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WO2024126649A1 - Gel injectable comprenant de la toxine botulique et ses utilisations - Google Patents

Gel injectable comprenant de la toxine botulique et ses utilisations Download PDF

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Publication number
WO2024126649A1
WO2024126649A1 PCT/EP2023/085754 EP2023085754W WO2024126649A1 WO 2024126649 A1 WO2024126649 A1 WO 2024126649A1 EP 2023085754 W EP2023085754 W EP 2023085754W WO 2024126649 A1 WO2024126649 A1 WO 2024126649A1
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WO
WIPO (PCT)
Prior art keywords
moieties
composition
fibroin
kda
weight
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2023/085754
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English (en)
Inventor
Radovan VUKICEVIC
Colin DRABE
Thomas Hengl
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merz Pharma GmbH and Co KGaA
Original Assignee
Merz Pharma GmbH and Co KGaA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Merz Pharma GmbH and Co KGaA filed Critical Merz Pharma GmbH and Co KGaA
Priority to CN202380080326.7A priority Critical patent/CN120225172A/zh
Priority to EP23829050.6A priority patent/EP4633596A1/fr
Priority to AU2023392523A priority patent/AU2023392523A1/en
Priority to KR1020257014907A priority patent/KR20250124803A/ko
Publication of WO2024126649A1 publication Critical patent/WO2024126649A1/fr
Priority to IL320943A priority patent/IL320943A/en
Priority to MX2025006389A priority patent/MX2025006389A/es
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
    • A61K38/4893—Botulinum neurotoxin (3.4.24.69)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1767—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/02—Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/04—Dispersions; Emulsions
    • A61K8/042—Gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/008—Preparations for oily skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/91—Injection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/95—Involves in-situ formation or cross-linking of polymers

Definitions

  • Injectable gel comprising botulinum toxin and uses thereof
  • the present invention relates to a composition
  • 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.
  • botulinum toxin also: botulinum neurotoxin, BoNT
  • botulinum neurotoxin BoNT
  • BoNT botulinum neurotoxin
  • 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.
  • 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.
  • commercial botulinum toxin is often stored as vacuum-dried (lyophilized) material and/or contains protecting and preserving excipients.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions 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
  • a composition comprising or consisting of:
  • botulinum toxin (B) botulinum toxin; (C) optionally one or more liquid or viscous cosmetically and/or pharmaceutically acceptable carriers;
  • (E) optionally one or more further cosmetically and/or pharmaceutically acceptable ingredients different from components (A) to (D).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • polysaccharides in particular hyaluronic acid moieties
  • fibroins or salts thereof one or more fibroins or salts thereof
  • crosslinker facilitating the reaction between the aforementioned components and forming amide bonds in between the polysaccharide (in particular hyaluronic acid) and the fibroin.
  • 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.
  • composition of the present invention may be used for any purpose.
  • the composition of the present invention may be injectable.
  • 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.
  • 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.
  • the one or more polysaccharide moieties each comprise one or more carboxylic acid residues or salts thereof.
  • 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.
  • 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:
  • (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
  • 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.
  • 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.
  • 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.
  • the one or more polysaccharide moieties comprise or consist of one or more glycosaminoglycan moieties.
  • 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.
  • 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.
  • 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).
  • 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
  • a composition comprising or consisting of:
  • (E) optionally one or more further cosmetically and/or pharmaceutically acceptable ingredients different from components (A) to (D).
  • the cross-linked material of the present invention is a gel.
  • the cross-linked material of the present invention is a polysaccharide/fibroin gel.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the one or more fibroin moieties have an average molecular weight of 10 to 400 kDa.
  • 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.
  • 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.
  • At least one of the one or more fibroin moieties may have an average molecular weight of 50 to 400 kDa.
  • 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.
  • the fibroin moieties are of a single type and have thus (essentially) one molecular weight.
  • the fibroin moieties have at least two different average molecular weights each comprising primary amino residues or salts thereof.
  • the fibroin moieties may also be a mixture of fibroin moieties of different average molecular weight.
  • 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.
  • 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.
  • 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.
  • 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.
  • a fibroin moiety is preferably derived from naturally occurring fibroin.
  • amide binds are understandable in the broadest sense.
  • 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.
