the cheek fat pads, improving tear troughs, improving nose appearance, lip augmentation, cheek augmentation, perioral augmentation, infraorbital augmentation, correcting facial asymmetry, improving jawline, chin augmentation, or a combination of two or more thereof, wherein a sufficient amount of the injectable composition according to the invention is administered to a subject in need.

[0213] As used herein, the subject (also: individual) can be any animal, typically a mammal, preferably a domesticated mammal or a human. The individual is particularly preferred to be a human. The person being treated may also be referred to as a patient, regardless of his / her health condition.

[0214] Administration may be performed in any manner. In a preferred embodiment, administration is via needle. In a preferred embodiment, application is performed via a syringe, particularly via intradermal or subcutaneous injection. Application can be manual, using a mechanical pump, or even automated. For example, the Syringe One system can be used for application. Specification 16 / 21 pages 19 CN 121285400 A

[0215] The crosslinked materials of the present invention and the injectable compositions of the present invention can be used for any purpose. Optionally, the crosslinked materials of the present invention and the injectable compositions of the present invention can be used for cosmetic and / or therapeutic purposes. The injectable compositions can be fillers, particularly soft tissue fillers, such as soft tissue fillers, particularly dermal fillers or connective tissue fillers.

[0216] The present invention also relates to the injectable compositions according to the present invention as fillers, such as soft tissue fillers, particularlyThis refers to the use of dermal fillers or connective tissue fillers. It can be used as a super-capacitor. In this context, it can be used as a hydrogel.

[0217] As used herein, the term "filler" can be understood in the broadest sense as any agent that can be used to fill cavities or as a soft tissue filler, preferably a soft tissue filler. Soft tissue fillers can be understood in the broadest sense as materials designed to increase the volume of areas of soft tissue defects. Fillers can be applied to any location and by any type of injection, and are suitable for cosmetic / aesthetic applications as well as therapeutic purposes. Generally, fillers can be any composition that adds, replaces, or increases subcutaneous volume, resulting in, for example, smoothing skin wrinkles, plumping lips, improving skin appearance, or treating scars. They are generally used in dermal areas, such as below the epidermis or above the subcutaneous layer, and can therefore be injected subcutaneously, hypodermically, or intradermally, or in combinations thereof.

[0218] Injectable compositions within the scope of this invention can be administered by syringe (dispensing from syringe) under normal conditions and pressure. Furthermore, the filler composition of the present invention is preferably (substantially) sterile. The injectable composition is preferably suitable for injection into mammals, particularly humans.

[0219] Remodeling can be performed for cosmetic purposes, or after tissue loss, such as due to an accident or surgical intervention. For example, a portion of the face may be injured due to an accident. On the other hand, the cheekbone area can be made prominent by subcutaneous filling. The breast or a portion thereof can be surgically removed. On the other hand, breast reconstruction or breast augmentation also has aesthetic reasons.

[0220] The injectable composition of the present invention is preferably administered to an individual in an effective amount by injection, such as by subcutaneous or intradermal injection. In a preferred embodiment, in the context of this use, the injectable composition is a filler. In a more preferred embodiment, in the context of this use, the injectable composition is a filler, particularly a super-capacitor, and this use includes compositions comprising the application of the cross-linked material of the present invention to the target tissue, particularly in a subcutaneous or intradermal manner. For example, the injectable composition can be injected intradermally or subcutaneously using a continuous puncture technique. Effective amount means the amount of (injectable) soft tissue filler composition sufficient to cause a beneficial or desired cosmetic (aesthetic) or therapeutic result.

[0221] In a particularly preferred embodiment, in the context of this use, the injectable composition is a filler, which may be a super-capacitor, particularly a soft tissue filler, and the use includes the subcutaneous or intradermal application of a composition comprising the cross-linked material of the present invention. For such uses, the cross-linked material according to the present invention is particularly advantageous because the cross-linked material is more stable in aqueous environments, such as bodily fluids, and can penetrate cells due to its structure and characteristics.

[0222] Another aspect of the present invention relates to the use of the cross-linked material or injectable composition according to the invention in a method for regenerating tissue of an individual in need.

