EP4633596A1

EP4633596A1 - Injectable gel comprising botulinum toxin and uses thereof

Info

Publication number: EP4633596A1
Application number: EP23829050.6A
Authority: EP
Inventors: Radovan VUKICEVIC; Colin DRABE; Thomas Hengl
Original assignee: Merz Pharma GmbH and Co KGaA
Current assignee: Merz Pharma GmbH and Co KGaA
Priority date: 2022-12-15
Filing date: 2023-12-14
Publication date: 2025-10-22
Also published as: MX2025006389A; KR20250124803A; CN120225172A; WO2024126649A1; AU2023392523A1; IL320943A; JP2025541064A

Abstract

The present invention relates to a composition comprising (A) a cross-linked material comprising one or more fibroin moieties and one or more polysaccharide moieties covalently conjugated with each other, (B) botulinum toxin. Furthermore, the present invention relates to therapeutic and aesthetic uses of such composition.

Description

Injectable gel comprising botulinum toxin and uses thereof

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

Botulinum toxin is however inherently instable, in particular under alkaline conditions. It is further heat-labile. For this purpose, commercial botulinum toxin is often stored as vacuum-dried (lyophilized) material and/or contains protecting and preserving excipients.

Several uses based on the injection of botulinum toxin in or under a subject’s skin or other soft tissue directly to the site of intended effect. In typical long-term therapeutic or aesthetic uses, botulinum toxin has to be administered again after certain time, often every few months, due to the reversibility of its inhibitory effect of acetylcholine release. As botulinum toxin directly affects the cellular endocytosis and exocytosis mechanism by inactivating the SNARE proteins, there is only a limited time period of botulinum toxin uptake for approximately one day before further uptake is inhibited by blocking of the endocytosis pathways. This is undesirable in many applications. Thus, it is desired to extend the duration of effect of a botulinum toxin injection, in other words to provide retarded and/or controlled release formulations thereof, which are able to adjust the retention and the release profile of the botulinum toxin to the uptake capacity of the target cell. This is partly achieved by preparing retarded and/or controlled release formulations, where botulinum toxin is embedded in a pharmaceutically acceptable matrix, which can provide an adapted and optionally extended duration of effect. For this purpose, biocompatible compositions were considered. US 9,044,477 and US 9,050,336 teach mixtures comprising chemical entities including hyaluronic acid, botulinum toxin, and albumin. Herein, viscous materials are formed by adaptable concentrations of hyaluronic acid, where the hyaluronic acid may also be crosslinked hyaluronic acid. WO 2020/056371 teaches mixtures of clostridial toxin and non-crosslinked hyaluronic acid or salt thereof. The components hyaluronic acid, botulinum toxin, and albumin are simply admixed in the compositions. WO 2011/119468 teaches a hydrogel for soft tissue augmentation comprising a polymer that can be hyaluronic acid, which can further comprise other ingredients. WO 2017/148915 teaches compositions comprising non-crosslinked hyaluronic and botulinum toxin. Some of these compositions are well compatible to be injected and generally usable for soft tissue augmentation.

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

It was considered to obtain a desirable shelf stability and improve adjustability of release kinetics by cross-linking components via chemical cross-linking structures. WO 2020/132331 describes conjugates of fibroin moieties, preferably cross-linked hyaluronic acid moieties and cross-linking moieties such as polyethylene glycol (PEG) and/or polypropylene glycol (PPG), usable as tissue fillers. Further, this document teaches that a large variety of different other ingredients can be comprised in a tissue filler. Such conjugates have the disadvantage that synthetic xenobiotic moieties are comprised in the structure such as PEG and/or PPG linkers. Such xenobiotic structures are often not fully biodegradable and/or bio-resorbable. Such moieties are often undesired in materials that are injected into a subject’s body. A bio-based material composed of fibroin moieties and hyaluronic acid moieties as such is described in PCT/EP2022/066989. There is, however, no teaching that such conjugate is usable in the context of a composition containing botulinum toxin.

In view of the above, it is still an unmet need to provide compositions that have a well-controlled and adjustable release of botulinum toxin while widely avoiding synthetic non-biodegradable moieties such as synthetic linker moieties. The main components of such compositions should preferably be widely biodegradable and/or bio-resorbable. The resources of the solid main components used for preparation of such compositions should preferably be bio-based, thus of biological or biotechnological origin, while optionally synthetic agents such as one or more anesthetics can be comprised. Particularly desirable is an injectable composition usable for treating or preventing a disease or condition associated with hyperactive cholinergic activity, in particular of muscles and/or glands, pain, or for rejuvenation and/or improvement of the skin quality of the face and/or body.

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

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

(A) a cross-linked material comprising or consisting of

(A-i) one or more fibroin moieties, and

(A-ii) one or more polysaccharide moieties; wherein the one or more fibroin moieties are covalently conjugated with 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 further cosmetically and/or pharmaceutically acceptable ingredients different from components (A) to (D).

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

It was surprisingly found that synthetic linker structures as commonly used in the prior art in the context of injectable hydrogels, such as reactive linkers, polyethylene glycol (PEG) and/or polypropylene glycol (PPG), are avoidable. The cross-linked material used in the composition of the present invention may essentially consist of amino acid moieties and polysaccharide moieties (in particular hyaluronic acid moieties), which can, as such as separated chemical moieties, also be found in nature. In aqueous environment, the obtained cross-linked material may form a gel such as, in particular, a hydrogel. The preparation of a (hydro)gel may be comparably easy and less laborious than other means of crosslinking in the art. Beside water or an aqueous buffer, only three raw materials were used in the synthesis of the cross-linked material, i.e., one or more polysaccharides (in particular hyaluronic acid moieties) or a salts thereof, one or more fibroins or salts thereof, and a crosslinker facilitating the reaction between the aforementioned components and forming amide bonds in between the polysaccharide (in particular hyaluronic acid) and the fibroin.

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

The composition of the present invention may be used for any purpose. Preferably, the composition of the present invention may be injectable. For instance, it may be used for various aesthetic and therapeutic purposes, which are exemplified below.

The cross-linked material of the composition of the present invention may also be designated by an abbreviation such as HA-fibroin, HA/fibroin, HA-Fib, HA/Fib, etc. The cross-linked material may also be usable as biopolymer filler such as, e.g., biopolymer dermal filler.

As used in the context of the present invention, the term “polysaccharide moiety” may be understood in the broadest sense as any moiety of a polysaccharide known in the art, which may be conjugated with one or more fibroin moieties without an interconnecting linker structure.

In a preferred embodiment, the one or more polysaccharide moieties each comprise one or more carboxylic acid residues or salts thereof. Preferably, the one or more polysaccharide moieties further comprises hydroxy groups. A polysaccharide may be a naturally occurring polysaccharide that may be modified or may be a synthetic polysaccharide. In this context, a polysaccharide may be branched or unbranched. It will be understood that the term “polysaccharide moiety” may also include salts and modified forms thereof. In a preferred embodiment, the polysaccharide has not been oxidized. The one or more polysaccharide moieties may comprise one or more types of sugar acid moieties or salts thereof. In a preferred embodiment, the one or more polysaccharide moieties comprise one or more types of sugar acid moieties or salts thereof, wherein the one or more types of sugar acid moieties are selected from the group consisting of:

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

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

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

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

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

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

In a preferred embodiment, the 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 sulphate, 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 comprising carboxylic acid groups are also commercially available (e.g., from HTL Biotechnology, Javene, France).

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

The present invention thus also relates to a composition comprising or consisting of:

(A) a cross-linked material comprising or consisting of

(A-i) one or more fibroin moieties, and

(A-ii) one or more hyaluronic acid moieties; wherein the one or more fibroin moieties are covalently conjugated with the one or more hyaluronic acid 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

In a preferred embodiment, the cross-linked material of the present invention is a gel. In a preferred embodiment, the cross-linked material of the present invention is a polysaccharide/fibroin gel. In a preferred embodiment, the cross-linked material of the present invention is a hyaluronic acid/fibroin gel (HA/fibroin gel).

The cross-linked material of the composition of the present invention may optionally have good shear-thinning properties. The composition may optionally have thixotropic properties. Thus, it may be less viscous when stressed. The composition of the present invention may be injected very well, while still being rather viscous in its target area (when e.g., administered in a subcutaneous area). Comparably low extrusion forces are required. Gels of high viscosity and low extrusion force are obtainable. In a preferred embodiment, the composition is injectable into a soft tissue, in particular injectable intradermally, subdermally, subcutaneously, and/or intramuscularly.

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

As used in the context of the present invention, the term “fibroin moiety” may be understood in the broadest sense as any moiety of fibroin known in the art. The fibroin moiety may also be designated by its abbreviation Fib or fib.

In a preferred embodiment, the one or more fibroin moieties have an average molecular weight of at least 1 kDa, or of at least 5 kDa, or of at least 10 kDa, or of at least 100 kDa, or of at least 200 kDa or more. Preferably, the one or more fibroin moieties are each polymeric moieties or a complex of polymeric moieties of a total molecular weight (Mw) of at least 5 kDa (5000 Dalton, 5 Kilodalton), more preferably at least 10 kDa (10000 Dalton), even more preferably at least 100 kDa, in particular at least 200 kDa or more. In one embodiment, the one or more fibroin moieties have an average molecular weight of 10 to 400 kDa.

In a preferred embodiment, the one or more fibroin moieties have an average molecular weight not more than 2000 kDa, not more than 1000 kDa, not more than 750 kDa, not more than 500 kDa, not more than 250 kDa, not more than 200 kDa, or not more than 150 kDa.

