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WO2024251576A1 - Timbre d'hydrogel fabriqué à partir d'une protéine de type collagène (clp) - Google Patents

Timbre d'hydrogel fabriqué à partir d'une protéine de type collagène (clp) Download PDF

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Publication number
WO2024251576A1
WO2024251576A1 PCT/EP2024/064702 EP2024064702W WO2024251576A1 WO 2024251576 A1 WO2024251576 A1 WO 2024251576A1 EP 2024064702 W EP2024064702 W EP 2024064702W WO 2024251576 A1 WO2024251576 A1 WO 2024251576A1
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WO
WIPO (PCT)
Prior art keywords
protein
collagen
patch
hydrogel
base layer
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Pending
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PCT/EP2024/064702
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English (en)
Inventor
Sven Weber
Thomas Endres
Maria MONTERO MIRABET
Maria Camilla OPERTI
Kathrin Nollenberger
Norbert Windhab
Andrea ENGEL
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Evonik Operations GmbH
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Evonik Operations GmbH
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Publication of WO2024251576A1 publication Critical patent/WO2024251576A1/fr
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    • 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
    • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
    • A61K9/7023—Transdermal patches and similar drug-containing composite devices, e.g. cataplasms
    • A61K9/703—Transdermal patches and similar drug-containing composite devices, e.g. cataplasms characterised by shape or structure; Details concerning release liner or backing; Refillable patches; User-activated patches
    • A61K9/7038—Transdermal patches of the drug-in-adhesive type, i.e. comprising drug in the skin-adhesive layer
    • A61K9/7046—Transdermal patches of the drug-in-adhesive type, i.e. comprising drug in the skin-adhesive layer the adhesive comprising macromolecular compounds
    • A61K9/7069—Transdermal patches of the drug-in-adhesive type, i.e. comprising drug in the skin-adhesive layer the adhesive comprising macromolecular compounds obtained otherwise than by reactions only involving carbon to carbon unsaturated bonds, e.g. polysiloxane, polyesters, polyurethane, polyethylene oxide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/5415—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with carbocyclic ring systems, e.g. phenothiazine, chlorpromazine, piroxicam
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3637—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the origin of the biological material other than human or animal, e.g. plant extracts, algae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)

Definitions

  • the present invention refers to a patch, comprising a hydrogel base layer obtained by reacting at least one collagen-like protein and at least one cross-linker selected from 4-(4,6-dimethoxy-1 ,3,5- triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM), glutaraldehyde, transglutaminase, diisocyanate, or a combination of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N- hydroxysuccinimide (NHS); optionally a further hydrogel layer obtained by reacting at least one collagen-like protein and at least one cross-linker comprising at least two groups each comprising a polyalkylene glycol moiety and a succinimidyl group; wherein the hydrogel base layer has a thickness of at most 8 mm and the patch has a thickness of at most 16 mm as well as a manufacturing method thereof. Furthermore, the present invention refers to the use of the patch
  • the inventors of the present invention have surprisingly found that one or more of these objects can be solved by using a specific patch made of a specific hydrogel base layer obtained by reacting at least one collagen-like protein and at least one cross-linker selected from 4-(4,6-dimethoxy-1 ,3,5- triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM), glutaraldehyde, transglutaminase, diisocyanate, or a combination of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N- hydroxysuccinimide (NHS).
  • the patch can contain one or more further hydrogel layer.
  • the hydrogel base layer has a thickness of at most 8 mm and the patch has a thickness of at most 16 mm in order to ensure suitable flexibility and stretchability.
  • the patch can be additionally loaded with at least one polymeric active agent delivery particle, at least one liposome or at least one polyplex.
  • the hydrogel patch can be used as is (non-dried). Optional heat-drying or lyophilization enables further control of morphology and properties. Dried patches can be re-hydrated prior to use.
  • the present invention refers to a patch, comprising or consisting of i) a hydrogel base layer obtained by reacting at least one collagen-like protein and at least one cross-linker selected from 4-(4,6- dimethoxy-1 ,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM), glutaraldehyde, transglutaminase, diisocyanate, or a combination of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS); ii) optionally a further hydrogel layer obtained by reacting at least one collagen-like protein and at least one cross-linker comprising at least two groups each comprising a polyalkylene glycol moiety and a succinimidyl group; wherein the hydrogel base layer has a thickness of at most 8 mm and the patch has a thickness of at most 16 mm.
