Self-crosslinked hydrogel combination with botulinum neurotoxin

Abstract

The present invention relates to a method for preparing a composition comprising (A) a cross-linked material comprising one or more polysaccharide moieties covalently cross-linked with each other through ester bonds using one or more triazine-based activating agents, and (B) at least one botulinum toxin. Furthermore, the present invention relates to the composition obtainable by said method, and therapeutic and aesthetic uses of such composition.

Description

[0001] Merz Pharma GmbH & Co. KGaA 4 November 2025

[0002] MP75182PC

[0003] Self-crosslinked hydrogel combination with Botulinum neurotoxin

[0004] The present invention relates to a method for preparing a composition comprising (A) a cross-linked material comprising one or more polysaccharide moieties covalently cross-linked with each other through ester bonds using one or more triazine-based activating agents, and (B) at least one botulinum toxin. Furthermore, the present invention relates to the composition obtainable by said method, and therapeutic and aesthetic uses of such composition.

[0005] Since the 1980s, botulinum toxin (also: botulinum neurotoxin, BoNT) is used for treating or preventing motoric disorders, such as, e.g., dystonia or spasticity, and / or wrinkles. It is used in pharmaceutical and aesthetic contexts. Botulinum toxin inhibits acetylcholine release, which leads to lower muscle contraction or even muscle paralysis. Botulinum toxin is known in the context of reduction and prevention of wrinkles that are due to muscle contraction. Furthermore, botulinum toxin can improve skin quality, improve skin laxity, and may have a lifting effect as summarized by Goldie et al. (Clinical, Cosmetic and Investigational Dermatology, 2021 , 14:643-654). One commercial product based on botulinum toxin is Xeomin® / Bocouture® (Merz Pharmaceuticals GmbH, Germany).

[0006] Botulinum toxin is, however, inherently instable, in particular under alkaline conditions. It is further heat-labile. For this reason, commercial botulinum toxin products are often stored as vacuum-dried (lyophilized) material and / or are combined with protecting and preserving excipients.

[0007] Several uses based on the injection of botulinum toxin in or under a subject’s skin or other soft tissue directly to the site of intended effect are known. In typical longterm therapeutic or aesthetic uses, botulinum toxin is administered again after a certain period of time, often every few months, due to the reversibility of its inhibitory effect of acetylcholine release.

[0008] As botulinum toxin directly affects the cellular endocytosis and exocytosis mechanism by inactivating the SNARE proteins, there is only a limited time period for botulinum toxin uptake of approximately one day before further uptake is inhibited by blocking of the endocytosis pathways. This is undesirable in many applications. Thus, there is a need to extend the duration of effect of a botulinum toxin injection, and to provide retarded and / or controlled release formulations, which are able to adjust the retention and the release profile of the botulinum toxin to the uptake capacity of the target cell. This is partly achieved by preparing retarded and / or controlled release formulations, where botulinum toxin is embedded in a pharmaceutically acceptable matrix, which can provide an adapted and optionally extended duration of effect. For this purpose, biocompatible compositions were considered, see e.g. US 9,044,477 and US 9,050,336, which teach mixtures comprising chemical entities including hyaluronic acid, botulinum toxin, and albumin. Herein, viscous materials are formed by adaptable concentrations of hyaluronic acid, where the hyaluronic acid may also be cross-linked hyaluronic acid.

[0009] WO 2020 / 056371 teaches mixtures of clostridial toxin and non-crosslinked hyaluronic acid or salt thereof. The components hyaluronic acid, botulinum toxin, and albumin are simply admixed in the compositions. WO 2011 / 119468 teaches a hydrogel for soft tissue augmentation comprising a polymer that can be hyaluronic acid, which can further comprise other ingredients. WO 2017 / 148915 teaches compositions comprising non-crosslinked hyaluronic and botulinum toxin. Some of these compositions are well compatible to be injected and generally usable for soft tissue augmentation.

[0010] However, stability, including shelf life and retarded and / or controlled release of botulinum toxin, is not as desired by users. In particular, the aforementioned compositions of the prior art can hardly be adapted to the desired release kinetics. This can be adapted merely roughly by the concentration and chain length of the hydrogel-forming component.

[0011] Attempts to obtain desirable shelf stability and improve adjustability of release kinetics by cross-linking components via chemical cross-linking structures have been described. WO 2020 / 132331 describes conjugates of fibroin moieties, preferably cross-linked hyaluronic acid moieties and cross-linking moieties such as polyethylene glycol (PEG) and / or polypropylene glycol (PPG), usable as tissue fillers.

[0012] Further, this document teaches that a large variety of different other ingredients can be comprised in a tissue filler. Such conjugates have the disadvantage that synthetic xenobiotic moieties are comprised in the structure such as PEG and / or PPG linkers. How to cross-link polysaccharides such as hyaluronic acid with synthetic xenobiotic cross-linking agents, such as, for example, the epoxide-based linker butanediol diglycidyl ether (BDDE) are described in US-A 2012 / 0190644, WO 2015 / 149941 and WO 2020 / 030629, or di- or multinucleophilic functional cross-linkers are described in US-A 2020 / 0140626. Such materials have several drawbacks.

[0013] For instance, residuals of such non-reacted or half-reacted bivalent linkers can be harmful and limit usability of the materials. There are maximally administrable contents of such reactive linkers and, thus, also for safety reasons also for filler materials prepared by using such components. When administered to a subject in need thereof and degraded in said subject, xenobiotic and non-degradable or poorly degradable metabolites can be generated. Xenobiotic structures are often not fully biodegradable and / or bio-resorbable. Such moieties are often undesired in materials that are injected into a subject’s body.

[0014] WO 2024 / 126649 teaches compositions comprising botulinum toxin and conjugates of hyaluronic acid with fibroin. Other conjugates of hyaluronic acid and proteins, peptides and amino acids are also known. For instance, hyaluronic acid cross-linked with elastin was described in WO 2011 / 119468. Furthermore, polysaccharides were cross-linked with fibroin, as described in WO 2022 / 268871 . CN-A 105713211 describes the linkage of hyaluronate with high contents of the amino acid lysine containing two amino groups.

[0015] Such methods have also the drawback that the presence of proteins, peptides or amino acids is not always desirable. The process is rather complex as it contains several components and, depending on the protein used, amide bonds within the protein sequence may be cleaved. In some cases, the proteins and peptides may bear undesired immunogenic responses.

[0016] Thus, it is desirable to cross-link polysaccharide such as hyaluronic acid without such interconnecting proteins, peptides and amino acids.

[0017] In this context, it was studied to cross-link polysaccharides via amide bonds wherein the polysaccharides bear both, carboxylic acid residues and amino groups, which react with each other. For this purpose, polysaccharides, such as glycosaminoglycans are modified to contain free amine groups which are conjugated to free carboxylic groups, see WO 2019 / 002369. Such processes, however, need an additional step of providing non-natural intermediate products such as provision of amino groups. Further, such obtained conjugates, undesirably contain amide bonds.

[0018] It is desirable to directly conjugate natural polysaccharides via ester bonds.

[0019] In the prior art, there is an apparent prejudice that the formation of ester bonds between polysaccharides requires harsh activating agents such as carbodiimide activating agents (comprising the structural motif -N=C=N-) or 2-chloro- methylpyridinium iodide.

[0020] For example, EP-A 0341745 teaches that esterification can be achieved by means of 2-chloro-methylpyridinium iodide. JP-A 2019019201 teaches the use of carbodiimide activating agents such as N,N’-dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), and 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). These reactive agents are undesirably irritant or even toxic and may bear further disadvantages.

[0021] When, on the other hand, such activating agents are omitted, esterification does either nor occur or is extremely slow and ineffective, even at harsh pH ranges as shown in JP-A 2003 / 252905.

[0022] In view of the above, it is still an unmet need to provide compositions that have a well-controlled and adjustable release of botulinum toxin while employing a crosslinked material prepared by a method associated with only low procedural efforts, that avoids additional (e.g. xenobiotic) linker moieties introduced into the hydrogel and minimizes the residuals of toxic reagents. It is further desired to obtain crosslinking of polysaccharides through ester bonds rather than amide bonds, while avoiding the use of irritant and toxic compounds.

[0023] The main components of such compositions preferably should be widely biodegradable and / or bio-resorbable.

[0024] The resources of solid main components used for preparation of such compositions should preferably be bio-based, thus of biological or biotechnological origin, while optionally synthetic agents, such as one or more anesthetics can be comprised.

[0025] Particularly desirable is an injectable composition usable for treating or preventing a disease or condition associated with hyperactive cholinergic activity, in particular of muscles and / or glands, pain, or for rejuvenation and / or improvement of the skin quality of the face and / or body. Surprisingly, it has been found that a composition comprising, or consisting of, a cross-linked material, comprising one or more polysaccharide moieties, in particular hyaluronic acid moieties, which are covalently conjugated with each other without an interconnecting linker structure, can be used in combination with botulinum toxin to provide a composition having desired properties.

[0026] Such composition can be obtained from a method comprising the reaction of at least some carboxylic acid residues of polysaccharide moieties with at least some hydroxy residues of polysaccharide moieties to form ester bonds cross-linking polysaccharide moieties covalently with each other, wherein the reaction is triggered by one or more triazine-based activating agents.

[0027] Such composition is injectable and usable for treating or preventing a disease or condition associated with hyperactive cholinergic activity, in particular of muscles and / or glands, pain, or for rejuvenation and / or improvement of the skin quality of the face and / or body. Such composition can serve as a biodegradable and bioresorbable retarded and / or controlled release formulation for botulinum toxin.

[0028] One first aspect of the invention relates to a method for preparing a composition comprising (or consisting of):

[0029] (A) a cross-linked material, comprising (or consisting of) one or more polysaccharide moieties covalently cross-linked with each other through ester bonds, wherein the cross-linked material comprises more ester bonds cross-linking polysaccharide moieties covalently with each other than amide bonds cross-linking polysaccharide moieties covalently with each other;

[0030] (B) at least one botulinum toxin;

[0031] (C) optionally one or more liquid cosmetically and / or pharmaceutically acceptable carriers;

[0032] (D) optionally one or more anesthetics; and

[0033] (E) optionally one or more further cosmetically and / or pharmaceutically acceptable ingredients different from components (A) to (D), wherein the method comprises (or consists of) the steps:

[0034] (i) combining the following components with each other: a) polysaccharide moieties comprising carboxylic acid residues or salts thereof and hydroxy residues, b) one or more triazine-based activating agents that effect reaction of carboxylic acid residues with hydroxy residues thereby forming ester bonds, and c) one or more solvents; and

[0035] (ii) allowing at least some of the carboxylic acid residues of the polysaccharide moieties to react with at least some of the hydroxy residues of the polysaccharide moieties, thereby forming ester bonds covalently crosslinking polysaccharide moieties with each other; and

[0036] (iii) obtaining cross-linked material from step (ii) as component (A) and optionally purifying the cross-linked material obtained from step (ii); and

[0037] (iv) adding: at least one botulinum toxin as component (B), and optionally one or more liquid cosmetically and / or pharmaceutically acceptable carriers as component (C), and / or optionally one or more anesthetics as component (D), and / or optionally one or more further cosmetically and / or pharmaceutically acceptable ingredients different from components (A) to (D) as component (E); with the proviso that step (ii) is conducted in the absence of carbodiimide activating agents, succinimidyl-based activating agents, glycidyl-based activating agents, paranitrophenol esters, and 2-chloro-methylpyridinium iodide.

[0038] A second aspect of the invention is a composition obtainable (or obtained) by the method of the invention. Yet another aspect is the use of a cross-linked material as obtained in step (iii) of the method for retarded and / or controlled release of one or more pharmaceutically and / or cosmetically active agents, such as botulinum toxin. Yet another aspect is the composition of the invention for use as a medicament, e.g., for treating or preventing a disease or condition associated with hyperactive cholinergic activity and / or pain. Yet another aspect is the use of the composition for cosmetic applications.

[0039] It has been found that such composition bears unexpectedly beneficial properties. The composition of the present invention may have a long shelf life and storability due to the avoidance of reactive groups. It is also thermally comparably stable.

[0040] Botulinum toxin may be stabilized in the cross-linked material and its retarded and / or controlled release can be adjusted well to the intended purposes. Optionally, a delayed release / depot function may be achieved. Further, in vivo longevity of the cross-linked material may be extended when injected into a subject’s tissue, which may be caused by limited muscle activity. Optionally, adverse events such as, e.g., eye ptosis that might be caused by botulinum toxin administration may be reduced due to the reduced spread. Reducing the spread upon injection may reduce the unintended distribution of an injected botulinum toxin composition in the surrounding tissue. This may reduce side effects and may reduce unintended dilution and thereby extend duration of the effect. A reduced diffusion of botulinum toxin may lead to a controlled (optionally slower) release / distribution and optimized cellular uptake of botulinum toxin. Lower content (e.g., less (enzymatic) units thereof) of botulinum toxin may be needed to achieve a comparable effect. Higher contents (e.g., more (enzymatic) units thereof) of botulinum toxin may have a longer duration of effect, but still bear an acceptable adverse effect profile.

[0041] It has been found that the cross-linked material used in the present invention bears unexpectedly beneficial properties. Viscosity may be widely controlled or maintained, and is not substantially affected by the presence of botulinum toxin.