[0223] In other words, the present invention also relates to a method for regenerating tissue of an individual in need, the method comprising applying the cross-linked material or injectable composition according to the invention to the individual in need. Tissue regeneration of the individual in need can be performed for therapeutic and / or cosmetic purposes.

[0224] It should be understood that the definitions and preferred embodiments shown in the context of the cross-linked materials, hydrogels, methods and injectable compositions described above are applicable to the use of individual tissue regeneration after necessary modifications.

[0225] The tissue to be regenerated can be any tissue. In a preferred embodiment, the tissue is soft tissue. In a more preferred embodiment, the tissue is soft tissue selected from the group consisting of dermal tissue (including dermal and subcutaneous tissue) and connective tissue. The method can then be used for reshaping and rejuvenation, including the uses described above. In another preferred embodiment of the invention, the tissue is joint tissue. Optionally, for this purpose, the crosslinking material may contain one or more cell proliferation factors that stimulate the proliferation of individual tissues.

[0226] In an alternative preferred embodiment, the tissue is bone tissue. The crosslinking material of the present invention can then be applied to the location of the bone tissue to which growth is desired, such as in a fracture gap or for bone elongation. Optionally, for this purpose, the crosslinking material may contain one or more cell proliferation factors that stimulate the proliferation of osteocytes.

[0227] Depending on the particular use, those skilled in the art will use the microparticle crosslinking material according to the present invention or will use the crosslinking material block according to the present invention.

[0228] The crosslinking material according to the present invention is particularly advantageous for the above-described therapeutic and cosmetic uses because the crosslinking material is more stable in aqueous environments, such as bodily fluids, and is able to penetrate cells due to its crosslinked structure and surface characteristics. As mentioned above, the crosslinking material of the present invention is obtained by conjugating the polysaccharide portion of the present invention. This conjugation itself also has unexpectedly beneficial properties.

[0229] As used herein, the terms “about” and “approximately” may be understood to include a range of up to + / - 10% deviation from individual values. It should be understood that specific values ​​have also been explicitly disclosed.

[0230] It should be further understood that the range covers the numerical values ​​provided by the general rounding value across the entire rounding limit. For example, the range of “1 mg” covers a range from 0.50 to 1.49 mg.

[0231] However, the numerical values ​​of the present invention also disclose more detailed values ​​of one or more orders of magnitude. Thus, for example, “1 mg” may also include the specific disclosed value of “1.0 mg”. Brief Description of the Drawings

[0232] Figure 1 shows an example of extrusion pressure measurement of a hyaluronic acid (HA) product crosslinked with 1.5 eq DMTMM. Measurements were performed using a TSK 27G.The 1 / 2” needle was used at 12.6 mm / min. The graph shows the overlay of multiple measurements. It can be seen that the measurement results are comparable and no significant inhomogeneity was found.

[0233] Figure 2 shows the degradation kinetics of the respective hyaluronic acid (HA) based crosslinking materials, which are crosslinked with different amounts of DMTMM. Degradation was carried out by contacting the corresponding crosslinking material with 150 units of hyaluronidase and incubating at 37°C. The upper graph shows HA obtained from the first supplier. The lower graph shows HA obtained from another supplier. Downward triangles indicate uncrosslinked HA. Squares indicate HA crosslinked with 0.5 equivalents (eq.) of DMTMM. Circles indicate HA crosslinked with 1.0 equivalents (eq.) of DMTMM. Upward triangles indicate HA crosslinked with 1.5 equivalents (eq.) of DMTMM. Detailed Description

[0234] Examples

[0235] Example 1

[0236] Method

[0237] Preparation of crosslinked hyaluronic acid materials according to the present invention

[0238] Dissolve 1.5 g of sodium hyaluronate (HA) (dry weight) with an intrinsic viscosity of 2.85 m³ / kg in 45 mL of phosphate-buffered saline (PBS) buffer at pH 7.4. Allow the resulting mixture to pre-swell for approximately 15 hours. Note that this is an optional step and can be omitted.