  • a hydrogel base layer obtained by react
  • the present invention refers to a method of preparing a patch according to the present invention comprising or consisting of the steps i) providing water, the at least one collagen-like protein and the at least one cross-linker and homogenizing the mixture, then transferring the mixture into a mold and allowing the mixture to gel in order to form the hydrogel base layer; ii) optionally providing water, the at least one collagen-like protein and the at least one crosslinker and homogenizing the mixture, then transferring the mixture into the mold containing the hydrogel base layer and allowing the mixture to gel in order to form the at least one further hydrogel layer; iii) thereafter optionally freeze or heat drying the hydrogel base layer and optionally the at least one further hydrogel layer to form the patch.
  • the present invention refers to a use of the patch according to the present invention for wound sealing, drug delivery, occlusive application, tissue regeneration, or forming a barrier membrane.
  • Figure 1 Freeze-dried single layer patches (left) and heat-dried double patch (right).
  • FIG. 2 Meloxicam-Resomer microparticles (A) incorporated in a heat dried CLP soft tissue patch crosslinked with 4-Arm-PEG-SG (B). Macroscopic observation of the undried (C) and heat dried CLP patches with Resomer particles when submerged in ddFW after heat drying (D). (E) shows the applied heat-dried soft tissue patch on a glove.
  • FIG. 3 Measured release kinetics of Meloxicam (M) which was encapsulated in Resomer microparticles (MPs) and subsequently immobilized in a single and a double layered CLP patch with identical formulation regarding the DMTMM layer.
  • M Meloxicam
  • MPs Resomer microparticles
  • Figure 4 HFF cells exposed to the hydrogel surface of different formulations after washing. The cells were cultured for 24 h under culture conditions. Brightfield pictures were taken afterwards.
  • Figure 5 (A) Prepared collagen soft tissue patches with different thicknesses after freeze-drying. (B) The material was bended by 180° in the dry state and after soaking for 10 min in ddFW.
  • Figure 6 Example formulation of soft tissue patch which can be rolled without breaking in dry and wet state (10 min soaking in ddFW).
  • “One or more”, as used herein, relates to at least one and comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9 or more of the referenced species. Similarly, “at least one” means one or more, i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or more. "At least one”, as used herein in relation to any component, refers to the number of chemically different molecules, i.e. to the number of different types of the referenced species, but not to the total number of molecules.
  • At least one surfactant means that at least one type of molecule falling within the definition for a surfactant is used but that also two or more different types of surfactants falling within this definition can be present but does not mean that only one or more molecules of one type of surfactant are present.
  • “Essentially free of’ according to the present invention with regard to compounds means that the compound can only be present in an amount, which does not influence the characteristics of the composition, in particular the respective compound is present in less than 3 wt.-%, preferably 1 wt.- %, more preferably 0.01 wt.-%, based on the total weight of the composition or is not present at all.
  • the weight average molecular weight Mw and the number average molecular weight Mn can be determined by GPC employing polystyrene standards.
  • a hydrogel is according to the present invention considered to be non-adhesive, if after 2D cell cultivation for 24 h on the hydrogel and subsequent washing (3x 1x PBS) not more than 10%, preferably 5% more preferably 0% of cells remain on the surface.
  • the percentage is calculated by comparing the number of cells after washing on the hydrogel with the number of cells on an optimized cell culture surface (plasma treated PS) also after washing.
  • the hydrogel area and the optimized plate surface were identical as well as the cell number during cell seeding.
  • At least one collagen-like protein is reacted with at least one specific crosslinker.
  • the patch has a thickness of at most 16 mm, preferably 2 to 8 mm.
  • the patch can be a sponge, a dried patch, e.g. heat dried or freeze dried, or a re-hydrated patch.
  • the patch according to the present invention is obtained by a method comprising or consisting of the steps i) providing water, the at least one collagen-like protein and the at least one cross-linker and homogenizing the mixture, then transferring the mixture into a mold and allowing the mixture to gel in order to form the hydrogel base layer; ii) optionally providing water, the at least one collagen-like protein and the at least one crosslinker and homogenizing the mixture, then transferring the mixture into the mold containing the hydrogel base layer and allowing the mixture to gel in order to form the at least one further hydrogel layer; iii) thereafter optionally freeze or heat drying the hydrogel base layer and optionally the at least one further hydrogel layer to form the final patch.