[0042] Likewise, also enzymatic degradability may be desirably diminished. It was surprisingly found that xenobiotic linker structures as commonly used in the prior art can be avoided. The cross-linked material used in the present invention may essentially consist of polysaccharide moieties, which can also be found in nature.

[0043] The cross-linked material used in the present invention may also be designated as “cross-linked polysaccharide”, “self-cross-linked material” or “self-cross-linked polysaccharide”. The composition obtainable from the method of the present invention may have a long-lasting stability. In aqueous environment, the obtained composition may form a hydrogel.

[0044] The composition of the present invention may optionally also be used to mimic an extracellular matrix and may, thus, induce cell proliferation and / or cellular migration in addition to the effect achieved by botulinum toxin such as decreasing muscle contraction or inducing muscle paralysis.

[0045] The composition of the present invention may have good shear-thinning properties. Preferably, it may have thixotropic properties. Thus, it may be less viscous when stressed. It may be injected very well, while still being rather viscous in its target area (when e.g., injected intramuscularly). Comparably low extrusion forces are required. Gels of high viscosity and low extrusion force are obtainable.

[0046] The composition of the present invention may be used for any purpose. Preferably, the composition of the present invention may be injectable. For instance, it may be used for various aesthetic and therapeutic purposes, which are exemplified below. In a preferred embodiment, the composition is injectable into a soft tissue, more preferably injectable intradermally, subdermally, subcutaneously, and / or intramuscularly. Particularly preferably, the composition is used for intramuscular injection.

[0047] A soft tissue may be any soft tissue. In a preferred embodiment, the soft tissue is selected from the group consisting of dermal tissue (including tissue of the dermis and the subcutis) and connective tissue, a muscle, or an articulation (joint) tissue. Preferably, a soft tissue is muscle tissue.

[0048] The process of the present invention can be used without undue burden and with comparably low technical efforts.

[0049] The claimed method is particularly beneficial compared to procedures described in the prior art because the preparation of the cross-linked material requires only two educts (raw / starting materials) in addition to a solvent, i.e. , polysaccharide moieties and one or more triazine-based activating agents. Further components such as linkers are not required.

[0050] The omittance of xenobiotic linker structures such as, e.g., BDDE, may enable higher amounts of material administered to a subject, e.g., injected intramuscularly. Furthermore, it may have longevity in a subject’s body after administration.

[0051] The obtainable cross-linked material of the present invention may have a long shelf life and storability due to the avoidance of reactive groups. It is also thermally comparably stable.

[0052] As used herein, ester bonds may be understood in the broadest sense as generally understood in the art. Typically, an ester bond has the structure -O-CO- or -CO-O-, including tautomeric structures thereof. In contrast, an amide bond typically has the structure -NH-CO- or -CO-NH- or a tautomeric structure thereof.

[0053] As indicated above, the polysaccharide moieties are preferably predominantly linked with each other via ester bonds. In other words, more than 50% by mole, more than 75% by mole, or even more than 80% by mole, based on all bonds between polysaccharide moieties (interconnecting groups between polysaccharide moieties) may preferably be ester bonds. In a preferred embodiment, more ester bonds than amide bonds are formed in the cross-linked material. In a preferred embodiment, the cross-linked material comprises a molar ratio of ester groups : amide groups of at least 25 : 1 , preferably at least 50 : 1 , in particular at least 100 : 1. In a preferred embodiment, the crosslinked material bears (essentially) no amine groups. Accordingly, the cross-linked material may comprise e.g., from 0 to 0.04%, preferably from 0 to 0.02%, more preferably from 0 to 0.01 %, such as 0%, based on the total number of ester bonds and amide bonds, of amide bonds.

[0054] As used herein, the terms “residue” and “group” may be understood interchangeably. The term “moiety” in the context of the present invention may be understood in the broadest sense as any molecular structure of the specified type. A moiety may be either a compound comprising or consisting of the respective structure or may form part of a larger chemical entity such as, e.g., the cross-linked material of the composition of the present invention. For example, “polysaccharide moiety” may be understood as any molecular structure that can be considered a polysaccharide or that comprises within its structure a polysaccharide structure.

[0055] As used herein, the terms “carboxylic acid residue” and “carboxy residue” may be understood interchangeably as generally understood in the art, i.e., as residue -COOH or the salt -COO’ thereof.

[0056] In the polysaccharide moieties used as educts of the method of the present invention, there are preferably more hydroxy residues (-OH) than primary amine groups (-NH2, including salts thereof, i.e., -NH3+(and counterions)).

[0057] In a preferred embodiment, the polysaccharide moieties comprise a molar ratio of hydroxy residues : primary amine groups of at least 25 : 1 , preferably at least 50 : 1 , in particular at least 100 : 1 . In a preferred embodiment, the polysaccharide moieties bears (essentially) no amine groups. Accordingly, the cross-linked material may comprise e.g., from 0 to 0.04%, preferably from 0 to 0.02%, more preferably from 0 to 0.01 %, such as 0%, based on the total number of hydroxy residues and amine residues, of amine residues.

[0058] As used in the context of the present invention, the term “polysaccharide moiety” may be understood the broadest sense as any moiety of a polysaccharide known in the art which comprises carboxylic acid residues or salts thereof and hydroxy residues. As used in the context of the present invention, the term “polysaccharide” may be understood in the broadest sense as any polysaccharide in the art. According to the present invention, at least one polysaccharide moiety comprises at least one carboxylic acid residue or salt thereof. The polysaccharide moieties further comprise hydroxy residues. A polysaccharide may be a naturally occurring polysaccharide that may be modified or may be a synthetic polysaccharide. In this context, a polysaccharide may be branched or unbranched. It will be understood that the term “polysaccharide moiety” may also include salts and modified forms thereof. In a preferred embodiment, the polysaccharide has not been oxidized.

[0059] Preferably, polysaccharide moieties are polymeric moieties of a weight average molecular weight (Mw) of at least 1 kDa (1000 Da), more preferably at least 5 kDa, even more preferably at least 10 kDa. In a preferred embodiment, 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 to 4000 kDa. In a preferred embodiment, the polysaccharide moieties have a weight average molecular weight (Mw) in the range of from 10 to 10000 kDa, more preferably in the range of from 25 to 7500 kDa, in particular in the range of 50 to 4000 kDa. Molecular weight values and distributions may be determined, e.g., by Gel Permeation Chromatography (GPC) calibrated to pullulan standards, using samples with a concentration of 5 mg / mL in PBS buffer (pH 7.4).

[0060] The polysaccharide moieties may have any intrinsic viscosity (e.g., determined by a rheometer such as, e.g., described in the experimental section below). In a preferred embodiment, the polysaccharide moieties may have an intrinsic viscosity of 1 to 4 m3 / kg (20°C, 1013 hPa, water). In a preferred embodiment, the polysaccharide moieties may have an intrinsic viscosity of 1 .0 to 3.3 m3 / kg (20°C, 1013 hPa, water). In a preferred embodiment, the polysaccharide moieties may have an intrinsic viscosity of 1 .1 to 3.2 m3 / kg, of 2.0 to 3.1 m3 / kg, or of 2.5 to 3.0 m3 / kg (each at 20°C, 1013 hPa, water).

[0061] In a preferred embodiment, the polysaccharide moieties have a weight average molecular weight of 1500 to 3500 kDa (1.5 and 3.5 MDa). More preferably, it may have a weight average molecular weight in the range of from 100 and 5000 kDa, of from 200 to 2000 kDa, of from 250 to 1500 kDa, of from 300 to 1000 kDa, of from 400 to 900 kDa or of from 500 to 900 kDa.

[0062] In a preferred embodiment, the polysaccharide moieties comprise (or consist of) one or more types of sugar acid moieties or salts thereof. In a preferred embodiment, the polysaccharide moieties comprise or consist of one or more types of sugar acid moieties or salts thereof, wherein the one or more types of sugar acid moieties are selected from the group consisting of:

[0063] (B1 ) one or more uronic acid moieties, in particular selected from the group consisting of glucuronic acid moiety, galacturonic acid moiety, iduronic acid moiety, and combinations of two or more thereof;

[0064] (B2) one or more aldonic acid moieties, in particular selected from the group consisting of glyceric acid moiety, xylonic acid moiety, gluconic acid moiety, ascorbic acid moiety, and combinations of two or more thereof;

[0065] (B3) one or more ulosonic acid moieties, in particular selected from the group consisting of neuraminic acid moiety, ketodeoxyoctulosonic acid moiety, and combinations thereof; and / or

[0066] (B4) one or more aldaric acid moieties, in particular selected from the group consisting of tartaric acid moiety, meso-galactaric acid moiety, glucaric acid moiety, and combinations of two or more thereof.

[0067] In a preferred embodiment, the polysaccharide moieties comprise uronic acid moieties. In a preferred embodiment, the polysaccharide moieties comprise glucuronic acid moieties. In a preferred embodiment, the polysaccharide moieties comprise D-glucuronic acid moieties.

[0068] In a preferred embodiment, the polysaccharide moieties comprise or consist of D- sugar moieties. In an alternative embodiment, the polysaccharide moieties comprise or consist of L-sugar moieties. In an alternative embodiment, the polysaccharide moieties comprise or consist of a combination of D-sugar moieties and L-sugar moieties. For instance, in such combination, racemic mixtures of sugar moieties may be comprised or specific sugar moieties are D-sugar moieties and others are L- sugar moieties. In a preferred embodiment, the polysaccharide moieties comprise or consist of one or more glycosaminoglycan moieties.

[0069] In a preferred embodiment, the polysaccharide moieties are selected from the group consisting of hyaluronic acid (HA) moieties, heparosan moieties, heparin, chondroitin sulphate, and mixtures of two or more thereof. In a preferred embodiment, the polysaccharide moieties comprise or consist of hyaluronic acid, glycerol-grafted hyaluronic acid, heparosan, chondroitin sulfate, and carboxymethyl cellulose. Such polysaccharides comprising carboxylic acid groups are also commercially available. In a preferred embodiment, the polysaccharide moieties comprise or consist of hyaluronic acid, glycerol-grafted hyaluronic acid, heparosan, chondroitin sulfate, and carboxymethyl cellulose. It will be understood that these may also comprise salts thereof.

[0070] In a preferred embodiment, the polysaccharide moieties comprise or consist of one or more hyaluronic acid moieties. It will be understood that this may also comprise salts thereof.

[0071] In a preferred embodiment, the cross-linked material obtained in the method of the present invention is a gel. In a preferred embodiment, the cross-linked material is a polysaccharide-based gel. In a preferred embodiment, the cross-linked material of the present invention is a hyaluronic acid-based gel (HA gel). Such material may optionally also be designated as self-cross-linked hyaluronic acid (HA).

[0072] Hyaluronic acid (also: HA, hyaluronate, or hyaluronan) may be understood in the broadest sense as any hyaluronic acid in the art. It may be a polysaccharide moiety that contains hyaluronic acid moieties (also hyaluronic acid units), preferably comprises at least 50 mol% of hyaluronic acid moieties, more preferably at least 75 mol%, even more preferably at least 80 mol%, even more preferably at least 90 mol%, based on the whole content of saccharide moieties in the polysaccharide, of hyaluronic acid moieties. Hyaluronic acid may optionally comprise one or more saccharide moieties other than hyaluronic acid.

[0073] In a preferred embodiment, hyaluronic acid is a natural glycosaminoglycan composed of linked repeating units of N-acetyl-D-glucosamine and D-glucuronic acid ([alpha-1 ,4-D-glucuronic acid-beta-1 ,3-N-acetyl-D-glucosamine]n). Accordingly, the repeating / monomeric unit of hyaluronic acid may be exemplarity the following or a salt thereof:

[0074] Accordingly, a repeating structure motif of hyaluronic acid may be exemplarity the following or a salt thereof:

[0075] Hyaluronic acid may also embrace glycerol-grafted hyaluronic acid.

[0076] Hyaluronic acid may be used as described in WO 2022 / 268871 . The weight average molecular weight (Mw) of hyaluronic acid in the context of the present invention 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 more.

[0077] In a preferred embodiment, the polysaccharide moieties have a weight average molecular weight as laid out above.

[0078] Heparosan may be understood in the broadest sense as any heparosan. In a preferred embodiment, it may be such as described in WO 2015 / 149941 . Heparosan (HEP) is a biopolymer belonging to the glycosaminoglycan (GAG) family of polysaccharides.

[0079] In humans, it is an intermediate product in the biosynthesis of heparin and heparin sulfate. The structure of heparosan is highly similar to that of hyaluronic acid (HA) since it has the same monosaccharide component sugars as hyaluronic acid and differs from HA only in that the beta-(1 ,3) glycosidic bond between the glucuronic acid (GlclIA) and the / V-acetylglucosamine (GIcNAc) in HA is replaced by a beta- (1 ,4) glycosidic bond in HEP and in that the beta-(1 ,4) glycosidic bond between / V- acetylglucosamine (GIcNAc) and the glucuronic acid (GlclIA) in HA is replaced by an alpha-(1 ,4) glycosidic bond in HEP:

[0080] GlcllA-beta-(1 -4)-[GlcNAc-alpha-(1 -4)-GlcUA-beta-(1 -4)]n-GlcNAc HEP

[0081] Typically, heparosan has excellent biocompatibility. Heparosan carries a high number of negative charges and hydroxy residues and is therefore highly hydrophilic, which increases tissue compatibility. Furthermore, due to the fact that heparosan polymers, even after modification, still comprise stretches that occur in natural heparan sulfate and heparin polymers, heparosan is typically non- immunogenic (e.g., does not induce antibodies). Moreover, due to the structural similarity between heparosan and hyaluronic acid, the same chemical modifications, including oxidation to aldehydes as that known for hyaluronic acid may be made on the functional groups. The molecular weight (Mw) of the heparosan polymers used in the context of the present invention may have any molecular weight.