In a preferred embodiment, the one or more fibroin moieties have an average molecular weight of 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 one or more fibroin moieties have an average molecular weight of in the range of 10 to 400 kDa. In another preferred embodiment, the one or more fibroin moieties have an average molecular weight of in the range of 100 to 150 kDa.

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

In one embodiment, the fibroin moieties are of a single type and have thus (essentially) one molecular weight.

In another embodiment, the fibroin moieties have at least two different average molecular weights each comprising primary amino residues or salts thereof. In other words, the fibroin moieties may also be a mixture of fibroin moieties of different average molecular weight. In a preferred embodiment, the fibroin moieties have at least two different molecular weights and at least one fibroin moiety has, preferably at least two fibroin moieties both have, in particular all fibroin moieties 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. In a preferred embodiment, the fibroin moieties have at least two different molecular weights and at least one fibroin moiety has, preferably at least two fibroin moieties both have, in particular all fibroin moieties each have, a molecular weight in the range of 50 to 400 kDa.

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

Preferably, a fibroin moiety comprises at least one backbone of a full-length fibroin polypeptide, in particular (essentially) consists of one or more backbones of one or more full-length fibroin polypeptides. In other words, a fibroin moiety is preferably derived from naturally occurring fibroin.

As used herein, amide binds are understandable in the broadest sense. Typically, an amide bond has the structure -NH-CO- or a tautomeric structure thereof. The amide bond formed between the fibroin moieties and the polysaccharide moieties, such as hyaluronic acid moieties, may have any chirality. In one embodiment, it is a racemic mixture. In one embodiment, the majority of amino acid moieties of fibroin are L-amino acid moieties. In one embodiment, (essentials) all amino acid moieties of fibroin are L-amino acid moieties.

In a preferred embodiment, the one or more fibroin moieties are silk fibroin moieties, more preferably silk fibroin moieties having at least 80% sequence homology to a natural insect or spider silk fibroin moiety. In a preferred embodiment, fibroin is silk fibroin. In an alternative preferred embodiment, 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, in particular identity, to one or more naturally occurring silk fibroin polypeptides. Silk fibroin may also include a truncated form thereof. Silk fibroin may be silkworm (Bombyx mori) fibroin and insect or spider silk fibroin. Alternatively, fibroin may be prepared from another natural source or by means of genetic engineering (also: bio-fermentation, biotechnological means) or synthetic engineering.

The term “moiety” in the context of the present invention may be understood in the broadest sense as any molecular structure.

A moiety may be either a compound comprising or consisting of the respective structure or may form part of a larger chemical entity such as, e.g., the cross-linked material of the composition of the present invention. For instance, a fibroin moiety may be a fibroin or a chemical entity comprising fibroin. A fibroin moiety may optionally comprise more than one fibroin backbone conjugated with each other. Optionally, one or more fibroin backbones may bind to one or more other structures such as, in particular, the one or more polysaccharide moieties (in particular hyaluronic acid moieties). It will be understood that the term “fibroin moiety” may also include salts and modified forms thereof.

According to the present invention, at least a part of the one or more fibroin moieties comprises primary amino residues or salts thereof. Amino groups can, for instance, form part of lysinyl residues of the one or more fibroin moieties. Preferably, at least a part of the one or more fibroin moieties comprises one or more lysinyl residues that may optionally be bound to the polysaccharide moiety (in particular hyaluronic acid moiety).

As used in the context of the present invention, the term “fibroin” may be understood the broadest sense as any fibroin known in the art. Fibroin may be obtained from a commercial supplier (e.g., Advanced BioMatrix, USA (e.g. product No. 5154-20ML); CareSilk, Italy (e.g. product No. CSK10-1051 )) or may be prepared from a natural source or by means of genetic engineering (also: bio-fermentation, biotechnological means) or rather synthetic engineering. For example, it may be fibroin of Bombyx mori or, alternatively, a species selected from the group consisting of Antheraea, Cricula, Sami, Gonometa an Nephila (e.g., Nephila clavipes) species or a homologue of having at least 80 %, more preferably at least 90 %, even more preferably at least 95 %, even more preferably at least 98 % sequence homology, in particular identity, to one of the aforementioned or a truncated form thereof. It will be understood that also mixtures of different fibroins may be used.

In a preferred embodiment, fibroin is ((essentially) complete) silkworm (Bombyx mori) fibroin. In a particularly preferred embodiment, fibroin is silkworm fibroin obtainable or obtained from Bombyx mori. Silkworm fibroin may be obtained from silkworm cocoons. The process of obtaining silk from silkworm is well-known in the art. For example, silkworm cocoons may be boiled for about 30 min (minutes) in an aqueous solution. Optionally, the aqueous solution may comprise about 0.02 M Na2COs. The cocoons may be rinsed with water or an aqueous buffer to extract the sericin proteins and the extracted fibroin may be dissolved in an aqueous buffer. Salts that may be used for this purpose may, exemplarily, include lithium bromide, lithium thiocyanate, calcium nitrate and mixtures thereof. Optionally, the extracted fibroin may be dissolved in about 9-12 M lithium bromide solution.

The salt may be removed by any means, e.g., dialysis. In a preferred embodiment, other components of the silkworm cocoons have been (essentially) removed such as, e.g., sericin. Thus, preferably, at least 50 wt.%, in more preferably at least 75 wt.%, even more preferably at least 80 wt.%, particular at least 90 wt.%, of the sericin initially contained in the silkworm cocoon have been removed. Silk fibroin may be type I, type II or type III silk fibroin or a mixture of two or more thereof. Preferably, fibroin is or comprises type I silk fibroin. Fibroin may bear the properties as described in the art such as described in US-A 2014/315828. Alternatively, one or more fibroin polypeptides, including silkworm fibroin polypeptides and complete silkworm fibroin, may also be obtained by means of genetic engineering. Genetically engineered fibroin may be, for example, obtained from bacteria, insect cells, spider cells, yeast, mammalian cells, transgenic animals, or transgenic plants.

The one or more fibroin moieties may be stored at any condition. For instance, the one or more fibroin moieties may be stored in a freezer or a liquid gas, e.g. in a temperature range of from -15°C to -200°C. For example, the one or more fibroin moieties may be stored at approximately -80°C or in liquid nitrogen (i.e., at approximately -196°C). When thawing a previously frozen solid fibroin or fibroin solution, preferably, the one or more fibroin moieties may be optionally protected from air. The one or more fibroin moieties may be stored in dry state as a powder or as a solution in water or a buffer (e.g., in a concentration in the range of from 10 to 100 mg/mL (e.g., approximately 50 mg/mL) at temperature between 4°C and ambient temperature. When thawing a previously frozen solid fibroin or fibroin solution, preferably, the one or more fibroin moieties may be optionally protected from air.

As used in the context of the present invention, the term “hyaluronic acid” (also: HA, hyaluronate, or hyaluronan) may be understood in the broadest sense as any moiety of hyaluronic acid known in the art.

Hyaluronic acid may be understood in the broadest sense as a polysaccharide moiety that contains hyaluronic acid moieties (also hyaluronic acid units), preferably comprises at least 50 mol% of hyaluronic acid moieties, more preferably at least 75 mol%, even more preferably at least 80 mol%, even more preferably at least 90 mol%, referred to the whole content of saccharide moieties in the polysaccharide, of hyaluronic acid moieties.

Hyaluronic acid may be used as described in WO 2017/162676 or in WO 2017/148915. Also cross-linked and optionally modified hyaluronic acid such as described in WO 2020/127407 may be used as hyaluronic acid in the context of the present invention. Polysaccharide, in particular hyaluronic acid, may optionally comprise one or more saccharide moieties other than hyaluronic acid. Polysaccharide, in particular hyaluronic acid, may optionally be partly modified. It may, for instance, be partly oxidized and may bear aldehyde groups and/or may be cross-linked. Such modifications are described, for instance in WO 2020/127407.

In a preferred embodiment, hyaluronic acid is a naturally glycosaminoglycan composed of linked repeating units of V-acetyl-D-glucosamine and D-glucuronic acid ([alpha-1 ,4-D-glucuronic acid-beta-1 ,3-N-acetyl-D-glucosamine]_n). Accordingly, the repeating unit of hyaluronic acid may be exemplarity the following: be understood that the hyaluronic acid also includes hydrates, salts and solvents of the above chemical structures.

Preferably, at least one polysaccharide moiety is a polymeric moiety of 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 more.

Preferably, at least one polysaccharide moiety is a hyaluronic acid moiety and the hyaluronic acid moiety is a polymeric moiety of 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 more.

Preferably, the one or more polysaccharide moieties (in particular hyaluronic acid moieties) have an average molecular weight (Mw) in the range of from 10 to 10000 kDa. In a preferred embodiment, the one or more polysaccharide moieties (in particular hyaluronic acid moieties) have an average molecular weight in the range of 50 to 4000 kDa. More preferably, the one or more polysaccharide moieties (in particular hyaluronic acid moieties) have an average Mw in the range of from 100 to 10000 kDa. In one embodiment, the at least one polysaccharide moiety (in particular hyaluronic acid moiety) is a polymeric moiety having an intrinsic viscosity of 1.0 to 3.3 m³/kg (20°C, 1013 hPa, water).

In a preferred embodiment, at least one of the one or more polysaccharide moieties, in particular all of the one or more polysaccharide 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 from 100 and 5000 kDa, of from 200 to 2000 kDa, of from 250 to 1500 kDa, of from 300 to 1000 kDa, of from 400 to 900 kDa or of from 500 to 900 kDa.

