Injectable gels comprising botulinum toxins and uses thereof

Abstract

The present invention relates to a composition comprising (A) a crosslinked material comprising one or more silk fibroin moieties and one or more polysaccharide moieties covalently bound to each other, (B) a botulinum toxin. Furthermore, the present invention relates to therapeutic and cosmetic uses of such a composition.

Description

[0001] The present invention relates to a composition comprising (A) a crosslinked material comprising one or more silk fibroin moieties covalently bound to one or more polysaccharide moieties, and (B) botulinum toxin. Furthermore, the present invention relates to the therapeutic and cosmetic use of such a composition.

[0002] Since the 1980s, botulinum toxin (also known as: botulinum neurotoxin, BoNT) has been used to treat or prevent movement disorders such as dystonia or spasticity, and / or wrinkles. Botulinum toxin is used in the pharmaceutical and cosmetic fields. Botulinum toxin inhibits the release of acetylcholine, which results in reduced muscle contraction or even muscle paralysis. Botulinum toxin is well known for reducing and preventing wrinkles caused by muscle contraction. In addition, 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). Commercial products based on botulinum toxin are (Merz Pharmaceuticals GmbH, Germany).

[0003] However, botulinum toxin is inherently unstable, especially under alkaline conditions. It is also heat intolerant. For this reason, commercial botulinum toxin is usually stored in the form of vacuum-dried (lyophilized) material and / or contains protective and preservative excipients.

[0004] Several uses are based on injecting botulinum toxin directly into the skin or other soft tissues of a subject, or under the skin or other soft tissues to the site of the desired effect. In typical long-term therapeutic or cosmetic uses, due to the reversibility of its inhibitory effect on acetylcholine release, botulinum toxin must be injected again after a certain time (usually every few months). Since botulinum toxin directly affects the endocytosis and exocytosis mechanisms of cells by inactivating SNARE proteins, the absorption time of botulinum toxin is limited, about one day, after which further absorption is inhibited by blocking the endocytic pathway. This is undesirable in many applications. Therefore, it is desirable to extend the duration of the effect of botulinum toxin injection, in other words, to provide sustained-release and / or controlled-release formulations thereof that can adjust the retention and release profiles of botulinum toxin according to the absorption capacity of the targeted cells. This is achieved in part by preparing sustained-release and / or controlled-release formulations in which 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 are considered. US 9,044,477 and US 9,050,336 teach mixtures comprising chemical entities including hyaluronic acid, botulinum toxin, and albumin. Among them, the viscous material is formed by hyaluronic acid at an adjustable concentration, where the hyaluronic acid can also be cross-linked hyaluronic acid. WO 2020 / 056371 teaches a mixture of clostridial toxin and non-cross-linked hyaluronic acid or its salt. The hyaluronic acid, botulinum toxin, and albumin components are simply mixed in the composition. WO 2011 / 119468 teaches hydrogels for soft tissue augmentation, which comprise polymers that can be hyaluronic acid and which can further comprise other components. WO 2017 / 148915 teaches a composition comprising non-cross-linked hyaluronic acid and botulinum toxin. Some of these compositions are well compatible with injection and are generally useful for soft tissue augmentation.

[0005] However, the stability (including shelf life and sustained-release and / or controlled-release of botulinum toxin) is not ideal. In particular, the compositions of the prior art mentioned above can hardly adapt to the desired release kinetics. This can only be roughly defined by the concentration and chain length of the hydrogel-forming components.

[0006] It is believed that crosslinking the components via a chemical crosslinked structure can achieve an ideal shelf stability and improve the adjustability of release kinetics. WO 2020 / 132331 describes conjugates of silk fibroin moieties, preferably crosslinked hyaluronic acid moieties and crosslinking moieties such as polyethylene glycol (PEG) and / or polypropylene glycol (PPG), which can be used as tissue fillers. In addition, this document teaches that various other components can be included in the tissue filler. A disadvantage of such conjugates is that synthetic exogenous moieties are included in the structure, such as PEG and / or PPG linkers. Such exogenous structures are generally not fully biodegradable and / or bioabsorbable. Such moieties are generally undesirable in materials injected into a subject.

[0007] A bio-based material composed of a silk fibroin moiety and a hyaluronic acid moiety is described in PCT / EP2022 / 066989. However, it is not taught that such conjugates can be used in a composition containing botulinum toxin.

[0008] In view of the above, there remains an unmet need to provide a composition having the following characteristics: having well-controlled and adjustable release of botulinum toxin while widely avoiding synthetic non-biodegradable moieties such as synthetic linker moieties. The main components of such a composition should preferably be widely biodegradable and / or bioabsorbable. The source of the solid main components for preparing such a composition should preferably be bio-based and thus of biological or biotechnological origin, and may optionally contain synthetic agents such as one or more anesthetics. Particularly desirable is an injectable composition that can be used to treat or prevent diseases or disorders associated with cholinergic hyperactivity, particularly hyperactivity of muscles and / or glands, pain, or for restoring and / or improving the skin quality of the face and / or body.

[0009] Surprisingly, we have found that a composition comprising or consisting of a crosslinked material can be used in combination with botulinum toxin to provide a composition having the desired properties, said crosslinked material comprising one or more silk fibroin moieties and one or more polysaccharide moieties, particularly hyaluronic acid moieties, covalently bound to each other without an interconnecting linker structure. Such a composition is injectable and can be used to treat or prevent diseases or disorders associated with cholinergic hyperactivity, particularly hyperactivity of muscles and / or glands, pain, or for restoring and / or improving the skin quality of the face and / or body. Such a composition can be used as a biodegradable and bioabsorbable sustained-release and / or controlled-release formulation of botulinum toxin.

[0010] A first aspect of the present invention relates to a composition comprising or consisting of:

[0011] (A) a crosslinked material comprising or consisting of:

[0012] (A-i) one or more fibroin protein moieties, and

[0013] (A-ii) one or more polysaccharide moieties;

[0014] wherein the one or more fibroin protein moieties are covalently bound to the one or more polysaccharide moieties without an interconnecting linker structure;

[0015] (B) botulinum toxin;

[0016] (C) optionally, one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers;

[0017] (D) optionally, one or more anesthetics; and

[0018] (E) optionally, one or more other cosmetically and / or pharmaceutically acceptable ingredients different from components (A) to (D).

[0019] It has been found that such a composition has unexpected beneficial properties. Due to the avoidance of reactive groups, the composition of the present invention can have a longer shelf life and storability. It also has relative thermal stability. Botulinum toxin can be stabilized in the crosslinked material, and its delayed and / or controlled release can be well adjusted to the intended purpose. Optionally, a delayed release / storage function can be achieved. In addition, when injected into the tissue of a subject, the in vivo lifetime of the crosslinked material can be extended, which may be due to restricted muscle activity. Optionally, due to reduced diffusion, adverse events (such as ptosis) caused by botulinum toxin administration can be reduced. Reducing diffusion during injection can reduce the accidental distribution of the injected botulinum toxin composition in the surrounding tissue. This can reduce side effects and can reduce accidental dilution, thereby prolonging the duration of the effect. Reducing the diffusion of botulinum toxin can result in a controlled (optionally slower) release / distribution of botulinum toxin and optimized cellular uptake. A lower content (e.g., fewer (enzyme) units) of botulinum toxin may be required to achieve comparable effects. A higher content (e.g., more (enzyme) units) of botulinum toxin may have a longer duration of effect but still have an acceptable adverse reaction profile.

[0020] Surprisingly, it has been found that in the context of injectable hydrogels, synthetic linker structures commonly used in the prior art, such as reactive linkers, polyethylene glycol (PEG) and / or polypropylene glycol (PPG), can be avoided. The crosslinking materials used in the compositions of the present invention can consist essentially of an amino acid moiety and a polysaccharide moiety (particularly a hyaluronic acid moiety), which can also exist as separate chemical moieties in nature. In an aqueous environment, the obtained crosslinking materials can form gels, such as hydrogels in particular. Compared with other crosslinking methods in the art, the preparation of (hydro)gels may be relatively easy and more labor-saving. In addition to water or an aqueous buffer, the synthesis of the crosslinking materials uses only three raw materials, namely one or more polysaccharides (particularly a hyaluronic acid moiety) or their salts, one or more fibroin proteins or their salts, and a crosslinking agent that promotes the reaction between the above components and forms an amide bond between the polysaccharide (particularly hyaluronic acid) and fibroin protein.

[0021] The compositions of the present invention can also optionally be used to mimic the extracellular matrix and can, therefore, induce cell proliferation and / or cell migration in addition to the effects achieved by botulinum toxin (such as reducing muscle contraction or inducing muscle paralysis).

[0022] The compositions of the present invention can be used for any purpose. Preferably, the compositions of the present invention can be injectable. For example, it can be used for various cosmetic and therapeutic purposes, as described below.

[0023] The crosslinking materials of the compositions of the present invention can also be represented by abbreviations, such as HA-fibroin, HA / fibroin, HA-Fib, HA / Fib, etc. The crosslinking materials can also be used as biopolymer fillers, such as biopolymer dermal fillers.

[0024] In the context of the present invention, the term "polysaccharide moiety" can be understood most broadly as any moiety of a polysaccharide known in the art, which can bind to one or more fibroin moieties without an interconnecting linker structure.

[0025] In a preferred embodiment, each of the one or more polysaccharide moieties contains one or more carboxylic acid residues or their salts. Preferably, the one or more polysaccharide moieties also contain hydroxyl groups. The polysaccharide can be a naturally occurring polysaccharide, which can be modified, or a synthetic polysaccharide. In this case, the polysaccharide can be branched or unbranched. It should be understood that the term "polysaccharide moiety" can also include salts and their modified forms. In a preferred embodiment, the polysaccharide is not oxidized. The one or more polysaccharide moieties can include one or more types of sugar acid moieties or their salts.

[0026] In a preferred embodiment, the one or more polysaccharide moieties include one or more types of sugar acid moieties or their salts, wherein the one or more types of sugar acid moieties are selected from the group consisting of:

[0027] (B1) one or more uronic acid moieties, in particular selected from the group consisting of glucuronic acid moieties, galacturonic acid moieties, iduronic acid moieties and combinations of two or more thereof;

[0028] (B2) one or more aldaric acid moieties, in particular selected from the group consisting of glyceric acid moieties, xylonic acid moieties, gluconic acid moieties, ascorbic acid moieties and combinations of two or more thereof;

[0029] (B3) one or more ulosonic acid moieties, in particular selected from the group consisting of neuraminic acid moieties, ketodeoxyoctulosonic acid moieties and combinations thereof; and / or

[0030] (B4) one or more aldaric acid moieties, in particular selected from the group consisting of tartaric acid moieties, meso-galactaric acid moieties, glucaric acid moieties and combinations of two or more thereof.

[0031] In a preferred embodiment, one or more polysaccharide moieties comprise uronic acid moieties. In a preferred embodiment, one or more polysaccharide moieties comprise glucuronic acid moieties. In a preferred embodiment, one or more polysaccharide moieties comprise D-glucuronic acid moieties.

[0032] In a preferred embodiment, one or more polysaccharide moieties comprise or consist of D-sugar moieties. In an alternative embodiment, one or more polysaccharide moieties comprise or consist of L-sugar moieties. In an alternative embodiment, one or more polysaccharide moieties comprise or consist of a combination of D-sugar moieties and L-sugar moieties. For example, in such a combination, a racemic mixture of sugar moieties may be included, or a particular sugar moiety is a D-sugar moiety while others are L-sugar moieties.

[0033] In a preferred embodiment, the one or more polysaccharide moieties comprise or consist of one or more glycosaminoglycan moieties. In a preferred embodiment, the one or more polysaccharide moieties comprise or consist of one or more hyaluronic acid (HA) moieties, heparosan moieties, chondroitin sulfate moieties, carboxymethyl cellulose moieties, or a combination of two or more thereof. In a preferred embodiment, the one or more polysaccharide moieties are selected from the group consisting of hyaluronic acid (HA) moieties, heparosan moieties, heparin, chondroitin sulfate, and mixtures of two or more thereof. In a preferred embodiment, the one or more polysaccharide moieties comprise or consist of hyaluronic acid, heparosan, chondroitin sulfate, and carboxymethyl cellulose. Such polysaccharides containing carboxylic acid groups are also commercially available (e.g., from HTL Biotechnology, Javene, France).

[0034] In a preferred embodiment, the one or more polysaccharide moieties comprise or consist of one or more hyaluronic acid moieties.

[0035] Accordingly, the present invention also relates to a composition comprising or consisting of:

[0036] (A) A crosslinked material comprising or consisting of:

[0037] (A-i) One or more fibroin moieties, and

[0038] (A-ii) One or more hyaluronic acid moieties;

[0039] wherein the one or more fibroin moieties are covalently conjugated to the one or more hyaluronic acid moieties without an interconnecting linker structure;

[0040] (B) Botulinum toxin;

[0041] (C) Optionally, one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers;

[0042] (D) Optionally, one or more anesthetics; and

[0043] (E) Optionally, one or more other cosmetically and / or pharmaceutically acceptable ingredients different from components (A) to (D).

[0044] In a preferred embodiment, the crosslinked material of the present invention is a gel. In a preferred embodiment, the crosslinked material of the present invention is a polysaccharide / fibroin gel. In a preferred embodiment, the crosslinked material of the present invention is a hyaluronic acid / fibroin gel (HA / fibroin gel).

[0045] The crosslinked material of the composition of the present invention may optionally have good shear thinning properties. The composition may optionally have thixotropy. Thus, when stressed, its viscosity may be low. The composition of the present invention can be well injected while still being quite viscous in its target area (e.g., when administered in the subcutaneous area). A relatively low extrusion pressure is required. A gel with high viscosity and low extrusion pressure can be obtained.