[0082] Chondroitin sulfate may be understood in the broadest sense as generally understood in the art. In an aqueous environment, it may be a structure comprising the following repeating / monomeric units (counterions are not depicted and may be any cations such as, e.g., sodium):

[0083] The polysaccharide moieties may be one or more types of polysaccharide moieties. These may be different in molecular size and / or may be different in the composition of monomeric units and / or may be different in chemical structure. Optionally, different polysaccharide moieties may be different types of polysaccharide moieties.

[0084] In one embodiment, the polysaccharide moieties, in particular hyaluronic acid moieties, have at least two different molecular weights. In other words, the polysaccharide moieties may also be a mixture of polysaccharide moieties of different molecular weight. In a preferred embodiment, the polysaccharide moieties have at least two different molecular weights and at least one polysaccharide moiety has, preferably at least two polysaccharide moieties, in particular all polysaccharide moieties, each have 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 from 100 to 3500 kDa, in the range of from 200 to 2000 kDa, in the range of from 250 to 1500 kDa, in the range of from 300 to 1000 kDa, in the range of from 400 to 900 kDa, or in the range of from 500 to 900 kDa. In a preferred embodiment, the polysaccharide moieties have at least two different molecular weights and at least one polysaccharide moiety has, preferably at least two polysaccharide moieties, in particular all polysaccharide moieties, each have a molecular weight in the range of 1500 to 3500 kDa.

[0085] Preferably, if the polysaccharide moieties comprise N-acetylamino residues, such as is the case in glycosaminoglycan moieties including hyaluronic acid moieties, heparin moieties, heparan sulfate moieties, chondroitin sulfate moieties, keratan sulfate moieties, in particular hyaluronic acid moieties, the polysaccharide moieties have not been subjected to any reactions resulting in deacetylation of the N- acetylamino residues, to ensure that the amount of amide bonds in the cross-linked material is kept at a minimum.

[0086] In contrast, if the polysaccharide moieties comprise substantial amounts of primary amino residues, it can be beneficial to protect the primary amino residues, e.g., by acetylation, to ensure that the cross-linked material comprises more ester bonds cross-linking polysaccharide moieties covalently with each other than amide bonds cross-linking polysaccharide moieties covalently with each other.

[0087] In a preferred embodiment, the total amount of polysaccharide moieties does not comprise more than 20% by weight, does not comprise more than 10% by weight, does not comprise more than 5% by weight, does not comprise more than 1 % by weight, does not comprise more than 0.5% by weight, or does not comprise more than 0.1 % by weight, based on the total amount of polysaccharide moieties, of polysaccharide moieties of molecular weights of less than 200 kDa.

[0088] In a preferred embodiment, the polysaccharide moieties comprise or consist of at least two hyaluronic acid moieties having at least two different molecular weights and at least one hyaluronic acid moiety has, preferably at least two hyaluronic acid moieties both have, in particular all hyaluronic acid moieties each have, 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 from 100 to 3500 kDa, in the range of from 200 to 2000 kDa, in the range of from 250 to 1500 kDa, in the range of from 300 to 1000 kDa, in the range of from 400 to 900 kDa, or in the range of from 500 to 900 kDa. In a preferred embodiment, the polysaccharide moieties comprise or consist of at least two hyaluronic acid moieties having at least two different molecular weights and at least one hyaluronic acid moiety has, preferably at least two hyaluronic acid moieties both have, in particular all hyaluronic acid moieties each have, a molecular weight in the range of 1500 to 3500 kDa.

[0089] The triazine-based activating agent may be used in the method of the invention in any content suitable for cross-linking carboxylic acid residues of the polysaccharide moieties with at least some of the hydroxy residues of the polysaccharide moieties to form ester bonds thereby cross-linking polysaccharide moieties covalently with each other.

[0090] Typically, a triazine-based activating agent activates the carboxylic acid residues of the polysaccharide moieties. Thus, the content of triazine-based activating agent may be reasonably defined as molar equivalents (eq.) related to the carboxylic acid residues of the polysaccharide moieties. It will be understood that, in this context, carboxylic acid residues also embrace salts thereof. In other words, molar equivalents (eq.) of the triazine-based activating agent is defined as molar ratio of [triazine-based activating agent] : [carboxylic acid residues of the polysaccharide moieties]. For this purpose, in the context of sodium salt of hyaluronic acid (HA), in an approximation, it may be assumed that each HA monomeric / repeating unit when used as sodium salt (having a molecular weight (MW) of approximately 402 g / mol) bears one carboxylic acid residue. The crosslinking degree can be adjusted with different molar equivalents (eq.) of triazine-based activating agent.

[0091] In a preferred embodiment, the molar ratio of [triazine-based activating agent] : [carboxylic acid residues of the polysaccharide moieties] 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 .

[0092] As used in the context of the present invention, a triazine-based activating agent may be any compound based on a triazine core structure that effects reaction of carboxylic acid residues with hydroxy residues thereby forming ester bonds. It will be understood that it is mainly meant that an activating agent is a compound that effects reaction of carboxylic acid residues of the polysaccharide moieties with hydroxy residues of the polysaccharide moieties thereby forming ester bonds.

[0093] In a preferred embodiment, an activating agent is typically not covalently included in the cross-linked material. Thus, it may be typically optionally removed from the cross-linked material of the present invention by any means such as, e.g., washing, filtration, etc. A triazine-based activating agent may have any structure that comprises a triazine core that is suitable for cross-linking carboxylic acid residues of the polysaccharide moieties with at least some of the hydroxy residues of the polysaccharide moieties to form ester bonds thereby cross-linking polysaccharide moieties covalently with each other. For instance, a triazine-based activating agent may have the structure: wherein R1, R2and R3are independently from each other any residue, such as, e.g., each independently a residue selected from the group consisting of hydrogen, deuterium, Ce-Cw-aryl, C2-Cio-heteroaryl, linear or branched Ci-Cio-(cyclo)alkyl, linear or branched Ci-Cio-(cyclo)alkoxy, linear or branched C1-C10- hetero(cyclo)alkyl, linear or branched C2-Cio-(cyclo)alkenyl, C2-C10- hetero(cyclo)alkenyl, linear or branched C2-Cio-(cyclo)alkinyl, and C2-C10- hetero(cyclo)alkenyl, wherein each of the aforementioned residues is optionally substituted with one or more residues selected from the group consisting of halogen, Ce-Cw-aryl, C2-Cw- heteroaryl, linear or branched Ci-Cw-(cyclo)alkyl, linear or branched Ci-Cw- hetero(cyclo)alkyl, linear or branched Ci-Cw-(cyclo)alkoxy, linear or branched C2- Cw-(cyclo)alkenyl, C2-Cw-hetero(cyclo)alkenyl, linear or branched C2-Cw- (cyclo)alkinyl, and C2-Cw-hetero(cyclo)alkenyl, or a salt thereof.

[0094] In a preferred embodiment, at least one or the residues R1, R2and R3is an optionally substituted morpholinium residue. In a preferred embodiment, at least one or the residues R1, R2and R3is a Ci-C4-alkyl-morpholinium residue. In a preferred embodiment, at least one or the residues R1, R2and R3is a methylmorpholinium residue. In a preferred embodiment, at least one or the residues R1, R2and R3is an optionally substituted linear or branched Ci-Cw-(cyclo)alkoxy residue. In a preferred embodiment, at least one or the residues R1, R2and R3is an optionally substituted linear or branched Ci-C4-alkoxy residue. In a preferred embodiment, at least one or the residues R1, R2and R3is an optionally substituted linear or branched methoxy residue. In a preferred embodiment, the triazine-based activating agent has a molecular weight of not more than 1500 g / mol, of not more than 1000 g / mol, of not more than 500 g / mol, of not more than 400 g / mol,

[0095] In a preferred embodiment, the one or more activating agents are selected from the group consisting of 4-(4,6-dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholinium (DMTMM) or a salt thereof, and / or 2-chloro-4,6,-dimethoxy-1 ,3,5-triazine or a salt thereof, and combinations thereof.

[0096] In a preferred embodiment, the activating agent is 4-(4,6-dimethoxy-1 ,3,5-triazin-2- yl)-4-methylmorpholinium (DMTMM) or a salt thereof,

[0097] A salt of DMTMM is preferably a salt wherein the counter-ion is an anion that is cosmetically and / or pharmaceutically acceptable such as, e.g., chloride, acetate, bicarbonate (hydrogen carbonate), or a mixture of two or more anions.

[0098] In a preferred embodiment, the activating agent is 4-(4,6-dimethoxy-1 ,3,5-triazin-2- yl)-4-methylmorpholinium chloride (CAS No. 3945-69-5).

[0099] DMTMM is considered as having a comparably low and essentially negligible toxicity, is not cancerogenic, not mutagenic, and not teratogenic / reprotoxic in the generally used amounts. Thus, it is particularly well usable for preparing a soft-tissue filler such as a dermal or connective tissue filler.

[0100] When using DMTMM or a salt thereof as activating agent, 4-methylmorpholine (NMM) and / or 4,6-dimethoxy-1 ,3-5-triazine-2-ol (DMT) may be formed as degradation product(s). For example, the chloride salt of DMTMM may be used as as activating agent:

[0101] When used e.g., with a hyaluronic acid moiety, the carboxylic acid residue of the hyaluronic acid moiety is activated and a reaction with a hydroxyl residue of the hyaluronic acid moiety is enabled. Typically, the carboxylic acid and the hydroxyl groups of hyaluronic acid encircled in the following structure are involved in the reaction.

[0102] In a preferred embodiment, the method is further characterized in that it does not comprise at least one, preferably at least two, more preferably at least three, more preferably all of the following:

[0103] (a) the use of interconnecting linker moieties inserted between the carboxylic acid residues or salts thereof and hydroxy residues of the polysaccharide moieties, in particular no peptidic or xenobiotic linker moieties;

[0104] (b) the use of a carbodiimide-based activating agent, in particular selected from the group consisting of N,N’-dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC), and combinations of two or more thereof;

[0105] (c) the use of reactive groups selected from the group consisting of glycidyl ethers, maleiimides, acid anhydrides, alkoxides and combinations of two or more thereof;

[0106] (d) polysaccharide moieties having primary amine groups, thiol groups, imide groups, imine groups, or epoxy groups.

[0107] The solvent used in the method of the invention may be any solvent that is suitable for reacting carboxylic acid residues or salts thereof and hydroxy residues of the polysaccharide moieties. In a preferred embodiment, the solvent is suitable for dissolving or suspending the polysaccharide moieties and dissolving the triazinebased activating agents. In a preferred embodiment, the solvent is suitable for dissolving the polysaccharide and the triazine-based activating agents. In a preferred embodiment, the solvent is a polar solvent. In a preferred embodiment, the solvent is a protic solvent. In a preferred embodiment, the solvent is a protic polar solvent. In a preferred embodiment, the solvent comprises more than 50 wt.%, of at least 60 wt.%, of at least 70 wt.%, of at least 80 wt.%, of at least 90 wt.%, of at least 95 wt.%, or even 100 wt.%, based on the total mass of the solvent, of one or more components selected from the group consisting of: water; one or more alcohols, preferably one or more Ci-Cs-alcohols, more preferably, one or more Ci-Cs-alcohols selected from the group consisting of methanol, ethanol, n- propanol, isopropanol, n-butanol (1 -butanol), sec-butanol (2-butanol) isobutanol, (2- methylpropan-1 -ol), tert-butanol (2-methylpropanol), pentan-1 -ol, 2-methylbutan-1- ol, 3-methylbutan-1 -ol, 2,2-dimethylpropan-1-ol, pentan-2-ol, 3-methylbutan-2-ol, pentan-3-ol, and / or 2-methylbutan-2-ol, and a combination of two or more thereof, in particular methanol and / or ethanol; one or more primary amines, in particular in particular one or more Ci-Cs-amines; one or more carbonic acids, preferably one or more Ci-Cs-carbonic acids selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, valerianic acid, isovalerianic acid, in particular formic acid and / or acetic acid; one or more primary or secondary amides, preferably one or more Ci-Cs-amides, in particular formamide; one or more sulfoxides, preferably one or more Ci-Cs-amides, in particular dimethyl sulfoxide (DMSO); and a combination of two or more thereof.