In a particularly preferred embodiment, the one or more polysaccharide moieties are one of the one or more hyaluronic acid moieties and the at least one of the one or more hyaluronic acid moieties, in particular 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 from 100 and 5000 kDa, of from 200 to 2000 kDa, of from 250 to 1500 kDa, of from 300 to 1000 kDa, of from 400 to 900 kDa or of from 500 to 900 kDa.

The average molecular weight (Mw) of 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 more. The average molecular weight (Mw) of polysaccharide in the context of the present invention is preferably in the range of from 10 to 10000 kDa, more preferably 100 to 10000 kDa, or 100 to 5000 kDa. In a more preferred embodiment, polysaccharide has an average molecular weight (Mw) in the range of from 50 to 4000 kDa. More preferably, polysaccharide has a average Mw in the range of from 100 to 3500 kDa, of from 200 to 2000 kDa, of from 250 to 1500 kDa, of from 300 to 1000 kDa, of from 400 to 900 kDa or of from 500 to 900 kDa. In a particularly preferred embodiment, all of the one or more polysaccharide moieties (in particular hyaluronic acid moieties) have an average molecular weight of 1500 to 3500 kDa.

In one embodiment, the polysaccharide moieties (in particular hyaluronic acid moieties) are of a single type and have thus (essentially) one molecular weight.

In another embodiment, the polysaccharide moieties (in particular hyaluronic acid moieties) have at least two different molecular weights each comprising primary amino residues or salts thereof. In other words, the polysaccharide moieties (in particular hyaluronic acid moieties) may optionally also be a mixture of polysaccharide moieties (in particular hyaluronic acid moieties) of different molecular weight. In a preferred embodiment, the polysaccharide moieties (in particular hyaluronic acid moieties) have at least two different molecular weights and at least one polysaccharide moiety (in particular hyaluronic acid moiety) has, preferably at least two polysaccharide moieties (in particular hyaluronic acid moieties), in particular all polysaccharide moieties (in particular hyaluronic acid moieties), each have a molecular weight in the range of 10 to 10000 kDa, in the range of 100 to 10000 kDa, or in the range of 100 to 5000 kDa. in the range of from 100 to 3500 kDa, in the range of from 200 to 2000 kDa, in the range of from 250 to 1500 kDa, in the range of from 300 to 1000 kDa, in the range of from 400 to 900 kDa, or in the range of from 500 to 900 kDa.

In a preferred embodiment, the polysaccharide moieties (in particular hyaluronic acid moieties) have at least two different molecular weights and at least one polysaccharide moiety (in particular hyaluronic acid moiety) has, preferably at least two polysaccharide moieties (in particular hyaluronic acid moieties), in particular all polysaccharide moieties (in particular hyaluronic acid moieties), each have a molecular weight in the range of 1500 to 3500 kDa.

In one embodiment, the polysaccharide moieties (in particular hyaluronic acid moieties) comprise or consist of at least two polysaccharide moieties (in particular hyaluronic acid moieties) having at least two different molecular weights and at least one polysaccharide moiety (in particular hyaluronic acid moiety) has, preferably at least two polysaccharide moieties (in particular hyaluronic acid moieties) both have, in particular all polysaccharide moieties (in particular hyaluronic acid moieties) each have, a molecular weight in the range of 10 to 10000 kDa, in the range of 100 to 10000 kDa, or in the range of 100 to 5000 kDa. in the range of from 100 to 3500 kDa, in the range of from 200 to 2000 kDa, in the range of from 250 to 1500 kDa, in the range of from 300 to 1000 kDa, in the range of from 400 to 900 kDa, or in the range of from 500 to 900 kDa. In a preferred embodiment, the polysaccharide moieties (in particular hyaluronic acid moieties) comprise or consist of at least two polysaccharide moieties (in particular hyaluronic acid moieties) having at least two different molecular weights and at least one polysaccharide moiety (in particular hyaluronic acid moiety) has, preferably at least two polysaccharide moieties (in particular hyaluronic acid moieties) both have, in particular all polysaccharide moieties (in particular all hyaluronic acid moieties) each have, a molecular weight in the range of 1500 to 3500 kDa.

The (mass) ratio between the total mass of the one or more fibroin moieties (component A-i) and the total mass of the one or more polysaccharide moieties (component A-ii), in particular hyaluronic acid moieties) may be any ratio. When particularly high water/buffer absorbance is desired, the polysaccharide moieties (in particular hyaluronic acid moieties) may be used in mass excess. When particularly high stability is desired, the fibroin moieties may be used in larger mass excess. Preferably in the (mass) ratio (A-i): (A-ii) in the range of from 1 :100 to 100:1 .

In a preferred embodiment, the mass ratio between the one or more fibroin moieties (A-i) and the 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, in particular in the range of 1 :1 to 1 :5. In a preferred embodiment, the mass ratio between the one or more fibroin moieties (A-i) and the 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, in particular in the range of 1 : 1 to 1 :5.

For instance, the mass ratio between the one or more fibroin moieties (A-i) and the one or more polysaccharide moieties (A-ii), i.e., (A-i):(A-ii), may be in the range of 1 :9 to 2: 1 , of 1 :8 to 1.5:1 , of 1 :7 to 1 : 1 , of 1 :6 to 1 :1 , of 1 :5 to 1 :1 , of 1 :4 to 1 : 1 , of 1 :3 to 1 : 1 , of 1 :2 to 1 : 1 , or of 1 : 1 .5 to 1 : 1 . For instance, the mass ratio between the one or more fibroin moieties (A-i) and the one or more hyaluronic acid moieties (A-ii), i.e., (A-i):(A-ii), may be in the range of 1 :9 to 2:1 , of 1 :8 to 1.5:1 , of 1 :7 to 1 :1 , of 1 :6 to 1 :1 , of 1 :5 to 1 :1 , of 1 :4 to 1 :1 , of 1 :3 to 1 :1 , of 1 :2 to 1 :1 , or of 1 :1.5 to 1 :1. In one embodiment, the fibroin moieties have at least two different molecular weights each comprising primary amino residues or salts thereof, and the polysaccharide moieties (in particular the hyaluronic acid moieties) have at least two different molecular weights each comprising primary amino residues or salts thereof.

In a preferred embodiment:

(a) the fibroin moieties have at least two different molecular weights and at least one fibroin moiety has, preferably at least two fibroin moieties both have, in particular all fibroin moieties 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

(b) the polysaccharide moieties have at least two different molecular weights and at least one polysaccharide moiety has, preferably at least two polysaccharide moieties both have, in particular all polysaccharide moieties each have, a molecular weight in the range of 10 to 10000 kDa, in the range of 100 to 10000 kDa, or in the range of 100 to 5000 kDa. in the range of from 100 to 3500 kDa, in the range of from 200 to 2000 kDa, in the range of from 250 to 1500 kDa, in the range of from 300 to 1000 kDa, in the range of from 400 to 900 kDa, or in the range of from 500 to 900 kDa.

In a preferred embodiment:

(b) the hyaluronic acid moieties have at least two different molecular weights and at least one polysaccharide moiety has, preferably at least two polysaccharide moieties both have, in particular all polysaccharide moieties each have, a molecular weight in the range of 10 to 10000 kDa, in the range of 100 to 10000 kDa, or in the range of 100 to 5000 kDa. in the range of from 100 to 3500 kDa, in the range of from 200 to 2000 kDa, in the range of from 250 to 1500 kDa, in the range of from 300 to 1000 kDa, in the range of from 400 to 900 kDa, or in the range of from 500 to 900 kDa.

In a preferred embodiment, the cross-linked material forms a gel in the liquid or viscous pharmaceutically acceptable carrier, in particular a hydrogel, and the botulinum toxin is dissolved in the liquid or viscous pharmaceutically acceptable carrier soaked in the gel, in particular hydrogel.

In a preferred embodiment, the cross-linked material of the present invention is a gel. In a preferred embodiment, the cross-linked material of the present invention is a hyaluronic acid/fibroin gel (HA/fibroin gel). In a preferred embodiment, the crosslinked material of the present invention is a hyaluronic acid/fibroin hydrogel (HA/fibroin hydrogel).

In one embodiment:

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

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

In one embodiment:

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

The one or more fibroin moieties and the one or more polysaccharide moieties may be conjugated with each other by any means. In particular, the one or more fibroin moieties and the one or more hyaluronic acid moieties may be conjugated with each other by any means.

For instance, these components may be conjugated with each other via one or more amide bonds (-NH-CO- or -CO-NH-). This conjugation reaction may be facilitated by any means such as by one or more activating agents, such as one or more activating agents that effect reaction of carboxylic acid residues with amino residues thereby forming amide bonds

As herein, an activating agent may be any compound that effects reaction of the one or more fibroin moieties and the one or more polysaccharide moieties (in particular hyaluronic acid moieties). Preferably, an activating agent may be any compound that effects reaction of carboxylic acid residues with amino residues thereby forming amide bonds. It will be understood that it is mainly meant that an activating agent is a compound that effects reaction of carboxylic acid residues of the one or more polysaccharide moieties in particular hyaluronic acid moieties) with amino residues of the one or more fibroin moieties thereby forming amide bonds.

In a preferred embodiment, the one or more activating agents are selected from the group consisting of

(C1 ) one or more triazine-based activating agents, in particular selected from the group consisting of 4-(4,6-dimethoxy-1 ,3,5-triazin-2-yl)~4- methylmorpholinium (DMTMM), a salt thereof, and/or 2-chloro-4,6,- dimethoxy-1 ,3,5-triazine (CDMT) and combinations thereof;

(C2) one or more carbodiimide activating agents, in particular selected from the group consisting of N,N’-dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC), and combinations of two or more thereof; and

(C3) combinations thereof.