[0046] In a preferred embodiment, the composition can be injected into soft tissue, particularly intradermally, subdermally, subcutaneously, and / or intramuscularly.

[0047] The soft tissue can be any soft tissue. In a preferred embodiment, the soft tissue is selected from the group consisting of dermal tissue (tissue including the dermis and subcutaneous tissue) and connective tissue, muscle, or joint tissue.

[0048] As used in the context of the present invention, the term "silk fibroin moiety" can be most broadly understood as any silk fibroin moiety known in the art. The silk fibroin moiety can also be represented by its abbreviation Fib or fib.

[0049] In a preferred embodiment, the average molecular weight of the one or more silk fibroin moieties is at least 1 kDa, or at least 5 kDa, or at least 10 kDa, or at least 100 kDa, or at least 200 kDa or higher. Preferably, each of the one or more silk fibroin moieties is a polymer moiety or a complex of polymer moieties, the total molecular weight (Mw) of which is at least 5 kDa (5000 daltons, 5 kilodaltons), more preferably at least 10 kDa (10000 daltons), even more preferably at least 100 kDa, particularly at least 200 kDa or higher. In one embodiment, the average molecular weight of the one or more silk fibroin moieties is from 10 to 400 kDa.

[0050] In a preferred embodiment, the average molecular weight of the one or more silk fibroin moieties does not exceed 2000 kDa, does not exceed 1000 kDa, does not exceed 750 kDa, does not exceed 500 kDa, does not exceed 250 kDa, does not exceed 200 kDa, or does not exceed 150 kDa.

[0051] In a preferred embodiment, the average molecular weight of the one or more silk fibroin moieties is at least 5 kDa, in the range of 5 to 1000 kDa, in the range of 5 to 400 kDa, in the range of 10 to 400 kDa, or in the range of 100 to 150 kDa. In a preferred embodiment, the average molecular weight of the one or more silk fibroin moieties is in the range of 10 to 400 kDa. In another preferred embodiment, the average molecular weight of the one or more silk fibroin moieties is in the range of 100 to 150 kDa.

[0052] In a particularly preferred embodiment, at least one of the one or more fibroin protein moieties, in particular all of the one or more fibroin protein moieties, may have an average molecular weight of 50 to 400 kDa. For example, the average molecular weight of the one or more fibroin protein moieties may be 10 to 100 kDa, 50 to 150 kDa, 100 to 150 kDa, 75 to 200 kDa, 100 to 250 kDa or 200 to 400 kDa.

[0053] In one embodiment, the fibroin protein moiety is of a single type and thus (substantially) has one molecular weight.

[0054] In another embodiment, the fibroin protein moiety has at least two different average molecular weights, each average molecular weight consisting of a primary amino residue or a salt thereof. In other words, the fibroin protein moiety may also be a mixture of fibroin protein moieties of different average molecular weights. In a preferred embodiment, the fibroin protein moiety has at least two different molecular weights, and at least one fibroin protein moiety has, preferably at least two fibroin protein moieties both have, in particular each of all fibroin protein moieties has a molecular weight in the range of 5 to 1000 kDa, in the range of 5 to 400 kDa, in the range of 10 to 400 kDa, in the range of 100 to 150 kDa, in the range of 10 to 100 kDa, in the range of 50 to 150 kDa, in the range of 100 to 150 kDa, in the range of 75 to 200 kDa, in the range of 100 to 250 kDa or in the range of 200 to 400 kDa. In a preferred embodiment, the fibroin protein moiety has at least two different molecular weights, and at least one fibroin protein moiety has, preferably at least two fibroin protein moieties both have, in particular all fibroin protein moieties have, a molecular weight in the range of 50 to 400 kDa.

[0055] As used in the present invention, the molecular weight (Mw) is preferably the average molecular weight of the species characterized. Each fibroin protein moiety may have one or more full-length fibroin protein polypeptides and / or one or more main chains (amide / protein main chains) of one or more fibroin protein polypeptides or a complex of two or more thereof.

[0056] Preferably, the fibroin protein moiety comprises at least one main chain of a full-length fibroin protein polypeptide, in particular (substantially) consisting of one or more main chains of one or more full-length fibroin protein polypeptides. In other words, the fibroin protein moiety is preferably derived from a naturally occurring fibroin.

[0057] As used herein, the amide bond can be understood in the broadest sense. Generally, the amide bond has the structure -NH-CO- or its tautomeric structure. The amide bond formed between the fibroin moiety and the polysaccharide moiety (such as the hyaluronic acid moiety) can have any chirality. In one embodiment, it is a racemic mixture. In one embodiment, most of the amino acid moieties of fibroin are L-amino acid moieties. In one embodiment, (substantially) all of the amino acid moieties of fibroin are L-amino acid moieties.

[0058] In a preferred embodiment, one or more fibroin moieties are silk fibroin moieties, more preferably silk fibroin moieties having at least 80% sequence homology with natural insect or spider fibroin moieties. In a preferred embodiment, the fibroin is silk fibroin. In an alternative preferred embodiment, the fibroin is a polypeptide or a complex of two or more polypeptides having at least 80%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98% sequence homology (especially identity) with one or more naturally occurring silk fibroin polypeptides. Silk fibroin can also include truncated forms thereof. The fibroin can be Bombyx mori silk fibroin and insect or spider fibroin. Alternatively, the fibroin can be prepared from another natural source or by genetic engineering (also known as: bioprocessing, biotechnological means) or synthetic engineering.

[0059] The term "moiety" in the context of the present invention can be understood most broadly as any molecular structure.

[0060] The moiety can be a compound containing or consisting of the corresponding structure, or can form part of a larger chemical entity (such as the crosslinked material of the composition of the present invention). For example, the fibroin moiety can be fibroin or a chemical entity containing fibroin. The fibroin moiety can optionally contain a plurality of fibroin backbones bound to each other. Optionally, one or more fibroin backbones can be bound to one or more other structures, such as, in particular, one or more polysaccharide moieties (especially hyaluronic acid moieties). It should be understood that the term "fibroin moiety" can also include its salts and modified forms.

[0061] According to the present invention, at least a portion of one or more fibroin moieties contains a primary amino residue or its salt. The amino group can, for example, form part of a lysinyl residue of one or more fibroin moieties. Preferably, at least a portion of one or more fibroin moieties contains one or more lysinyl residues, which can optionally be bound to a polysaccharide moiety (especially a hyaluronic acid moiety).

[0062] In the context of the present invention, the term "fibroin" can be most broadly understood as any fibroin known in the art. Fibroin can be obtained from commercial suppliers (e.g., Advanced BioMatrix, USA (e.g., product number 5154 - 20ML); CareSilk, Italy (e.g., product number CSK10 - 1051)), or can be prepared from natural sources, or by genetic engineering (also known as bioprocessing, biotechnological means) or synthetic engineering. For example, it can be fibroin from the silkworm Bombyx mori, or, alternatively, fibroin from a species selected from the group consisting of species of the genera Antheraea, Cricula, Sami, Gonometa, and Nephila (e.g., Nephila clavipes), or a homolog having at least 80%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98% sequence homology (especially identity) with one of the above species or a truncated form thereof. It should be understood that mixtures of different fibroins can also be used.

[0063] In a preferred embodiment, the fibroin is ((substantially) intact) silkworm (Bombyx mori) fibroin. In a particularly preferred embodiment, the fibroin is fibroin obtained from or derived from the silkworm. Fibroin from silkworms can be obtained from cocoons. The process of obtaining silk from silkworms is well known in the art. For example, the cocoons can be boiled in an aqueous solution for about 30 min (minutes). Optionally, the aqueous solution can contain about 0.02 M Na2CO3. The cocoons can be rinsed with water or an aqueous buffer to extract sericin, and the extracted fibroin can be dissolved in an aqueous buffer. The salts that can be used for this purpose can include, for example, lithium bromide, lithium thiocyanate, calcium nitrate, and mixtures thereof. Optionally, the extracted fibroin can be dissolved in a solution of about 9 - 12 M lithium bromide.

[0064] The salts can be removed by any means, such as dialysis. In a preferred embodiment, other components of the cocoons have been (substantially) removed, such as sericin. Thus, preferably, at least 50% by weight, more preferably at least 75% by weight, even more preferably at least 80% by weight, especially at least 90% by weight of the sericin initially present in the cocoons has been removed. Silk fibroin from silkworms can be type I, type II, or type III silk fibroin from silkworms or a mixture of two or more thereof. Preferably, the fibroin is or comprises type I silk fibroin from silkworms. The fibroin can have the properties described in the art, such as those described in US A 2014 / 315828.

[0065] Alternatively, one or more fibroin polypeptides, including silk fibroin polypeptides and intact silk fibroin, can also be obtained by genetic engineering. Genetically engineered fibroin can be obtained, for example, from bacteria, insect cells, spider cells, yeast, mammalian cells, transgenic animals, or transgenic plants.

[0066] One or more fibroin moieties can be stored under any conditions. For example, one or more fibroin moieties can be stored in a refrigerator or in liquefied gas, for example, in the temperature range of -15°C to -200°C. For example, one or more fibroin moieties can be stored at about -80°C or in liquid nitrogen (i.e., at about -196°C). When thawing a previously frozen solid fibroin or fibroin solution, preferably, one or more fibroin moieties can be optionally isolated from air. One or more fibroin moieties can be stored in a dry state in the form of a powder or an aqueous solution or a buffer solution (for example, in a concentration range of 10 to 100 mg / mL (for example, about 50 mg / mL), at a temperature between 4°C and ambient temperature). When thawing a previously frozen solid fibroin or fibroin solution, preferably, one or more fibroin moieties can be optionally isolated from air.

[0067] As used in the context of the present invention, the term "hyaluronic acid" (also referred to as: HA, hyaluronate, or hyaluronic acid) can be understood in the broadest sense as any hyaluronic acid moiety known in the art.

[0068] Hyaluronic acid can be understood in the broadest sense as a polysaccharide moiety containing hyaluronic acid moieties (also referred to as hyaluronic acid units), preferably containing at least 50 mol% of hyaluronic acid moieties, more preferably the content of hyaluronic acid moieties is at least 75 mol% of the total content of sugar moieties in the polysaccharide, more preferably at least 80 mol%, more preferably at least 90 mol%.

[0069] Hyaluronic acid can be used as described in WO 2017 / 162676 or WO 2017 / 148915. In addition, crosslinked and optionally modified hyaluronic acid (as described in WO 2020 / 127407) can also be used as hyaluronic acid in the context of the present invention.

[0070] The polysaccharide, especially hyaluronic acid, can optionally contain one or more sugar moieties other than hyaluronic acid. The polysaccharide, especially hyaluronic acid, can be optionally partially modified. For example, it can be partially oxidized and can carry aldehyde groups and / or can be crosslinked. Such modifications are described, for example, in WO 2020 / 127407.

[0071] In a preferred embodiment, hyaluronic acid is a natural glycosaminoglycan composed of repeating units linked by N-acetyl-D-glucosamine and D-glucuronic acid ([α-1,4-D-glucuronic acid-β-1,3-N-acetyl-D-glucosamine] n ). Thus, examples of repeating units of hyaluronic acid can be as follows:

[0072]

[0073] It should be understood that hyaluronic acid also includes hydrates, salts, and solvents of the above chemical structures.

[0074] Preferably, at least one polysaccharide moiety is a polymeric moiety having an average molecular weight (Mw) of at least 1 kDa (1000 Da), more preferably at least 5 kDa, even more preferably at least 10 kDa, even more preferably at least 50 kDa, even more preferably at least 100 kDa, even more preferably at least 200 kDa, even more preferably at least 300 kDa or higher.

[0075] Preferably, at least one polysaccharide moiety is a hyaluronic acid moiety, and the hyaluronic acid moiety is a polymeric moiety having an average molecular weight (Mw) of at least 1 kDa (1000 Da), more preferably at least 5 kDa, even more preferably at least 10 kDa, even more preferably at least 50 kDa, even more preferably at least 100 kDa, even more preferably at least 200 kDa, even more preferably at least 300 kDa or higher.

[0076] Preferably, one or more polysaccharide moieties (especially hyaluronic acid moieties) have an average molecular weight (Mw) in the range of 10 to 10000 kDa. In a preferred embodiment, one or more polysaccharide moieties (especially hyaluronic acid moieties) have an average molecular weight in the range of 50 to 4000 kDa. More preferably, one or more polysaccharide moieties (especially hyaluronic acid moieties) have an average Mw of 100 to 10000 kDa. In one embodiment, at least one polysaccharide moiety (especially hyaluronic acid moiety) is a polymeric moiety having an intrinsic viscosity of 1.0 to 3.3 m 3 / kg (20 °C, 1013 hPa, water).

[0077] In a preferred embodiment, at least one of one or more polysaccharide moieties, especially all of one or more polysaccharide moieties, can have an average molecular weight of 100 to 3500 kDa (1.5 and 3.5 MDa). More preferably, it can have an average molecular weight in the range of 100 to 5000 kDa, 200 to 2000 kDa, 250 to 1500 kDa, 300 to 1000 kDa, 400 to 900 kDa, or 500 to 900 kDa.

[0078] In a particularly preferred embodiment, the one or more polysaccharide moieties are one of the one or more hyaluronic acid moieties, and at least one of the one or more hyaluronic acid moieties, particularly all of the one or more hyaluronic acid moieties, may have an average molecular weight of 100 to 3500 kDa (1.5 and 3.5 MDa). More preferably, it may have an average molecular weight in the range of 100 to 5000 kDa, 200 to 2000 kDa, 250 to 1500 kDa, 300 to 1000 kDa, 400 to 900 kDa or 500 to 900 kDa.