[0239] Dissolve different amounts (e.g., approximately 0, 0.2, 0.5, 1.0, 1.5, or 2.0 molar equivalents (eq.) of the triazine activator 4-(4,6-dimethoxy-1,35-triazin-2-yl)-4-methylmorpholine chloride (DMTMM)) in 5 mL of PBS buffer at pH 7.4. Use 1.03 g of DMTMM to obtain approximately 1 eq (relative to the carboxyl group of HA).

[0240] 45 mL of PBS buffer containing HA and 5 mL of PBS buffer containing DMTMM were mixed together. This yielded an HA concentration of 30 mg / mL. Mixing was continued until a visually homogeneous composition was obtained. This was typically achieved after about 10 min.

[0241] In the experiments described herein, the samples were held at 40°C for 3 hours. However, it was noted that the reaction proceeded well at other temperatures, such as ambient temperatures of about 20°C–22°C. Further observation showed that shorter reaction times also produced viable crosslinked materials.

[0242] Subsequently, the crosslinked material (in hydrogel form) was homogenized again for 30 min and sieved. It was noted that this was an optional step and could be omitted.

[0243] Subsequently, the crosslinked material (in hydrogel form) was placed on a dialysis membrane (molecular weight cutoff (MWCO) 12–14 kDa).The membrane was purified for approximately 2 days to remove DMTMM byproducts. The swelling ratio was controlled by the weight of the membrane, and dialysis was stopped once the product showed the desired concentration of crosslinking material. Note that this is an optional step and can be omitted.

[0244] Optionally, a local anesthetic, such as lidocaine, was added to the crosslinking material (in hydrogel form). Optionally, the crosslinking material (in hydrogel form) was sterilized and optionally filled into a syringe.

[0245] Determination of Properties

[0246] Rheology (including intrinsic viscosity) was determined using a 302 modular simplified rheometer (AntonPaar GmbH, Graz, Austria). Briefly, plates / plates with a 1.0 mm gap size were used in oscillating mode (PP20) at 25°C; Tau (stress) was set to 10 Pa, and the oscillation frequency range was 10 to 0.1 Hz. The reported G' value corresponds to 1 Hz.

[0247] Extrusion pressure was measured using a TA.XT. (Stable Micro Systems) Plus tissue analyzer. In short, the syringe was squeezed at a constant speed (12.6 mm / min), and the squeezing force (in N) was measured over time. A 1 / 2-inch 27G TSK (TSK Laboratory, Japan) needle was used.

[0248] Enzymatic degradation: Material degradation was assessed at 36°C via an Anton Paar MCR 302 rheometer and a CP50-1 plate in oscillating mode. Measurements were recorded every minute for 1 hour. The contents of a syringe (1 mL) were deposited onto the measuring plate, and 150 μL of 150 U (active units) of hyaluronidase solution was mixed in the gel for 30 seconds. The G' value at 1 Hz was converted to degradation % and reported.

[0249] Molecular weight and distribution were determined by gel permeation chromatography (GPC) calibrated with pullulan standards using a sample at a concentration of 5 mg / mL in PBS buffer (pH 7.4).

[0250] Results

[0251] It was found that crosslinked materials with beneficial properties could be obtained at various different content ratios. The results of the small-scale experiments are summarized in Table 1 below. The rheological parameters are consistent with other polysaccharide-based hydrogels (e.g., HA fillers), and the G' value decreases by approximately 50% after sterilization. Representative extrusion curves of gels crosslinked with 1.5 eq activator (DMTMM) are shown in Figure 1. Unbound by this theory, the crosslinked materials appear to exhibit a shear-thinning effect due to low extrusion pressure.

[0252] Further investigation was conducted on the enzymatic degradation of different products. The results are depicted in Figure 2. It can be seen that a wide range of polysaccharide fractions and activators can be used.

[0253] It was found that crosslinked materials using less activator (DMTMM) equivalents exhibited a lower degree of crosslinking.The degree of cross-linking material can be completely degraded by enzymes after one hour, while the cross-linking material with a higher degree of cross-linking is more stable. This shows that the method of the present invention is very suitable for purposefully adjusting the degree of cross-linking to achieve a desired purpose.