According to the present invention, an activating agent is typically not covalently included in the cross-linked material. Thus, it can be typically optionally removed from the cross-linked material of the present invention by any means such as, e.g., washing, filtration, etc. In a preferred embodiment, a triazine-based activating agent 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. A salt of DMTMM is preferably a salt wherein the counter-ion is an anion that is cosmetically and/or pharmaceutically acceptable such as, e.g., chloride, acetate, bicarbonate (hydrogen carbonate), or a mixture of two or more anions.

In a preferred embodiment, a triazine-based activating agent is 4-(4,6-dimethoxy- 1 ,3,5-triazin-2-yl)-4-methylmorpholinium chloride.

In a preferred embodiment, a carbodiimide activating agent is N,N’- dicyclohexylcarbodiimide (DCC), or 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC).

In a preferred embodiment, the activating agent is 4-(4,6-dimethoxy-1 ,3,5-triazin-2- yl)-4-methylmorpholinium (DMTMM) or a salt thereof. Preferably, it is a 4-(4,6- dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholinium salt, in particular 4-(4,6- dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholinium chloride (CAS No. 3945-69-5).

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

When using DMTMM as activating agent, 4-methylmorpholine (NMM) and/or 4,6- dimethoxy-1 ,3-5-triazine-2-ol (DMT) may be formed as degradation product(s).

As used herein, the term “without an interconnecting linker structure” may be understood in the broadest sense in that no further chemical moiety that does not originate from (also: is not present in) fibroin moieties or polysaccharide moieties (in particular hyaluronic acid moieties) is introduced into the chemical structure that conjugates one or more fibroin moieties covalently with one or more polysaccharide moieties (in particular hyaluronic acid moieties), preferably via amide bonds. In other words, the amide bonds are preferably formed from inclusion of a nitrogen atom originating from fibroin (e.g., a lysinyl side chain) and from inclusion of a carbon atom originating from polysaccharide moieties (in particular hyaluronic acid moieties).

In a preferred embodiment, the one or more fibroin moieties are covalently conjugated with the one or more polysaccharide moieties via amide bonds. In a preferred embodiment, the one or more fibroin moieties are covalently conjugated with the one or more hyaluronic acid moieties via amide bonds.

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

In a preferred embodiment, the cross-linked material is further characterized in that it does not comprise:

(a) imide groups;

(b) imine groups;

(c) epoxy groups; and/or

(d) xenobiotic linker moieties, interconnecting one or more fibroin moieties with one or more polysaccharide moieties.

(a) imide groups;

(b) imine groups;

(c) epoxy groups; and/or

(d) xenobiotic linker moieties, interconnecting one or more fibroin moieties with one or more hyaluronic acid moieties.

In a preferred embodiment, the cross-linked material is further characterized in that it does as a whole not comprise: (a) imide groups;

(b) imine groups;

(c) epoxy groups; and

(d) xenobiotic linker moieties.

A cross-linked material may be prepared by any means. The skilled artisan will be aware of means for preparing such. For example, it may be prepared as described in PCT/EP2022/066989.

For instance, it may be prepared by a method comprising:

(i) contacting the following components with each other:

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

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

(A-iii) one or more activating agents that effect reaction of carboxylic acid residues with amino residues thereby forming amide bonds, and

(A-iv) one or more solvents; and

(ii) allowing a reaction of at least some of the carboxylic acid residues with at least some of the primary amino residues to form amide bonds conjugating the one or more fibroin moieties covalently with the one or more polysaccharide moieties (in particular hyaluronic acid moieties); and

(iii) optionally purifying the cross-linked material obtained from step (ii).

In a preferred embodiment, the method is further characterized in that it comprises a step (iii) of purifying the cross-linked material by filtration, washing and/or dialysis, in particular crossflow filtration, diafi Itration and/or dead-end filtration. Such optional purification steps, including filtration steps, are further exemplified in PCT/EP2022/066989.

Step (ii) may be conducted for any time suitable for this purpose. Optionally, step (ii) may be conducted for 1 min to 1 week or longer, 2 min to 5 days, 3 min to 4 days, 5 min to 72 hours, 5 min to 24 hours, 10 min to 12 hours, 30 min to 6 hours, 1 hour to 5 hours, or 2 to 4 hours. Steps (ii) and/or (iii) may be conducted for at any temperature suitable for this purpose such as, e.g., at 0°C to 100°C, at 4°C to 95°C, at 10°C to 70°C, at 15°C to 30°C, at 18 to 25°C, at 20°C to 70°C, at 20°C to 40°C, or at 60°C to 70°C. Steps (i) and (ii) and optional step (iii) may be conducted at any pressure. For example, pressure may be ambient pressure (e.g, often approximately 970 to 1100 hPa outer pressure).

In a preferred embodiment, the method is further characterized in that steps (i) and (ii) are conducted in a single batch.

In a preferred embodiment, the one or more polysaccharide moieties (as component A-ii, in particular hyaluronic acid moieties) and the one or more activating agents (as component A-iii) are dissolved in one or more solvents (as component A-iv) in a first step without the one or more fibroin moieties (as component A-i) and incubated. This may activate the carboxylic groups of the polysaccharide moieties (in particular hyaluronic acid moieties). Incubation may be conducted for any time sufficient for such purpose. In a preferred embodiment, in a first sub-step, the one or more polysaccharide moieties (in particular hyaluronic acid moieties) are dissolved in the one or more solvents and in a subsequent sub-step, the one or more activating agents are added, together representing the activation step. After an incubation, the one or more fibroin moieties (as component A-i) may be added. This may be further incubated to conduct step (ii) of the method of the present invention. Step (ii) may be conducted using any suitable solvent such as water or an aqueous buffer. Optionally, the solution may be stirred during the reaction step.

Accordingly, in a preferred embodiment, step (i) of the method comprises the following sub-steps:

(ia) contacting the following components with each other:

(A-iv) one or more solvents, preferably wherein the one or more polysaccharide moieties (in particular hyaluronic acid moieties) are first dissolved in the one or more solvents and subsequently the one or more activating agents are added;

(ib) allowing a reaction of at least some of the carboxylic acid residues with one or more activating agents thereby forming one or more activated polysaccharide moieties (also: polysaccharide-activating agent conjugates, in particular hyaluronic acid moieties, i.e.. hyaluronic acid-activating agent conjugates); and

(ic) adding to the activated polysaccharide moieties (in particular hyaluronic acid moieties) of sub-step (ib):

(A-i) one or more fibroin moieties comprising primary amino residues or salts thereof.

In another preferred embodiment, the components and optionally one or more further components are all mixed at once.

In a preferred embodiment, the method comprises:

(i) contacting the following components with each other:

(A-i) one or more silk fibroin moieties having an average molecular weight of at least 5 kDa that comprises primary amino residues or salts thereof,

(A-ii) one or more polysaccharide moieties (in particular hyaluronic acid moieties) having an average molecular weight of at least 50 kDa that comprises carboxylic acid residues or salts thereof,

(A-iii) one or more triazine-based activating agents that effect reaction of carboxylic acid residues with amino residues thereby forming amide bonds, in particular wherein the activating agent is 4-(4,6-dimethoxy- 1 ,3,5-triazin-2-yl)-4-methylmorpholinium or a salt thereof; and

(A-iii) one or more solvents; and

(ii) allowing a reaction of at least some of the carboxylic acid residues with at least some of the primary amino residues to form amide bonds conjugating the one or more silk fibroin moieties covalently with the one or more polysaccharide moieties (in particular hyaluronic acid moieties); and

(iii) optionally purifying the cross-linked material obtained from step (ii).

Any solvent usable as component A-iv for the method of the present invention may 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. In an embodiment of the present invention, the method of the invention composes the following (preferably sequential) step: dissolution of polysaccharide (in particular hyaluronic acid), in particular polysaccharide sodium salt (in particular hyaluronic acid sodium salt), in water or buffer; addition of activator (preferably DMTMM) to the polysaccharide solution (in particular hyaluronic acid solution); allowing activation (preferably for some hours, e.g. at a temperature of 18 to 22°C); adding fibroin solution to the activated polysaccharide (in particular activated hyaluronic acid); stirring and allowing the formation of the cross-linked material (preferably for some hours, e.g. at a temperature of 18 to 22°C); purifying the cross-linked material (e.g. removal of DMTMM and degradation products thereof, e.g., by means of filtration and/or dialysis, e.g. at a temperature of 18 to 22°C); optionally adding an anesthetic; and optionally sterilizing.

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

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

In a preferred embodiment, the botulinum toxin is botulinum neurotoxin serotype A, in particular botulinum neurotoxin serotype A1 . The term "pure botulinum neurotoxin", as used herein, means the botulinum neurotoxin free of complexing proteins (sometimes also referred to as the “neurotoxic component”), or more precisely, the botulinum neurotoxin without neurotoxin-associated complexing proteins (NAPs). The pure botulinum neurotoxin is the (active) neurotoxic polypeptide that ultimately inhibits acetylcholine release. It is a di-chain protein comprised of a light chain (LC; about 50 kDa) and a heavy chain (HC; about 100 kDa), held together by a disulfide bond. The active neurotoxic polypeptide may therefore also be referred to herein as the "150 kDa neurotoxin", “Clostridium botulinum neurotoxin (150 kD)” or "neurotoxic component". Preferably, the botulinum toxin is the (pure) botulinum neurotoxin contained in Xeomin® or is Xeomin®.