[0108] In a preferred embodiment, the solvent is an aqueous solution. An aqueous solvent may be understood in the broadest sense as a solvent that comprises a water content by weight of more than 50 wt.%, of at least 60 wt.%, of at least 70 wt.%, of at least 80 wt.%, of at least 90 wt.%, of at least 95 wt.%, or even 100 wt.%, based on the total mass of the solvent. In one embodiment of the present invention, an aqueous buffer comprises, in addition to water, one or more components selected from the group consisting of one or more alcohols (in particular one or more Ci-Cs- alcohols such as, e.g., methanol, ethanol, n-propanol, isopropanol, n-butanol (1 - butanol), sec-butanol (2-butanol) isobutanol, (2-methyl-propan-1 -ol), tert-butanol (2-methylpropanol), pentan-1 -ol, 2-methylbutan-1-ol, 3-methylbutan-1 -ol, 2,2- dimethylpropan-1 -ol, pentan-2 -ol, 3-methylbutan-2-ol, pentan-3-ol, and / or 2- methylbutan-2-ol), one or more primary amines (in particular one or more C1-C5- amines), one or more carbonic acids (in particular one or more Ci-Cs-carbonic acids such as., e.g., formic acid, acetic acid, propionic acid, butyric acid, valerianic acid, isovalerianic acid), one or more primary or secondary amides (in particular one or more Ci-Cs-amides such as, e.g., formamide), one or more sulfoxides (in particular one or more Ci-Cs-amides such as, e.g., dimethyl sulfoxide (DMSO)), one or more inorganic or organic cations (in particular one or more inorganic or organic cations of a molecular weight or less than 1000 Da, in particular alkali cations or earth alkali cations, other metal cations, protons, ammonium cations, etc.), one or more inorganic or organic anions (in particular one or more inorganic or organic anions of a molecular weight or less than 1000 Da, in particular chlorine, sulfate, etc.), one or more silicates, and a combination of two or more thereof.

[0109] In a preferred embodiment, the solvent is an aqueous buffer (e.g., phosphate buffered saline (PBS), Tris buffer, borate buffer, acetic acid-buffered buffer, etc.). In a preferred embodiment, a hydroalcoholic solvent is used such as, e.g., a mixture of water with ethanol, methanol, propanol, butanol, and / or pentanol. In a preferred embodiment, water is used as solvent. In a preferred embodiment, the solvent is an aqueous buffer having a pH in the range of 5 to 9, in particular 6 to 8. For example, the solvent may be an aqueous buffer having a pH in the range of 6.1 to 7.9, of 6.5 to 7.7, of 6.7 to 7.5, or of 7.0 to 7.5,

[0110] It was surprisingly found that the reaction steps, do not need alkalization and the reaction takes place at essentially neutral pH. This rebutted the prejudice in the art indicating that either strongly acidic or basic pH ranges were required.

[0111] Water as used herein, may be understood in the broadest sense. Preferably, water is deionized water, distilled water, or tap water, in particular deionized water or distilled water.

[0112] In a preferred embodiment, the step (iii) of the method according to the invention does not comprise a step of purification of the cross-linked material obtained from step (ii). In another embodiment, step (iii) of the method comprises at least one purification step. In one embodiment, the method comprises at least one step of purifying the cross-linked material by filtration, washing and / or dialysis, in particular crossflow filtration, diafiltration and / or dead-end filtration.

[0113] Dialysis may be conducted by any means. In a preferred embodiment, dialysis is using dialysis membranes having a molecular weight cut-off (MWCO) of 1 to 25 kDa, of 5 to 20 kDa, or of 10 to 15 kDa. Purification time may be adapted to the molecule structures and may be in the range of at least 1 hour, at least 6 hours, 12 to 78 hours, 1 to 7 days, or 1 to 3 days. The swelling ratio may be optionally controlled by the weight of the membranes and the dialysis may be stopped once the product shows the desired cross-linked material concentration.

[0114] It will be understood that filtration may be crossflow filtration, dead-end filtration, or a combination of both. Filtration may be performed by any means. In the context of filtration, the a filter may have any pore size suitable for purifying the cross-linked material, i.e., preferably withholding the cross-linked material and allowing the passage of reactants and optionally of non-reacted polysaccharides. Optionally, the pore size may be in a range of 5 nm to 2 pm, more particularly a pore size of 30 nm to 600 nm, more particularly a pore size of 80 nm to 300 nm, particularly a pore size of 5 nm to 60 nm. A filter may be of any material such as, e.g., ceramic, metal, polymer material, or a combination thereof.

[0115] Optionally, filtration may be dynamic filtration such as, e.g., described in WO 2020 / 030629. Accordingly, step (iii) may optionally comprise dynamic filtration of the cross-linked material, optionally, comprising the following steps: a) transferring the cross-linked material in a dynamic filtration device which is equipped with semipermeable filter disc(s) and diaf iltrating the gel comprising the steps of: i) concentrating the cross-linked material by applying a rotational speed within the range of 20 1 / min to 500 1 / min and a overpressure within the range of 0.5 to 6 bar to a predetermined concentration; or pumping the cross-linked material directly into the process chamber of the dynamic filtration device; ii) conducting a diafiltration to reduce unwanted molecules by applying a rotational speed within the range of 20 1 / min to 500 1 / min and a overpressure within the range of 0.5 to 6 bar; b) optionally adding a mixture comprising a non-cross-linked material and water to the cross-linked material.

[0116] In one embodiment, the dynamic filtration device is equipped with 1 to 10 semipermeable filter disc(s). In DCF, any rotational speed and pressure may be used, such as, e.g., a rotational speed within the range of 20 1 / min to 500 1 / min and a pressure within the range of 0.5 to 3 bar. In DCF, any concentration may be used such as e.g., 10 to 70 mg / g.

[0117] In an embodiment of the present invention, crossflow filtration (also: cross-flow filtration) is dynamic crossflow filtration (DCF). Thus, in an embodiment, the method may be further characterized in that step (iii) comprises purifying the cross-linked material by DCF. For example, DCF may be such as described in WO 2020 / 030629.

[0118] In one embodiment, steps (i) and (ii) are conducted in a single batch. In another embodiment, steps (i) and (ii) are conducted in separate batches.

[0119] Steps (i) and (ii) and (iii) (optionally including purification) may be each conducted at any temperature range. In a preferred embodiment, step (i) is conducted at a temperature in the range of from 5 to 90°C, preferably 18 to 60°C, in particular more preferably 18 to 25°C or 20°C to 50°C. In a preferred embodiment, step (ii) is conducted at a temperature in the range of from 5 to 90°C, preferably 18 to 60°C, in particular 20°C to 50°C. In a preferred embodiment, step (iii) is conducted at a temperature in the range of from 5 to 90°C, or 18 to 60°C, or 20°C to 50°C. In a preferred embodiment, steps (i), (ii) and (iii) are conducted at a temperature in the range of from 5 to 90°C, preferably 18 to 60°C, in particular 20°C to 50°C. In a preferred embodiment, steps (i) and (ii) are conducted at temperatures that do not differ by more than 10°C, do not differ by more than 5°C, or do not differ by more than 2°C. Steps (i), (ii) and (iii) (optionally including purification) may be conducted at any pressure. For example, pressure may be ambient pressure (e.g., often approximately 970 to 1100 hPa outer pressure).

[0120] Step (i) of combining the components with each other may be conducted by any means. In a preferred embodiment, the method is further characterized in that step (i) involves the mixing of the components, i.e., the polysaccharide and the one or more activating agents, and one or more solvents, and optional one or more further components. Such mixing may be conducted by any means such as, e.g., by means of stirring and / or shaking.

[0121] In a preferred embodiment, the polysaccharide moieties are mixed with the solvents or parts of the solvent before contacting it with the one or more activating agents. This step may contain an incubation of the mixture of the polysaccharide moieties and the solvents or parts of the solvent (also: pre-swelling). For instance, such optional incubation step may be conducted (e.g., at a temperature of 4 to 40°C) for at least 10 min, for 30 min to 7 days, for 1 hour to 2 days, for 6 to 36 hours, or for 12 to 24 hours. The one or more activating agents may be optionally premixed with parts of the solvent and subsequently added to the mixture of the polysaccharide moieties and the solvents or parts of the solvent. The reaction step (ii) may be conducted as long as suitable for achieving the desired reaction of the carboxylic acid residues or salts thereof with the hydroxyl residues. Step (ii) may be conducted for any time suitable for this purpose. Optionally, step (ii) may be conducted 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) may be conducted for at any temperature suitable for this purpose such as, e.g., 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 to 25°C, at 20°C to 70°C, at 20°C to 40°C, or at 60°C to 70°C.

[0122] In a preferred embodiment, the step (ii) is conducted for not more than 72 h, for not more than 48 h, or for not more than 24 h. In a preferred embodiment, the step (ii) is conducted for at least 10 min, preferably for 15 min to 48 h, in particular for 30 min to 18 h. In a preferred embodiment, the method is further characterized in that the step (ii) is conducted at a temperature in the range of from 5 to 90°C, preferably 18 to 60°C, in particular 20°C to 50°C, for at least 10 min, preferably for 15 min to 48 h, in particular for 30 min to 18 h. In a preferred embodiment, the method is further characterized in that the step (ii) is conducted at a temperature in the range of from 5 to 90°C for at least 10 min. In a preferred embodiment, the method is further characterized in that the step (ii) is conducted at a temperature in the range of 18 to 60°C for 30 min to 18 h. Step (ii) may be conducted at any pH. For example, it may be conducted at a pH in the range of 5 to 9, in particular 6 to 8, of 6.1 to 7.9, of 6.5 to 7.7, of 6.7 to 7.5, or of 7.0 to 7.5,

[0123] During reaction step (ii), the polysaccharide moieties may be used at any concentration range. In a preferred embodiment, 0.01 to 800 mg / mL, or 0.1 to 500 mg / mL, or .5 to 200 mg / mL of polysaccharide moieties is used. In a preferred embodiment, 1 to 100 mg / mL, or 2 to 75 mg / mL, or 5 to 50 mg / mL, or 20-25 mg / mL, or 25 to 35 mg / mL, of polysaccharide moieties is used.

[0124] The optional purification in step (iii), if conducted, may be conducted for any time suitable for this purpose. Optionally, purification in step (iii) may be conducted 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. Purification in step (iii) may be conducted at any temperature suitable for this purpose such as, e.g., 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 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, the method is further characterized in that:

[0125] (a) step (iii) comprises purifying the cross-linked material by filtration, washing and / or dialysis, in particular crossflow filtration, diafiltration and / or dead-end filtration;

[0126] (b) steps (i) and (ii) are conducted in a single batch;

[0127] (c) steps (i), (ii) and (iii) are conducted at a temperature in the range of from 5 to 90°C, preferably 18 to 60°C, in particular 20°C to 50°C; and / or

[0128] (d) the step (ii) is conducted for at least 10 min, preferably for 15 min to 48 h, in particular for 30 min to 18 h.

[0129] In a preferred embodiment, the method comprises:

[0130] (i) contacting the following components with each other: a) polysaccharide moieties comprising carboxylic acid residues or salts thereof and hydroxy residues having a weight average molecular weight of at least 50 kDa, b) one or more triazine-based activating agents that effect reaction of carboxylic acid residues with hydroxy residues thereby forming ester bonds, in particular wherein the activating agent 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 6 to 8; and

[0131] (ii) allowing a reaction of at least some of the carboxylic acid residues with at least some of the hydroxy residues to form ester bonds cross-linking polysaccharide moieties covalently with each other; and

[0132] (iii) obtaining cross-linked material from step (ii) as component (A) and optionally purifying the cross-linked material obtained from step (ii);

[0133] (iv) adding: at least one botulinum toxin as component (B), and optionally one or more liquid cosmetically and / or pharmaceutically acceptable carriers as component (C), and / or optionally one or more anesthetics as component (D), and / or optionally one or more further cosmetically and / or pharmaceutically acceptable ingredients different from components (A) to (D) as component (E);

[0134] In an optional further step, the cross-linked material obtained in step (iii) may be treated further. For instance, it may be optionally homogenized and / or passed through a sieve (screening). In this context, the terms sieve, filter and mesh may be understood interchangeably. This may result in a particularly homogeneous material (e.g., hydrogel). Optionally, the method may include a step of sterilization before adding botulinum toxin and optional other components susceptible to degradation in the sterilization process. Components not susceptible to degradation, e.g., certain anesthetics (D), can be added to the composition before a step of sterilization.

[0135] In a step (iv), component (B) and optionally (C), (D) and / or (E) are added to the cross-linked material obtained in step (iii).

[0136] In a preferred embodiment, step (iv) comprises adding botulinum toxin (component (B)) and one or more liquid cosmetically and / or pharmaceutically acceptable carriers (component (C)) and optionally one or more components (D) and / or (E). Such composition is preferably an injectable composition.

[0137] In a preferred embodiment, step (iv) comprises adding botulinum toxin (component

[0138] (B)) dissolved in one or more liquid cosmetically and / or pharmaceutically acceptable carriers (component (C)) and optionally one or more components (D) and / or (E). Such step may also comprise mixing the components and optionally suspending component (A) in component (C) in which component (B) and optionally one or more further components may be dissolved. Such composition is preferably an injectable composition.

[0139] In a preferred embodiment, step (iv) comprises admixing botulinum toxin (component (B)) and at least one anesthetic (component (D)) dissolved in one or more liquid cosmetically and / or pharmaceutically acceptable carriers (component

[0140] (C)) and optionally one or more components (E). Such step may also comprise mixing the components and optionally suspending component (A) in component (C) in which components (B) and (D) and optionally one or more further components (E) may be dissolved. Such composition is preferably an injectable composition.