[0254] Example 2

[0255] Preparation of cross-linked chondroitin sulfate material according to the present invention

[0256] In the same manner as in Example 1, 2.0 g of chondroitin sulfate (CS) with a weight average molecular weight of 100 kDa was dissolved in PBS buffer (pH 7.4). Different amounts (approximately 0.0, 0.5, 1.0 or 1.5 molar equivalents (eq.) relative to the carboxyl group of CS) of DMTMM were dissolved in PBS buffer at pH 7.4 and mixed with PBS buffer containing CS until a visually homogeneous composition was obtained, and the mixture was reacted at 23°C for 17 h. Subsequently, the cross-linked hydrogel was purified by dialysis using PBS buffer. 1.15 g DMTMM was used to obtain approximately 1 eq (carboxyl group relative to CS). Rheological properties were determined as described in Example 1 above and are shown in Table 2.

[0257] Table 1. Summary of results using two different batches of hyaluronic acid (HA) (30 mg / mL per batch) and different molecular equivalents (eq.) of the activator DMTMM. The results show the rheological values ​​of sterilized and non-sterilized products.

[0258] Specification 20 / 21 pages 23 CN 121285400 A

[0259] Specification 21 / 21 pages 24 CN 121285400 A Figure 1 Specification Figure 1 / 2 pages 25 CN 121285400 A Figure 2 Specification Figure 2 / 2 pages 26 CN 121285400 A

Claims

1. A method of preparing a crosslinked material, the method comprising: (i) contacting with each other: (A) polysaccharide moieties comprising carboxylic acid residues or salts thereof and hydroxyl residues, (B) one or more triazine-based activating agents that react carboxylic acid residues with hydroxyl residues to form ester bonds, and (C) one or more solvents; and (ii) reacting at least some of the carboxylic acid residues of the polysaccharide moieties with at least some of the hydroxyl residues of the polysaccharide moieties to form ester bonds covalently crosslinking the polysaccharide moieties to each other; and (iii) optionally purifying the crosslinked material obtained from step (ii), wherein the ester bonds covalently crosslinking the polysaccharide moieties to each other are more numerous than amide bonds covalently crosslinking the polysaccharide moieties to each other, wherein step (ii) is performed in the absence of any activating agents other than triazine-based activating agents, in particular wherein step (ii) is performed in the absence of carbodiimide activating agents, succinimidyl-based activating agents, glycidoxypropyl-based activating agents, p-nitrophenol esters, and 2-chloromethylpyridine iodide.

2. The method of claim 1, wherein the polysaccharide moieties comprise a molar ratio of hydroxyl residues: primary amino groups of at least 25: 1, preferably at least 50: 1, in particular at least 100: 1, preferably wherein the polysaccharide moieties comprise or consist of hyaluronic acid, glycerol-grafted hyaluronic acid, heparan sulfate, chondroitin sulfate, and carboxymethylcellulose.

3. The method of any one of claims 1 or 2, wherein the polysaccharide moieties comprise or consist of one or more hyaluronic acid moieties.

4. The method of any one of claims 1 to 3, wherein the polysaccharide moieties have a weight average molecular weight of at least 50 kDa, preferably at least 200 kDa, in particular in the range of 500 kDa to 4000 kDa.

5. The method of any one of claims 1 to 4, wherein the one or more activating agents are selected from the group consisting of 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4-methylmorpholinium or a salt thereof, and / or 2-chloro-4,6,-dimethoxy-l,3,5-triazine or a salt thereof, and combinations thereof.

6. The method of any one of claims 1 to 5, wherein the activating agent is 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4-methylmorpholinium or a salt thereof, in particular 4-(4,6-dimethoxy-l,3,5-triazin-2-yl)-4-methylmorpholinium chloride.

7. The method of any one of claims 1 to 6, wherein the solvent is an aqueous solution, in particular an aqueous buffer, preferably an aqueous buffer having a pH in the range of 5 to 9, in particular in the range of 6 to 8.