  • the majority of amino acid moieties of fibroin are L-amino acid moieties.
  • (essentials) all amino acid moieties of fibroin are L-amino acid moieties.
  • 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.
  • fibroin is silk fibroin.
  • 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.
  • fibroin may be prepared from another natural source or by means of genetic engineering (also: bio-fermentation, biotechnological means) or synthetic engineering.
  • molecular structure 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.
  • 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.
  • 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).
  • the term “fibroin moiety” may also include salts and modified forms thereof.
  • 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.
  • 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).
  • 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.
  • a commercial supplier e.g., Advanced BioMatrix, USA (e.g. product No. 5154-20ML); CareSilk, Italy (e.g. product No. CSK10-1051 )
  • genetic engineering also: bio-fermentation, biotechnological means
  • 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.
  • fibroin is ((essentially) complete) silkworm (Bombyx mori) fibroin.
  • 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.
  • other components of the silkworm cocoons have been (essentially) removed such as, e.g., sericin.
  • sericin e.g., 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.
  • 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.
  • one or more fibroin polypeptides 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.
  • 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.
  • the one or more fibroin moieties may be stored at approximately -80°C or in liquid nitrogen (i.e., at approximately -196°C).
  • 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.
  • 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.
  • the one or more fibroin moieties may be optionally protected from air.
  • hyaluronic acid also: HA, hyaluronate, or hyaluronan
  • HA hyaluronic acid
  • 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.
  • 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.
  • 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 ).
  • 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.
  • 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.
  • Mw average molecular weight
  • 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.
  • Mw average molecular weight
  • the one or more polysaccharide moieties have an average molecular weight (Mw) in the range of from 10 to 10000 kDa.
  • Mw average molecular weight
  • the one or more polysaccharide moieties have an average molecular weight in the range of 50 to 4000 kDa.
  • the one or more polysaccharide moieties have an average Mw in the range of from 100 to 10000 kDa.
  • 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 m 3 /kg (20°C, 1013 hPa, water).
  • At least one 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.
  • 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.
  • 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.
  • Mw average molecular weight
  • the polysaccharide moieties are of a single type and have thus (essentially) one molecular weight.
  • the polysaccharide moieties (in particular hyaluronic acid moieties) have at least two different molecular weights each comprising primary amino residues or salts thereof.
  • the polysaccharide moieties may optionally also be a mixture of polysaccharide moieties (in particular hyaluronic acid moieties) of different molecular weight.
  • the polysaccharide 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.
  • the polysaccharide 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.
  • the polysaccharide 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.
  • the polysaccharide 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.
  • the polysaccharide moieties (in particular hyaluronic acid moieties) may be used in mass excess.
  • 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 .
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • the cross-linked material of the present invention is a gel.
  • the cross-linked material of the present invention is a hyaluronic acid/fibroin gel (HA/fibroin gel).
  • the crosslinked material of the present invention is a hyaluronic acid/fibroin hydrogel (HA/fibroin hydrogel).
  • 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
  • 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.
  • 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
  • the one or more fibroin moieties and the one or more polysaccharide moieties may be conjugated with each other by any means.
  • the one or more fibroin moieties and the one or more hyaluronic acid moieties may be conjugated with each other by any means.
  • 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
  • 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
  • DCC N,N’-dicyclohexylcarbodiimide
  • DIC diisopropylcarbodiimide
  • EDC 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimid
  • an activating agent is typically not covalently included in the cross-linked material.
  • 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.
  • 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.
  • a triazine-based activating agent is 4-(4,6-dimethoxy- 1 ,3,5-triazin-2-yl)-4-methylmorpholinium chloride.
  • a carbodiimide activating agent is N,N’- dicyclohexylcarbodiimide (DCC), or 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimid (EDC).
  • the activating agent is 4-(4,6-dimethoxy-1 ,3,5-triazin-2- yl)-4-methylmorpholinium (DMTMM) or a salt thereof.
  • DTMM 4-(4,6-dimethoxy-1 ,3,5-triazin-2- yl)-4-methylmorpholinium
  • 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.
  • DMTMM 4-methylmorpholine
  • DMT 4,6- dimethoxy-1 ,3-5-triazine-2-ol
  • 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.
  • 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).
  • 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.
  • 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.