[0141] In a preferred embodiment, the cross-linked material (A) forms a gel (in particular a hydrogel) in a liquid pharmaceutically acceptable carrier (C), and the botulinum toxin (B) is dissolved in the liquid pharmaceutically acceptable carrier (C) soaked in the gel (in particular hydrogel).

[0142] The solvent used in steps (i) and (ii) and / or optionally in step (iii) may optionally be partly or completely removed. As the used components, including the activating agent, may bear a comparably low toxicity, the solvent used in steps (i) and (ii) and / or optionally in step (iii) may alternatively optionally also be maintained in the cross-linked material. If removed, the solvent used in steps (i) and (ii) and / or optionally in step (iii) may be removed by any means such as, e.g., by replacing it by another solvent (e.g., another aqueous buffer) and / or by evaporation.

[0143] The composition of the invention, which is obtainable by the method of the invention, comprises or consists of:

[0144] (A) a cross-linked material comprising one or more polysaccharide moieties covalently cross-linked with each other through ester bonds, wherein the crosslinked material comprises more ester bonds cross-linking polysaccharide moieties covalently with each other than amide bonds cross-linking polysaccharide moieties covalently with each other;

[0145] (B) at least one botulinum toxin;

[0146] (C) optionally one or more liquid cosmetically and / or pharmaceutically acceptable carriers;

[0147] (D) optionally one or more anesthetics; and

[0148] (E) optionally one or more further cosmetically and / or pharmaceutically acceptable ingredients different from components (A) to (D), and is free of carbodiimide activating agents, succinimidyl-based activating agents, glycidyl-based activating agents, paranitrophenol esters, 2-chloro-methylpyridinium iodide, and their reaction products. Preferably, the composition obtained from the method of the invention is free of any activating agents and reaction products thereof other than triazine-based activating agents or reaction products thereof.

[0149] It will be understood that the definitions and preferred embodiments as laid out in the context of the method of the present invention mutatis mutandis apply to the cross-linked material of the present invention.

[0150] The composition obtained from the method of the invention will often contain residual amounts of the at least one triazine-based activating agent used in the method of preparation, or a reaction product thereof.

[0151] For example, the amount of the triazine-based activating agent or one or more reaction products thereof may be at least 1 ppb, e.g. 1 ppb to 1000 ppm, 10 ppb to 500 ppm, 100 ppb to 100 ppm, or 1 ppm to 10 ppm (w / w), based on the total weight of the composition. If the step (iii) of the method of the invention involved purification of the cross-linked material obtained from step (ii), the amount of the activating agent or reaction products thereof will typically be in the lower range, e.g. from 1 ppb to 1 ppm. If no purification is involved, the amount may be in the upper portion of the range, e.g. from 1 ppm to 1000 ppm.

[0152] In an embodiment of the present invention, the composition of the invention has been prepared by using DMTMM as activating agent in step (i). In an embodiment of the present invention, the composition, may comprise residual amounts of:

[0153] (a) 4-(4,6-dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholinium (DMTMM) or a salt thereof,

[0154] (b) N-methylmorpholinium or salt thereof (NMM); and / or

[0155] (c) 4,6-dimethoxy-1 , 3, 5-triazin-2-ol (DMT) or a tautomer or salt thereof.

[0156] In an embodiment of the present invention, the composition comprises residual NMM and / or DMT. In an embodiment of the present invention, the composition comprises up to 0.1 % by weight, preferably 0.01 to 1000 ppm, 0.1 to 100 ppm, or 1 to 50 ppm of residual NMM and / or DMT, referred to the total weight of the composition.

[0157] The cross-linked material (A) in the composition of the invention may comprise or consist of one or more polysaccharide moieties, in particular hyaluronic acid moieties, having a weight average molecular weight of at least 50 kDa, wherein the polysaccharide moieties, in particular hyaluronic acid moieties, are covalently crosslinker with each other via ester bonds without an interconnecting linker structure, further characterized in that the cross-linked material does not comprise diimide groups, epoxy groups or xenobiotic linker moieties. In a particular embodiment, the cross-linked material (A) does not comprise fibroin moieties.

[0158] As used herein, the term “without an interconnecting linker structure” may be understood in the broadest sense in that no further chemical moiety that does not originate from (also: is not present in) polysaccharide moieties (e.g., hyaluronic acid moieties) is introduced into the chemical structure that conjugates polysaccharide moieties with ester bonds. In other words, the ester bonds are preferably formed from inclusion of an oxygen atom originating from polysaccharide moieties (e.g., hyaluronic acid moieties) and from inclusion of a carbon atom originating from polysaccharide moieties (e.g., hyaluronic acid moieties).

[0159] In a preferred embodiment, the cross-linked material is further characterized in that it does not comprise imide groups or diimide groups. In a preferred embodiment, the cross-linked material is further characterized in that it does not comprise imine groups. In a preferred embodiment, the cross-linked material is further characterized in that it does not comprise epoxy groups. In a preferred embodiment, the crosslinked material is further characterized in that it does not comprise xenobiotic linker moieties groups.

[0160] In a preferred embodiment, the cross-linked material is further characterized in that it does not comprise any one or more, preferably any of the following:

[0161] (a) diimide groups

[0162] (b) imide groups;

[0163] (c) imine groups;

[0164] (d) epoxy groups; and / or

[0165] (e) xenobiotic linker moieties, interconnecting polysaccharide moieties.

[0166] As used in the context of the present invention, the term “botulinum toxin” may be understood in the broadest sense as any type of botulinum toxin known in the art.

[0167] The term “botulinum toxin”, as used herein, is not particularly limited and includes botulinum toxin of any serotype (e.g., BoNT / A-H. For example, the botulinum toxin may be of serotype A or B (BoNT / A, BoNT / B). Preferably, the botulinum toxin is of serotype A, more preferably of serotype A1 (BoNT / A1 ), and most preferably BoNT / A1 produced by Clostridium botulinum Hall strain. Furthermore, as used herein, the term “botulinum toxin” (“BT”) and the synonymously used term "botulinum neurotoxin" (“BoNT”) are intended to refer to the pure botulinum neurotoxin and / or any complex thereof, i.e., any complex of the pure botulinum neurotoxin and complexing proteins (referred to as the “toxin complex”). Preferably, the botulinum toxin is the pure botulinum neurotoxin of serotype A.

[0168] In a preferred embodiment, the botulinum toxin is botulinum neurotoxin serotype A, in particular botulinum neurotoxin serotype A1 .

[0169] The term "pure botulinum neurotoxin", as used herein, means the botulinum neurotoxin free of complexing proteins (sometimes also referred to as the “neurotoxic component”), or more precisely, the botulinum neurotoxin without neurotoxin-associated complexing proteins (NAPs). The pure botulinum neurotoxin is the (active) neurotoxic polypeptide that ultimately inhibits acetylcholine release. It is a di-chain protein comprised of a light chain (LC; about 50 kDa) and a heavy chain (HC; about 100 kDa), held together by a disulfide bond. The active neurotoxic polypeptide may therefore also be referred to herein as the "150 kDa neurotoxin", “Clostridium botulinum neurotoxin (150 kD)” or "neurotoxic component". Preferably, the botulinum toxin is the (pure) botulinum neurotoxin contained in Xeomin® or is Xeomin®.

[0170] The term “toxin complex”, as used herein, refers to a high-molecular complex of the neurotoxic component and a set of complexing proteins (NAPs), such as the 900 kDa, 500 kDa, and 300 kDa C. botulinum type A toxin complexes. The complexing proteins are nontoxic nonhaemagglutinin (NTNHA) and, in strains of serotype A-D, different haemagglutinins (HAs). For example, the 900 kDa complex is included in onabotulinumtoxin A (Botox®A / istabel®, Allergan, Inc., Irvine, CA, USA), and abobotulinumtoxin A (Dysport®, Azzalure®, Ipsen, Paris, France), Alluzience® (Ipsen / Galderma) and Innotox® (Medytox) also contain a toxin complex as active agent. Preferably, the botulinum toxin is, besides the pure botulinum neurotoxin that is contained in Xeomin® or is Xeomin®, the toxin complex contained in Botox® or Dysport®, or is Botox® or Dysport®.

[0171] A botulinum toxin may also be such as described in WO2017 / 148915.

[0172] The botulinum toxin may be a natural neurotoxin obtainable from Clostridium botulinum or any other botulinum toxin such as a botulinum toxin obtainable from alternative sources, including recombinant technologies and genetic or chemical modification. Chimeric or genetically modified botulinum toxins, i.e., botulinum toxins containing mutations including substitutions, deletions and insertions, are also encompassed by the terms “botulinum toxin”, “neurotoxic component” and the like. Preferably, the mutation does not compromise any of the biological activities of botulinum toxin. However, it is also envisaged to use mutations to modulate the biological activity of the botulinum toxin. Also included are botulinum toxins containing chemically modified amino acids, for example one or more amino acids which are glycosylated, acetylated or otherwise modified.

[0173] This may be beneficial to the uptake or stability of the toxin. Particularly preferred is the lipidation of the neurotoxic component.

[0174] Within the present invention, the dose is expressed in biological (enzymatic) units because the used botulinum toxin may contain, for example, variable percentages of inactive toxin that contribute to the overall protein load without contributing to efficacy. Within the context of the present invention, the biological potency of botulinum toxin is determined using the mouse bioassay (MBA). The MBA determines the mean lethal dose (LD50) of toxin / neurotoxin after intraperitoneal injection in mice, i.e. , the dose of toxin / neurotoxin capable of killing 50% of a group of mice. On this basis, 1 unit (U) of toxin / neurotoxin, as used herein, is defined as one mouse LD50 (1.0 LD50 = 1.0 U). The LD50 mouse bioassay is the gold standard among various biological, chemical or immunological detection methods for botulinum toxin and is known to those skilled in the art (see, e.g., Pearce, L.B.; Borodic, G.E.; First, E.R.; MacCallum, R.D. Measurement of botulinum toxin activity: Evaluation of the lethality assay. Toxicol. Appl. Pharmacol. 1994, 128, 69-77).

[0175] Another useful method for determining the biological activity (biological potency) of a botulinum neurotoxin is a cell-based potency assay which is disclosed, for example, in WO 2009 / 114748, WO 2013 / 049508 or WO 2014 / 207109. The activity results obtained with such cell-based assays correspond to the activity values obtained in the mouse i.p. LD50 assay because the values are calibrated using the LD50 reference standard.

[0176] Due to differences in the LD50 tests used by manufacturers of commercial botulinum toxin formulations, the unit potencies indicated by the manufacturers for their commercial botulinum toxin formulations is proprietary and cannot easily be compared. Therefore, within the framework of the present invention, the conversion rates provided below are used to establish the comparative potencies of incobotulinumtoxinA ("INCO"; Xeomin®, Bocouture®; botulinum toxin serotype A, free of complexing proteins; Merz Pharmaceuticals GmbH), onabotulinumtoxinA ("ONA"; Botox®, Vistabel®; botulinum toxin complex of serotype A; Allergan Inc.), abobotulinumtoxinA ("ABO"; Dysport®, Azzalure®; botulinum toxin complex of serotype A; Medicis Pharmaceutical Corp., Galderma Lab.), rimabotulinumtoxinB ("RIM"; Myobloc®, NeuroBloc®; botulinum toxin serotype B; Solstice Neurosciences Inc.), and PurTox® ("TBD"; botulinum toxin serotype A; Mentor Worldwide LLC).

[0177] For use herein, the conversion rate of ONA and INCO is 1 :1 . The conversion rate of ONA / INCO:ABO is 1 :2.5. The conversion rate of ONA / INCO:RIM is 1 :50, and the conversion rate of ONA / INCO:TBD is 1 :1.5. Furthermore and preferably, within the context of the present invention, 1 U of INCO (Xeomin®) and 1 U of onabotulinumtoxinA ("ONA"; Botox®) shall be deemed to correspond to one mouse LD50 (1 .0 LD50), or 1 U, measured using the MBA as described above.

[0178] In a preferred embodiment, the botulinum toxin is not covalently bound with the cross-linked material. As used herein, a liquid cosmetically and / or pharmaceutically acceptable carrier may be any component being usable as such. It may be an easily flowing liquid carrier or a viscous liquid carrier.

[0179] Preferably, a liquid carrier as comprised in the composition may be any injectable carrier. Typically, the liquid carrier is a carrier that is non-toxic to the mammal, in particular a human, when administered to the mammal in the sense of the present invention. The liquid carrier may preferably comprise or consist of one or more solvents such as, e.g., water, an aqueous buffer, glycerol, dimethyl sulfoxide (DMSO), ethanol, vegetable oil, paraffin oil or combinations thereof. More preferably, the liquid carrier comprises or consists of a non-pyrogenic isotonic buffer, more particularly a physiological saline solution or a buffered physiological saline solution. A saline may, e.g., have a sodium chloride concentration of 0.9 % by weight.

