Uses of jellyfish collagen

Abstract

Collagen compositions and methods of manufacture for use in treating wounds are provided. A composition for use in treating wounds is provided, the composition comprising jellyfish collagen, the jellyfish collagen being in the form of a micronized powder.

Description

[Technical Field]

[0001] The present invention relates to jellyfish collagen and its preparation for use in the treatment of wounds. [Background technology]

[0002] Wound healing is a complex process involving coordinated interactions between various immune and biological systems. Long-term wounds remain a challenging clinical problem, affecting approximately 6 million patients per year and with a significant economic impact.

[0003] Wound healing is the process by which skin (or other organ tissues) repair themselves after injury. In normal skin, the epidermis (outermost layer) and dermis (inner or deeper layer) are in a steady state of equilibrium and are insulated from the external environment. When the skin is broken, the normal (physiological) process of wound healing begins. The classical model of wound healing consists of three or four sequential and overlapping stages: Stage 1: Stop the bleeding. Stage 2: Inflammation. Stage 3: Proliferation. Phase 4: Reconstruction.

[0004] When skin is injured, a series of complex biochemical events occur in a tightly orchestrated cascade to repair the injury. Several potential stimuli (e.g., local tissue ischemia, bioburden, necrotic tissue, and repetitive trauma) can cause the wound to stall at the inflammatory stage, contributing to chronicity. One key component of chronic wounds is elevated levels of matrix metalloproteinases (MMPs). At elevated levels, MMPs degrade not only nonviable collagen but also viable collagen. In addition, fibroblasts in chronic wounds may not secrete tissue inhibitors of MMPs (TIMPs) at sufficient levels to control MMP activity. These events prevent the formation of the scaffolding necessary for cell migration and ultimately prevent the formation of extracellular matrix (ECM) and granulation tissue. In chronic wounds, including diabetic foot ulcers, the associated chronic microbial infection in combination with inflammation can lead to capillary degradation (reduced blood flow) and amputation. Therefore, new drugs targeting diabetic foot ulcers (DFUs) may advantageously offer the ability to stimulate blood flow to the affected area and promote wound healing.

[0005] Collagen-based wound dressings have been shown to be uniquely suited to address the problem of elevated MMP levels by acting as a "sacrificial matrix" in wounds.It has also been demonstrated that collagen degradation products are chemotactic for various cell types required for granulation tissue formation.In addition, collagen-based dressings have the ability to absorb exudate from wounds and maintain a moist wound environment.

[0006] Several different collagen dressings are available, using a variety of carriers / binders, such as gels, pastes, polymers, oxidized regenerated cellulose (ORC), and ethylenediaminetetraacetic acid (EDTA). The collagen contained in these products tends to be derived from bovine, porcine, equine, or avian sources that have been purified to render them non-antigenic. However, there are several drawbacks associated with the use of these types of collagen, such as poor angiogenic properties, increased risk of disease and viral transmission, and significant costs associated with obtaining these collagens.

[0007] Therefore, a source of collagen that would be suitable for use in treating wounds without exhibiting the above drawbacks would be particularly advantageous. Summary of the Invention

[0008] The present invention relates to jellyfish collagen for use in wound treatment. As evidenced by the in vivo and in vitro data presented below, the inventors have surprisingly found that compositions comprising jellyfish collagen are useful in wound treatment as an alternative to mammalian collagen (e.g., bovine), while providing comparable results and offering several superior properties, such as angiogenic properties, increased stability, reduced immunogenicity, and reduced risk of viral and disease / prion transmission. This is a completely unexpected finding, given the vastly different physicochemical and amino acid profiles of jellyfish collagen compared to mammalian collagen.

[0009] Thus, a first aspect of the present invention relates to a composition for use in the treatment of wounds, the composition comprising jellyfish collagen.

[0010] A second aspect of the present invention is a method for producing the aforementioned jellyfish collagen, comprising at least: i) extracting acid-soluble collagen from a jellyfish collagen source; ii) purifying the jellyfish collagen to provide a solution of purified jellyfish collagen. [Brief explanation of the drawings]