  • the cross-linked material is further characterized in that it does not comprise:
  • xenobiotic linker moieties interconnecting one or more fibroin moieties with one or more polysaccharide moieties.
  • the cross-linked material is further characterized in that it does not comprise:
  • the cross-linked material is further characterized in that it does as a whole not comprise: (a) imide groups;
  • 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.
  • it may be prepared by a method comprising:
  • step (iii) optionally purifying the cross-linked material obtained from step (ii).
  • 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.
  • 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.
  • 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.
  • pressure may be ambient pressure (e.g, often approximately 970 to 1100 hPa outer pressure).
  • the method is further characterized in that steps (i) and (ii) are conducted in a single batch.
  • 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.
  • the one or more polysaccharide 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.
  • the one or more fibroin moieties 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.
  • the solution may be stirred during the reaction step.
  • step (i) of the method comprises the following sub-steps:
  • the components and optionally one or more further components are all mixed at once.
  • the method comprises:
  • 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
  • step (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.
  • a polar solvent is used.
  • a protic solvent is used.
  • a protic polar solvent is used.
  • 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.
  • 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.
  • 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 e.g. at a temperature of 18 to 22°C
  • botulinum toxin may be understood in the broadest sense as any type of botulinum toxin known in the art.
  • botulinum toxin is not particularly limited and includes botulinum toxin of any serotype (e.g., BoNT/A-H.
  • the botulinum toxin may be of serotype A or B (BoNT/A, BoNT/B).
  • 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.
  • 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”).
  • the botulinum toxin is the pure botulinum neurotoxin of serotype A.
  • 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.
  • the active neurotoxic polypeptide may therefore also be referred to herein as the "150 kDa neurotoxin", “Clostridium botulinum neurotoxin (150 kD)” or "neurotoxic component”.
  • the botulinum toxin is the (pure) botulinum neurotoxin contained in Xeomin® or is Xeomin®.
  • toxin complex 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.
  • NAPs complexing proteins
  • the complexing proteins are nontoxic nonhaemagglutinin (NTNHA) and, in strains of serotype A-D, different haemagglutinins (HAs).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the botulinum toxin is not covalently bound with the cross-linked material.
  • a viscous cosmetically and/or pharmaceutically acceptable carrier may be any component being usable as such.
  • a liquid or viscous carrier according the present invention as comprised in the injectable composition may be any injectable carrier.
  • 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.
  • 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.
  • 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.
  • an aqueous buffer is a saline or a phosphate buffered saline.
  • 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.
  • a buffer may have a pH in a pharmaceutically and/or cosmetically acceptable when being injected in a subject.
  • 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.
  • an anesthetic may be any anesthetic component.
  • 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, le
  • an anesthetic is lodicaine.
  • 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.
  • 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.
  • a further component may be a filling or bulking agent.
  • such further component may be a salt (e.g., sodium chloride, NaCI).
  • such further component may be a stabilizing agent (e.g., a protein (e.g., albumin (e.g., human albumin)).
  • 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.)).
  • a sugar e.g., sucrose, glucose, trehalose, galactose, fructose, maltose, lactose, starch, cellulose, etc.
  • such further component may be a crystallization inhibitor.
  • such further component may be a scavenging agent.
  • such further component may be a cell proliferation factor may improve cellular invasion into an administered cross-linked material of the present invention.
  • 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.
  • 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.
  • one or more further components may be added during conducting a purifying.
  • one or more further components may be added to the prepared and optionally purified cross-linked material.
  • composition of the present invention may comprise the components in any concentration and content ranges.
  • 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.
  • the composition of the present invention comprises at least 0.1 % by weight, referred to the composition, of the cross-linked material.
  • the composition of the present invention 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.
  • the composition of the present invention 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.
  • 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.
  • the composition of the present invention 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.
  • 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.
  • the composition of the present invention comprises at least 0.1 units/g, referred to the composition, of botulinum toxin.
  • 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.
  • the composition of the present invention comprises at least 0.1 units/mL, referred to the composition, of botulinum toxin.
  • 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.
  • one injectable dose of the composition of the present invention comprises at least 0.1 units, referred to the composition, of botulinum toxin.
  • 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.
  • the composition of the present invention comprises not more than 1000 units/g, referred to the composition, of botulinum toxin.