[0180] An aqueous buffer may be any buffer comprising water that is pharmaceutically and / or cosmetically acceptable. Preferably, the buffer is pharmaceutically and / or cosmetically acceptable when being injected into a subject. Such buffer may, for instance, be a buffer selected from the group consisting of phosphate buffer, citrate buffer, citrate-phosphate buffer, lactate buffer, acetate buffer, and combinations of two or more thereof. It will be understood that such buffer may optionally also include further ingredients such as, e.g., one or more pharmaceutically and / or cosmetically acceptable salts and / or one or more pharmaceutically and / or cosmetically acceptable sugars. In a preferred embodiment, an aqueous buffer is a saline or a phosphate buffered saline.

[0181] In a preferred embodiment, the liquid pharmaceutically acceptable carrier comprises or consists of water, an aqueous buffer, glycerol or a combination of two or more thereof.

[0182] A buffer may have any pH. Preferably, a buffer may have a pH in a pharmaceutically and / or cosmetically acceptable when being injected in a subject. In a preferred embodiment, the buffer may have a pH stabilized in the range of 6.0 to 8.0, 6.5 to 7.5, 6.5 to 7.0, 7.0 to 7.5, 7.1 to 7.5, or 7.2 to 7.4.

[0183] As used herein, an anesthetic may be any anesthetic component. Preferably, an anesthetic is a local anesthetic. A local anesthetic may make injection into an individual more comfortable. Suitable local anesthetics for use herein include, but are not limited to, ambucaine, amolanone, amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine, butacaine, butamben, butanilicaine, butethamine, butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine, dibucaine, dimethysoquin, dimethocaine, diperodon, dycyclonine, ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine, euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracaine, isobutyl p- aminobenzoate, leucinocaine mesylate, levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, myrtecaine, naepaine, octacaine, orthocaine, oxethazaine, parethoxycaine, phenacaine, phenol, piperocaine, piridocaine, polidocanol, pramoxine, prilocaine, procaine, propanocaine, proparacaine, propipocaine, propoxycaine, psuedococaine, pyrrocaine, ropivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine, zolamine, and salts thereof. In a preferred embodiment, an anesthetic is lodicaine. Optionally, a combination of two or more of the mentioned anesthetic agents, for example a combination of lidocaine and other "caine"-anesthetic(s) like prilocaine, may also be used herein.

[0184] A further cosmetically and / or pharmaceutically acceptable ingredients different from components (A) to (D) may be any component that is usable for such purpose.

[0185] For instance, such further component may be a further carrier, e.g., solid cosmetically and / or pharmaceutically acceptable carrier that is optionally soluble in the liquid cosmetically and / or pharmaceutically acceptable carrier. For instance, such further component may be a filling or bulking agent. For instance, such further component may be a salt (e.g., sodium chloride, NaCI). For instance, such further component may be a stabilizing agent (e.g., a protein (e.g., albumin (e.g., human albumin)). For instance, such further component may be a component that slows down the degradation of botulinum toxin (e.g., a radical scavenging agent, (e.g., a polyol such as glycerol, a sugar alcohol (e.g., selected from the group consisting of mannitol, inositol, lactilol, xylitol, erythriol, sorbitol, etc.)). For instance, such further component may be a sugar (e.g., sucrose, glucose, trehalose, galactose, fructose, maltose, lactose, starch, cellulose, etc.).

[0186] For instance, such further component may be a crystallization inhibitor. For instance, such further component may be a scavenging agent. For instance, such further component may be a cell proliferation factor that may improve cellular invasion into an administered cross-linked material of the present invention. For instance, such further component may be a dye that may, e.g., either facilitate localized administration (e.g., injection), may improve localization of the injection (e.g., a pharmaceutically acceptable fluorescent dye like fluorescein or rhodamine), or may improve invisibility of the otherwise whitish cross-linked material (e.g., by rendering it flesh-colored). Any other pharmaceutically active compound may also be added. Then, the composition of the present invention may optionally also serve as a retarded and / or controlled release form for administration.

[0187] The other components may be added at any time such as before, during or after purifying the cross-linked material. For instance, one or more further components may be added during conducting a purification. In another embodiment of the present invention, one or more further components may be added to the prepared and optionally purified cross-linked material.

[0188] The composition of the present invention may comprise the components in any concentration and content ranges.

[0189] In one embodiment, the composition of the present invention is a (hydro)gel, liquid, or viscous composition. In one embodiment, the composition of the present invention is an injectable composition.

[0190] In a preferred embodiment, the composition of the present invention comprises at least 0.1 % by weight, based on the total weight of the composition, of the crosslinked material. In a preferred embodiment, in particular when the composition of the present invention is a (hydro)gel or a liquid, the composition comprises at least 0.2 % by weight, at least 0.5 % by weight, at least 1 % by weight, at least 1 .5 % by weight, at least 2 % by weight, at least 5 % by weight, or at least 10 % by weight, based on the total weight of the composition, of the cross-linked material.

[0191] In a preferred embodiment, in particular when the composition of the present invention is a (hydro)gel or a liquid, the composition comprises not more than 3.5 % by weight, based on the total weight of the composition, of the cross-linked material. In a preferred embodiment, in particular when the composition of the present invention is a (hydro)gel or a liquid composition, the composition comprises not more than 20 % by weight, not more than 10 % by weight, not more than 5 % by weight, not more than 3.5 % by weight, or not more than 2 % by weight, or not more than 1 % by weight, based on the total weight of the composition, of the cross-linked material.

[0192] In a preferred embodiment, the composition of the present invention comprises 0.01 to 99.9 % by weight, 0.1 to 99.9 % by weight, 0.15 to 90 % by weight, 0.2 to 80 % by weight, 0.5 to 70 % by weight, 1 to 50 % by weight, 5 to 40 % by weight, or 10 to 25 % by weight, based on the total weight of the composition, of the cross-linked material. In a preferred embodiment, in particular when the composition of the present invention is a (hydro)gel or a liquid composition, the composition comprises 0.1 to 3.5 % by weight, based on the total weight of the composition, of the crosslinked material. In a preferred embodiment, in particular when the composition of the present invention is a (hydro)gel, a liquid, or a viscous composition, the composition comprises 0.11 to 3.0 % by weight, 0.12 to 2.5 % by weight, 0.15 to 2.0 % by weight, 0.2 to 1 .5 % by weight, 0.5 to 1 .0 % by weight, 0.5 to 2.0 % by weight, or 1 .0 to 3.0 % by weight, based on the total weight of the composition, of the crosslinked material.

[0193] In a preferred embodiment, the composition of the present invention comprises at least 0.1 units of botulinum toxin per gram of the composition (which has the same meaning as 0.1 units / g, based on the total weight of the composition). In a preferred embodiment, the composition of the present invention comprises at least 0.2 units / g, at least 0.5 units / g, at least 1 unit / g, at least 3 units / g, at least 5 units / g, at least 10 units / g, at least 25 units / g, at least 50 units / g, at least 75 units / g, at least 100 units / g, at least 150 units / g, at least 200 units / g, at least 250 units / g, or at least 500 units / g, based on the total weight of the composition, of botulinum toxin. In a preferred embodiment, in particular when the composition of the present invention is a (hydro)gel or a liquid composition, the composition comprises at least 0.1 units of botulinum toxin per mL of the composition (which has the same meaning as 0.1 units / mL, based on the total volume of the composition).

[0194] In a preferred embodiment, the composition of the present invention comprises at least 0.2 units / mL, at least 0.5 units / mL, at least 1 unit / mL, at least 3 units / mL, at least 5 units / mL, at least 10 units / mL, at least 25 units / mL, at least 50 units / mL, at least 75 units / mL, at least 100 units / mL, at least 150 units / mL, at least 200 units / mL, at least 250 units / mL, or at least 500 units / mL, based on the total volume of the composition, of botulinum toxin. In a preferred embodiment, one injectable dose of the composition of the present invention comprises at least 0.1 units of botulinum toxin. In a preferred embodiment, an injectable dose of the composition of the present invention comprises at least 0.2 units, at least 0.5 units, at least 1 unit, at least 3 units, at least 5 units, at least 10 units, at least 25 units, at least 50 units, at least 75 units, at least 100 units, at least 150 units, at least 200 units, at least 250 units, or at least 500 units of botulinum toxin.

[0195] In a preferred embodiment, the composition of the present invention comprises not more than 1000 units / g, based on the total weight of the composition, of botulinum toxin. In a preferred embodiment, the composition of the present invention comprises not more than 500 units / g, not more than 250 units / g, not more than 200 units / g, not more than 150 units / g, not more than 100 units / g, not more than 50 units / g, or not more than 10 units / g based on the total weight of the composition, of botulinum toxin. In a preferred embodiment, in particular when the composition of the present invention is a (hydro)gel, a liquid or a viscous composition, the composition comprises not more than 1000 units / mL, based on the total volume of the composition, of botulinum toxin. In a preferred embodiment, the composition of the present invention comprises not more than 500 units / mL, not more than 250 units / mL, not more than 200 units / mL, not more than 150 units / mL, not more than 100 units / mL, not more than 50 units / mL, or not more than 10 units / mL based on the total volume of the composition, of botulinum toxin. In a preferred embodiment, an injectable dose of the composition of the present invention comprises not more than 1000 units, of botulinum toxin.

[0196] In a preferred embodiment, the composition of the present invention comprises not more than 500 units, not more than 250 units, not more than 200 units, not more than 150 units, not more than 100 units, not more than 50 units, or not more than 10 units of botulinum toxin.

[0197] In a preferred embodiment, the composition of the present invention comprises 0.1 to 1000 units / g, based on the total weight of the composition, of botulinum toxin.

[0198] In a preferred embodiment, the composition of the present invention comprises 0.1 to 500 units / g, 0.1 to 250 units / g, 0.2 to 200 units / g, 0.5 to 150 units / g, 1 to 100 units / g, 10 to 100 units / g, 20 to 75 units / g, or 10 to 50 units / g, based on the total weight of the composition, of botulinum toxin. In a preferred embodiment, in particular when the composition of the present invention is a (hydro)gel, a liquid or a viscous composition, the composition comprises 0.1 to 500 units / mL, 0.1 to 250 units / mL, 0.1 to 150 units / mL, 0.1 to 100 units / mL, 0.2 to 200 units / mL, 0.5 to 150 units / mL, 1 to 100 units / mL, 10 to 100 units / mL, 20 to 75 units / mL, or 10 to 50 units / mL, based on the total volume of the composition, of botulinum toxin. In a preferred embodiment, an injectable dose of the composition of the present invention comprises 0.1 to 500 units, 0.1 to 250 units, 0.2 to 200 units, 0.5 to 150 units, 1 to 100 units, 10 to 100 units, 20 to 75 units, 10 to 50 units, 20 to 40 units, or 40 to 60 units of botulinum toxin.

[0199] The composition of the present invention may comprise 0 to 99.9 % by weight, based on the total weight of the composition, of one or more liquid cosmetically and / or pharmaceutically acceptable carriers. In a preferred embodiment, the composition of the present invention comprises 0.1 to 99.9 % by weight, 1 to 99.5 % by weight. 5 to 99 % by weight, 10 to 95 % by weight, 20 to 92 % by weight, 30 to 90 % by weight, 50 to 85 % by weight, 60 to 80 % by weight, 65 to 75 % by weight, 65 to 99 % by weight, or 70 to 99 % by weight, based on the total weight of the composition, of one or more liquid cosmetically and / or pharmaceutically acceptable carriers.

[0200] In a preferred embodiment, the composition of the present invention comprises 0 to 1 % by weight, based on the total weight of the composition, of one or more anesthetics. In a preferred embodiment, the composition of the present invention comprises 0.01 to 1 % by weight, 0.05 to 0.9 % by weight, 0.1 to 0.8 % by weight, 0.1 to 0.7 % by weight, 0.2 to 0.5 % by weight, 0.2 to 0.4 % by weight, based on the total weight of the composition, of one or more one or more anesthetics.

[0201] In a preferred embodiment, the composition of the present invention comprises 0 to 50 % by weight, based on the total weight of the composition, of one or more further cosmetically and / or pharmaceutically acceptable ingredients. In a preferred embodiment, the composition of the present invention comprises up to 40 % by weight, up to 30 % by weight, up to 20 % by weight, up to 10 % by weight, up to 5 % by weight, up to 1 % by weight, up to 0.5 % by weight, or up to 0.1 % by weight, based on the total weight of the composition, of one or more further cosmetically and / or pharmaceutically acceptable ingredients. In a preferred embodiment, the composition of the present invention comprises 0.1 to 50 % by weight, 0.1 to 40 % by weight, 0.1 to 30 % by weight, 0.1 to 20 % by weight, 0.1 to 10 % by weight, 0.1 to 5 % by weight, 0.1 to 1 % by weight, 0.2 to 50 % by weight, 0.5 to 40 % by weight, 1 to 30 % by weight, 2 to 20 % by weight, 5 to 10 % by weight, 10 to 20 % by weight, or 20 to 50 % by weight, based on the total weight of the composition, of one or more further cosmetically and / or pharmaceutically acceptable ingredients. In a preferred embodiment, the composition, which preferably is an injectable composition, comprising as component (B) 1 to 150 units of at least one botulinum toxin per mL of the composition, and further comprises or consists of:

[0202] (A) 0.1 to 99.9 % by weight, preferably 0.1 to 10 % by weight, more preferably

[0203] 0.1 to 3.5 % by weight, based on the total weight of the composition, of the cross-linked material;

[0204] (C) 0 to 99.8 % by weight, preferably 50 to 99.8 % by weight, based on the total weight of the composition, of one or more liquid cosmetically and / or pharmaceutically acceptable carriers;

[0205] (D) 0 to 10 % by weight, preferably 0 to 5 % by weight, more preferably 0 to

[0206] 1 % by weight, based on the total weight of the composition, one or more anesthetics; and

[0207] (E) 0 to 50 % by weight, preferably 0 to 49.8 % by weight, based on the total weight of the composition, of one or more further cosmetically and / or pharmaceutically acceptable ingredients.