  • Freeze and heat drying is known to the person skilled in the art. For example, freeze drying can be performed at -40 to -60 °C and heat drying can be performed at 30 to 45 °C.
  • the hydrogel base layer is obtained by reacting at least one collagen-like protein and at least one cross-linker selected from 4-(4,6-dimethoxy-1 ,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM), glutaraldehyde, transglutaminase, diisocyanate, or a combination of 1 -ethyl-3-(3- dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS).
  • the hydrogel base layer has a thickness of at most 8 mm, preferably 0.1 to 8, more preferably 0.5 to 6, most preferably 1 to 2 mm.
  • collagen-like proteins are suitable.
  • the collagen-like protein is a collagen-like protein from Streptococcus pyogenes, which is preferably the Scl2 protein from Streptococcus pyogenes.
  • the at least one collagen-like protein is a bacterial collagen-like protein, preferably produced by fermentation in Pichia, Brevibacillus, Bacillus, Escherichia or Corynebacterium, preferably Pichia pastoris, Brevibacillus choshinensis or Corynebacterium glutamicum.
  • the collagen-like proteins may be expressed in Corynebacterium, preferably in Corynebacterium glutamicum.
  • One particularly suitable collagen-like protein is derivable from following polynucleotide.
  • amino acid sequence that is at least > 60%, identical to the amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
  • amino acid sequence that is at least > 65%, or > 70%, or > 75%, or > 80%, or > 85% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
  • the polynucleotide encodes an amino acid sequence that is at least > 90%, > 92%, > 94%, > 96%, > 97%, > 98%, > 99% or 100%, preferably > 97%, particularly preferably > 98%, very particularly preferably > 99%, and extremely preferably 100%, identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
  • the polynucleotide is a replicable nucleotide sequence encoding the collagen-like protein from Streptococcus pyogenes.
  • polypeptide variants of SEQ ID NO:1 to 4 which contain one or more insertion(s) or deletion(s) are suitable as well.
  • the polypeptide contains a maximum of 5, a maximum of 4, a maximum of 3, or a maximum of 2, insertions or deletions of amino acids.
  • Polypeptides comprising one of the polypeptide variants of SEQ ID NO:1 to 4 and on or more of the truncated variants of the collagen-like protein of SEQ ID NO:5 to 12 can be used as well.
  • the vector comprising the nucleotide sequences according to the present invention is suitable for replication in yeast of the genus Pichia pastoris.
  • Microorganisms of the genera Pichia, Corynebacterium, Pseudomonas or Escherichia that comprise the polynucleotides, vectors and polypeptides according to the invention are suitable as well.
  • Preferred microorganisms are Pichia pastoris, Brevibacillus choshinensis or Corynebacterium glutamicum.
  • Microorganism of the species P. pastoris, E. coli, P. putida or C. glutamicum comprising any of the nucleotide sequences according to the present invention any of the polypeptides or any of the vectors according to the present invention are suitable.
  • the microorganism may be a microorganism in which the nucleotide sequence is present in overexpressed form.
  • Overexpression means, generally, an increase in the intracellular concentration or activity of a ribonucleic acid, a protein (polypeptide) or an enzyme, compared with the starting strain (parent strain) or wild-type strain, if this is the starting strain.
  • a starting strain (parent strain) is taken to mean the strain on which the measure leading to the overexpression was carried out.
  • the methods of recombinant overexpression are preferred. These include all methods in which a microorganism is produced using a DNA molecule provided in vitro.
  • DNA molecules comprise, for example, promoters, expression cassettes, genes, alleles, encoding regions etc. These are converted into the desired microorganism by methods of transformation, conjugation, transduction or like methods.
  • the extent of the expression or overexpression can be established by measuring the amount of the mRNA transcribed by the gene, by determining the amount of the polypeptide, and by determining the enzyme activity.
  • the bacterial collagen-like protein can be obtained in a fermentative process comprising the following steps: a) fermentation of a microorganism according to the present invention in a medium, b) accumulation of the bacterial collagen-like protein in the medium, wherein a fermentation broth is obtained.