[0079] The average molecular weight (Mw) of the polysaccharide 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 greater. The average molecular weight (Mw) of the polysaccharide in the context of the present invention is preferably 10 to 10000 kDa, more preferably 100 to 10000 kDa, or 100 to 5000 kDa. In a more preferred embodiment, the average molecular weight (Mw) of the polysaccharide is 50 to 4000 kDa. More preferably, the average molecular weight of the polysaccharide is in the range of 100 to 3500 kDa, 200 to 2000 kDa, 250 to 1500 kDa, 300 to 1000 kDa, 400 to 900 kDa or 500 to 900 kDa.

[0080] In a particularly preferred embodiment, the average molecular weight of all of the one or more polysaccharide moieties (particularly hyaluronic acid moieties) is 1500 to 3500 kDa.

[0081] In one embodiment, the polysaccharide moiety (particularly the hyaluronic acid moiety) is of a single type and thus (substantially) has one molecular weight.

[0082] In another embodiment, the polysaccharide moiety (particularly the hyaluronic acid moiety) has at least two different molecular weights, each molecular weight comprising a primary amino residue or a salt thereof. In other words, the polysaccharide moiety (particularly the hyaluronic acid moiety) can also optionally be a mixture of polysaccharide moieties (particularly hyaluronic acid moieties) of different molecular weights. In a preferred embodiment, the polysaccharide moiety (particularly the hyaluronic acid moiety) has at least two different molecular weights, and at least one polysaccharide moiety (particularly the hyaluronic acid moiety) has, preferably at least two polysaccharide moieties (particularly the hyaluronic acid moieties), particularly all polysaccharide moieties (particularly the hyaluronic acid moieties), each having a molecular weight in the range of 10 to 10,000 kDa, in the range of 100 to 10,000 kDa, or in the range of 100 to 5,000 kDa, in the range of 100 to 3,500 kDa, in the range of 200 to 2,000 kDa, in the range of 250 to 1,500 kDa, in the range of 300 to 1,000 kDa, in the range of 400 to 900 kDa, or in the range of 500 to 900 kDa.

[0083] In a preferred embodiment, the polysaccharide moiety (particularly the hyaluronic acid moiety) has at least two different molecular weights, and at least one polysaccharide moiety (particularly the hyaluronic acid moiety) has, preferably at least two polysaccharide moieties (particularly the hyaluronic acid moieties), particularly all polysaccharide moieties (particularly the hyaluronic acid moieties) each having a molecular weight in the range of 1,500 to 3,500 kDa.

[0084] In one embodiment, the polysaccharide moiety (particularly the hyaluronic acid moiety) comprises or consists of at least two polysaccharide moieties (particularly hyaluronic acid moieties) having at least two different molecular weights, and at least one polysaccharide moiety (particularly the hyaluronic acid moiety) has, preferably at least two polysaccharide moieties (particularly the hyaluronic acid moieties) both have, particularly all polysaccharide moieties (particularly the hyaluronic acid moieties) each have, a molecular weight in the range of 10 to 10,000 kDa, in the range of 100 to 10,000 kDa, or in the range of 100 to 5,000 kDa, in the range of 100 to 3,500 kDa, in the range of 200 to 2,000 kDa, in the range of 250 to 1,500 kDa, in the range of 300 to 1,000 kDa, in the range of 400 to 900 kDa, or in the range of 500 to 900 kDa. In a preferred embodiment, the polysaccharide moiety (particularly the hyaluronic acid moiety) comprises or consists of at least two polysaccharide moieties (particularly hyaluronic acid moieties) having at least two different molecular weights, and at least one polysaccharide moiety (particularly the hyaluronic acid moiety) has, preferably at least two polysaccharide moieties (particularly the hyaluronic acid moieties) both have, particularly all polysaccharide moieties (particularly all hyaluronic acid moieties) each have, a molecular weight in the range of 1,500 to 3,500 kDa.

[0085] The (mass) ratio between the total mass of one or more fibroin moieties (Component A-i) and the total mass of one or more polysaccharide moieties (Component A-ii), particularly a hyaluronic acid moiety, can be any ratio. When particularly high water / buffer absorbance is required, a polysaccharide moiety in mass excess, particularly a hyaluronic acid moiety, can be used. When particularly high stability is required, a fibroin moiety in even greater mass excess can be used. Preferably, the (mass) ratio (A-i):(A-ii) is in the range of 1:100 to 100:1.

[0086] In a preferred embodiment, the mass ratio between one or more fibroin moieties (A-i) and one or more polysaccharide moieties (A-ii), i.e., (A-i):(A-ii), is in the range of 5:1 to 1:20, preferably in the range of 1:1 to 1:10, particularly in the range of 1:1 to 1:5. In a preferred embodiment, the mass ratio between one or more fibroin moieties (A-i) and one or more hyaluronic acid moieties (A-ii), i.e., (A-i):(A-ii), is in the range of 5:1 to 1:20, preferably in the range of 1:1 to 1:10, particularly in the range of 1:1 to 1:5.

[0087] For example, the mass ratio between one or more fibroin moieties (A-i) and one or more polysaccharide moieties (A-ii), i.e., (A-i):(A-ii), can be in the range of 1:9 to 2:1, 1:8 to 1.5:1, 1:7 to 1:1, 1:6 to 1:1, 1:5 to 1:1, 1:4 to 1:1, 1:3 to 1:1, 1:2 to 1:1, or 1:1.5 to 1:1. For example, the mass ratio between one or more fibroin moieties (A-i) and one or more hyaluronic acid moieties (A-ii), i.e., (A-i):(A-ii), can be in the range of 1:9 to 2:1, 1:8 to 1.5:1, 1:7 to 1:1, 1:6 to 1:1, 1:5 to 1:1, 1:4 to 1:1, 1:3 to 1:1, 1:2 to 1:1, or 1:1.5 to 1:1.

[0088] In one embodiment, the fibroin moiety has at least two different molecular weights, each molecular weight containing a primary amino residue or its salt, and the polysaccharide moiety, particularly a hyaluronic acid moiety, has at least two different molecular weights, each molecular weight containing a primary amino residue or its salt.

[0089] In a preferred embodiment:

[0090] (a) The fibroin portion has at least two different molecular weights, and at least one fibroin portion has, preferably at least two fibroin portions both have, particularly all fibroin portions each have, a molecular weight in the range of 5 to 1000 kDa, in the range of 5 to 400 kDa, in the range of 10 to 400 kDa, in the range of 100 to 150 kDa, in the range of 10 to 100 kDa, in the range of 50 to 150 kDa, in the range of 100 to 150 kDa, in the range of 75 to 200 kDa, in the range of 100 to 250 kDa or in the range of 200 to 400 kDa; and

[0091] (b) The polysaccharide portion has at least two different molecular weights, and at least one polysaccharide portion has, preferably at least two polysaccharide portions both have, particularly all polysaccharide portions 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 100 to 3500 kDa, in the range of 200 to 2000 kDa, in the range of 250 to 1500 kDa, in the range of 300 to 1000 kDa, in the range of 400 to 900 kDa, or in the range of 500 to 900 kDa.

[0092] In a preferred embodiment:

[0093] (a) The fibroin portion has at least two different molecular weights, and at least one fibroin portion has, preferably at least two fibroin portions both have, particularly all fibroin portions each have, a molecular weight in the range of 5 to 1000 kDa, in the range of 5 to 400 kDa, in the range of 10 to 400 kDa, in the range of 100 to 150 kDa, in the range of 10 to 100 kDa, in the range of 50 to 150 kDa, in the range of 100 to 150 kDa, in the range of 75 to 200 kDa, in the range of 100 to 250 kDa or in the range of 200 to 400 kDa; and

[0094] (b) The hyaluronic acid portion has at least two different molecular weights, and at least one polysaccharide portion has, preferably at least two polysaccharide portions both have, particularly all polysaccharide portions 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 100 to 3500 kDa, in the range of 200 to 2000 kDa, in the range of 250 to 1500 kDa, in the range of 300 to 1000 kDa, in the range of 400 to 900 kDa, or in the range of 500 to 900 kDa.

[0095] In a preferred embodiment, the crosslinked material forms a gel, particularly a hydrogel, in a liquid or viscous pharmaceutically acceptable carrier, and the botulinum toxin is dissolved in the liquid or viscous pharmaceutically acceptable carrier that soaks in the gel, particularly the hydrogel.

[0096] In a preferred embodiment, the crosslinked material of the present invention is a gel. In a preferred embodiment, the crosslinked material of the present invention is a hyaluronic acid / silk fibroin gel (HA / silk fibroin gel). In a preferred embodiment, the crosslinked material of the present invention is a hyaluronic acid / silk fibroin hydrogel (HA / silk fibroin hydrogel).

[0097] In one embodiment:

[0098] (a) The silk fibroin moiety has at least two different molecular weights, and at least one silk fibroin moiety has, preferably at least two silk fibroin moieties both have, particularly all silk fibroin moieties have, a molecular weight in the range of 5 to 1000 kDa, in the range of 50 to 400 kDa; and

[0099] (b) The polysaccharide moiety has at least two different molecular weights, and at least one polysaccharide moiety has, preferably at least two polysaccharide moieties both have, particularly all polysaccharide moieties have, a molecular weight in the range of 1500 to 3500 kDa.

[0100] In one embodiment:

[0101] (a) The silk fibroin moiety has at least two different molecular weights, and at least one silk fibroin moiety has, preferably at least two silk fibroin moieties both have, particularly all silk fibroin moieties have, a molecular weight in the range of 5 to 1000 kDa, in the range of 50 to 400 kDa; and

[0102] (b) The hyaluronic acid moiety has at least two different molecular weights, and at least one hyaluronic acid moiety has, preferably at least two hyaluronic acid moieties both have, particularly each hyaluronic acid moiety has, a molecular weight in the range of 1500 to 3500 kDa.

[0103] One or more silk fibroin moieties and one or more polysaccharide moieties can be combined with each other in any way. Specifically, one or more silk fibroin moieties and one or more hyaluronic acid moieties can be combined with each other in any way.

[0104] For example, these components can be combined with each other through one or more amide bonds (-NH-CO- or -CO-NH-). This binding reaction can be promoted in any way, for example, by one or more activators, such as one or more activators prompting the reaction of carboxylic acid residues with amino residues to form amide bonds.

[0105] As described herein, the activator can be any compound that promotes the reaction of one or more fibroin moieties with one or more polysaccharide moieties (especially hyaluronic acid moieties). Preferably, the activator can be any compound that promotes the reaction of carboxylic acid residues with amino residues to form amide bonds. It should be understood that this mainly means that the activator is a compound that promotes the reaction of carboxylic acid residues of one or more polysaccharide moieties (especially hyaluronic acid moieties) with amino residues of one or more fibroin moieties to form amide bonds.

[0106] In a preferred embodiment, one or more activators are selected from the group consisting of:

[0107] (C1) One or more triazine-based activators, especially selected from the group consisting of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine (DMTMM), its salts and / or 2-chloro-4,6,-dimethoxy-1,3,5-triazine (CDMT) and combinations thereof;

[0108] (C2) One or more carbodiimide activators, especially selected from the group consisting of N,N'-dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and combinations of two or more thereof; and

[0109] (C3) Combinations thereof.

[0110] According to the present invention, the activator is generally not included in the crosslinked material in a covalent bond form. Therefore, generally, optionally, it can be removed from the crosslinked material of the present invention by any means (such as washing, filtration, etc.).

[0111] In a preferred embodiment, the triazine-based activator is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium (DMTMM) or its salt, preferably 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium salt. The salt of DMTMM is preferably a salt in which the counterion is a cosmetically and / or pharmaceutically acceptable anion, such as chloride, acetate, bicarbonate (hydrogen carbonate) or a mixture of two or more anions.

[0112] In a preferred embodiment, the triazine-based activator is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride.

[0113] In a preferred embodiment, the carbodiimide activator is N,N'-dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0114] In a preferred embodiment, the activator is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium (DMTMM) or a salt thereof. Preferably, it is a 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium salt, especially 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (CAS No. 3945-69-5).

[0115] DMTMM is considered to have relatively low and substantially negligible toxicity and is not carcinogenic, mutagenic, or teratogenic / reproductive toxic at the amounts commonly used. Thus, it is particularly suitable for the preparation of soft tissue fillers, such as dermal fillers or connective tissue fillers.

[0116] When DMTMM is used as the activator, 4-methylmorpholine (NMM) and / or 4,6-dimethoxy-1,3,5-triazin-2-ol (DMT) may be formed as degradation products.

[0117] As used herein, the term "without an interconnecting linker structure" can be understood in the broadest sense, i.e., no other chemical moieties that are not derived from (i.e., not present in) the fibroin moiety or the polysaccharide moiety (especially the hyaluronic acid moiety) are introduced into the chemical structure that covalently binds (preferably via an amide bond) one or more fibroin moieties to one or more polysaccharide moieties (especially the hyaluronic acid moiety). In other words, the amide bond is preferably formed by the inclusion of a nitrogen atom derived from the fibroin (e.g., a lysyl side chain) and the inclusion of a carbon atom derived from the polysaccharide moiety (especially the hyaluronic acid moiety).

[0118] In a preferred embodiment, one or more fibroin moieties are covalently bound to one or more polysaccharide moieties via an amide bond. In a preferred embodiment, one or more fibroin moieties are covalently bound to one or more hyaluronic acid moieties via an amide bond.

[0119] In a preferred embodiment, the crosslinked material is further characterized in that it does not contain imide groups. In a preferred embodiment, the crosslinked material is further characterized in that it does not contain imine groups. In a preferred embodiment, the crosslinked material is further characterized in that it does not contain epoxy groups. In a preferred embodiment, the crosslinked material is further characterized in that it does not contain exogenous linker moiety groups.

[0120] In a preferred embodiment, the crosslinked material is further characterized in that it does not contain:

[0121] (a) an imide group;

[0122] (b) an imine group;

[0123] (c) an epoxy group; and / or

[0124] (d) an exogenous linker moiety,

[0125] interconnecting one or more silk fibroin moieties with one or more polysaccharide moieties.