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

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

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

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

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

Due to differences in the LD50 tests used by manufacturers of commercial botulinum toxin formulations, the unit potencies indicated by the manufacturers for their commercial botulinum toxin formulations is proprietary and cannot easily be compared. Therefore, within the framework of the present invention, the conversion rates provided below are used to establish the comparative potencies of incobotulinumtoxinA ("INCO"; Xeomin®, Bocouture®; botulinum toxin serotype A, free of complexing proteins; Merz Pharmaceuticals GmbH), onabotulinumtoxinA ("ONA"; Botox®, Vistabel®; botulinum toxin complex of serotype A; Allergan Inc.), abobotulinumtoxinA ("ABO"; Dysport®, Azzalure®; botulinum toxin complex of serotype A; Medicis Pharmaceutical Corp., Galderma Lab.), rimabotulinumtoxinB ("RIM"; Myobloc®, NeuroBloc®; botulinum toxin serotype B; Solstice Neurosciences Inc.), and PurTox® ("TBD"; botulinum toxin serotype A; Mentor Worldwide LLC). For use herein, the conversion rate of ONA and INCO is 1 :1. The conversion rate of ONA/INCO:ABO is 1 :2.5. The conversion rate of ONA/INCO:RIM is 1 :50, and the conversion rate of ONA/INCO:TBD is 1 :1.5. Furthermore and preferably, within the context of the present invention, 1 U of INCO (Xeomin®) and 1 U of onabotulinumtoxinA ("ONA"; Botox®) shall be deemed to correspond to one mouse LD50 (1 .0 LD50), or 1 U, measured using the MBA as described above.

In a preferred embodiment, the botulinum toxin is not covalently bound with the cross-linked material.

As used herein, a viscous cosmetically and/or pharmaceutically acceptable carrier may be any component being usable as such.

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

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

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

As used herein, an anesthetic may be any anesthetic component.

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

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

For instance, such further component may be a further carrier, e.g. solid cosmetically and/or pharmaceutically acceptable carrier that is optionally soluble in the liquid or viscous cosmetically and/or pharmaceutically acceptable carrier. For instance, such further component may be a filling or bulking agent. For instance, such further component may be a salt (e.g., sodium chloride, NaCI). For instance, such further component may be a stabilizing agent (e.g., a protein (e.g., albumin (e.g., human albumin)). For instance, such further component may be a component that slows down the degradation of botulinum toxin (e.g., a radical scavenging agent, (e.g., a polyol such as glycerol, a sugar alcohol (e.g. selected from the group consisting of mannitol, inositol, lactilol, xylitol, erythriol, sorbitol, etc.)). For instance, such further component may be a sugar (e.g., sucrose, glucose, trehalose, galactose, fructose, maltose, lactose, starch, cellulose, etc.). For instance, such further component may be a crystallization inhibitor. For instance, such further component may be a scavenging agent. For instance, such further component may be a cell proliferation factor may improve cellular invasion into an administered cross-linked material of the present invention. For instance, such further component may be a dye that may, e.g., either localized administration (e.g., injection), may improve localization of the injection (e.g., a pharmaceutically acceptable fluorescent dye like fluorescein or rhodamine), or may improve invisibility of the otherwise whitish cross-linked material (e.g., by rendering it flesh-colored). Any other pharmaceutically active compound may also be added. Then, the composition of the present invention may optionally also serve as a retarded and/or controlled release form for administration.

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

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

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.

In a preferred embodiment, the composition of the present invention comprises at least 0.1 % by weight, referred to the composition, of the cross-linked material. In a preferred embodiment, in particular when the composition of the present invention is a (hydro)gel, a liquid, the composition comprises at least 0.2 % by weight, at least 0.5 % by weight, at least 1 % by weight, at least 1.5 % by weight, at least 2 % by weight, at least 5 % by weight, or at least 10 % by weight, referred to the composition, of the cross-linked material.

In a preferred embodiment, in particular when the composition of the present invention is a (hydro)gel, a liquid, or a viscous composition, the composition comprises not more than 3.5 % by weight, referred to the composition, of the crosslinked material. In a preferred embodiment, in particular when the composition of the present invention is a (hydro)gel, a liquid, or a viscous composition, the composition comprises not more than 20 % by weight, not more than 10 % by weight, not more than 5 % by weight, not more than 3.5 % by weight, or not more than 2 % by weight, or not more than 1 % by weight, referred to the composition, of the cross-linked material.

In a preferred embodiment, the composition of the present invention comprises 0.01 to 99.9 % by weight, 0.1 to 99.9 % by weight, 0.15 to 90 % by weight, 0.2 to 80 % by weight, 0.5 to 70 % by weight, 1 to 50 % by weight, 5 to 40 % by weight, or 10 to 25 % by weight, referred to the composition, of the cross-linked material. In a preferred embodiment, in particular when the composition of the present invention is a (hydro)gel, a liquid, or a viscous composition, the composition comprises 0.1 to 3.5 % by weight, referred to the composition, of the cross-linked material. In a preferred embodiment, in particular when the composition of the present invention is a (hydro)gel, a liquid, or a viscous composition, the composition comprises 0.11 to 3.0 % by weight, 0.12 to 2.5 % by weight, 0.15 to 2.0 % by weight, 0.2 to 1.5 % by weight, 0.5 to 1 .0 % by weight, 0.5 to 2.0 % by weight, or 1 .0 to 3.0 % by weight, referred to the composition, of the cross-linked material.

In a preferred embodiment, the composition of the present invention comprises at least 0.1 units/g, referred to the composition, of botulinum toxin. In a preferred embodiment, the composition of the present invention comprises at least 0.2 units/g, at least 0.5 units/g, at least 1 unit/g, at least 1 units/g, at least 5 units/g, at least 10 units/g, at least 25 units/g, at least 50 units/g, at least 75 units/g, at least 100 units/g, at least 150 units/g, at least 200 units/g, at least 250 units/g, or at least 500 units/g, referred to the composition, of botulinum toxin. In a preferred embodiment, in particular when the composition of the present invention is a (hydro)gel, a liquid or a viscous composition, the composition comprises at least 0.1 units/mL, referred to the composition, of botulinum toxin. In a preferred embodiment, the composition of the present invention comprises at least 0.2 units/mL, at least 0.5 units/mL, at least 1 unit/mL, at least 3 units/mL, at least 5 units/mL, at least 10 units/mL, at least 25 units/mL, at least 50 units/mL, at least 75 units/mL, at least 100 units/mL, at least 150 units/mL, at least 200 units/mL, at least 250 units/mL, or at least 500 units/mL, referred to the composition, of botulinum toxin. In a preferred embodiment, one injectable dose of the composition of the present invention comprises at least 0.1 units, referred to the composition, of botulinum toxin. In a preferred embodiment, an injectable dose of the composition of the present invention comprises at least 0.2 units, at least 0.5 units, at least 1 unit, at least 1 units, at least 5 units, at least 10 units, at least 25 units, at least 50 units, at least 75 units, at least 100 units, at least 150 units, at least 200 units, at least 250 units, or at least 500 units, referred to the composition, of botulinum toxin.

In a preferred embodiment, the composition of the present invention comprises not more than 1000 units/g, referred to the composition, of botulinum toxin. In a preferred embodiment, the composition of the present invention comprises not more than 500 units/g, not more than 250 units/g, not more than 200 units/g, not more than 150 units/g, not more than 100 units/g, not more than 50 units/g, or not more than 10 units/g referred to the composition, of botulinum toxin. In a preferred embodiment, in particular when the composition of the present invention is a (hydro)gel, a liquid or a viscous composition, the composition comprises not more than 1000 units/mL, referred to the composition, of botulinum toxin. In a preferred embodiment, the composition of the present invention comprises not more than 500 units/mL, not more than 250 units/mL, not more than 200 units/mL, not more than 150 units/mL, not more than 100 units/mL, not more than 50 units/mL, or not more than 10 units/mL referred to the composition, of botulinum toxin. In a preferred embodiment, an injectable dose of the composition of the present invention comprises not more than 1000 units, referred to the composition, of botulinum toxin. In a preferred embodiment, the composition of the present invention comprises not more than 500 units, not more than 250 units, not more than 200 units, not more than 150 units, not more than 100 units, not more than 50 units, or not more than 10 units referred to the composition, of botulinum toxin. In a preferred embodiment, the composition of the present invention comprises 0.1 to 1000 units/g, referred to the composition, of botulinum toxin.

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

The composition of the present invention may comprise 0 to 99.9 % by weight, referred to 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 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, referred to 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 0 to 1 % by weight, referred to the composition, of one or more anesthetics. In a preferred embodiment, the composition of the present invention comprises 0.01 to 1 % by weight, 0.05 to 0.9 % by weight, 0.1 to 0.8 % by weight, 0.1 to 0.7 % by weight, 0.2 to 0.5 % by weight, 0.2 to 0.4 % by weight, referred to the composition, of one or more one or more anesthetics.

In a preferred embodiment, the composition of the present invention comprises 0 to 50 % by weight, referred to the composition, of one or more further cosmetically and/or pharmaceutically acceptable ingredients. In a preferred embodiment, the composition of the present invention comprises up to 40 % by weight, up to 30 % by weight, up to 20 % by weight, up to 10 % by weight, up to 5 % by weight, up to 1 % by weight, up to 0.5 % by weight, or up to 0.1 % by weight, referred to the composition, of one or more further cosmetically and/or pharmaceutically acceptable ingredients. In a preferred embodiment, the composition of the present invention comprises 0.1 to 50 % by weight, 0.1 to 40 % by weight, 0.1 to 30 % by weight, 0.1 to 20 % by weight, 0.1 to 10 % by weight, 0.1 to 5 % by weight, 0.1 to 1 % by weight, 0.2 to 50 % by weight, 0.5 to 40 % by weight, 1 to 30 % by weight, 2 to 20 % by weight, 5 to 10 % by weight, 10 to 20 % by weight, or 20 to 50 % by weight, referred to the composition, of one or more further cosmetically and/or pharmaceutically acceptable ingredients.