  • the culture medium or fermentation medium that is to be used must appropriately satisfy the demands of the respective strains. Descriptions of culture media of various microorganisms are contained in the handbook "Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981). The terms culture medium and fermentation medium or medium are mutually exchangeable.
  • sugars and carbohydrates can be used, such as, e.g., glucose, sucrose, lactose, fructose, maltose, molasses, sucrose-containing solutions from beet sugar or sugar cane processing, starch, starch hydrolysate and cellulose, oils and fats, such as, for example, soybean oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as, for example, palmitic acid, stearic acid and linoleic acid, alcohols such as, for example, glycerol, methanol and ethanol, and organic acids, such as, for example, acetic acid or lactic acid.
  • oils and fats such as, for example, soybean oil, sunflower oil, groundnut oil and coconut fat
  • fatty acids such as, for example, palmitic acid, stearic acid and linoleic acid
  • alcohols such as, for example, glycerol, methanol and ethanol
  • organic acids such as, for example, acetic acid or
  • nitrogen source organic nitrogen compounds such as peptones, yeast extract, meat extract, malt extract, corn-steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used.
  • the nitrogen sources can be used individually or as a mixture.
  • phosphoric acid potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts
  • the culture medium must, in addition, contain salts, for example in the form of chlorides or sulphates of metals such as, for example, sodium, potassium, magnesium, calcium and iron, such as, for example, magnesium sulphate or iron sulphate, which are necessary for growth.
  • salts for example in the form of chlorides or sulphates of metals such as, for example, sodium, potassium, magnesium, calcium and iron, such as, for example, magnesium sulphate or iron sulphate, which are necessary for growth.
  • essential growth substances such as amino acids, for example homoserine and vitamins, for example thiamine, biotin or pantothenic acid, can be used in addition to the above-mentioned substances.
  • Said starting materials can be added to the culture in the form of a single batch or supplied in a suitable manner during the culturing.
  • Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acid compounds such as phosphoric acid or sulphuric acid, are used in a suitable manner for pH control of the culture.
  • the pH is generally adjusted to 6.0 to 8.5, preferably 6.5 to 8.
  • antifoams can be used, such as, for example, polyglycol esters of fatty acids.
  • suitable selectively acting substances such as, for example, antibiotics, can be added to the medium.
  • the fermentation is preferably carried out under aerobic conditions. In order to maintain said aerobic conditions, oxygen or oxygen-containing gas mixtures such as, for example, air, are introduced into the culture.
  • liquids that are enriched with hydrogen peroxide are likewise possible.
  • the fermentation is carried out at superatmospheric pressure, for example at a superatmospheric pressure of 0.03 to 0.2 MPa.
  • the temperature of the culture is usually 20°C to 45°C, and preferably 25°C to 40°C, particularly preferably 30°C to 37°C.
  • the culturing is preferably continued until an amount sufficient for the measure of obtaining the desired organic chemical compound has formed. This goal is usually reached within 10 hours to 160 hours. In continuous processes, longer culture times are possible. Due to the activity of the microorganisms, enrichment (accumulation) of the fine chemicals in the fermentation medium and/or in the cells of the microorganisms occurs.
  • the process may be characterized by a process which is selected from the group consisting of batch process, fed-batch process, repetitive fed-batch process, and continuous process.
  • the performance of the processes or fermentation processes according to the invention with respect to one or more of the parameters selected from the group of concentration (compound formed per volume), yield (compound formed per carbon source consumed), volumetric productivity (compound formed per volume and time) and biomass-specific productivity (compound formed per cell dry mass or bio dry mass and time or compound formed per cell protein and time) or other process parameters and combinations thereof, is increased by at least 0.5%, at least 1 %, at least 1.5% or at least 2%, based on processes or fermentation processes with microorganisms in which the promoter variant according to the invention is present.
  • a fermentation broth which contains the desired collagen-like protein, and preferably amino acid or organic acid.
  • a fermentation broth means, in a preferred embodiment, a fermentation medium or nutrient medium in which a microorganism was cultured for a certain time and at a certain temperature.
  • the fermentation medium, or the media used during the fermentation contains/contain all substances or components that ensure production of the desired collagen-like protein and typically ensure growth and/or viability.