[0126] In a preferred embodiment, the crosslinked material is further characterized in that it does not contain:

[0127] (a) an imide group;

[0128] (b) an imine group;

[0129] (c) an epoxy group; and / or

[0130] (d) an exogenous linker moiety,

[0131] interconnecting one or more silk fibroin moieties with one or more hyaluronic acid moieties.

[0132] In a preferred embodiment, the crosslinked material is further characterized in that it does not contain, as a whole:

[0133] (a) an imide group;

[0134] (b) an imine group;

[0135] (c) an epoxy group; and

[0136] (d) an exogenous linker moiety.

[0137] The crosslinked material can be prepared by any means. Those skilled in the art will know the methods for preparing such materials. For example, it can be prepared as described in PCT / EP2022 / 066989.

[0138] For example, it can be prepared by the following method:

[0139] (i) bringing into contact the following components:

[0140] (A-i) one or more silk fibroin moieties comprising primary amino residues or salts thereof,

[0141] (A-ii) one or more polysaccharide moieties (in particular hyaluronic acid moieties) comprising carboxylic acid residues or salts thereof,

[0142] (A-iii) one or more activators that promote the reaction of carboxylic acid residues with amino residues to form amide bonds, and

[0143] (A-iv) one or more solvents; and

[0144] (ii) reacting at least some of the carboxylic acid residues with at least some of the primary amino residues to form amide bonds that covalently link one or more fibroin moieties to one or more polysaccharide moieties (particularly hyaluronic acid moieties); and

[0145] (iii) optionally purifying the crosslinked material obtained from step (ii).

[0146] In a preferred embodiment, the method is further characterized in that it includes step (iii) of purifying the crosslinked material by filtration, washing, and / or dialysis, particularly crossflow filtration, diafiltration, and / or dead-end filtration. Such optional purification steps (including the filtration step) are further illustrated in PCT / EP2022 / 066989.

[0147] Step (ii) can be carried out for any time suitable for this purpose. Optionally, step (ii) can be carried out for 1 minute to 1 week or longer, 2 minutes to 5 days, 3 minutes to 4 days, 5 minutes to 72 hours, 5 minutes to 24 hours, 10 minutes to 12 hours, 30 minutes to 6 hours, 1 hour to 5 hours, or 2 hours to 4 hours. Step (ii) and / or (iii) can be carried out at any temperature suitable for this purpose, for example, 0°C to 100°C, 4°C to 95°C, 10°C to 70°C, 15°C to 30°C, 18°C to 25°C, 20°C to 70°C, 20°C to 40°C, or 60°C to 70°C. Steps (i) and (ii) and the optional step (iii) can be carried out at any pressure. For example, the pressure can be ambient pressure (e.g., typically about 970 to 1100 hPa external pressure).

[0148] In a preferred embodiment, the method is further characterized in that steps (i) and (ii) are carried out in a single batch.

[0149] In a preferred embodiment, in the first step, one or more polysaccharide moieties (as component A-ii, particularly hyaluronic acid moieties) and one or more activators (as component A-iii) are dissolved in one or more solvents (as component A-iv) without one or more fibroin moieties (as component A-i) and incubated. This can activate the carboxyl groups of the polysaccharide moieties (particularly hyaluronic acid moieties). The incubation can be carried out for any time sufficient to achieve this purpose. In a preferred embodiment, in the first sub-step, one or more polysaccharide moieties (particularly hyaluronic acid moieties) are dissolved in one or more solvents, and one or more activators are added in a subsequent sub-step, which together represent the activation step. After incubation, one or more fibroin moieties (as component A-i) can be added. This can be further incubated for step (ii) of the method of the present invention. Step (ii) can be carried out using any suitable solvent (such as water or an aqueous buffer). Optionally, the solution can be stirred during the reaction step.

[0150] Thus, in a preferred embodiment, step (i) of the method comprises the following sub-steps:

[0151] (ia) bringing into contact the following components:

[0152] (A-ii) one or more polysaccharide moieties (particularly hyaluronic acid moieties), including carboxylic acid residues or their salts,

[0153] (A-iii) one or more activators that cause the carboxylic acid residues to react with amino residues to form amide bonds, and

[0154] (A-iv) one or more solvents,

[0155] Preferably, wherein first one or more polysaccharide moieties (particularly hyaluronic acid moieties) are dissolved in one or more solvents, and then one or more activators are added;

[0156] (ib) reacting at least some of the carboxylic acid residues with one or more activators to form one or more activated polysaccharide moieties (also referred to as: polysaccharide-activator conjugates, particularly hyaluronic acid moieties, i.e., hyaluronic acid-activator conjugates); and

[0157] (ic) adding to the activated polysaccharide moiety (particularly hyaluronic acid moiety) of sub-step (ib):

[0158] (A-i) one or more fibroin moieties containing primary amino residues or their salts.

[0159] In another preferred embodiment, all the components and optionally one or more other components are mixed simultaneously.

[0160] In a preferred embodiment, the method comprises:

[0161] (i) bringing into contact the following components:

[0162] (A-i) one or more silk fibroin moieties having an average molecular weight of at least 5 kDa and comprising primary amino residues or salts thereof,

[0163] (A-ii) one or more polysaccharide moieties (especially hyaluronic acid moieties) having an average molecular weight of at least 50 kDa and comprising carboxylic acid residues or salts thereof,

[0164] (A-iii) one or more triazine-based activators that cause the carboxylic acid residues to react with the amino residues to form amide bonds, especially where the activator is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium or a salt thereof; and

[0165] (A-iii) one or more solvents; and

[0166] (ii) reacting at least some of the carboxylic acid residues with at least some of the primary amino residues to form amide bonds that covalently link one or more silk fibroin moieties to one or more polysaccharide moieties (especially hyaluronic acid moieties); and

[0167] (iii) optionally purifying the crosslinked material obtained from step (ii).

[0168] Any solvent that can be used as component A-iv in the method of the present invention can be used. In a preferred embodiment, a polar solvent is used. In a preferred embodiment, a protic solvent is used. In a preferred embodiment, a protic polar solvent is used.

[0169] In one embodiment of the present invention, the method of the present invention comprises the following (preferably consecutive) steps:

[0170] Dissolve the polysaccharide (especially hyaluronic acid), especially the polysaccharide sodium salt (especially sodium hyaluronate), in water or a buffer;

[0171] Add the activator (preferably DMTMM) to the polysaccharide solution (especially the hyaluronic acid solution);

[0172] Allow activation (preferably for several hours, for example at a temperature of 18 to 22 °C);

[0173] Add the silk fibroin solution to the activated polysaccharide (especially the activated hyaluronic acid);

[0174] Stir and allow the formation of the crosslinked material (preferably for several hours, for example at a temperature of 18 to 22 °C);

[0175] Purify the cross-linked material (e.g., remove DMTMM and its degradation products, e.g., by filtration and / or dialysis, e.g., at a temperature of 18 to 22 °C);

[0176] Optionally add an anesthetic; and

[0177] Optionally sterilize.

[0178] As used in the context of the present invention, the term "botulinum toxin" can be understood most broadly to mean any type of botulinum toxin known in the art.

[0179] The term "botulinum toxin" as used herein is not particularly limited and includes botulinum toxins of any serotype (e.g., BoNT / A-H. For example, the botulinum toxin can be serotype A or B (BoNT / A, BoNT / B). Preferably, the botulinum toxin is serotype A, more preferably serotype A1 (BoNT / A1), and most preferably BoNT / A1 produced by the Hall strain of Clostridium botulinum. In addition, the term "botulinum toxin" ("BT") and the term "botulinum neurotoxin" ("BoNT") used synonymously herein are intended to refer to pure botulinum neurotoxin and / or any complex thereof, i.e., any complex of pure botulinum neurotoxin and complex proteins (referred to as "toxin complex"). Preferably, the botulinum toxin is pure serotype A botulinum neurotoxin.

[0180] In a preferred embodiment, the botulinum toxin is serotype A botulinum neurotoxin, particularly serotype A1 botulinum neurotoxin.

[0181] The term "pure botulinum neurotoxin" as used herein refers to a botulinum neurotoxin that does not contain complex proteins (sometimes also referred to as the "neurotoxic component"), or more precisely, a botulinum neurotoxin that does not contain neurotoxin-associated complex proteins (NAP). The pure botulinum neurotoxin is the (active) neurotoxic polypeptide that ultimately inhibits the release of acetylcholine. It is a double-chain protein composed of a light chain (LC; approximately 50 kDa) and a heavy chain (HC; approximately 100 kDa), and the light chain and the heavy chain are linked together by a disulfide bond. Therefore, the active neurotoxic polypeptide can 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

[0182] As used herein, the term "toxin complex" refers to a high molecular weight complex of a neurotoxic component and a group of complex proteins (NAP), such as 900 kDa, 500 kDa, and 300 kDa botulinum toxin type A complexes. The complex proteins are non-toxic non-hemagglutinins (NTNHA), and in strains of serotypes A-D, are different hemagglutinins (HA). For example, onabotulinumtoxin A( Allergan, Inc., Irvine, California, USA) contains the 900 kDa complex, while abobotulinumtoxin A( Ipsen, Paris, France), (Ipsen / Galderma), and (Medytox) also contain the toxin complex as an active agent. Preferably, in addition to the pure botulinum neurotoxin contained in or , the botulinum toxin is the toxin complex contained in or , or is the botulinum toxin described in or

[0183] The botulinum toxin can also be of the kind described in WO2017 / 148915.

[0184] The botulinum toxin can be a natural neurotoxin obtained from Clostridium botulinum, or any other botulinum toxin, such as a botulinum toxin obtained from other sources, including recombinant technology and genetic or chemical modification. The terms "botulinum toxin", "neurotoxic component", etc. also include chimeric or genetically modified botulinum toxins, i.e., botulinum toxins containing mutations (including substitutions, deletions, and insertions). Preferably, the mutations do not impair any biological activity of the botulinum toxin. However, the use of mutations to modulate the biological activity of the botulinum toxin is also contemplated. Also included are botulinum toxins containing chemically modified amino acids, such as glycosylated, acetylated, or otherwise modified one or more amino acids. This may be beneficial for the absorption or stability of the toxin. Particularly preferred is the lipidation of the neurotoxic component.

[0185] In the present invention, the dose is expressed in biological (enzymatic) units because the botulinum toxin used may contain, for example, different percentages of inactive toxin, which contribute to the total protein load but not to the efficacy. In the context of the present invention, the biological potency of the botulinum toxin is determined using the mouse bioassay (MBA). The MBA determines the median lethal dose (LD 50)), i.e., the dose of toxin / neurotoxin that can kill 50% of a group of mice. On this basis, 1 unit (U) of toxin / neurotoxin used in this article is defined as the LD50 of one mouse (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 lethality assays. Toxicol. Appl. Pharmacol. 1994, 128, 69 - 77).

[0186] Another useful method for determining the biological activity (biopotency) of botulinum toxin is the cell - based potency assay, which is disclosed in, for example, WO 2009 / 114748, WO 2013 / 049508, or WO 2014 / 207109. The activity results obtained by such cell - based assays correspond to the activity values obtained in the mouse intraperitoneal injection LD50 assay because these values are calibrated using the LD50 reference standard.

[0187] Due to the differences in the LD50 tests used by manufacturers of commercial botulinum toxin preparations, the unit potencies labeled by manufacturers for their commercial botulinum toxin preparations are proprietary and cannot be easily compared. Therefore, within the framework of the present invention, the conversion factors provided below are used to determine incobotulinumtoxinA (“INCO”; botulinum toxin serotype A, without complex proteins; Merz Pharmaceuticals GmbH), onabotulinumtoxinA (“ONA”; botulinum toxin serotype A complex; Allergan Inc.), abobotulinumtoxinA (“ABO”; botulinum toxin serotype A complex; Medicis Pharmaceutical Corp., Galderma Lab.), rimabotulinumtoxinB (“RIM”; botulinum toxin serotype B; Solstice Neurosciences Inc.) and Comparative potencies of (“TBD”; botulinum toxin serotype A; Mentor Worldwide LLC). 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. Additionally, preferably, within the scope of the present invention, 1U INCO and 1U onabotulinumtoxinA (“ONA”; ) shall be considered to correspond to 1 mouse LD50 (1.0 LD50), or 1U, measured using the above MBA.

[0188] In a preferred embodiment, the botulinum toxin is not covalently bound to a crosslinking material.

[0189] As used herein, a viscous cosmetically and / or pharmaceutically acceptable carrier can be any component that can be used as a carrier.

[0190] Preferably, the liquid or viscous carrier according to the present invention comprised in the injectable composition can be any injectable carrier. Generally, the liquid or viscous carrier is a carrier that is non-toxic to mammals, particularly humans, when administered in the context of the present invention. The liquid or viscous carrier can preferably comprise or consist of one or more solvents, such as water, aqueous buffers, glycerol, dimethyl sulfoxide (DMSO), ethanol, vegetable oils, paraffin oil, or combinations thereof. More preferably, the liquid or viscous carrier comprises or consists of a pyrogen-free isotonic buffer, more specifically a saline solution or a buffered saline solution. The saline can have, for example, a sodium chloride concentration of 0.9% by weight.

[0191] An aqueous buffer can be any buffer containing water that is pharmaceutically and / or cosmetically acceptable. Preferably, it is pharmaceutically and / or cosmetically acceptable when injected into a subject. Such buffers can be, for example, buffers selected from the group consisting of phosphate buffers, citrate buffers, citrate-phosphate buffers, lactate buffers, acetate buffers, and combinations of two or more thereof. It should be understood that such buffers can also optionally include other components, such as one or more pharmaceutically and / or cosmetically acceptable salts and / or one or more pharmaceutically and / or cosmetically acceptable sugars. In a preferred embodiment, the aqueous buffer is saline or phosphate buffered saline.