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

(A) 0.1 to 99.9 % by weight, preferably 0.1 to 10 % by weight, referred to the composition, of the cross-linked material;

(B) 0.1 to 150 units/mL, preferably 0.1 to 100 units/mL, referred to the composition, of botulinum toxin;

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

(D) 0 to 10 % by weight, preferably 0 to 5 % by weight, referred to the composition, one or more anesthetics; and

(E) 0 to 50 % by weight, preferably 0 to 49.9 % by weight, referred to the composition, of one or more further cosmetically and/or pharmaceutically acceptable ingredients.

In a preferred embodiment, the composition is an injectable composition comprises or consists of:

(A) 0.1 to 3.5 % by weight, referred to the composition, of the cross-linked material;

(B) 1 to 150 units/mL, referred to the composition, of botulinum toxin;

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

(D) 0 to 1 % by weight, referred to the composition, one or more anesthetics; and (E) 0 to 50 % by weight, referred to the composition, of one or more further cosmetically and/or pharmaceutically acceptable ingredients.

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

(B) 1 to 150 units/mL, referred to the composition, of botulinum toxin;

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

(D) 0 to 1 % by weight, referred to the composition, one or more anesthetics; and

(E) 0 to 89.9 % by weight, referred to the composition, of one or more further cosmetically and/or pharmaceutically acceptable ingredients.

(B) 1 to 150 units/mL, referred to the composition, of botulinum toxin;

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

(D) 0 to 1 % by weight, referred to the composition, one or more anesthetics; and (E) 0 to 49.9 % by weight, preferably 0 to 34.9 % by weight, referred to the composition, of one or more further cosmetically and/or pharmaceutically acceptable ingredients.

(A) 0.5 to 3.5 % by weight, referred to the composition, of the cross-linked material;

(B) 1 to 150 units/mL, referred to the composition, of botulinum toxin;

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

(E) 0 to 49.5 % by weight, preferably 0 to 34.9 % by weight, referred to the composition, of one or more further cosmetically and/or pharmaceutically acceptable ingredients.

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

(A) 0.1 to 3.5 % by weight, referred to the composition, of a cross-linked 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, in particular hyaluronic acid moieties, having an average molecular weight in the range of 50 to 4000 kDa, wherein the one or more fibroin moieties are covalently conjugated with the one or more polysaccharide moieties, in particular hyaluronic acid moieties, without an interconnecting linker structure, preferably via amide bonds, preferably having a mass ratio (A i) to (A-ii) of 5:1 to 1 :20,

(B) 1 to 150 units/mL, referred to the composition, botulinum toxin not covalently bound with the cross-linked material;

(C) 50 to 99.9 % by weight, more preferably 65 to 99.9 % by weight, referred to the composition, 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;

(D) 0 to 1 % by weight, referred to the composition, of one or more anesthetics; and

(E) 0 to 49.9 % by weight, more preferably 0 to 34.9 % by weight, referred to the composition, of one or more further cosmetically and/or pharmaceutically acceptable ingredients different from components (A) to (D).

(A-ii) one or more hyaluronic acid moieties having an average molecular weight in the range of 50 to 4000 kDa, wherein the one or more fibroin moieties are covalently conjugated with the one or more hyaluronic acid moieties without an interconnecting linker structure, preferably via amide bonds, preferably having a mass ratio (A i) to (A-ii) of 5:1 to 1 :20,

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

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

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

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

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

A further aspect of the present invention relates to a method for preparing a composition of the present invention, comprising the steps:

(i) contacting the following components with each other:

(A-ii) one or more polysaccharide moieties comprising carboxylic acid residues or salts thereof,

(A-iv) one or more solvents;

(ii) allowing a reaction of at least some of the carboxylic acid residues with at least some of the primary amino residues to form amide bonds conjugating the one or more fibroin moieties covalently with the one or more polysaccharide moieties; (iii) obtaining cross-linked material (component (A)) from step (ii) and optionally purifying the cross-linked material; and

(iv) adding: 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 further cosmetically and/or pharmaceutically acceptable ingredients different from components (A) to (D) (component (E)).

It will be understood that the definitions and preferred embodiments as laid out in the context of composition of the present invention and method for preparing a cross-linked material comprised therein mutatis mutandis apply to the method for preparing a composition of the present invention.

In particular, it will be understood that the any one of steps (i) to (iii) may preferably be conducted as laid out in the context of preparing a cross-linked material comprised in the composition of the present invention.

In a preferred embodiment, the method for preparing a composition of the present invention, comprising the steps:

(i) contacting the following components with each other:

(A-ii) one or more hyaluronic acid moieties comprising carboxylic acid residues or salts thereof,

(A-iv) one or more solvents;

(ii) allowing a reaction of at least some of the carboxylic acid residues with at least some of the primary amino residues to form amide bonds conjugating the one or more fibroin moieties covalently with the one or more hyaluronic acid moieties;

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

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

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 composition is preferably an injectable composition.

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

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

As indicated above, the cross-linked material as described and defined herein may have various functionalities, optionally including allowing the retarded and/or controlled release of one or more pharmaceutically and/or cosmetically active agents, such as, e.g., botulinum toxin. It will be understood that the retarded and/or controlled release (e.g., depot) function of the hydrogel can be adopted by means of adapting the cross-linking range, the content of the cross-linked material in the composition and the ratio between the botulinum toxin and the cross-linked material. This may address the needs of the relevant indication (e.g. reducing adverse effects, elongating the duration of botulinum effect, etc.).

Accordingly, a further aspect of the present invention relates to the use of a crosslinked material comprising or consisting of (A-i) one or more fibroin moieties, and (A-ii) one or more polysaccharide moieties, wherein the one or more fibroin moieties are covalently conjugated with the one or more polysaccharide moieties without an interconnecting linker structure, preferably by forming amide bonds, for retarded and/or controlled release of one or more pharmaceutically and/or cosmetically active agents, in particular botulinum toxin, preferably wherein the cross-linked material forms a gel a liquid or viscous pharmaceutically acceptable carrier, in particular wherein the cross-linked material forms part of a composition of the present invention.

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

The present invention relates to the use of a cross-linked material comprising or consisting of (A-i) one or more fibroin moieties, and (A-ii) one or more hyaluronic acid moieties, wherein the one or more fibroin moieties are covalently conjugated with the one or more hyaluronic acid moieties without an interconnecting linker structure, preferably by forming amide bonds, for retarded and/or controlled release of one or more pharmaceutically and/or cosmetically active agents, in particular botulinum toxin, preferably wherein the cross-linked material forms a gel a liquid or viscous pharmaceutically acceptable carrier, in particular wherein the cross-linked material forms part of a composition of the present invention.

In a preferred embodiment, the cross-linked material forms part of a composition of the present invention. In a preferred embodiment, the cross-linked material forms gel, in particular in combination with one or more liquid or viscous cosmetically and/or pharmaceutically acceptable carriers.

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

Accordingly, a further aspect of the present invention relates to the composition of the present invention for use as a medicament.

Accordingly, the present invention further relates to a medicament comprising the composition of the present invention.

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

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

Accordingly, in other words, the present invention relates to a method of treating or preventing a disease or condition associated with hyperactive cholinergic activity, comprising a step of administering a sufficient amount of the composition of the present invention to a subject in need thereof.

It will be understood that the definitions and preferred embodiments as laid out in the context of the composition of the present invention and the use of the present invention mutatis mutandis apply to the use in treating or preventing and a method of treating or preventing of the present invention. As used herein, the term “hyperactive cholinergic innervation” may be understood in the broadest sense as generally understood in the art. It may be understood as relating to a synapse, which is characterized by an unusually high amount of acetylcholine release into the synaptic cleft. In this context, “unusually high” may relate to an increase of, e.g., up to 25%, up to 50% or more with respect to a reference activity which may be obtained, for example, by comparing the release with the release at a synapse of the same type but which is not in a hyperactive state, wherein muscle dystonia may be indicative of the hyperactive state. In this context, “up to 25%” may, for example, be understood as >0% to about 25%. Methods for performing the measurements of synaptic activities are known in the art.

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

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

For instance, administration may be in the dermis area, such as below the epidermis or above the hypodermis and as such the composition may be injected subcutaneously, hypodermically, intradermally, subdermally, or some combinations. In another embodiment, the composition may be administered intramuscularly. Hyperactive cholinergic activity may be such of any part of the subject’s body. In a preferred embodiment, the hyperactive cholinergic activity is such of muscles and/or glands.

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

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

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

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

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

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

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

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

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

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

In a preferred embodiment, the rejuvenation and/or improvement of the skin quality of the face and/or body comprises improving and/or reducing and/or filling and/or preventing wrinkles, in particular of wrinkles resulting from muscular activity such as mimic activity, skin smoothing, improving skin laxity, lifting effect, moisturizing and/or softening the skin, improving and/or reducing and/or filling facial lines, soft-tissue augmentation, improving subdermal support of the brows, malar and buccal fat pads, improving tear troughs, improving nose appearance, resolving facial asymmetries, improving jawlines, or a combination of two or more thereof. According to general understanding, lines may also be understood as wrinkles and vice versa. The terms “lines” and “wrinkles” may be used interchangeably. As used herein, improvable and/or reducable and/or fillable facial lines may exemplarily be selected from the group consisting of horizontal forehead lines, glabellar lines (e.g., glabellar frown lines), periorbital lines, crow’s feet, bunny lines, nasolabial folds, peri-lip lines, upper radial lip lines, lower radial lip lines, corner of the mouth lines, marionette lines, perioral lip lines, oral commissures, labiomental crease and cobblestone chin. In a preferred embodiment, improvable facial lines may be horizontal forehead lines and/or glabellar lines and/or periorbital lines including crow’s feet.