  • 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.
  • the composition of the present invention 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.
  • 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.
  • an injectable dose of the composition of the present invention comprises not more than 1000 units, referred to the composition, of botulinum toxin.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • composition which preferably is an injectable composition, comprises or consists of:
  • composition 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;
  • composition is an injectable composition comprises or consists of:
  • (B) 1 to 150 units/mL, referred to the composition, of botulinum toxin;
  • composition 0 to 99.9 % by weight, referred to the composition, of one or more liquid or viscous cosmetically and/or pharmaceutically acceptable carriers;
  • composition 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.
  • the composition comprises at least 10 % by weight, referred to the composition, of a liquid or viscous pharmaceutically acceptable carrier.
  • 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.
  • composition is an injectable composition comprises or consists of:
  • (B) 1 to 150 units/mL, referred to the composition, of botulinum toxin;
  • composition 10 to 99.9 % by weight, referred to the composition, of one or more liquid or viscous cosmetically and/or pharmaceutically acceptable carriers;
  • composition 0 to 1 % by weight, referred to the composition, one or more anesthetics.
  • composition 0 to 89.9 % by weight, referred to the composition, of one or more further cosmetically and/or pharmaceutically acceptable ingredients.
  • composition is an injectable composition comprises or consists of:
  • (B) 1 to 150 units/mL, referred to the composition, of botulinum toxin;
  • composition 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;
  • composition 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.
  • composition is an injectable composition comprises or consists of:
  • (B) 1 to 150 units/mL, referred to the composition, of botulinum toxin;
  • composition 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;
  • composition 0 to 1 % by weight, referred to the composition, one or more anesthetics.
  • the composition is an injectable composition comprising or consisting of:
  • composition 0.1 to 3.5 % by weight, referred to the composition, of a cross-linked material comprising or consisting of
  • (B) 1 to 150 units/mL, referred to the composition, botulinum toxin not covalently bound with the cross-linked material;
  • composition 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;
  • the composition is an injectable composition comprising or consisting of:
  • composition 0.1 to 3.5 % by weight, referred to the composition, of a cross-linked material comprising or consisting of
  • (B) 1 to 150 units/mL, referred to the composition, botulinum toxin not covalently bound with the cross-linked material;
  • composition 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;
  • the components (A) and (B), optionally (C), optionally (D), and optionally (E), may be combined with each other by any means.
  • 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.
  • the one or more components (B), optionally (D), and optionally (E) are dissolved or suspended in component (C).
  • a (hydro)gel of the cross-linked material (A) is formed in component (C).
  • the sequential order of mixing may be freely chosen.
  • 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 (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)).
  • component (A), (B) and (C), and optionally (D) and/or (E) may be mixed with each other by any means.
  • component (A) is suspended and component (B)
  • component (B) is dissolved in one or more liquid or viscous cosmetically and/or pharmaceutically acceptable carriers (component (C)).
  • component (C) liquid or viscous cosmetically and/or pharmaceutically acceptable carriers
  • component (D) and/or component (E) may be further be present in this composition.
  • 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.
  • the materials may be mixed.
  • 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:
  • step (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
  • botulinum toxin component (B)
  • optionally one or more liquid or viscous cosmetically and/or pharmaceutically acceptable carriers component (C)
  • optionally one or more anesthetics component (D)
  • optionally one or more further cosmetically and/or pharmaceutically acceptable ingredients different from components (A) to (D) (component (E)).
  • 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.
  • the method for preparing a composition of the present invention comprising the steps:
  • step (iii) obtaining cross-linked material (component (A)) from step (ii) and optionally purifying the cross-linked material;
  • botulinum toxin component (B)
  • optionally one or more liquid or viscous cosmetically and/or pharmaceutically acceptable carriers component (C)
  • optionally one or more anesthetics component (D)
  • optionally one or more further cosmetically and/or pharmaceutically acceptable ingredients different from components (A) to (D) (component (E)).
  • adding of component (B) and optionally (C), (D) and/or (E) is adding to the cross-linked material of step (iii).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.).
  • 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.
  • 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.
  • the cross-linked material forms part of a composition of the present invention.
  • the cross-linked material forms gel, in particular in combination with one or more liquid or viscous cosmetically and/or pharmaceutically acceptable carriers.