[0192] In a preferred embodiment, the liquid or viscous pharmaceutically acceptable carrier comprises or consists of water, an aqueous buffer, glycerol, or a combination of two or more thereof.

[0193] The buffering agent can have any pH. Preferably, the buffering agent can have a pharmaceutically and / or cosmetically acceptable pH when injected into a subject. In a preferred embodiment, the pH of the buffer solution can be stabilized within a 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.

[0194] As used herein, the anesthetic agent can be any anesthetic component.

[0195] Preferably, the anesthetic agent is a local anesthetic. The local anesthetic can make the injection into an individual more comfortable. Local anesthetics suitable for use herein include, but are not limited to, ambucaine, amolocaine, amilocaine, butoxyprocaine, benzocaine, betoxycaine, benzoylmetanilide, bupivacaine, butacaine, butamben, butanilicaine, butethamine, butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine, dibucaine, dimethisoquin, dimethocaine, diparocaine, dycyclonine, ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine, euphroeine, phenacaine, formocaine, hexycaine, oxybuprocaine, isobutyl p-aminobenzoate, leucomycin methanesulfonate, levoxadrol, lidocaine, mepivacaine, meprylcaine, meprylcaine, methoxyflurane, metacaine, napacaine, orthocaine, oxethazaine, parethoxycaine, phenacaine, phenol, piridocaine, polidocanol, promecaine, prilocaine, procaine, propipocaine, proxymetacaine, pseudococaine, pyrrocaine, ropivocaine, salicyl alcohol, tetracaine, trimecaine, trimetozine, zoalamide, and their salts. In a preferred embodiment, the anesthetic agent is lidocaine. Optionally, a combination of two or more of the aforementioned anesthetic agents can also be used herein, such as a combination of lidocaine and other "caine" anesthetics (such as prilocaine).

[0196] Other cosmetically and / or pharmaceutically acceptable components different from components (A) to (D) can be any components useful for this purpose.

[0197] For example, such further components can be other carriers, such as solid cosmetically and / or pharmaceutically acceptable carriers optionally soluble in a liquid or viscous cosmetically and / or pharmaceutically acceptable carriers. For example, such further components can be fillers or swelling agents. For example, such further components can be salts (e.g., sodium chloride, NaCl). For example, such further components can be stabilizers (e.g., proteins (e.g., albumin (e.g., human albumin))). For example, such further components can be components that slow down the degradation of botulinum toxin (e.g., free radical scavengers, (e.g., polyols such as glycerol, sugar alcohols (e.g., selected from the group consisting of mannitol, inositol, lactitol, xylitol, erythritol, sorbitol, etc.))). For example, such further components can be sugars (e.g., sucrose, glucose, trehalose, galactose, fructose, maltose, lactose, starch, cellulose, etc.). For example, such further components can be crystallization inhibitors. For example, such further components can be scavengers. For example, such further components can be cell proliferation factors, which can improve cell invasion into the cross-linked material of the present invention being administered. For example, such further components can be dyes, which can, for example, be administered locally (e.g., by injection), can improve the localization of the injection (e.g., pharmaceutically acceptable fluorescent dyes such as fluorescein or rhodamine), or can improve the invisibility of the otherwise white cross-linked material (e.g., by making it flesh-colored). Any other pharmaceutically active compound can also be added. Then, the composition of the present invention can also optionally be in the form of a sustained-release and / or controlled-release form for administration.

[0198] Other components can be added at any time, for example, before, during, or after the purification of the cross-linked material. For example, one or more further components can be added during the purification process. In another embodiment of the present invention, one or more further components can be added to the prepared and optionally purified cross-linked material.

[0199] The composition of the present invention can include components in any concentration and content range.

[0200] 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.

[0201] In a preferred embodiment, the composition of the present invention contains at least 0.1% by weight of the cross-linked material based on the composition. In a preferred embodiment, especially when the composition of the present invention is a (hydro)gel or liquid, the composition contains 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 of the cross-linked material based on the composition.

[0202] In a preferred embodiment, particularly when the composition of the present invention is a (hydro)gel, liquid or viscous composition, the crosslinking material contained in the composition does not exceed 3.5% by weight based on the composition. In a preferred embodiment, particularly when the composition of the present invention is a (hydro)gel, liquid or viscous composition, the crosslinking material contained in the composition does not exceed 20% by weight based on the composition, does not exceed 10% by weight based on the composition, does not exceed 5% by weight based on the composition, does not exceed 3.5% by weight based on the composition, or does not exceed 2% by weight based on the composition, or does not exceed 1% by weight based on the composition.

[0203] In a preferred embodiment, the composition of the present invention contains 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 of crosslinking material based on the composition. In a preferred embodiment, particularly when the composition of the present invention is a (hydro)gel, liquid or viscous composition, the composition contains 0.1 to 3.5% by weight of crosslinking material based on the composition. In a preferred embodiment, particularly when the composition of the present invention is a (hydro)gel, liquid or viscous composition, the composition contains 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 of crosslinking material.

[0204] In a preferred embodiment, the composition of the present invention comprises at least 0.1 unit / g of botulinum toxin based on the composition. In a preferred embodiment, the composition of the present invention comprises at least 0.2 unit / g, at least 0.5 unit / g, at least 1 unit / g, at least 1 unit / g, at least 5 unit / g, at least 10 unit / g, at least 25 unit / g, at least 50 unit / g, at least 75 unit / g, at least 100 unit / g, at least 150 unit / g, at least 200 unit / g, at least 250 unit / g or at least 500 unit / g of botulinum toxin based on the composition. In a preferred embodiment, especially when the composition of the present invention is a (hydro)gel, liquid or viscous composition, the composition comprises at least 0.1 unit / mL of botulinum toxin based on the composition. In a preferred embodiment, the composition of the present invention comprises at least 0.2 unit / mL, at least 0.5 unit / mL, at least 1 unit / mL, at least 3 unit / mL, at least 5 unit / mL, at least 10 unit / mL, at least 25 unit / mL, at least 50 unit / mL, at least 75 unit / mL, at least 100 unit / mL, at least 150 unit / mL, at least 200 unit / mL, at least 250 unit / mL or at least 500 unit / mL of botulinum toxin based on the composition. In a preferred embodiment, an injectable dose of the composition of the present invention comprises at least 0.1 unit of botulinum toxin based on the composition. In a preferred embodiment, the injectable dose of the composition of the present invention comprises at least 0.2 unit, at least 0.5 unit, at least 1 unit, at least 1 unit, at least 5 unit, at least 10 unit, at least 25 unit, at least 50 unit, at least 75 unit, at least 100 unit, at least 150 unit, at least 200 unit, at least 250 unit or at least 500 unit of botulinum toxin based on the composition.

[0205] In a preferred embodiment, the composition of the present invention comprises no more than 1000 units / g of botulinum toxin, based on the composition. In a preferred embodiment, the composition of the present invention comprises no more than 500 units / g, no more than 250 units / g, no more than 200 units / g, no more than 150 units / g, no more than 100 units / g, no more than 50 units / g or no more than 10 units / g of botulinum toxin, based on the composition. In a preferred embodiment, especially when the composition of the present invention is a (hydro)gel, liquid or viscous composition, the composition comprises no more than 1000 units / mL of botulinum toxin, based on the composition. In a preferred embodiment, the composition of the present invention comprises no more than 500 units / mL, no more than 250 units / mL, no more than 200 units / mL, no more than 150 units / mL, no more than 100 units / mL, no more than 50 units / mL or no more than 10 units / mL of botulinum toxin, based on the composition. In a preferred embodiment, the injectable dose of the composition of the present invention comprises no more than 1000 units of botulinum toxin, based on the composition. In a preferred embodiment, the composition of the present invention comprises no more than 500 units, no more than 250 units, no more than 200 units, no more than 150 units, no more than 100 units, no more than 50 units or no more than 10 units of botulinum toxin, based on the composition.

[0206] In a preferred embodiment, the composition of the present invention comprises 0.1 to 1000 units / g of botulinum toxin, based on the composition.

[0207] 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 of botulinum toxin, based on the composition. In a preferred embodiment, especially when the composition of the present invention is a (hydro)gel, liquid or 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 of botulinum toxin, based on the composition. In a preferred embodiment, the 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, based on the composition.

[0208] The composition of the present invention may comprise from 0 to 99.9% by weight, based on the composition, of one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers. In a preferred embodiment, the composition of the present invention comprises from 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 composition, of one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers.

[0209] In a preferred embodiment, the composition of the present invention comprises from 0 to 1% by weight, based on the composition, of one or more anesthetics. In a preferred embodiment, the composition of the present invention comprises from 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 composition, of one or more anesthetics.

[0210] In a preferred embodiment, the composition of the present invention comprises from 0 to 50% by weight, based on the composition, of one or more other 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 composition, of one or more other cosmetically and / or pharmaceutically acceptable ingredients. In a preferred embodiment, the composition of the present invention comprises from 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%, 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 composition, of one or more other cosmetically and / or pharmaceutically acceptable ingredients.

[0211] In a preferred embodiment, the composition is preferably an injectable composition, which comprises or consists of:

[0212] (A) from 0.1 to 99.9% by weight, preferably from 0.1 to 10% by weight, of a crosslinking material, based on the composition;

[0213] (B) from 0.1 to 150 units / mL, preferably from 0.1 to 100 units / mL, of botulinum toxin, based on the composition;

[0214] (C) 0 to 99.9% by weight, preferably 50 to 99.9% by weight, of one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers, based on the composition;

[0215] (D) 0 to 10% by weight, preferably 0 to 5% by weight, of one or more anesthetics, based on the composition; and

[0216] (E) 0 to 50% by weight, preferably 0 to 49.9% by weight, of one or more other cosmetically and / or pharmaceutically acceptable ingredients, based on the composition.

[0217] In a preferred embodiment, the composition is an injectable composition comprising or consisting of:

[0218] (A) 0.1 to 3.5% by weight of a crosslinking material, based on the composition;

[0219] (B) 1 to 150 units / mL of botulinum toxin, based on the composition;

[0220] (C) 0 to 99.9% by weight of one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers, based on the composition;

[0221] (D) 0 to 1% by weight of one or more anesthetics, based on the composition; and

[0222] (E) 0 to 50% by weight of one or more other cosmetically and / or pharmaceutically acceptable ingredients, based on the composition.

[0223] In a preferred embodiment, the composition comprises at least 10% by weight of a liquid or viscous pharmaceutically acceptable carrier, based on the composition. In a preferred embodiment, the composition comprises at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% by weight of a liquid or viscous pharmaceutically acceptable carrier, based on the composition.

[0224] In a preferred embodiment, the composition is an injectable composition which comprises or consists of:

[0225] (A) 0.1 to 3.5% by weight of a crosslinking material, based on the composition;

[0226] (B) 1 to 150 units / mL of botulinum toxin, based on the composition;

[0227] (C) Based on the composition, 10 to 99.9% by weight of one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers;

[0228] (D) Based on the composition, 0 to 1% by weight of one or more anesthetics; and

[0229] (E) Based on the composition, 0 to 89.9% by weight of one or more other cosmetically and / or pharmaceutically acceptable ingredients.

[0230] In a preferred embodiment, the composition is an injectable composition comprising or consisting of:

[0231] (A) Based on the composition, 0.1 to 3.5% by weight of a crosslinking material;

[0232] (B) Based on the composition, 1 to 150 units / mL of botulinum toxin;

[0233] (C) Based on the composition, 50 to 99.9% by weight, preferably 65 to 99.9% by weight of one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers;

[0234] (D) Based on the composition, 0 to 1% by weight of one or more anesthetics; and

[0235] (E) Based on the composition, 0 to 49.9% by weight, preferably 0 to 34.9% by weight of one or more other cosmetically and / or pharmaceutically acceptable ingredients.

[0236] In a preferred embodiment, the composition is an injectable composition comprising or consisting of:

[0237] (A) Based on the composition, 0.5 to 3.5% by weight of a crosslinking material;

[0238] (B) Based on the composition, 1 to 150 units / mL of botulinum toxin;

[0239] (C) Based on the composition, 50 to 99.5% by weight, preferably 65 to 99.5% by weight of one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers;

[0240] (D) Based on the composition, 0 to 1% by weight of one or more anesthetics; and

[0241] (E) Based on the composition, 0 to 49.5% by weight, preferably 0 to 34.9% by weight of one or more other cosmetically and / or pharmaceutically acceptable ingredients.

[0242] In a preferred embodiment, the composition is an injectable composition, which comprises or consists of:

[0243] (A) Based on the composition, 0.1 to 3.5% by weight of a crosslinked material, which comprises or consists of:

[0244] (A-i) one or more fibroin protein moieties having an average molecular weight of at least 5 to 400 kDa, and

[0245] (A-ii) one or more polysaccharide moieties, particularly hyaluronic acid moieties, having an average molecular weight in the range of 50 to 4000 kDa,

[0246] wherein one or more fibroin protein moieties are covalently bound to one or more polysaccharide moieties, particularly hyaluronic acid moieties, without an interconnecting linker structure, preferably by an amide bond;

[0247] preferably, the mass ratio of (A-i):(A-ii) is 5:1 to 1:20;

[0248] (B) Based on the composition, 1 to 150 units / mL of botulinum toxin not covalently bound to the crosslinked material;

[0249] (C) Based on the composition, 50 to 99.9% by weight, more preferably 65 to 99.9% by weight, of one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers, including or consisting of water, an aqueous buffer, glycerol, or a combination of two or more thereof;

[0250] (D) Based on the composition, 0 to 1% by weight of one or more anesthetics; and

[0251] (E) Based on the composition, 0 to 49.9% by weight, more preferably 0 to 34.9% by weight, of one or more other cosmetically and / or pharmaceutically acceptable ingredients different from components (A) to (D).