[0208] In a preferred embodiment, the composition comprises at least 10 % by weight, based on the total weight of the composition, of a liquid pharmaceutically acceptable carrier. In a preferred embodiment, the composition comprises at least 20 % by weight, at least 30 % by weight, at least 40 % by weight, at least 50 % by weight, at least 60 % by weight, at least 65 % by weight, at least 70 % by weight, at least 75 % by weight, at least 80 % by weight, at least 85 % by weight, at least 90 % by weight, or at least 95 % by weight, based on the total weight of the composition, of a liquid pharmaceutically acceptable carrier.

[0209] In a preferred embodiment, the composition is an injectable composition, comprising as component (B) 1 to 150 units of at least one botulinum toxin per mL of the composition, and further comprises or consists of:

[0210] (A) 0.1 to 3.5 % by weight, preferably 0.5 to 3.5% by weight, based on the total weight of the composition, of the cross-linked material;

[0211] (C) 10 to 99.8 % by weight, preferably 50 to 99.8% by weight, more preferably 65 to 99.4% by weight, based on the total weight of the composition, of one or more liquid cosmetically and / or pharmaceutically acceptable carriers;

[0212] (D) 0 to 1 % by weight, based on the total weight of the composition, one or more anesthetics; and

[0213] (E) 0 to 89.8 % by weight, preferably 0 to 49.8% by weight, more preferably 0 to 49.4% by weight, more preferably 0 to 34.8% by weight, based on the total weight of the composition, of one or more further cosmetically and / or pharmaceutically acceptable ingredients.

[0214] The components (A) and (B), optionally (C), optionally (D), and optionally (E), may be combined with each other by any means. For instance, the components (B), optionally (C), optionally (D), and optionally (E) are dissolved or suspended in a liquid form and mixed with the cross-linked material (A) which is also forming a (hydro)gel. Optionally, the one or more components (B), optionally (D), and optionally (E) are dissolved or suspended in component (C). Optionally, a (hydro)gel of the cross-linked material (A) is formed in component (C). The sequential order of mixing may be freely chosen. Optionally, there may be any premixes of two or more components.

[0215] Component (B) and optionally one or more of components (C), (D) and / or (E) may be mixed with at least one cross-linked material (component (A)). The components

[0216] (A), (B) and (C), and optionally (D) and / or (E) may be mixed with each other by any means. In a preferred embodiment, component (A) is suspended and component

[0217] (B) is dissolved in one or more liquid cosmetically and / or pharmaceutically acceptable carriers (component (C)). Optionally, further component (D) and / or component (E) may be further be present in this composition.

[0218] For example, for mixing, component (A) may be present in one syringe and component (B) dissolved in component (C) optionally further comprising one or more of components (D) and / or (E) may be present in second syringe. The two syringes may be connected (e.g., by a luer lock) with each other. By pushing content of the second syringe into the first syringe, the materials may be mixed. Optionally, the syringes are pushed from one syringe to the other several times.

[0219] It will be understood that the cross-linked material obtainable instep (iii) of the method of the present invention bears special technical characteristics such as structural characteristics and the absence of harsh (bi)functional linkers and activating agents. As indicated above, the cross-linked material as described and defined herein may have various functionalities, optionally including allowing the retarded and / or controlled release of one or more pharmaceutically and / or cosmetically active agents, such as, e.g., botulinum toxin. It will be understood that the retarded and / or controlled release (e.g., depot) function of the hydrogel can be adopted by means of adapting the cross-linking range, the content of the crosslinked material in the composition and the ratio between the botulinum toxin and the cross-linked material. This may address the needs of the relevant indication (e.g. reducing adverse effects, elongating the duration of botulinum effect, etc.).

[0220] Accordingly, a further aspect of the present invention relates to the use of a crosslinked material for retarded and / or controlled release of one or more pharmaceutically and / or cosmetically active agents, in particular botulinum toxin, wherein the cross-linked material is a cross-linked material obtained in step (iii) of the method according to the invention. Preferably, the cross-linked material forms a gel with a liquid pharmaceutically acceptable carrier. More preferably, the crosslinked material forms part of a composition according to the invention. In a preferred embodiment, the cross-linked material forms gel, in particular in combination with one or more liquid cosmetically and / or pharmaceutically acceptable carriers.

[0221] It will be understood that the definitions and preferred embodiments as laid out in the context of the cross-linked material comprisable in the composition of the present invention mutatis mutandis apply to the use of the cross-linked material of the present invention.

[0222] The composition of the present invention may be used for any purpose. For instance, it may be used for therapeutic purposes and / or for cosmetic purposes. In other words, it may be used for aesthetic or cosmetic indications.

[0223] Accordingly, a further aspect of the present invention relates to the composition of the present invention for use as a medicament. Accordingly, the present invention further relates to a medicament comprising the composition of the present invention. Accordingly, the present invention further relates to the use of the composition of the present invention for the preparation of a medicament.

[0224] It will be understood that the definitions and preferred embodiments as laid out in the context of the composition of the present invention mutatis mutandis apply to the use of the composition for any purpose including a use in a therapeutic context as well as to a medicament comprising the composition of the present invention.

[0225] A further aspect of the present invention relates to the composition of the present invention for use in a method of treating or preventing a disease or condition associated with hyperactive cholinergic activity.

[0226] Accordingly, in other words, the present invention relates to a method of treating or preventing a disease or condition associated with hyperactive cholinergic activity, comprising a step of administering a sufficient amount of the composition of the present invention to a subject in need thereof. Moreover, the present invention relates to a medicament for treating or preventing a disease or condition associated with hyperactive cholinergic activity, comprising the composition of the present invention. Accordingly, the present invention further relates to the use of the composition of the present invention for the preparation of a medicament for treating or preventing a disease or condition associated with hyperactive cholinergic activity.

[0227] It will be understood that the definitions and preferred embodiments as laid out in the context of the composition of the present invention and the use of the present invention mutatis mutandis apply to the use in treating or preventing and a method of treating or preventing of the present invention.

[0228] As used herein, the term “hyperactive cholinergic innervation” may be understood in the broadest sense as generally understood in the art. It may be understood as relating to a synapse, which is characterized by an unusually high amount of acetylcholine release into the synaptic cleft. In this context, “unusually high” may relate to an increase of, e.g., up to 25%, up to 50% or more with respect to a reference activity which may be obtained, for example, by comparing the release with the release at a synapse of the same type but which is not in a hyperactive state, wherein muscle dystonia may be indicative of the hyperactive state. In this context, “up to 25%” may, for example, be understood as >0% to about 25%. Methods for performing the measurements of synaptic activities are known in the art.

[0229] As used herein, a subject (also: an individual) may be any animal, typically a mammal, preferably a domestic mammal or a human. Particularly preferably, an individual is a human. A treated human can also be designated as a patient, independent on his / her health state.

[0230] Administration may be conducted by any means. In a preferred embodiment, administration is administration via a syringe, in particular intradermal, subdermal, subcutaneous or intramuscular administration via a syringe. Preferably, administration is intramuscular administration. An injectable composition within the meaning of the present invention may be administered by means of (dispensed from) syringes under normal conditions under normal pressure. Moreover, the composition of the present invention is preferably (essentially) sterile. Preferably, the injectable composition is suitable for injection into a mammal, in particular a human. Administration may be manual administration, administration using a mechanical pump, or even automated administration. For instance, a 1 mL syringe may be used for administration. As an example, an injection may be carried out with an injection needle, such as, e.g., with an injection needle size in a range of 20 to 40 gauge, 27 to 35 gauge, or 30 to 33 gauge.

[0231] For instance, administration may be in the dermis area, such as below the epidermis or above the hypodermis and as such the composition may be injected subcutaneously, hypodermically, intradermally, subdermally, or some combinations. In another embodiment, the composition may be administered intramuscularly.

[0232] Hyperactive cholinergic activity may be such of any part of the subject’s body. In a preferred embodiment, the hyperactive cholinergic activity is such of muscles and / or glands.

[0233] The disease or condition treated in the context of the present invention may be any disease or condition associated with hyperactive cholinergic activity. In a preferred embodiment, the disease or condition is selected from the group consisting of a motoric disorder, in particular dystonia and / or spasticity, post-stroke spasticity, cervical dystonia, a neuromuscular diseases such as dystonia, spasm, cramps, blepharospasm, tremor, hyperkinetic movement disorders, and cerebral palsy, pain such as diabetic neuropathy, peripheral neuropathy, neuropathic pain and trigeminal pain, pathologically high saliva production sialorrhea, hyperhidrosis, urological disorders such as bladder dysfunction, overactive bladder, detrusor overactivity, neurogenic bladder and interstitial cystitis, sphincter-oddi dysfunction, treatment of vulvodynia and chronic pelvic pain, prostate disorder such as benign prostate hyperplasia (BPH) and detrusor sphincter dyssynergia (DSD), neurological disorders such as chronic or episodic migraines, strabismus, (tension) headaches, achalasia, Hirschsprung's disease, anal fissure, a dermatological or aesthetical problem improvable by muscle relaxation, articular pathology, arthritis, and a combination of two or more thereof.

[0234] In a preferred embodiment, the disease or condition is selected from the group consisting of neuromuscular diseases such as dystonia, spasm, tremor, hyperkinetic movement disorders, and cerebral palsy, pain such as diabetic neuropathy, peripheral neuropathy, neuropathic pain and trigeminal pain, sialorrhea, hyperhidrosis (also: hyperhydrosis), urological disorders such as detrusor overactivity, overactive bladder, neurogenic bladder and interstitial cystitis, treatment of vulvodynia and chronic pelvic pain, prostate disorder such as benign prostate hyperplasia (BPH) and detrusor sphincter dyssynergia (DSD), and neurological disorders such as chronic or episodic migraines, cervical dystonia, post-stroke spasticity, and blepharospasm, and a combination of two or more thereof.

[0235] As used herein, dystonia may be understood in the broadest sense as any dystonia. It may, for instance, be focal dystonia (typically affecting just one part of the subject’s body). Segmental dystonia (typically affecting more than one part of the subject’s body), or generalized dystonia (typically affecting (nearly) the whole body). Preferably, dystonia is focal dystonia or segmental dystonia. Examples of focal dystonia are laryngeal dystonia (voice dystonia) or cervical dystonia (misalignment of the head / neck) or, wherein cervical dystonia might be also considered as segmental dystonia, blepharospasm (uncontrolled blinking of a lid), oromandibular dystonia (affection of the mouth area and / or masticatory) and spasmodic dysphonia (affection of the vocal cords). An Example of generalized dystonia is the Segawa syndrome, which is characterized by anomalies of the leg positions. In a preferred embodiment, when treating dystonia, the region affected by dystonia is administered parenterally, preferably in form of an injection. Such injection may be preferably carried out in or close to the affected body part, wherein the injection is preferably subcutaneous or intramuscular, in particular intramuscular.

[0236] Spasticity may be understood in the broadest sense as generally understood in the art. It may be understood as an enhanced internal tension of the (skeletal) muscles. The origin for spasticity is a damage of the areas of the central nervous system responsible for motions, wherein these areas are brain and spinal cord, in particular the pyramidal tract of the first motoneuron. A common reason for spasticity is a hypoxic damage of the motoric brain area by a cerebral infarct. A monospasticity is a spastic paralysis of one muscle or one extremity. Examples of muscles which may be spastically paralyzed are flexor carpi radialis, flexor carpi ulnaris, flexor, digitorium superficalis, flexor digitorium profundis, brachioradialis, biceps brachial is, pronator quadratus, pronator teres, flexor pollicis longus, flexor pollicis brevis, opponens pollicis. A paraspasticity is characterized by the paralysis of both legs. A hemispasticity describes the paralysis of the extremities of one half of the body or one half of the face (hemifacial spasm). Finally, a tetraspasticity relates to the spastic paralysis of all four extremities, wherein further also the neck and core muscles may be affected. In one embodiment, the present composition for use in the treatment of spasticity is administered parenterally, preferably in form of an injection. Such injection may be preferably carried out in or close to the affected body part, wherein the injection is preferably subcutaneous or intramuscular, in particular intramuscular.

[0237] The composition of the present invention can also be used as a filler composition such as a soft tissue filler, in particular a dermal filler or connective tissue filler. Thus, the present invention also refers to the use of the composition of the present invention as filler composition such as a soft tissue filler, in particular a dermal filler or connective tissue filler.

[0238] A further aspect of the present invention relates to the use of a composition of the present invention for cosmetic applications comprising rejuvenation and / or improvement of the skin quality of the face and / or body.