  • 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.
  • composition of the present invention for use as a medicament.
  • the present invention further relates to a medicament comprising the composition of the present invention.
  • 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.
  • 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.
  • hypoactive 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.
  • “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.
  • “up to 25%” may, for example, be understood as >0% to about 25%.
  • a subject 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.
  • 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.
  • the composition of the present invention is preferably (essentially) sterile.
  • 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.
  • 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.
  • 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.
  • the composition may be administered intramuscularly.
  • Hyperactive cholinergic activity may be such of any part of the subject’s body.
  • 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.
  • 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
  • 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.
  • neuromuscular diseases such as dystonia, spasm, tremor, hyperkinetic movement disorders, and cerebral palsy
  • pain such as diabetic neuropathy, peripheral neuropathy, neuropathic pain
  • 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.
  • focal dystonia examples include 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.
  • 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.
  • 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).
  • 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.
  • a filler composition such as a soft tissue filler, in particular a dermal filler or connective tissue filler.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • lines may also be understood as wrinkles and vice versa.
  • 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.
  • improvable facial lines may be horizontal forehead lines and/or glabellar lines and/or periorbital lines including crow’s feet.
  • 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.
  • ambient temperature e.g., 18 to 30°C, preferably 18 to 25°C
  • a fridge e.g., at 0 to 15°C, preferably 3 to 10°C
  • a freezer e.g., -30 to 0°C, preferably
  • 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.
  • scope embraces the number values provided as commonly rounded values that embrace the whole rounding limits.
  • scope of “1 mg” embraces the range of from 0.50 to 1 .49 mg.
  • the number values of the present invention 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”.
  • Hyaluronic acid different intrinsic viscosities (HTL Biotechnology, Javene, France); 5% aqueous solution of fibroin from silk worm (CareSilk s.r.l.s., Lecce, Italia);
  • DTMM 4-(4,6-Dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholinium chloride
  • Lidocaine hydrochloride (Albemarle Corp., Charlotte, USA)
  • Xeomin® botulinum neurotoxin formulation
  • ambient conditions i.e., ambient temperature (such as 18 to 25°C, in particular approximately 20°C) and ambient/atmospheric pressure.
  • Extrusion Force 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 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 m 3 /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
  • 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.
  • 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).
  • 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.
  • 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.
  • EF extrusion force
  • 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.
  • EF extrusion force
  • Belotero Volume Lidocaine cross-linked hyaluronan
  • EF extrusion force
  • Hyaluronic acid (HA) with intrinsic viscosity of 2.8 m 3 /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
  • 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.
  • 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.
  • 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 m 3 /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
  • 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.
  • 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.
  • cross-linked materials herein exemplified as HA/fibroin gels
  • 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.
  • 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
  • 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.
  • 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
  • HA hyaluronic acid

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Abstract

La présente invention concerne une composition comprenant (A) un matériau réticulé comprenant une ou plusieurs fractions de fibroïne et une ou plusieurs fractions de polysaccharide conjuguées de manière covalente l'une à l'autre, (B) une toxine botulique. En outre, la présente invention concerne des utilisations thérapeutiques et esthétiques d'une telle composition
PCT/EP2023/085754 2022-12-15 2023-12-14 Gel injectable comprenant de la toxine botulique et ses utilisations Ceased WO2024126649A1 (fr)

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CN202380080326.7A CN120225172A (zh) 2022-12-15 2023-12-14 包含肉毒杆菌毒素的可注射凝胶及其用途
EP23829050.6A EP4633596A1 (fr) 2022-12-15 2023-12-14 Gel injectable comprenant de la toxine botulique et ses utilisations
AU2023392523A AU2023392523A1 (en) 2022-12-15 2023-12-14 Injectable gel comprising botulinum toxin and uses thereof
KR1020257014907A KR20250124803A (ko) 2022-12-15 2023-12-14 보툴리눔 독소를 포함하는 주사가능한 겔 및 이의 용도
IL320943A IL320943A (en) 2022-12-15 2025-05-15 Injectable gel containing botulinum toxin and its uses
MX2025006389A MX2025006389A (es) 2022-12-15 2025-05-30 Gel inyectable que comprende toxina botulinica y usos del mismo

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