[0252] In a preferred embodiment, the composition is an injectable composition, which comprises or consists of:

[0253] (A) Based on the composition, 0.1 to 3.5% by weight of a crosslinked material, the crosslinked material comprising or consisting of:

[0254] (A-i) one or more silk fibroin protein moieties having an average molecular weight of at least 5 to 400 kDa, and

[0255] (A-ii) one or more hyaluronic acid moieties having an average molecular weight in the range of 50 to 4000 kDa,

[0256] One or more silk fibroin moieties are covalently bound to one or more hyaluronic acid moieties without an interconnecting linker structure, preferably by an amide bond.

[0257] Preferably, the mass ratio (A-i):(A-ii) is from 5:1 to 1:20.

[0258] (B) Based on the composition, 1 to 150 units / mL of botulinum toxin not covalently bound to a crosslinking material.

[0259] (C) Based on the composition, 50 to 99.9% by weight, more preferably 65 to 99.9% by weight, of one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers comprising or consisting of water, an aqueous buffer, glycerol, or a combination of two or more thereof.

[0260] (D) Based on the composition, 0 to 1% by weight of one or more anesthetics; and

[0261] (E) Based on the composition, 0 to 49.9% by weight, more preferably 0 to 34.9% by weight, of one or more other cosmetically and / or pharmaceutically acceptable ingredients different from components (A) to (D).

[0262] Components (A) and (B), optional (C), optional (D), and optional (E) can be combined with each other in any manner. For example, components (B), optional (C), optional (D), and optional (E) are dissolved or suspended in a liquid or viscous form and mixed with the crosslinking material (A) that also forms a (hydro)gel. Optionally, one or more of components (B), optional (D), and optional (E) are dissolved or suspended in component (C). Optionally, a (hydro)gel of the crosslinking material (A) is formed in component (C). The order of mixing can be freely chosen. Optionally, any premixes of two or more components can be present.

[0263] One or more of component (B) and optional components (C), (D), and / or (E) can be mixed with at least one crosslinking material (component (A)). Components (A), (B), and (C), and optional (D) and / or (E) can be mixed with each other in any manner. In a preferred embodiment, component (A) is suspended and component (B) is dissolved in one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers (component (C)). Optionally, further components (D) and / or component (E) can further be present in the composition.

[0264] For example, for mixing, component (A) can be present in one syringe, while component (B) is dissolved in component (C), which optionally further comprises one or more of component (D) and / or (E). Component (B) can be present in a second syringe. The two syringes can be connected to each other (e.g., by a Luer lock). By pushing the contents of the second syringe into the first syringe, the materials can be mixed. Optionally, the syringes are pushed from one syringe to the other a few times.

[0265] Another aspect of the present invention relates to a method for preparing the composition of the present invention, comprising the following steps:

[0266] (i) bringing the following components into contact with each other:

[0267] (A-i) one or more fibroin protein moieties comprising a primary amino residue or a salt thereof,

[0268] (A-ii) one or more polysaccharide moieties comprising a carboxylic acid residue or a salt thereof,

[0269] (A-iii) one or more activators that promote the reaction of the carboxylic acid residue with the amino residue to form an amide bond, and

[0270] (A-iv) one or more solvents;

[0271] (ii) reacting at least some of the carboxylic acid residues with at least some of the primary amino residues to form amide bonds that covalently link one or more fibroin protein moieties with one or more polysaccharide moieties;

[0272] (iii) obtaining the crosslinked material (component (A)) from step (ii) and optionally purifying the crosslinked material; and

[0273] (iv) adding:

[0274] botulinum toxin (component (B)), and

[0275] optionally one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers (component (C)), and

[0276] optionally, one or more anesthetics (component (D)), and / or

[0277] optionally, one or more other cosmetically and / or pharmaceutically acceptable ingredients different from components (A) to (D) (component (E)).

[0278] It should be understood that the definitions and preferred embodiments set forth in the context of the composition of the present invention and the method for preparing the crosslinked material contained therein apply, mutatis mutandis, to the method for preparing the composition of the present invention.

[0279] In particular, it should be understood that any one of steps (i) to (iii) can preferably be carried out as set out in the context of preparing the cross-linked material comprised in the compositions of the present invention.

[0280] In a preferred embodiment, the method for preparing the composition of the present invention comprises the following steps:

[0281] (i) bringing into contact the following components:

[0282] (A-i) one or more fibroin protein moieties comprising a primary amino residue or a salt thereof,

[0283] (A-ii) one or more hyaluronic acid moieties comprising a carboxylic acid residue or a salt thereof,

[0284] (A-iii) one or more activators which cause the carboxylic acid residue to react with the amino residue to form an amide bond, and

[0285] (A-iv) one or more solvents;

[0286] (ii) reacting at least some of the carboxylic acid residues with at least some of the primary amino residues to form amide bonds which covalently link one or more fibroin protein moieties with one or more hyaluronic acid moieties;

[0287] (iii) obtaining the cross-linked material (component (A)) from step (ii) and optionally purifying the cross-linked material; and

[0288] (iv) adding:

[0289] botulinum toxin (component (B)), and

[0290] optionally, one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers (component (C)), and

[0291] optionally, one or more anaesthetics (component (D)), and / or optionally one or more other cosmetically and / or pharmaceutically acceptable ingredients different from components (A) to (D) (component (E)).

[0292] In a preferred embodiment, the addition of component (B) and optionally (C), (D) and / or (E) is added to the cross-linked material of step (iii).

[0293] In a preferred embodiment, step (iv) comprises adding botulinum toxin (component (B)) and one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers (component (C)) and optionally one or more components (D) and / or (E). Such a composition is preferably an injectable composition.

[0294] In a preferred embodiment, step (iv) comprises adding botulinum toxin (component (B)) dissolved in one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers (component (C)) and optionally one or more components (D) and / or (E). This step may also include mixing the components and optionally suspending component (A) in component (C), wherein component (B) and optionally one or more other components may be dissolved in this component (C). Such a composition is preferably an injectable composition.

[0295] In a preferred embodiment, step (iv) comprises mixing botulinum toxin (component (B)) dissolved in one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers (component (C)) and at least one anesthetic (component (D)) and optionally one or more components (E). This step may also include mixing the components and optionally suspending component (A) in component (C), wherein components (B) and (D) and optionally one or more other components may be dissolved in component (C). Such a composition is preferably an injectable composition.

[0296] As described above, the crosslinked materials described and defined herein can have various functions, optionally including allowing for the delayed and / or controlled release of one or more pharmaceutically and / or cosmetically active agents (such as botulinum toxin). It should be understood that the delayed and / or controlled release (e.g., depot) function of the hydrogel can be employed by adjusting the crosslinking range, the content of the crosslinked material in the composition, and the ratio between the botulinum toxin and the crosslinked material. This can meet the requirements of related indications (such as reducing adverse reactions, prolonging the duration of the action of botulinum toxin, etc.).

[0297] Accordingly, another aspect of the present invention relates to the use of a crosslinked material comprising or consisting of:

[0298] (A-i) one or more silk fibroin moieties, and

[0299] (A-ii) one or more polysaccharide moieties,

[0300] wherein one or more silk fibroin moieties are covalently bound to one or more polysaccharide moieties without an interconnecting linker structure, preferably by forming amide bonds,

[0301] for the delayed and / or controlled release of one or more pharmaceutically and / or cosmetically active agents, particularly botulinum toxin,

[0302] preferably wherein the crosslinked material forms a gel, liquid or viscous pharmaceutically acceptable carrier,

[0303] particularly wherein the crosslinked material forms part of the composition of the present invention.

[0304] It should be understood that the definitions and preferred embodiments listed in the context of the crosslinking materials that can be included in the compositions of the present invention apply, mutatis mutandis, to the uses of the crosslinking materials of the present invention.

[0305] The present invention relates to the use of a crosslinking material comprising or consisting of:

[0306] (A-i) one or more fibroin protein moieties, and

[0307] (A-ii) one or more hyaluronic acid moieties,

[0308] wherein one or more fibroin protein moieties are covalently bound to one or more hyaluronic acid moieties without an interconnecting linker structure, preferably by forming amide bonds,

[0309] for the delayed and / or controlled release of one or more pharmaceutically and / or cosmetically active agents, in particular botulinum toxin,

[0310] preferably wherein the crosslinking material forms a gelling, liquid or viscous pharmaceutically acceptable carrier,

[0311] in particular wherein the crosslinking material forms part of a composition of the present invention.

[0312] In a preferred embodiment, the crosslinking material forms part of a composition of the present invention. In a preferred embodiment, the crosslinking material forms a gel, in particular in combination with one or more liquid or viscous cosmetic and / or pharmaceutically acceptable carriers.

[0313] The compositions of the present invention can be used for any purpose. For example, it can be used for therapeutic purposes and / or cosmetic purposes. In other words, it can be used for cosmetic or aesthetic indications.

[0314] Accordingly, another aspect of the present invention relates to a composition of the present invention for use as a medicament.

[0315] Accordingly, the present invention also relates to a medicament comprising a composition of the present invention.

[0316] It should be understood that the definitions and preferred embodiments listed in the context of the compositions of the present invention apply, mutatis mutandis, to the uses of the compositions for any purpose, including uses in a therapeutic setting and medicaments comprising a composition of the present invention.

[0317] Another aspect of the present invention relates to a method for treating or preventing a disease or disorder associated with cholinergic hyperactivity using a composition of the present invention.

[0318] Thus, in other words, the present invention relates to a method of treating or preventing a disease or disorder associated with cholinergic hyperactivity, comprising the step of administering to a subject in need thereof an effective amount of a composition of the present invention.

[0319] It is to be understood that the definitions and preferred embodiments set forth in the context of the compositions of the present invention and the uses of the present invention apply mutatis mutandis to the therapeutic or prophylactic uses and methods of treatment of the present invention.

[0320] As used herein, the term "cholinergic hyperinnervation" can be understood in the broadest sense as commonly understood in the art. It can be understood as relating to synapses, which are characterized by the release of an abnormally large amount of acetylcholine into the synaptic cleft. In this context, "abnormally high" may refer to an increase relative to a reference activity of, for example, up to 25%, up to 50% or more, which is obtained by comparing the release with the release at synapses of the same type that are not in a hyperactive state, where dystonia may indicate a hyperactive state. In this context, "up to 25%" can be understood as, for example, >0% to about 25%. Methods for measuring synaptic activity are known in the art.

[0321] As used herein, a subject (also referred to as an individual) can be any animal, typically a mammal, preferably a domestic mammal or a human. Particularly preferably, the individual is a human. A human being being treated can also be designated a patient, regardless of his / her state of health.

[0322] Administration can be effected by any means. In a preferred embodiment, administration is by syringe, particularly by intradermal, subdermal, subcutaneous or intramuscular injection using a syringe. Injectable compositions within the meaning of the present invention can be administered (dispensed from a syringe) under normal conditions and at normal pressure. Furthermore, the compositions of the present invention are preferably (substantially) sterile. Preferably, the injectable composition is suitable for injection into a mammal, particularly a human. Administration can be manual, using a mechanical pump, or even automated. For example, administration can be effected using a 1 mL syringe. For example, injection can be effected using an injection needle, for example, using an injection needle size in the range of 20 to 40 gauge, 27 to 35 gauge or 30 to 33 gauge.

[0323] For example, administration can be effected in the dermal area, for example, beneath the epidermis or above the subcutaneous tissue, and thus the composition can be injected subcutaneously, subcutaneously (hypodermically), intradermally, subdermally or by some combination. In another embodiment, the composition can be administered intramuscularly.

[0324] Cholinergic hyperactivity can be cholinergic hyperactivity in any part of the subject's body. In a preferred embodiment, the cholinergic hyperactivity is cholinergic hyperactivity in muscles and / or glands.

[0325] The disease or disorder to be treated in the context of the present invention can be any disease or disorder associated with cholinergic hyperactivity. In a preferred embodiment, the disease or disorder is selected from the group consisting of: movement disorders of muscles, in particular dystonia and / or spasm, post-stroke spasticity, cervical dystonia, neuromuscular diseases such as dystonia, spasm, cramps, blepharospasm, tremors, hyperkinetic movement disorders and cerebral palsy, pain such as diabetic neuropathy, peripheral neuropathy, neuropathic pain and trigeminal neuralgia, pathological hypersecretion of saliva (sialorrhea), hyperhidrosis, urinary system diseases such as bladder dysfunction, overactive bladder, detrusor overactivity, neurogenic bladder and interstitial cystitis, sphincter of Oddi dysfunction, vulvodynia and treatment of chronic pelvic pain, prostate diseases such as benign prostatic hyperplasia (BPH) and detrusor sphincter dyssynergia (DSD), nervous system diseases such as chronic or episodic migraine, strabismus, (tension) headache, achalasia, Hirschsprung's disease, anal fissure, skin diseases or cosmetic problems that can be improved by muscle relaxation, joint lesions, arthritis and combinations of two or more thereof.

[0326] In a preferred embodiment, the disease or disorder is selected from the group consisting of: neuromuscular diseases such as dystonia, spasm, tremors, hyperkinetic movement disorders and cerebral palsy, pain such as diabetic neuropathy, peripheral neuropathy, neuropathic pain and trigeminal neuralgia, sialorrhea, hyperhidrosis (also known as: excessive sweating), urinary system diseases such as detrusor overactivity, overactive bladder, neurogenic bladder and interstitial cystitis, vulvodynia and treatment of chronic pelvic pain, prostate diseases such as benign prostatic hyperplasia (BPH) and detrusor sphincter dyssynergia (DSD), and nervous system diseases such as chronic or episodic migraine, cervical dystonia, post-stroke spasticity and blepharospasm. And combinations of two or more thereof.