[0239] In other words, the present invention relates to a method of rejuvenation and / or improvement of the skin quality of the face and / or body, comprising a step of administering a sufficient amount of the composition of the present invention to a subject in need thereof.

[0240] In other words, the present invention relates to the composition of the present invention for use in a method of rejuvenation and / or improvement of the skin quality of the face and / or body.

[0241] As noted above, the definitions and preferred embodiments as laid out in the context of the composition of the present invention mutatis mutandis apply to the use of the composition and any method of rejuvenation and / or improvement of the skin quality of the face and / or body.

[0242] In an embodiment, the use of the present invention may be a cosmetic use, thus may also be a non-therapeutic use. The use of the present invention may be conducted by cosmetics, cosmetic professionals or health care professionals.

[0243] In a preferred embodiment, the rejuvenation and / or improvement of the skin quality of the face and / or body comprises improving and / or reducing and / or filling and / or preventing wrinkles, in particular of wrinkles resulting from muscular activity such as mimic activity, skin smoothing, improving skin laxity, lifting effect, moisturizing and / or softening the skin, improving and / or reducing and / or filling facial lines, soft-tissue augmentation, improving subdermal support of the brows, malar and buccal fat pads, improving tear troughs, improving nose appearance, resolving facial asymmetries, reducing the prominence of the masseter muscle, improving jawlines, or a combination of two or more thereof. According to general understanding, lines may also be understood as wrinkles and vice versa. The terms “lines” and “wrinkles” may be used interchangeably.

[0244] As used herein, improvable and / or reducible and / or fillable facial lines may exemplarily be selected from the group consisting of horizontal forehead lines, glabellar lines (e.g., glabellar frown lines), periorbital lines, crow’s feet, bunny lines, nasolabial folds, peri-lip lines, upper radial lip lines, lower radial lip lines, corner of the mouth lines, marionette lines, perioral lip lines, oral commissures, labiomental crease and cobblestone chin. In a preferred embodiment, improvable facial lines may be horizontal forehead lines and / or glabellar lines and / or periorbital lines including crow’s feet.

[0245] In a further preferred embodiment, the improvement of the skin quality of the face and / or body comprises the reduction of skin pore size and / or sebum production. In a further preferred embodiment, the rejuvenation comprises the treatment of platysma bands and / or platysma muscles.

[0246] The composition of the present invention may also be used for any other purpose. It may be used for any purpose described in WO 2017 / 148915.

[0247] The composition of the present invention may be provided in any package. Depending on the intended use of the composition of the present invention, it can be provided in different packaging. It may be stored at any condition suitable for this purpose such as, e.g., at ambient temperature (e.g., 18 to 30°C, preferably 18 to 25°C), in a fridge (e.g., at 0 to 15°C, preferably 3 to 10°C), in a freezer (e.g., -30 to 0°C, preferably -25 to -10°C), in a deep freezer (e.g., -100 to -300°C, preferably -90 to -55°C), on liquid nitrogen, on dry ice, or even one or more liquid noble gases. For instance, it may be provided in a vial, in a syringe. It may be administered to a subject via injection (e.g., via a syringe or a drip). It may be stored as a hydrogel, as a gel containing other non-aqueous solvents, and / or as a suspension, emulsion, colloid or solution.

[0248] For the above therapeutic and cosmetic uses, the composition of the is particularly beneficial because the cross-linked material present therein is rather stable in aqueous environments such as body fluids and enables invasion of cells due to its cross-linked structure and surface characteristics.

[0249] As used herein, the terms “approximately” and “about” may be understood as a scope including a deviation of up + / - 10% of the respective number value. It will be understood that the specific values are also explicitly disclosed.

[0250] It will be further understood that the scope embraces the number values provided as commonly rounded values that embrace the whole rounding limits. For example, the scope of “1 mg” embraces the range of from 0.50 to 1 .49 mg.

[0251] The number values of the present invention, however, also disclose the more detailed values of one or more orders of magnitude more in detail. Accordingly, for example, “1 mg” may also include the specific disclosure of “1 .0 mg”.

[0252] The examples and claims illustrate embodiments of the present invention.

[0253] Examples

[0254] Example 1

[0255] Preparation of cross-linked hyaluronic acid material

[0256] 6.96 g hyaluronic acid (HA) (dry weight) with an intrinsic viscosity of 2.96 m3 / kg was solubilized in 160 mL of phosphate buffered saline (PBS) buffer of pH 7.4. After this, 2.07 g of the triazin-based activating agent 4-(4,6-dimethoxy-1 ,3,5-triazin-2-yl)-4- methylmorpholinium chloride (DMTMM) (0.5 molar equivalents (eq.) based on the carboxyl groups of HA) were dissolved in 40 mL PBS buffer and the DMTMM solution was mixed with the HA solution. Mixing was conducted until a visually homogeneous composition was achieved. This was typically achieved after approximately 10 min. The resultant mixture was allowed to react for 16 h at 23 °C.

[0257] Subsequently, the cross-linked material (present as a hydrogel) was placed in dialysis membranes (molecular weight cut-off (MWCO) 12-14 kDa) and was purified using PBS buffer for approximately 2 days to substantially remove the side products of the DMTMM. The final product was placed in 1 mL syringes and sterilized at 131 °C for 1 min. After sterilization, 50 units of BoNT (botulinum neurotoxin serotype A1 ) were added to 1 mL of the prepared hydrogel.

[0258] Rheology of the hydrogel with and without BoNT was determined using a 302 Modular Compact Rheometer (Anton Paar GmbH, Graz, Austria). Briefly, cone / plate in oscillation mode (CP50-1 ) at 25 °C was used; Deformation was set to 0.1 %, and the oscillation frequency ranged from 10 to 0.1 Hz. Reported G' values correspond to 1 Hz. Storage modulus (G’), loss factor (tan(5)) and complex viscosity at 1 Hz were reported as the measurement results.

[0259] It was found that the cross-linked material and the composition comprising BoNT both had beneficial properties, and the presence of BoNT did not substantially affect rheology.

[0260] A summary of the results is shown in the Table 1 below. The rheological parameters were consistent with other polysaccharide-based hydrogels (e.g., HA fillers).

[0261] Table 1. Summary of rheology measurements for the hydrogel without and with

[0262] BoNT

[0263] These properties can be conveniently adapted by changing the number of molar equivalents of DMTMM based on the carboxyl groups of HA. Similar observations are made when other polysaccharide moieties such as chondroitin sulfate or heparosan are used.

Claims

Merz Pharma GmbH & Co. KGaA 4 November 2025MP75182PCPatent claims1 . A method for preparing a composition comprising:(A) a cross-linked material comprising one or more polysaccharide moieties covalently cross-linked with each other through ester bonds, wherein the cross-linked material comprises more ester bonds cross-linking polysaccharide moieties covalently with each other than amide bonds crosslinking polysaccharide moieties covalently with each other;(B) at least one botulinum toxin;(C) optionally one or more liquid cosmetically and / or pharmaceutically acceptable carriers;(D) optionally one or more anesthetics; and(E) optionally one or more further cosmetically and / or pharmaceutically acceptable ingredients different from components (A) to (D), wherein the method comprises the steps:(i) combining the following components with each other: a) polysaccharide moieties comprising carboxylic acid residues or salts thereof and hydroxy residues, b) one or more triazine-based activating agents that effect reaction of carboxylic acid residues with hydroxy residues thereby forming ester bonds, and c) one or more solvents; and(ii) allowing at least some of the carboxylic acid residues of the polysaccharide moieties to react with at least some of the hydroxy residues of the polysaccharide moieties, thereby forming ester bonds covalently crosslinking polysaccharide moieties with each other; and(iii) obtaining cross-linked material from step (ii) as component (A) and optionally purifying the cross-linked material obtained from step (ii); and(iv) adding: at least one botulinum toxin as component (B), and optionally one or more liquid cosmetically and / or pharmaceutically acceptable carriers as component (C), and / or optionally one or more anesthetics as component (D), and / oroptionally one or more further cosmetically and / or pharmaceutically acceptable ingredients different from components (A) to (D) as component (E); with the proviso that step (ii) is conducted in the absence of carbodiimide activating agents, succinimidyl-based activating agents, glycidyl-based activating agents, paranitrophenol esters, and 2-chloro-methylpyridinium iodide.

2. The method according to claim 1 , wherein the polysaccharide moieties comprise hyaluronic acid moieties, glycerol-grafted hyaluronic acid moieties, heparosan moieties, chondroitin sulfate moieties, and carboxymethyl cellulose moieties, preferably hyaluronic acid moieties.

3. The method according to claim 1 or 2, wherein the botulinum toxin is botulinum neurotoxin serotype A, in particular botulinum neurotoxin serotype A1 .

4. The method according to any one of claims 1 to 3 for preparing an injectable composition, comprising as component (B) 1 to 150 units of at least one botulinum toxin per mL of the composition, and further comprising(A) 0.1 to 3.5 % by weight, based on the total weight of the composition, of the cross-linked material;(C) 50 to 99.8 % by weight, preferably 59 to 99.8 % by weight, based on the total weight of the composition, of one or more liquid cosmetically and / or pharmaceutically acceptable carriers, preferably comprising water, an aqueous buffer, glycerol or a combination of two or more thereof;(D) 0 to 1 % by weight, based on the total weight of the composition, of one or more anesthetics;(E) 0 to 49.8 % by weight, based on the total weight of the composition, one or more further cosmetically and / or pharmaceutically acceptable ingredients.

5. The method according to any one of claims 1 to 4, wherein the cross-linked material comprises from 0 to 0.04%, preferably from 0 to 0.02%, more preferably from 0 to 0.01 %, based on the total number of ester bonds and amide bonds, of amide bonds.

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

7. A composition obtainable by the method according to any one of claims 1 to 6, wherein the composition is free of carbodiimide activating agents, succinimidyl-based activating agents, glycidyl-based activating agents, paranitrophenol esters, 2-chloro-methylpyridinium iodide, and their reaction products.

8. The composition according to claim 7, wherein the composition comprises at least one triazine-based activating agent or a reaction product thereof.

9. The composition according to claim 7 or 8, wherein the botulinum toxin is not covalently bound with the cross-linked material, and / or wherein the cross-linked material forms a gel in the liquid pharmaceutically acceptable carrier, in particular a hydrogel, and the botulinum toxin is dissolved in the liquid pharmaceutically acceptable carrier soaked in the gel, in particular hydrogel.

10. The composition according to any one of claims 7 to 9, wherein the composition comprises at least 50 % by weight, based on the total weight of the composition, of a liquid pharmaceutically acceptable carrier, preferably wherein the composition is injectable in a soft tissue, in particular injectable intradermally, subdermally, subcutaneously, and / or intramuscularly.11 . The use of a cross-linked material for retarded and / or controlled release of one or more pharmaceutically and / or cosmetically active agents, in particular botulinum toxin, wherein the cross-linked material is a cross-linked material obtained in step iii) of the method according to any one of claims 1 to 5, preferably wherein the cross-linked material forms a gel with a liquid pharmaceutically acceptable carrier, in particular wherein the cross-linked material forms part of a composition according to any one of claims 7 to 10.

12. The composition according to any one of claims 7 to 10 for use as a medicament.

13. The composition according to any one of claims 7 to 10 for use in a method of treating or preventing a disease or condition associated with hyperactive cholinergic activity, in particular of muscles and / or glands, and / or pain, preferably wherein the disease or condition is selected from the group consisting of a motoric disorder, in particular dystonia and / or spasticity, a neuromuscular diseases such as dystonia, spasm, tremor, hyperkinetic movement disorders, and cerebral palsy, pain such as diabetic neuropathy, peripheral neuropathy, neuropathic pain and trigeminal pain, sialorrhea, hyperhidrosis, urological disorders such as detrusor overactivity, neurogenic bladder and interstitial cystitis, treatment of vulvodynia and chronic pelvic pain, prostate disorder such as benign prostate hyperplasia (BPH) and detrusor sphincter dyssynergia (DSD), migraine, neurological disorders such as chronic or episodic migraines, cervical dystonia, post-stroke spasticity, blepharospasm, strabismus, (tension) headaches, pathologically high saliva production, achalasia, sphincter-oddi dysfunction, Hirschsprung's disease, anal fissure, bladder dysfunction such as overactive bladder, a dermatological or aesthetical problem improvable by muscle relaxation, articular pathology, arthritis, and a combination of two or more thereof.

14. The use of a composition according to any one of claims 7 to 10 for cosmetic applications comprising rejuvenation and / or improvement of the skin quality of the face and / or body, preferably including improving and / or reducing and / or filling and / or preventing wrinkles, in particular of wrinkles resulting from muscular activity such as mimic activity, skin smoothing, improving skin laxity, lifting effect, moisturizing and / or softening the skin, improving and / or reducing and / or filling facial lines, soft- tissue augmentation, improving subdermal support of the brows, malar and buccal fat pads, improving tear troughs, improving nose appearance, resolving facial asymmetries, reducing the prominence of the masseter muscle, improving jawlines, or a combination of two or more thereof.

15. The use according to claim 14, wherein the improvement of the skin quality of the face and / or body comprises the reduction of skin pore size and / or sebum production, and / or the rejuvenation comprises the treatment of platysma bands and / or platysma muscles.

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