  • the resultant fermentation broth accordingly contains a) the biomass (cell mass) of the microorganism resulting from growth of the cells of the microorganism, b) the desired collagen-like protein formed in the course of the fermentation, c) the organic by-products possibly formed in the course of the fermentation, and d) the components of the fermentation medium used, or of the starting materials, that are not consumed by the fermentation, such as, for example, vitamins such as biotin, or salts such as magnesium sulphate.
  • the organic by-products include substances which are generated in addition to the respective desired compound by the microorganisms used in the fermentation and are possibly secreted.
  • the fermentation broth is withdrawn from the culture vessel or the fermentation container, optionally collected, and used for providing a product in liquid or solid form containing the collagen-like protein.
  • the expression "obtaining the collagen-like protein-containing product” is also used therefor.
  • the collagen-like protein-containing fermentation broth withdrawn from the fermentation container is itself the product obtained.
  • the collagen like protein can preferably be present in the aqueous solution with a concentration range from 2.5 to 100 mg/ml.
  • the collagen-like protein concentration in the hydrogel base layer is 10 to 40 mg/cm 3 .
  • the at least one cross-linker is provided in an aqueous solution having a pH value of 6 to 8, preferably 6.8 to 7.4.
  • At least one additive can be optionally present.
  • the at least one additive is a growth factor, for example a fibroblast growth factor, epidermal growth factor, nerve growth factor or connective tissue growth factor or a recombinant human bone morphogenesis protein.
  • the at least one additive is selected from thrombin, fibrinogen, chitosan, silicic acid precursors, heparin, heparin derived oligosaccharides, hyaluronic acid, and glycosaminoglycans.
  • the hydrogel base layer is a sponge.
  • the sponge can be obtained for example as disclosed in the method of the WO application PCT/EP2023/053300, which is incorporated by reference.
  • the sponge has a water uptake capacity of 800 to 3000%, based on the total dry weight of the sponge.
  • the sponge has a pore size of 15 to 300 pm.
  • the sponge has a total porosity of 75 to 99%, and an open porosity of 20 to 85%.
  • the sponge has a Young’s modulus of 45 to 250 kPa in dry form.
  • the sponge has a Young’s modulus of 4 to 35 kPa in wet form.
  • the hydrogel base layer can comprise at least one polymeric active agent delivery particle, at least one liposome or at least one polyplex.
  • the at least one polymeric active agent delivery particle, at least one liposome or at least one polyplex can be evenly distributed throughout the layer or at the surface of one layer or at the surface of all layers.
  • polymeric active agent delivery particles are suitable and a skilled person in the field of delivery particles knows how to obtain such particles.
  • Exemplarily polymeric active agent delivery particles and their production method are described in WO 2021063813 A1 and WO 2015181138 A1 which are incorporated by reference.
  • the at least one polymeric active agent delivery particle has a particle size of 1 to 250 pm, preferably measured by dynamic light scattering, more preferably according to DIN ISO 22412:2018-09 for example by using a Mastersizer 3000 (Malvern Panalytical Ltd., Malvern, UK).
  • polyplexes are suitable.
  • suitable polyplexes and their method of manufacturing are disclosed in WO 2016161345 A1 which is incorporated by reference.
  • liposomes are suitable.
  • suitable liposomes and their method of manufacturing are disclosed in WO 2012031043 A1 which is incorporated by reference.
  • Protein sequences are disclosed in WO 2012031043 A1 which is incorporated by reference.
  • SEQ ID NO:1 Streptomyces pyogenes Collagen-like protein (CLP), full length protein
  • the at least one further hydrogel layer is obtained by reacting at least one collagen-like protein and at least one cross-linker comprising at least two groups each comprising a polyalkylene glycol moiety and a succinimidyl group.
  • collagen-like proteins are suitable.
  • Preferred embodiments of the at least one collagen-like protein are the same as the abovedescribed embodiments for the at least one collagen-like protein of the hydrogel base layer.
  • cross-linker has one of following formulae (I) or (II): wherein
  • R 1 is a linear or branched alkyl group having up to 12 carbon atoms, preferably up to 8 carbon atoms, more preferably having five carbon atoms;
  • Aik is -CH 2 -, -CH2-CH2- or -CH 2 -CH 2 -CH 2 -, preferably -CH2-CH2-;
  • n is an integer from 1 to 1350, preferably, 50 to 1000, more preferably 125 to 660;
  • Aik is -CH2-, -CH2-CH2- or -CH 2 -CH 2 -CH 2 -, preferably -CH2-CH2-; and n is an integer from 1 to 1350, preferably 50 to 1000, more preferably 125 to 660.