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

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

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

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

It will be further understood that the scope embraces the number values provided as commonly rounded values that embrace the whole rounding limits. For example, the scope of “1 mg” embraces the range of from 0.50 to 1 .49 mg. The number values of the present invention, however, also disclose the more detailed values of one or more orders of magnitude more in detail. Accordingly, for example, “1 mg” may also include the specific disclosure of “1 .0 mg”.

The examples and claims illustrate embodiments of the present invention.

Examples

Materials and Methods

Raw materials

Hyaluronic acid, different intrinsic viscosities (HTL Biotechnology, Javene, France); 5% aqueous solution of fibroin from silk worm (CareSilk s.r.l.s., Lecce, Italia);

4-(4,6-Dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) (Sigma Aldrich, Darmstadt, Germany);

Water (internal system for desalted water);

Saline (B. Braun, Melsungen, Germany)

Lidocaine hydrochloride (Albemarle Corp., Charlotte, USA)

Xeomin® (botulinum neurotoxin formulation)

As far as not specified otherwise, all syntheses and measurements are carried out at ambient conditions, i.e., ambient temperature (such as 18 to 25°C, in particular approximately 20°C) and ambient/atmospheric pressure.

Extrusion Force (EF)

Extrusion Force (EF) was measured using the instrument- TA.XT Plus Texture Analyzer (Stable Micro Systems Ltd., Surrey, UK). A syringe equipped with 30G TSK needle (TSK Laboratory Europe, Oisterwijk, The Netherlands) was placed in the instrument and then the instrument pressed the syringe plunger at constant speed of 0.21 mm/s (approximately 1.26 cm/min) over a distance of 30 mm. The force required to press out the content of the syringe through the needle was recorded, mean value was calculated and this was reported as the Extrusion Force. Rheology

Rheology was measured using the instrument Anton Paar MCR 302 with cone-plate (CP50-1 , of 50 mm diameter) or plate-plate (PP20, of 20 mm diameter) geometry (Anton Paar GmbH, Graz, Austria). The measurement was performed in oscillation mode with frequency sweep from 0.1 Hz to 10 Hz at constant deformation of 0.1 % at 25°C. Storage modulus (G’) and loss factor (tanb) at 1 Hz were reported as the measurement results.

Example 1 - Preparation of cross-linked material and influence of the amount of lubrication phase on the properties of the material

Hyaluronic acid (HA) with intrinsic viscosity of 2.8 m³/kg (4.6 g corresponding to 4.0 g of dry polymer) was dissolved in 200 g of water (polymer concentration was 20 mg/g). DMTMM (3.3 g corresponding to 2.8 g of dry material - 1 eq. regarding the amount of HA) was added and the mixture was stirred for 1 hour. Thereafter, 100 mL of fibroin solution (concentration was 20 mg/g, HA/fibroin weight ratio was 2/1 ) was added: The mixture was stirred for 2 hours and then stirring was stopped. On the next day, the cross-linked material (here exemplified as a HA/fibroin gel) was purified using dynamic crossflow filtration (DCF Andritz, Membrane d= 152 mm (Andritz AG, Graz, Austria)) and 5 mM phosphate-buffered saline (PBS) solution at room temperature. The purified cross-linked material (here exemplified as a HA/fibroin gel) was then mixed with different amounts of lubrication phase (lubrication phase had concentration of 30 mg/g). Finally, the cross-linked material (here exemplified as a HA/fibroin gel) was filled into 1 mL syringes (Syringe One) and sterilized at 127°C for 8 min. Results are depicted in Table 1 below.

Table l. Properties of the obtained cross-linked material (here exemplified as a

G’ drop was calculated using the following formula:

G’ drop = (G’ at 1 Hz before sterilization - G’ at 1 Hz after sterilization) / G’ at 1 Hz before sterilization

Lubrication phase may be added to gels to reduce the extrusion force, however, in case of the cross-linked materials (here exemplified as a HA/fibroin gels) as contained in the composition of the present invention, the lubrication phase does surprisingly not cause a decrease of the extrusion force. Therefore, other materials might be prepared without lubrication phase.

It was found that a cross-linked material (here exemplified as a HA/fibroin gel) without lubrication phase may have G’ comparable to or even higher than the commercially available cross-linked hyaluronan product Belotero Volume Lidocaine (Anteis S.A., Plan-les-Ouates, Switzerland). While one of the herein exemplified HA/fibroin gels had a G’ of approximately 278 Pa after sterilization (Fib01 A), comparable a hyaluronan product Belotero Volume Lidocaine had a G’ of approximately 270 Pa. But extrusion force (EF) through 30G TSK needle was found to be significantly lower. The investigated HA/fibroin gel had an extrusion force (EF) of approximately 12 N, while cross-linked hyaluronan (Belotero Volume Lidocaine) had an extrusion force (EF) of approximately 22 N. This may support that HA/fibroin gels could offer similar lifting effect like the commercially available cross-linked hyaluronan product Belotero Volume Lidocaine (because of similar G’), while offering even better injection performance for the practitioner.

Furthermore, in this set of experiments the G’ of the cross-linked materials (here exemplified as a HA/fibroin gels) decreases by 10-25% after sterilization, which is significantly lower than the decrease of G’ of 78% in case of the control experiment (pure non-cross-linked hyaluronan). The results are also shown in Table 1 above. Example 2 - Preparation of cross-linked material and influence of HA/fibroin ratio on material properties)

Hyaluronic acid (HA) with intrinsic viscosity of 2.8 m³/kg (3.5 g corresponding to 3.0 g of dry polymer) was dissolved in 150 g of water (polymer concentration was 20 mg/g). DMTMM (2.5 g corresponding to 2.1 g of dry material - 1 eq. in regard to the amount of HA) was added and the mixture was stirred for 1 hour. Thereafter, 150 mL of fibroin solution (concentration was 20 mg/g, HA/fibroin weight ratio was 1/1 ) was added. The mixture was stirred for 2 hours and then stirring was stopped. On the next day, the cross-linked material (here exemplified as a HA/fibroin gel) was purified using dynamic crossflow filtration (DCF Andritz, Membrane d= 152 mm (Andritz AG, Graz, Austria)) and 5 mM phosphate-buffered saline (PBS) solution at room temperature. No lubrication phase was added. Finally, the cross-linked material (here exemplified as a HA/fibroin gel) was filled into 1 mL syringes (Syringe One) and sterilized at 127°C for 8 min. Results are depicted in Table 2 below.

Table 2. Properties of the obtained cross-linked material (here exemplified as a HA/fibroin gel). All given values are mean of double determination

It was found that an increase of the amount of fibroin in the cross-linked material, leads to cross-linked materials (here exemplified as a HA/fibroin gels) with higher G’.

Notably, this cross-linked material has lower extrusion force (even though it has higher G’). Explanation for this could be thixotropic (shear-thinning) behavior of fibroin. Cross-linked materials with comparably high and adjustable G’ and comparably low and adjustable extrusion force can be obtained. Example 3 - Preparation of cross-linked material and influence of intrinsic viscosity (IV) of hyaluronic acid (HA) on the material properties

Hyaluronic acid (HA) with intrinsic viscosity of 1.5 m³/kg (3.5 g corresponding to 3.0 g of dry polymer) was dissolved in 150 g of water (polymer concentration was 20 mg/g). DMTMM (2.5 g corresponding to 2.1 g of dry material - 1 eq. in regard to the amount of HA) was added and the mixture was stirred for 1 hour. Thereafter, 150 mL of fibroin solution (concentration was 20 mg/g, HA/fibroin weight ratio was 1/1 ) was added. The mixture was stirred for 2 hours and then stirring was stopped. On the next day, the cross-linked material (here exemplified as a HA/fibroin gel) was purified using dynamic crossflow filtration (DCF ANDRITZ, Membrane d= 152 mm (Andritz AG, Graz, Austria)) and 5 mM phosphate-buffered saline (PBS) solution at room temperature. No lubrication phase was added. Finally, the cross-linked material (here exemplified as a HA/fibroin gel) was filled into 1 mL syringes (Syringe One) and sterilized at 127°C for 8 min. Results are depicted in Table 3 below.

Table 3. Properties of the obtained cross-linked material (here exemplified as a HA/fibroin gel). All given values are mean of double determination

Example 4 - Enzymatic degradation of HA/fibroin gels

In order to investigate whether cross-linked materials (herein exemplified as HA/fibroin gels) can also be used as a reversible filler, the materials were treated with the enzyme hyaluronidase from ovine testes. Namely, approximately 0.50 g of gel was weighed via differential weighting and placed on the plate of the CP50-1 (cone-plate) system of Anton Paar MCR 302 rheometer (Anton Paar GmbH, Graz, Austria). A homogenous aqueous solution of 150 pL WFI containing 50 U hyaluronidase was added on top of the hydrogel on the plate. The hyaluronidase hydrogel mixture was homogenized manually by, e.g., the pipette tip for approximately 10 seconds. Thereafter, measurement was performed at 37°C in the oscillation mode at a deformation of 0.1 % and a frequency of 1 Hz. Measurement duration was 60 minutes with recording 1 point/min.