8. The method of any one of claims 1 to 7, wherein the method is further characterized in that it does not comprise at least one of: (a) using an interconnecting linker moiety interposed between the carboxylic acid residues or salts thereof and the hydroxyl residues of the polysaccharide moieties, in particular without a peptide linker moiety or a heterobiomass linker moiety; (b) using a carbodiimide-based activator; (c) using a reactive group selected from the group consisting of glycidyl ether, maleimide, anhydride, alkoxide, and combinations of two or more thereof; (d) a polysaccharide moiety having a primary amino group, a thiol group, an imide group, an imine group, or an epoxy group.

9. The method of any one of claims 1 to 8, wherein the method is further characterized by: (a) it comprises step (iii) of purifying the crosslinked material by filtration, washing, and / or dialysis, in particular by cross-flow filtration, diafiltration, and / or dead-end filtration; (b) steps (i) and (ii) are performed in a single batch; (c) steps (i) and (ii) and, optionally, step (iii) are performed at a temperature in the range of 5 °C to 90 °C, preferably 18 °C to 60 °C, in particular 20 °C to 50 °C; and / or (d) step (ii) is performed for at least 10 min, preferably 15 min to 48 h, in particular 30 min to 18 h.

10. The method of any one of claims 1 to 9, wherein the method comprises: (i) bringing into contact with each other: (A) polysaccharide moieties having a weight average molecular weight of at least 50 kDa, which comprise carboxylic acid residues or salts thereof and hydroxyl residues; (B) one or more triazine-based activators that react carboxylic acid residues with hydroxyl residues to form ester bonds, in particular wherein the activator is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium or a salt thereof; and (C) an aqueous solvent, preferably an aqueous solvent having a pH in the range of 5 to 9, in particular in the range of 6 to 8, and (ii) reacting at least some of the carboxylic acid residues with at least some of the hydroxyl residues to form ester bonds that covalently crosslink the polysaccharide moieties to each other; and (iii) optionally purifying the crosslinked material obtained from step (ii).

11. A crosslinked material obtainable from the method of any one of claims 1 to 10.

12. A crosslinked material comprising or consisting of one or more hyaluronic acid moieties having a weight average molecular weight of at least 50 kDa, wherein the hyaluronic acid moieties are covalently crosslinked to each other via ester bonds without an interconnecting linker structure, the crosslinked material is further characterized in that the crosslinked material does not comprise a diimide group, an epoxy group, or an isobiotin linker moiety, preferably wherein the crosslinked material comprises: (a) 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium or a salt thereof, (b) N-methylmorpholinium or a salt thereof; and / or (c) 4,6-dimethoxy-1,3,5-triazin-2-ol or a tautomer or salt thereof, and preferably wherein the crosslinked material is obtained from the method of any one of claims 1 to 10.

13. A hydrogel comprising: (A) the crosslinked material of any one of claims 11 or 12; and (B) an aqueous solution; and (C) optionally one or more additional pharmaceutically or cosmetically acceptable agents, in particular one or more local anesthetics.

14. The hydrogel of claim 13, wherein the hydrogel is a dermal filler.

14. An injectable composition comprising the crosslinked material of any one of claims 11 to 12 or the hydrogel of claim 13, wherein the crosslinked material is a super- viscosifier.

15. Use of an injectable composition according to claim 14 for cosmetic applications, wherein the cosmetic applications comprise facial and body remodelling and rejuvenation, preferably comprising filling of wrinkles, improving facial lines, breast reconstruction or augmentation, skin rejuvenation, buttock augmentation, malar remodelling, soft tissue augmentation, filling of facial wrinkles, improving glabellar lines, improving marionette lines, improving puppet lines, improving perioral lines, improving crow's feet, improving eyebrows, subcutaneous support of malar cheek fat pads, improving tear troughs, improving nasal appearance, lip augmentation, cheek augmentation, perioral area augmentation, infraorbital area augmentation, addressing facial asymmetry, improving jawline contour, chin augmentation, or a combination of two or more thereof.

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