[0327] As used herein, dystonia can be understood in the broadest sense as any dystonia. For example, it can be focal dystonia (usually affecting only a part of the subject's body), segmental dystonia (usually affecting multiple parts of the subject's body), or generalized dystonia (usually affecting (almost) the entire body). Preferably, the dystonia is focal dystonia or segmental dystonia. Examples of focal dystonia are laryngeal dystonia (vocal dystonia) or cervical dystonia (head / neck dystonia), or, where cervical dystonia may also be considered segmental dystonia, blepharospasm (uncontrolled eyelid blinking), oromandibular dystonia (affecting the oral region and / or chewing), and spasmodic dysphonia (affecting the vocal cords). An example of generalized dystonia is Segawa syndrome, which is characterized by abnormal leg positioning. In a preferred embodiment, when treating dystonia, the area affected by dystonia is administered parenterally, preferably in the form of an injection. This injection is preferably performed within or near the affected body part, where the injection is preferably subcutaneous or intramuscular, particularly intramuscular.

[0328] Spasm can be understood in the broadest sense as is commonly understood in the art. It can be understood as an increased internal tension of (skeletal) muscle. The origin of spasm is damage to the areas of the central nervous system responsible for movement, where these areas are the brain and spinal cord, particularly the pyramidal tract of the first motor neurons. A common cause of spasm is hypoxic damage to the motor brain areas caused by a cerebral infarction. Monospasm is spastic paralysis of a single muscle or a limb. Examples of muscles that may be spastically paralyzed are the flexor carpi radialis, flexor carpi ulnaris, flexor digitorum superficialis, flexor digitorum profundus, brachioradialis, biceps brachii, pronator quadratus, pronator teres, flexor pollicis longus, flexor pollicis brevis, and opponens pollicis. Paraplegic spasm is characterized by paralysis of both legs. Hemispasticity describes paralysis of the limbs on one half of the body or one half of the face (hemifacial spasm). Finally, tetraplegic spasm involves spastic paralysis of all four limbs, where the neck and core muscles may also be affected. In one embodiment, the composition of the present invention for treating spasm is administered parenterally, preferably in the form of an injection. This injection can preferably be performed within or near the affected body part, where the injection is preferably subcutaneous or intramuscular, particularly intramuscular.

[0329] The composition of the present invention can also be used as a filling composition, such as a soft tissue filler, particularly a dermal filler or a connective tissue filler. Accordingly, the present invention also relates to the use of the composition of the present invention as a filling composition, such as a soft tissue filler, particularly a dermal filler or a connective tissue filler.

[0330] Another aspect of the present invention relates to the use of the compositions of the present invention for cosmetic applications, including restoring and / or improving the skin quality of the face and / or body.

[0331] In other words, the present invention relates to a method for restoring and / or improving the skin quality of the face and / or body, comprising the step of administering to a subject in need thereof an effective amount of the composition of the present invention.

[0332] In other words, the present invention relates to the composition of the present invention for use in a method for restoring and / or improving the skin quality of the face and / or body.

[0333] As described above, the definitions and preferred embodiments set forth in the context of the composition of the present invention apply, mutatis mutandis, to the uses of the composition and to any method for restoring and / or improving the skin quality of the face and / or body.

[0334] In one embodiment, the use of the present invention may be a cosmetic use and thus may also be a non-therapeutic use. The use of the present invention may be carried out by a cosmetician, a beauty professional or a healthcare professional.

[0335] In a preferred embodiment, the restoration and / or improvement of the facial and / or body skin quality includes improving and / or reducing and / or filling and / or preventing wrinkles, especially wrinkles caused by muscle activity (such as facial expressions), 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 the subcutaneous support of the eyebrows, cheekbones and buccal fat pads, improving the tear trough, improving the appearance of the nose, resolving facial asymmetry, improving the jawline, or a combination of two or more thereof. As is generally understood, lines may also be understood as wrinkles and vice versa. The terms "lines" and "wrinkles" may be used interchangeably.

[0336] As used herein, facial lines that can be improved and / or reduced and / or filled may be exemplarily selected from the group consisting of: horizontal forehead lines, glabellar lines (such as glabellar frown lines), periorbital lines, crow's feet, bunny lines, nasolabial folds, perioral lines, upper lip lines, lower lip lines, commissure lines, marionette lines, perioral lip lines, oral commissures, mentolabial folds and oval chins. In a preferred embodiment, the facial lines that can be improved may be horizontal forehead lines and / or glabellar lines and / or periorbital lines including crow's feet.

[0337] In a further preferred embodiment, the improvement of the facial and / or body skin quality includes reducing the size of skin pores and / or sebum secretion. In a further preferred embodiment, the restoration includes treatment of the platysma band and / or the platysma muscle.

[0338] The composition of the present invention can also be used for any other purpose. It can be used for any purpose described in WO 2017 / 148915.

[0339] The composition of the present invention can be provided in any packaging. Depending on the intended use of the composition of the present invention, it can be provided in different packagings. It can be stored under any conditions suitable for this purpose, for example, at ambient temperature (e.g., 18 to 30 °C, preferably 18 to 25 °C), in a refrigerator (e.g., 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), in liquid nitrogen, in dry ice, or even in one or more liquid rare gases. For example, it can be contained in vials, syringes. It can be administered to a subject by injection (e.g., via a syringe or dropper). It can be stored as a hydrogel, a gel containing other non-aqueous solvents and / or a suspension, an emulsion, a colloid or a solution.

[0340] As used herein, the terms "about" and "approximately" can be understood to include ranges with deviations of up to + / - 10% of the corresponding numerical value. It should be understood that the specific values are also explicitly disclosed.

[0341] It should be further understood that the ranges include the numerical values provided in commonly rounded values, which include the entire rounding limits. For example, the range of "1 mg" includes the range of 0.50 to 1.49 mg.

[0342] However, the numerical values of the present invention also more specifically disclose more detailed values of one or more orders of magnitude. Thus, for example, "1 mg" can also include the specific disclosure of "1.0 mg".

[0343] The examples and claims illustrate the embodiments of the present invention. Examples

[0344] Materials and Methods

[0345] Raw Materials

[0346] Hyaluronic acid, different intrinsic viscosities (HTL Biotechnology, Javene, France);

[0347] 5% aqueous solution of silk fibroin from silkworms (CareSilk s.r.l.s., Lecce, Italy);

[0348] 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) (SigmaAldrich, Darmstadt, Germany);

[0349] Water (internal demineralized water system);

[0350] Saline solution (B.Braun, Melsungen, Germany)

[0351] Lidocaine hydrochloride (Albemarle Corp., Charlotte, USA)

[0352] (Botulinum neurotoxin preparation)

[0353] Unless otherwise stated, all syntheses and measurements were carried out under ambient conditions, i.e., ambient temperature (e.g., 18 to 25 °C, especially about 20 °C) and ambient / atmospheric pressure.

[0354] Extrusion force (EF)

[0355] The extrusion force (EF) was measured using an instrument TA.XT Plus texture analyzer (Stable Micro Systems Ltd., Surrey, UK). A syringe equipped with a 30G TSK needle (TSK Laboratory Europe, Oosterhout, Netherlands) was placed in the instrument, and then the instrument pressed the syringe plunger at a constant speed of 0.21 mm / s (about 1.26 cm / min) over a distance of 30 mm. The force required to extrude the syringe contents through the needle was recorded, the average value was calculated and reported as the extrusion force.

[0356] Rheology

[0357] Rheology was measured using an instrument AntonPaar MCR 302 (Anton Paar GmbH, Graz, Austria) with a cone-plate (CP50-1, diameter 50 mm) or plate-plate (PP20, diameter 20 mm) geometry. The measurements were carried out in oscillatory mode with a frequency sweep range from 0.1 Hz to 10 Hz, at a temperature of 25 °C and a constant deformation of 0.1%. The measurement results were reported as the storage modulus (G') and loss factor (tanδ) at 1 Hz.

[0358] Example 1 - Preparation of crosslinked materials and effect of the amount of lubricating phase on material properties

[0359] With an inherent viscosity of 2.8 m 3Hyaluronic acid (HA) (4.6 g, equivalent to 4.0 g of dry polymer) was dissolved in 200 g of water (polymer concentration of 20 mg / g). DMTMM (3.3 g, equivalent to 2.8 g of dry material - 1 equivalent relative to the amount of HA) was added, and the mixture was stirred for 1 hour. Subsequently, 100 mL of a silk fibroin solution (concentration of 20 mg / g, HA / silk fibroin weight ratio of 2 / 1) was added: the mixture was stirred for 2 hours and then stirring was stopped. The next day, the crosslinked material (here, HA / silk fibroin gel as an example) was purified at room temperature using dynamic crossflow filtration (DCF Andritz, membrane d = 152 mm (Andritz AG, Graz, Austria)) and a 5 mM phosphate buffered saline (PBS) solution. Then the purified crosslinked material (here, HA / silk fibroin gel as an example) was mixed with different amounts of a lubricating phase (concentration of the lubricating phase of 30 mg / g). Finally, the crosslinked material (here, HA / silk fibroin gel as an example) was loaded into a 1 mL syringe (syringe No. 1) and sterilized at 127 °C for 8 minutes. The results are shown in Table 1 below.

[0360] Table 1. Properties of the resulting crosslinked material (here, HA / silk fibroin gel as an example). All given values are the average of duplicate determinations

[0361]

[0362] The G' decrease was calculated using the following formula:

[0363] G' decrease = (G' at 1 Hz before sterilization - G' at 1 Hz after sterilization) / G' at 1 Hz before sterilization

[0364] The lubricating phase can be added to the gel to reduce the extrusion force. However, in the case of the crosslinked material (here, HA / silk fibroin gel as an example) contained in the composition of the present invention, the lubricating phase surprisingly does not result in a reduction in the extrusion force. Thus, other materials can be prepared without the lubricating phase.

[0365] We found that the G' of the cross-linked material without a lubricating phase (here exemplified by the HA / silk fibroin gel) may be comparable to or even higher than that of the commercially available cross-linked hyaluronic acid product Belotero Volume Lidocaine (Anteis S.A., Plan-les-Ouates, Switzerland). Although the G' of one HA / silk fibroin gel exemplified here was approximately 278 Pa (Fib01A) after sterilization, the G' of the similar hyaluronic acid product Belotero Volume Lidocaine was approximately 270 Pa. However, it was found that the extrusion force (EF) through a 30G TSK needle was significantly lower. The extrusion force (EF) of the studied HA / silk fibroin gel was approximately 12 N, while the extrusion force (EF) of the cross-linked hyaluronic acid (Belotero Volume Lidocaine) was approximately 22 N. This may indicate that the HA / silk fibroin gel can provide a lifting effect similar to that of the commercially available cross-linked hyaluronic acid product Belotero Volume Lidocaine (due to similar G'), while providing better injection performance for practitioners.

[0366] Furthermore, in this set of experiments, the G' of the cross-linked material (here exemplified by the HA / silk fibroin gel) decreased by 10 - 25% after sterilization, which was significantly lower than the 78% decrease in G' in the control experiment (pure non-cross-linked hyaluronic acid). The results are also shown in Table 1 above.

[0367] Example 2 - Preparation of Cross-Linked Materials and Influence of HA / Silk Fibroin Ratio on Material Properties

[0368] Dissolve hyaluronic acid (HA) with an intrinsic viscosity of 2.8 m 3 / kg (3.5 g, equivalent to 3.0 g of dry polymer) in 150 g of water (polymer concentration of 20 mg / g). Add DMTMM (2.5 g, equivalent to 2.1 g of dry material - 1 equivalent relative to the amount of HA), and stir the mixture for 1 hour. Then add 150 mL of silk fibroin solution (concentration of 20 mg / g, HA / silk fibroin weight ratio of 1 / 1). Stir the mixture for 2 hours, and then stop stirring. The next day, purify the cross-linked material (here exemplified by the HA / silk fibroin gel) at room temperature using dynamic cross-flow filtration (DCF Andritz, membrane d = 152 mm (Andritz AG, Graz, Austria)) and 5 mM phosphate buffered saline (PBS) solution. No lubricating phase was added. Finally, load the cross-linked material (here exemplified by the HA / silk fibroin gel) into a 1 mL syringe (syringe No. 1) and sterilize it at 127 °C for 8 minutes. The results are shown in Table 2 below.

[0369] Table 2. Properties of the obtained crosslinked materials (here HA / silk fibroin gel is taken as an example). All given values are the averages of duplicate determinations

[0370]

[0371]

[0372] The study found that an increase in the silk fibroin content in the crosslinked material results in a higher G' for the crosslinked material (here HA / silk fibroin gel is taken as an example).

[0373] It is worth noting that this crosslinked material has a low extrusion force (even though its G' is high). The explanation for this may be the thixotropic (shear thinning) behavior of silk fibroin. Crosslinked materials with a high and adjustable G' value and a low and adjustable extrusion force can be obtained.

[0374] Example 3 - Preparation of crosslinked materials and the effect of the intrinsic viscosity (IV) of hyaluronic acid (HA) on the material properties

[0375] Dissolve hyaluronic acid (HA) with an intrinsic viscosity of 1.5 m 3 / kg (3.5 g, equivalent to 3.0 g of dry polymer) in 150 g of water (polymer concentration of 20 mg / g). Add DMTMM (2.5 g, equivalent to 2.1 g of dry material - 1 equivalent relative to the amount of HA) and stir the mixture for 1 hour. Then add 150 mL of silk fibroin solution (concentration of 20 mg / g, HA / silk fibroin weight ratio of 1 / 1). Stir the mixture for 2 hours and then stop stirring. The next day, the crosslinked material (here HA / silk fibroin gel is taken as an example) is purified using dynamic crossflow filtration (DCFANDRITZ, membrane d = 152 mm (Andritz AG, Graz, Austria)) and 5 mM phosphate buffered saline (PBS) solution at room temperature. No lubricating phase is added. Finally, the crosslinked material (here HA / silk fibroin gel is taken as an example) is loaded into a 1 mL syringe (syringe No. 1) and sterilized at 127 °C for 8 minutes. The results are shown in Table 3 below.