A gel prepared of non-cross-linked HA was degraded fastest. Thereafter, was the gel consisting of cross-linked HA (Belotero Volume, Anteis S.A., Plan-les-Ouates, Switzerland) and the gels containing fibroin degraded slowest. This could be indication of prolonged longevity of a gel which contains fibroin compared to typical HA cross-linked gels. Results are depicted in Table 4 below.

Table 4. Enzymatic degradation of hydrogels (here exemplified as a HA/fibroin gel).

G’ drop was calculated using the following formula:

G’ drop = (G’ at 1 Hz before treatment - G’ at 1 Hz after treatment with hyaluronidase (at 10 min)) / G’ at 1 Hz before treatment

Example 5 - Accelerated stability study

To test stability, batch Fib05 (see above) was mixed with lidocaine (0.3% of lidocaine to prepare new batch: FibO5L) and placed in a climate chamber at 40°C (accelerated conditions). Characterization was carried out by measuring rheological properties and extrusion force (using 30G TSK needle) of the gel at different time points (at week 4, 8 and 12). All measurements were performed in triplicates. The results are depicted in Table 5 below.

Table 5. Properties of the obtained cross-linked material (here exemplified as a HA/fibroin gel) over time, incubated at 40°C at 75% relative humidity found that the cross-linked materials (herein exemplified as HA/fibroin gels) are comparably stable over time. Even after 12 weeks at 40°C at 75% relative humidity, no significant degradation or deterioration of properties were found.

In summary, it was found that cross-linked materials as contained in the composition of the present invention can be very well and efficiently prepared, optionally in a single batch, without burden. The materials have good properties to be injectable and appear to have shear-thinning I thixotropic properties.

Example 6 - Low viscous HA/fibroin gels mixed with solid botulinum toxin

3.2 g hyaluronic acid (HA) with intrinsic viscosity of 2.8 m³/kg was dissolved in 178 g of buffer. 4.3 g DMTMM was added and the mixture was stirred for 1 hour. Thereafter, 62.5 mL of fibroin solution containing 2 g of fibroin was added. The mixture was stirred for 15 min. The cross-linked material was purified using dialysis. Thereafter, the material was filled into 1 mL syringes and sterilized at 131 °C for 1 min. 50 units (U) of solid botulinum toxin formulation (Xeomin) was added to 1 mL of the resulting cross-linked hyaluronic acid-fibroin (HA/Fib) matrix (as cross-linked material according to the present invention). Results are depicted in Table 6 below.

Table 6. HA/fibroin gels with and without botulinum toxin (BoNT). Example 7 - HA/fibroin gels mixed with botulinum toxin solution

3.2 g hyaluronic acid (HA) with intrinsic viscosity of 2.8 m³/kg was dissolved in 178 g of buffer. 4.3 g DMTMM was added and the mixture was stirred for 1 hour. Thereafter, 62.5 mL of fibroin solution containing 2 g of fibroin was added. The mixture was stirred for 15 min. The crosslinked material was purified using dialysis. The material was filled into 1 mL syringes and sterilized at 131 °C for 1 min. The gel (1 mL) was mixed 1 :1 with 50 Units of botulinum toxin formulation (Xeomin previously dissolved in 1 mL saline). Results are depicted in Table 7 below.

Table 7. HA/fibroin gels with and without botulinum toxin (BoNT).

Claims

1 . A composition comprising or consisting of:

(A) a cross-linked material comprising or consisting of (A-i) one or more fibroin moieties, and

(B) botulinum toxin;

(D) optionally one or more anesthetics; and

2. The composition of claim 1 , wherein the one or more polysaccharide moieties each comprise one or more carboxylic acid residues or salts thereof, preferably wherein the one or more polysaccharide moieties comprise or consist of one or more hyaluronic acid moieties, heparosan moieties, chondroitin sulfate moieties, carboxymethyl cellulose moieties, or a combination of two or more thereof.

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

4. The composition of any of claims 1 to 3, wherein:

(a) the one or more fibroin moieties have an average molecular weight 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 moieties are silk fibroin moieties, more preferably silk fibroin moieties having at least 80% sequence homology to a natural insect or spider silk fibroin moiety;

(b) the one or more polysaccharide moieties have an average molecular weight in the range of at least 50 kDa, preferably of 50 to 4000 kDa; and/or

(c) the mass ratio between the components (A-i) : (A-ii) is in the range of 5:1 to 1 :20.

5. The composition of any of claims 1 to 4, wherein the one or more fibroin moieties are covalently conjugated with the one or more polysaccharide moieties via amide bonds, in particular wherein the cross-linked material is further characterized in that it does not comprise:

(a) imide groups;

(b) imine groups;

(c) epoxy groups; and

(d) xenobiotic linker moieties.

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

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

8. The composition of any of claims 1 to 7, wherein the composition is an injectable composition comprising or consisting of:

(A) 0.1 to 3.5 % by weight, referred to the composition, of the crosslinked material;

(B) 1 to 150 units/mL, referred to the composition, of botulinum toxin; (C) 50 to 99.9 % by weight, preferably 59 to 99.9 % by weight, referred to the composition, of one or more liquid or viscous cosmetically and/or pharmaceutically acceptable carriers;

(E) 0 to 49.9 % by weight, referred to the composition, optionally one or more further cosmetically and/or pharmaceutically acceptable ingredients.

9. The composition of any of claims 1 to 8, wherein the composition comprises at least 50 % by weight, referred to the composition, of a liquid or viscous pharmaceutically acceptable carrier, preferably wherein the liquid or viscous pharmaceutically acceptable carrier comprises or consists of water, an aqueous buffer, glycerol or a combination of two or more thereof, in particular wherein the composition is injectable in a soft tissue, in particular injectable intradermally, subdermally, subcutaneously, and/or intramuscularly.

10. The composition of any of claims 1 to 9, wherein the composition is an injectable composition comprising or consisting of:

(A-ii) one or more polysaccharide moieties, in particular hyaluronic acid moieties, having an average molecular weight in the range of 50 to 4000 kDa, wherein the one or more fibroin moieties are covalently conjugated with the one or more polysaccharide moieties, in particular hyaluronic acid moieties, without an interconnecting linker structure, preferably via amide bonds, preferably having a mass ratio (A-i) to (A-ii) of 5:1 to 1 :20,

(B) 1 to 150 units/mL, referred to the composition, botulinum toxin not covalently bound with the cross-linked material; (C) 50 to 99.9 % by weight, referred to the composition, 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;

(E) 0 to 49.9 % by weight, referred to the composition, of one or more further cosmetically and/or pharmaceutically acceptable ingredients different from components (A) to (D). A method for preparing a composition of any one of claims 1 to 10, comprising the steps:

(i) contacting the following components with each other:

(A-iv) one or more solvents;

(ii) allowing a reaction of at least some of the carboxylic acid residues with at least some of the primary amino residues to form amide bonds conjugating the one or more fibroin moieties covalently with the one or more polysaccharide moieties;

(iv) adding: 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 further cosmetically and/or pharmaceutically acceptable ingredients different from components (A) to (D) (component (E)). Use of a cross-linked material comprising or consisting of (A-i) one or more fibroin moieties, and

(A-ii) one or more polysaccharide moieties, wherein the one or more fibroin moieties are covalently conjugated with the one or more polysaccharide moieties without an interconnecting linker structure, for retarded and/or controlled release of one or more pharmaceutically and/or cosmetically active agents, in particular botulinum toxin, preferably wherein the cross-linked material forms a gel a liquid or viscous pharmaceutically acceptable carrier, in particular wherein the cross-linked material forms part of a composition of any one of claims 1 to 10 is used.

13. The composition of any of claims 1 to 10 for use as a medicament.

14. The composition of any of claims 1 to 10 for use in a method of treating or preventing a disease or condition associated with hyperactive cholinergic activity, in particular of muscles and/or glands, and/or pain, preferably wherein the disease or condition is selected from the group consisting of a motoric disorder, in particular dystonia and/or spasticity, a neuromuscular diseases such as dystonia, spasm, tremor, hyperkinetic movement disorders, and cerebral palsy, pain such as diabetic neuropathy, peripheral neuropathy, neuropathic pain and trigeminal pain, sialorrhea, hyperhidrosis, urological disorders such as detrusor overactivity, neurogenic bladder and interstitial cystitis, treatment of vulvodynia and chronic pelvic pain, prostate disorder such as benign prostate hyperplasia (BPH) and detrusor sphincter dyssynergia (DSD), migraine, neurological disorders such as chronic or episodic migraines, cervical dystonia, post-stroke spasticity, blepharospasm, strabismus, (tension) headaches, pathologically high saliva production, achalasia, sphincter-oddi dysfunction, Hirschsprung's disease, anal fissure, bladder dysfunction such as overactive bladder, a dermatological or aesthetical problem improvable by muscle relaxation, articular pathology, arthritis, and a combination of two or more thereof.

15. Use of a composition of any one or claims 1 to 10 for cosmetic applications comprising rejuvenation and/or improvement of the skin quality of the face and/or body, preferably including improving and/or reducing and/or filling and/or preventing wrinkles, in particular of wrinkles resulting from muscular activity such as mimic activity, skin smoothing, improving skin laxity, lifting effect, moisturizing and/or softening the skin, improving and/or reducing and/or filling facial lines, soft-tissue augmentation, improving subdermal support of the brows, malar and buccal fat pads, improving tear troughs, improving nose appearance, resolving facial asymmetries, improving jawlines, or a combination of two or more thereof. 16. The use of claim 15, wherein the improvement of the skin quality of the face and/or body comprises the reduction of skin pore size and/or sebum production.

17. The use of claim 15, wherein the rejuvenation comprises the treatment of platysma bands and/or platysma muscles.