  • cross-linker has following formula (III) wherein
  • R 1 is a linear or branched alkyl group having up to 12 carbon atoms, preferably up to 8 carbon atoms, more preferably having five carbon atoms, most preferably is
  • Aik is -CH 2 -, -CH2-CH2- or -CH 2 -CH 2 -CH 2 -, preferably -CH2-CH2-; n is an integer from 1 to 1350, preferably, 50 to 1000, more preferably 125 to 660; m is an integer from 2 to 8, preferably 4 to 8, most preferably 4.
  • the cross-linker has a molecular weight of 2.000 to 40.000 g/mol.
  • the reaction takes place in an aqueous medium, preferably a buffer, more preferably 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES), having a pH value of 6 to 8, preferably 7 to 8, more preferably 8.
  • HEPES 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid
  • the collagen-like protein concentration in the hydrogel is 10 to 100 mg/ml, preferably 2.5 - 40 mg/ml.
  • the collagen-like protein concentration in the further hydrogel layer is 10 to 40 mg/cm 3 .
  • the molar ratio of lysine groups in the collagen-like protein to the succinimidyl groups in the cross-linker is 1 :0.01 to 1 :1 .6.
  • the hydrogel is non-adhesive.
  • At least one additive can be optionally present.
  • the at least one additive is selected from thrombin, fibrinogen, chitosan, silicic acid precursors, heparin, heparin derived oligosaccharides, hyaluronic acid, and glycosaminoglycans.
  • the at least one polymeric active agent delivery particle, at least one liposome or at least one polyplex can be evenly distributed throughout the layer or at the surface of the layer.
  • the at least one polymeric active agent delivery particle has a particle size of 1 to 250 pm, preferably measured by dynamic light scattering, more preferably according to DIN ISO 22412:2018-09.
  • Preferred embodiments of the at least one polymeric active agent delivery particle, at least one liposome or at least one polyplex are the same as the above-described embodiments for the at least one polymeric active agent delivery particle, at least one liposome or at least one polyplex of the hydrogel base layer.
  • the bacterial collagen-like protein was produced in different host cells by fermentation.
  • the protein could be produced under similar conditions using either E. coll or C. glutamicum.
  • the CL single strand is secreted by the cell. No cell lysis is needed as an initial purification step in this approach.
  • a cell lysis is mandatory to remove the product from the cell.
  • the B. choshinensis strains were analyzed for their ability to produce the different collagen proteins in batch cultivations at 33°C and pH 7 using the DASGIP® parallel bioreactor system from Eppendorf (Hamburg, Germany). The fermentation was performed using 1 L reactors.
  • the production medium (TM medium, Biomed Res Int 2017, 2017: 5479762) contained 10 g/L glucose. Upon fermentation, supernatant has been separated from biomass by centrifugation and was used for SDS PAGE analysis. For all three variants, collagen-like protein was produced.
  • the full-length collagen-like protein and the no-V-domain variant were also expressed in Corynebacterium glutamicum. Therefore, the corresponding DNA sequences were cloned together with an upstream located signal peptide for protein secretion into a shuttle vector for C. glutamicum (Biotechnology Techniques 1999, 13: 437- 441 .).
  • the C. glutamicum strain ATCC 13032 was transformed with the new constructed plasmids by means of electroporation as described by Ruan et al. (Biotechnology Letters 2015, 37: 2445- 2452).
  • the C. glutamicum strains were analysed for their ability to produce the different collagen proteins in fed-batch cultivations at 30°C and pH 7 using the DASGIP® parallel bioreactor system from Eppendorf (Hamburg, Germany).
  • the fermentation was performed using 1 L reactors.
  • the production medium contained 20 g/L glucose in the batch phase and the fed-batch phase was run with a glucose feed of 4 g/L*h.
  • supernatant has been separated from biomass by centrifugation and was used for HPLC analysis.
  • collagen protein was produced.
  • product titer was higher as for the full-length variant.