[0376] Table 3. Properties of the obtained crosslinked materials (here HA / silk fibroin gel is taken as an example). All given values are the averages of duplicate determinations

[0377]

[0378] Example 4 - Enzymatic degradation of HA / silk fibroin gel

[0379] To investigate whether cross-linked materials (here HA / silk fibroin gels are taken as an example) can also be used as reversible fillers, these materials were treated with the enzyme hyaluronidase from sheep testes. That is, approximately 0.50 g of the gel was weighed by differential weighing and placed on the plate of the CP50-1 (cone-plate) system of an Anton Paar MCR 302 rheometer (Anton Paar GmbH, Graz, Austria). A homogeneous aqueous solution of 150 μL of WFI containing 50 U of hyaluronidase was added to the top of the hydrogel on the plate. The hyaluronidase hydrogel mixture was manually homogenized (e.g., by a pipette tip) for approximately 10 seconds. Thereafter, measurements were carried out in oscillatory mode at 37 °C with a strain of 0.1% and a frequency of 1 Hz. The measurement duration was 60 minutes, and 1 point was recorded per minute.

[0380] The gels prepared from non-cross-linked HA degraded the fastest. This was followed by the gels composed of cross-linked HA (Belotero Volume, Anteis S.A., Plan-les-Ouates, Switzerland), and the gels containing silk fibroin degraded the slowest. This may indicate that the gels containing silk fibroin have a longer lifespan compared to typical HA cross-linked gels. The results are shown in Table 4 below.

[0381] Table 4. Enzymatic degradation of hydrogels (here HA / silk fibroin gels are taken as an example). All given values are the averages of duplicate determinations

[0382]

[0383] The decrease in G' was calculated using the following formula:

[0384] Decrease in G' = (G' at 1 Hz before treatment - G' at 1 Hz after treatment with hyaluronidase (at 10 minutes)) / G' at 1 Hz before treatment

[0385] Example 5 - Accelerated stability study

[0386] To test the stability, batch Fib05 (see above) was mixed with lidocaine (0.3% lidocaine to prepare a new batch: Fib05L), and placed in a climatic chamber at 40 °C (accelerated conditions). It was characterized by measuring the rheological properties and the extrusion force (using a 30G TSK needle) of the gel at different time points (weeks 4, 8, and 12). All measurements were repeated three times. The results are shown in Table 5 below.

[0387] Table 5. Characteristics of the resulting cross-linked materials (here HA / silk fibroin gels are taken as an example) over time after incubation at 40 °C at 75% relative humidity

[0388] Parameter T0 4 weeks 8 weeks 12 weeks G’ [Pa] 440 442 483 434 tanδ 0.251 0.193 0.212 0.217 EF (30G TSK needle) [N] 9.5 9.3 9.2 8.0

[0389] The results show that the crosslinked materials (here exemplified by HA / silk fibroin gels) are quite stable over time. Even after incubation at 40 °C and 75% relative humidity for 12 weeks, no significant decrease or deterioration in performance was found.

[0390] In summary, it was found that the crosslinked materials contained in the compositions of the present invention can be prepared very well and efficiently, optionally in a single batch, without burden. These materials have good injectability properties and appear to be shear thinning / thixotropic.

[0391] Example 6 - Low-viscosity HA / silk fibroin gel mixed with solid botulinum toxin

[0392] 3.2 g of hyaluronic acid (HA) with an intrinsic viscosity of 2.8 m 3 / kg was dissolved in 178 g of buffer. 4.3 g of DMTMM was added and the mixture was stirred for 1 hour. Then, 62.5 mL of a silk fibroin solution containing 2 g of silk fibroin was added. The mixture was stirred for 15 minutes. The crosslinked material was purified using dialysis. Then, the material was loaded into a 1 mL syringe and sterilized at 131 °C for 1 minute. 50 units (U) of a solid botulinum toxin preparation (Xeomin) was added to 1 mL of the resulting crosslinked hyaluronic acid-silk fibroin (HA / Fib) matrix (as the crosslinked material of the present invention). The results are shown in Table 6 below.

[0393] Table 6. HA / silk fibroin gels with and without botulinum toxin (BoNT).

[0394]

[0395] Example 7 - HA / silk fibroin gel mixed with botulinum toxin solution

[0396] 3.2 g of hyaluronic acid (HA) (with an intrinsic viscosity of 2.8 m 3 / kg) was dissolved in 178 g of buffer. 4.3 g of DMTMM was added and the mixture was stirred for 1 hour. Then, 62.5 mL of a silk fibroin solution containing 2 g of silk fibroin was added. The mixture was stirred for 15 minutes. The crosslinked material was purified using dialysis. The material was loaded into a 1 mL syringe and sterilized at 131 °C for 1 minute. The gel (1 mL) was mixed with 50 units of a botulinum toxin preparation (Xeomin previously dissolved in 1 mL of saline) in a 1:1 ratio. The results are shown in Table 7 below.

[0397] Table 7. HA / silk fibroin gels with and without botulinum toxin (BoNT).

[0398]

Claims

1. A composition comprising or consisting of: (A) A crosslinked material comprising or consisting of: (A-i) One or more fibroin protein moieties, and (A-ii) One or more polysaccharide moieties; wherein the one or more fibroin protein moieties are covalently bound to the one or more polysaccharide moieties without an interconnecting linker structure; (B) Botulinum toxin; (C) Optionally, one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers; (D) Optionally, one or more anesthetics; and (E) Optionally, one or more other cosmetically and / or pharmaceutically acceptable ingredients different from components (A) to (D).

2. The composition according to claim 1, wherein each of the one or more polysaccharide moieties comprises one or more carboxylic acid residues or salts thereof, Preferably, wherein said one or more polysaccharide moieties comprise or consist of: one or more hyaluronic acid moieties, heparin precursor moieties, chondroitin sulfate moieties, carboxymethyl cellulose moieties, or a combination of two or more thereof.

3. The composition according to any one of claims 1 or 2, wherein the one or more polysaccharide moieties are one or more hyaluronic acid moieties.

4. The composition according to any one of claims 1 to 3, wherein: (a) The average molecular weight of the one or more fibroin protein moieties is in the range of at least 5 kDa, preferably 5 to 1000 kDa, in the range of 5 to 400 kDa, in the range of 10 to 400 kDa, or in the range of 100 to 150 kDa, preferably, wherein the one or more fibroin protein moieties are silk fibroin protein moieties from silkworms, more preferably silk fibroin protein moieties from silkworms having at least 80% sequence homology with natural insect or spider fibroin protein moieties; (b) The average molecular weight of the one or more polysaccharide moieties is in the range of at least 50 kDa, preferably 50 to 4000 kDa; and / or (c) The mass ratio between component (A-i):(A-ii) is in the range of 5:1 to 1:

20.

5. The composition according to any one of claims 1 to 4, wherein the one or more fibroin protein moieties are covalently bound to the one or more polysaccharide moieties via amide bonds, in particular wherein the crosslinked material is further characterized by not containing: (a) Imide groups; (b) Imine groups; (c) Epoxy groups; and (d) Exogenous linker moieties.

6. The composition according to any one of claims 1 to 5, wherein the botulinum toxin is botulinum neurotoxin serotype A, in particular botulinum neurotoxin serotype A1.

7. The composition according to any one of claims 1 to 6, wherein the botulinum toxin is not covalently bound to the crosslinked material, and / or wherein the crosslinked material forms a gel, in particular a hydrogel, in a liquid or viscous pharmaceutically acceptable carrier, and the botulinum toxin is dissolved in the liquid or viscous pharmaceutically acceptable carrier that is soaked in the gel, in particular the hydrogel.

8. The composition according to any one of claims 1 to 7, wherein the composition is an injectable composition comprising or consisting of: (A) Based on the composition, 0.1% to 3.5% by weight of a crosslinking material; (B) Based on the composition, 1 to 150 units / mL of botulinum toxin; (C) Based on the composition, 50% to 99.9% by weight, preferably 59% to 99.9% by weight, of one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers; (D) Based on the composition, 0 to 1% by weight of one or more anesthetics; and (E) Based on the composition, 0 to 49.9% by weight of optionally one or more other cosmetically and / or pharmaceutically acceptable ingredients.

9. The composition according to any one of claims 1 to 8, wherein the composition comprises at least 50% by weight, based on the composition, of a liquid or viscous pharmaceutically acceptable carrier, Preferably, the liquid or viscous pharmaceutically acceptable carrier comprises or consists of: water, an aqueous buffer, glycerol, or a combination of two or more thereof, especially wherein the composition is injectable into soft tissue, especially intradermal injection, subdermal injection, subcutaneous injection, and / or intramuscular injection.

10. The composition according to any one of claims 1 to 9, wherein the composition is an injectable composition comprising or consisting of: (A) Based on the composition, 0.1% to 3.5% by weight of a crosslinking material, the crosslinking material comprising or consisting of: (A-i) one or more silk fibroin moieties having an average molecular weight of at least 5 to 400 kDa, and (A-ii) one or more polysaccharide moieties, especially hyaluronic acid moieties, having an average molecular weight in the range of 50 to 4000 kDa, wherein the one or more silk fibroin moieties are covalently bound to the one or more polysaccharide moieties, especially hyaluronic acid moieties, without an interconnecting linker structure, preferably by an amide bond, preferably with a mass ratio of (A-i):(A-ii) of 5:1 to 1:20, (B) Based on the composition, 1 to 150 units / mL of botulinum toxin not covalently bound to the crosslinking material; (C) Based on the composition, 50% to 99.9% by weight of one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers, including water, an aqueous buffer, glycerol, or a combination of two or more thereof, or consisting of water, an aqueous buffer, glycerol, or a combination of two or more thereof; (D) Based on the composition, 0 to 1% by weight of one or more anesthetics; and (E) Based on the composition, 0 to 49.9% by weight of one or more other cosmetically and / or pharmaceutically acceptable ingredients different from components (A) to (D).

11. A method for preparing the composition according to any one of claims 1 to 10, comprising the steps of: (i) bringing the following components into contact with each other: (A-i) one or more silk fibroin moieties containing primary amino residues or salts thereof, (A-ii) one or more polysaccharide moieties containing carboxylic acid residues or salts thereof, (A-iii) one or more activators that cause the carboxylic acid residues to react with the amino residues to form an amide bond, and (A-iv) one or more solvents; (ii) React at least some of the carboxylic acid residues with at least some of the primary amino residues to form amide bonds that covalently link one or more fibroin portions to one or more polysaccharide portions; (iii) Obtain the crosslinked material (component (A)) from step (ii) and optionally purify the crosslinked material; and (iv) Add: Botulinum toxin (component (B)), and Optionally, one or more liquid or viscous cosmetically and / or pharmaceutically acceptable carriers (component (C)), and Optionally, one or more anesthetics (component (D)), and / or Optionally, one or more other cosmetically and / or pharmaceutically acceptable ingredients different from components (A) to (D) (component (E)).

12. Use of a crosslinked material for delaying and / or controlling the release of one or more pharmaceutically and / or cosmetically active agents, in particular botulinum toxin, said crosslinked material comprising or consisting of: (A-i) One or more fibroin portions, and (A-ii) One or more polysaccharide portions, wherein the one or more fibroin portions are covalently linked to the one or more polysaccharide portions without an interconnecting linker structure, Preferably, wherein the crosslinked material forms a gelled, liquid or viscous pharmaceutically acceptable carrier, In particular, wherein the crosslinked material forms part of a composition used according to any one of claims 1 to 10.

13. Use of the composition according to any one of claims 1 to 10 as a medicament.

14. Method of using the composition according to any one of claims 1 to 10 for treating or preventing a disease or disorder associated with cholinergic hyperactivity, in particular muscle and / or glandular hyperactivity, and / or pain, preferably, wherein the disease or disorder is selected from the group consisting of: movement disorders, in particular dystonia and / or spasm, neuromuscular diseases such as dystonia, spasm, tremor, hyperkinetic movement disorders and cerebral palsy, pain such as diabetic neuropathy, peripheral neuropathy, neuropathic pain and trigeminal neuralgia, sialorrhea, hyperhidrosis, urinary system diseases such as detrusor overactivity, neurogenic bladder and interstitial cystitis, vulvodynia and treatment of chronic pelvic pain, prostate diseases such as benign prostatic hyperplasia (BPH) and detrusor sphincter dyssynergia (DSD), migraine, nervous system diseases such as chronic or episodic migraine, cervical dystonia, post-stroke spasm, blepharospasm, strabismus, (tension) headache, pathological hypersecretion of saliva, achalasia, sphincter of Oddi dysfunction, Hirschsprung's disease, anal fissure, bladder dysfunction such as overactive bladder, skin diseases or cosmetic problems that can be improved by muscle relaxation, joint lesions, arthritis and combinations of two or more thereof.

15. Use of the composition according to any one of claims 1 to 10 for cosmetic applications, including restoring and / or improving the skin quality of the face and / or body, Preferably includes improving and / or reducing and / or filling and / or preventing wrinkles, especially wrinkles caused by muscle activity, such as facial expressions, 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 the subcutaneous support of the eyebrows, cheekbones and buccal fat pads, improving the tear trough, improving the appearance of the nose, resolving facial asymmetry, improving the jawline, or a combination of two or more thereof.

16. The use according to claim 15, wherein the improvement of the skin quality of the face and / or body includes reducing the size of skin pores and / or sebum secretion.

17. The use according to claim 15, wherein the restoration includes the treatment of the platysma band and / or the platysma muscle.

Images