  • the bacterial collagen-like protein was purified using precipitation with 2-Propanol at 15 v%. After precipitation of the Scl2 protein a centrifugation was performed. The pellet was dissolved in water, the triple helical Scl2 protein was unfolded at 40°C and filtered through a 100 kD membrane. This step serves to remove large sized impurities. The collected permeate was then concentrated in the consecutive 10 kD filtration. The retentate was washed to remove small sized impurities.
  • the collagen-like protein is referred to as Vecollan® in the following experiments as well.
  • Example 1 CLP hydrogel base layer preparation
  • Table 1 Pipetting scheme for the preparation of the hydrogel base patch using the previously described DMTMM technology.
  • the molar ratio (MR) described the ratio of functional side chains in CLP compared to the functional side chains in the crosslinker.
  • a MR of 1 :1 was applied.
  • Vecollan® CLP from Evonik Operations GmbH
  • 4-(4,6-Dimethoxy-1 ,3,5-triazin-2-yl)-4- methylmorpholinium chloride (short: DMTMM, TCI Chemicals, order number: D2919, Mw: 276.72 g/mol)
  • 4-arm PEG Succinimidyl Glutaramide (short: 4-Arm-PEG-SG, 10 kDa, JenKem Technology USA, order number: A7110-1* / 4ARM-SGA-10K,1g)
  • HEPES buffer pH 8.0 (1 M) sterile PanReac AppliChem; order number: A6906
  • a 10% (w/v) CLP stock solution was prepared. The mixture was incubated at room temperature (20 °C to 25 °C) on an orbital shaker (450 rpm) until a homogenous solution formed.
  • the DMTMM stock solution (196.25 mg/ml) as well as the 4-Arm-PEG-SG stock solution (201 .37 mg/ml) was prepared fresh (max. 1 h prior to the experiment).
  • Double layer preparation Components were mixed in a vessel with sufficient volume capacity by added them in the following order: ddH2O; CLP stock solution; HEPES buffer (final concentration: 0.1 M) and 4-Arm-PEG-SG stock solution. Before and after adding the crosslinker, the mixture was homogenized. 3.2 ml formulation was transferred into a silicon mold with 4x4x0.4 cm 3 and incubated under humid conditions for 1 h at RT (20 °C - 25 °C).
  • the second layer was molded on top of the first layer by mixing the following ingredients in the following order in a vessel with sufficient volume capacity: ddH2O; CLP stock solution and DMTMM stock solution. Before and after adding the crosslinker, the mixture was homogenized. 3.2 ml formulation was transferred into the silicon mold on top of the already prepared CLP hydrogel base layer. The hydrogel bilayer was incubated under humid conditions for 24 h at RT (20 °C - 25 °C).
  • Table 2 Pipetting scheme for the preparation CLP-based layer crosslinked with DMTMM or 4-Arm-PEG- SG (short: PEG-SG).
  • the molar ratio (MR) describes the ratio of functional side chains in CLP compared to the functional side chains in the crosslinker.
  • a MR of 1 :0.2 was applied for the CLP/PEG composite and a MR of 1 :1 was applied for the DMTMM crosslinked CLP base layer.
  • the applied 1 M HEPES buffer pH 8.0 was abbreviated with “buffer”.
  • the patches described in examples 1 and 2 were placed in a heat cabinet and exposed to 40 °C hot air for 16 h.
  • the resulting film-like patches have a transparent shape and increase in flexibility by swelling in aqueous solution.
  • the incubation time strongly depends on the patch thickness as well as its formulation. Also, the loading density is crucial for the drying process duration.
  • the patches described in examples 1 and 2 were placed in a freeze dryer and processed via the following program resulting in white sponge-like patches. Resulting patches are depicted in figure 1 .
  • Table 3 Freeze drying protocol to prepare sponge-like CLP patches.
  • hydrogels were prepared with 2 mm layer height in silicon molds by using the described formulations in table 4. Only the hydrogel base layer was used for this experiment. Resulting patches were analyzed regarding their breaking behavior when bended by 180° in dry and wet state (5 min soaking in 1 xPBS). When bending by 180° was possible, patches were enrolled to demonstrate increased flexibility. Results were documented in table 5. One example with pictures was given in figure 6.
  • Table 4 Different formulations for the preparation of hydrogel base layer crosslinked by DMTMM.
  • Example 6 CLP patch crosslinked with DMTMM and loaded with PLGA-based microparticles
  • Microparticles were added from a particle suspension in ddFW spiked into the water fraction of the formulation shown in example 1 .
  • An example formulation with particles is given in table 6.
  • Interface Applying the DMTMM crosslinked hydrogel base layer on top of the crosslinked PEG/CLP layer. Particles will sediment into the interphase. This protects the user from direct particle contact. This is especially important for enclosed high potent drugs to avoid direct contact when used by e.g., health-care personnel when applied.
  • Rotation Alternatively, the patch was prepared in a special mold under constant rotation until gelation occurred.
  • Viscous molding Third, the formulation was incubated at RT before molding until a significant viscosity increase was observed. After a final homogenization the viscous formulation was molded.
  • Table 6 Pipetting scheme for CLP crosslinked with DMTMM or 4-Arm-PEG-SG (short: PEG-SG) by incorporating microparticles w/ or w/o drug load.
  • the molar ratio (MR) described the ratio of functional side chains in CLP compared to the functional side chains in the crosslinker.
  • a MR of 1 :0.2 was applied forthe CLP/PEG composite and a MR of 1 :1 was applied forthe DMTMM crosslinked CLP layer.
  • the applied 1 M HEPES buffer pH 8.0 was abbreviated with “buffer”. If the double layer patch was produced, microparticles were incorporated in one layer only but incorporated in both layers worked comparably well.
  • Example 7 Meloxicam release from incorporated microparticles
  • the patches were submersed in 1x PBS buffer and incubated in a 37 °C warm water bath on an orbital shaker (85 rpm) in 50 ml Greiner tubes.
  • the volume of extraction medium was chosen in alignment with the solubility limit of the encapsulated Meloxicam in water. The chosen volume ensures that all of the encapsulated Meloxicam can be dissolved during the trial to avoid drawback effects due to solubility limitations.
  • 1 ml samples was taken from the supernatant. The reduced volume was replenished by 1 ml fresh 1 xPBS and the sample was analyzed using a Tecan multiplate reader with an absorbance wavelength of 365 nm.
  • HFF human fibroblasts
  • Hydrogels were washed (3x 440 pl 1xPBS, 1x 440 pl full culture medium w/ FBS; 1 h each on an orbital shaker (300 rpm)). After washing, detached and washed cells were added (300 pl of a 1 x10 5 cells/ml suspension). The plate was incubated under culture conditions (37 °C, 5% CO2, humid atmosphere) for 24 h. Then, the cell morphology was visualized using a transmission light microscope and pictures were taken (see figure 4).

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Abstract

La présente invention concerne un timbre comprenant une couche de base d'hydrogel obtenue par réaction d'au moins une protéine de type collagène et d'au moins un agent de réticulation choisi parmi le chlorure de 4-(4,6-diméthoxy-1,3,5-triazin-2-yl)-4-méthyl-morpholinium (DMTMM), le glutaraldéhyde, la transglutaminase, le diisocyanate ou une combinaison de 1-éthyl-3-(3-diméthylaminopropyl)carbodiimide (EDC) et de N-hydroxysuccinimide (NHS) ; éventuellement une autre couche d'hydrogel obtenue par réaction d'au moins une protéine de type collagène et d'au moins un agent de réticulation comprenant au moins deux groupes comprenant chacun un fragment polyalkylène glycol et un groupe succinimidyle ; la couche de base d'hydrogel présentant une épaisseur au plus de 8 mm le timbre présentant une épaisseur au plus de 16 mm ainsi que sa méthode de fabrication. En outre, la présente invention concerne l'utilisation du timbre selon la présente invention pour la fermeture de plaies, l'administration de médicaments, l'application occlusive, la régénération tissulaire ou la formation d'une membrane barrière.
PCT/EP2024/064702 2023-06-06 2024-05-29 Timbre d'hydrogel fabriqué à partir d'une protéine de type collagène (clp) Pending WO2024251576A1 (fr)

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WO2016161345A1 (fr) 2015-04-02 2016-10-06 The Regents Of The University Of Michigan Polymères hyper-ramifiés et polyplexes et systèmes d'administration d'adn ou d'arn en comportant
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