[0011] Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Figure 1] Shown is the change in group mean width (mm) from day 3 to day 7 as determined by caliper measurements. [Figure 2] The change in group mean width (mm) from day 3 to day 7, as determined by morphometry, is shown. [Figure 3] The change in group mean re-epithelialization rate (%) from day 3 to day 7 is shown. [Figure 4] The change in group mean granulation tissue formation score (FIG. 4A) and mean wound area granulation tissue formation rate (%) (FIG. 4B) from day 3 to day 7 is shown. [Figure 5] Representative images of H&E-stained wound sections from the Tegaderm-only group are shown. Panels A), B), and C) represent images of wound sections collected on day 3, and panels D), E), and f) represent images of wound sections collected on day 7. [Figure 6] Representative images of H&E-stained wound sections from the jellyfish collagen sponge group are shown. Panels A, B, and C represent images of wound sections collected on day 3, and panels D, E, and f represent images of wound sections collected on day 7. [Figure 7] Representative images of H&E-stained wound sections from the chemically modified (thiolated) jellyfish collagen paste group are shown. Panels A, B, and C show images of wound sections collected on day 3, and panels D, E, and f show images of wound sections collected on day 7. [Figure 8] Representative images of H&E-stained wound sections from the cross-linked jellyfish collagen sponge group are shown. Panels A, B, and C show images of wound sections collected on day 3, and panels D, E, and f show images of wound sections collected on day 7. [Figure 9]Representative images of H&E-stained wound sections from the Purocol® group are shown. Panels A), B), and C) represent images of wound sections collected on day 3, and panels D), E), and f) represent images of wound sections collected on day 7. [Figure 10] Representative images of collagen I-stained wound sections from different treatment groups are shown on days 3 and 7. Panel A) shows a wound section from the Tegaderm group on day 3, panel B) shows a wound section from the Tegaderm group on day 7, panel C) shows a wound section from the jellyfish collagen sponge group on day 3, panel D) shows a wound section from the jellyfish collagen sponge group on day 7, panel E) shows a wound section from the chemically modified (thiolated) jellyfish collagen paste group on day 3, panel F) shows a wound section from the chemically modified (thiolated) collagen paste group on day 7, panel G) shows a wound section from the cross-linked jellyfish collagen sponge group on day 3, panel H) shows a wound section from the cross-linked jellyfish collagen sponge group on day 7, panel I) shows a wound section from the Puracol® group on day 3, and panel L) shows a wound section from the Puracol® group on day 7. [Figure 11] Representative images of wound sections (day 3) from the Tegaderm only group labeled with the specific endothelial cell marker CD31 are shown. [Figure 12] Representative images of wound sections (day 7) from the Tegaderm only group labeled with the specific endothelial cell marker CD31 are shown. [Figure 13] Representative images of wound sections (day 3) from the jellyfish collagen sponge group labeled with the specific endothelial cell marker CD31 are shown. [Figure 14] Representative images of wound sections (day 7) from the jellyfish collagen sponge group labeled with the specific endothelial cell marker CD31 are shown. [Figure 15] Representative images of wound sections (day 3) from the chemically modified (thiolated) jellyfish collagen paste group labeled with the specific endothelial cell marker CD31 are shown. [Figure 16]Representative images of wound sections (day 7) from the chemically modified (thiolated) jellyfish collagen paste group labeled with the specific endothelial cell marker CD31 are shown. [Figure 17] Representative images of wound sections (day 3) from the cross-linked jellyfish collagen sponge group labeled with the specific endothelial cell marker CD31 are shown. [Figure 18] Representative images of wound sections (day 7) from the cross-linked jellyfish collagen sponge group labeled with the specific endothelial cell marker CD31 are shown. [Figure 19] Representative images of wound sections (day 3) from the Puracol® group labeled with the specific endothelial cell marker CD31 are shown. [Figure 20] Representative images of wound sections (day 7) from the Puracol® group labeled with the specific endothelial cell marker CD31 are shown. [Figure 21] Wound closure profiles of treatment groups in the db / db (BKS.CG-m Dock 7m+ / _+Leprdb / J) diabetic mouse model are shown as "% remaining wound area over time" data. These experiments were performed as described in Example 3, and the test groups were control (film dressing only), non-crosslinked sponge, 0.5% EDC crosslinked powder, 1.0% EDC crosslinked powder, and Promogran™. [Figure 22] The wound contraction profiles of the treatment groups in the db / db (BKS.CG-m Dock 7m+ / _+Leprdb / J) diabetic mouse model are shown as "% Wound Contraction" data. These experiments were performed as described in Example 3, and the test groups were control (film dressing only), non-crosslinked sponge, 0.5% EDC crosslinked powder, 1.0% EDC crosslinked powder, and Promogran™. [Figure 23]The wound re-epithelialization profile among different treatment groups in the db / db (BKS.CG-m Dock 7m+ / _+Leprdb / J) diabetic mouse model, which was first measurable on day 4 after wounding, is shown as "% wound re-epithelialization." These experiments were performed as described in Example 3, and the test groups were control (film dressing only), non-crosslinked sponge, 0.5% EDC crosslinked powder, 1.0% EDC crosslinked powder, and Promogran™. [Figure 24] Representative examples of the histological appearance of wounds from each experimental group in the db / db (BKS.CG-m Dock 7m+ / _+Leprdb / J) diabetic mouse model are shown (approximately to scale). Higher magnification views of the center of each wound can be found in Figure 25. [Figure 25] High magnification images of the central section of a representative example of the histological appearance of the wound displayed in Figure 24 are shown. [Figure 26] 1 shows the effect of 1% EDC cross-linked jellyfish collagen powder on (A) day 8, (B) day 12, and (C) day 16, showing the development of vascularization (V) of the hydrated mass. [Figure 27] Wound closure profiles for all treatment groups in the db / db (BKS.CG-m Dock 7m+ / _+Leprdb / J) diabetic mouse model are shown as "% remaining wound area over time" data. These experiments were performed as described in Example 4, and the test groups were control (film dressing only), 1.0% EDC crosslinked powder, thiolated powder, and Integra® Flowable Matrix. Results for all animals in the test groups are shown as mean ± standard error (n = 12 through day 35, n = 4 from days 42 to 63). [Figure 28] Wound closure profiles for all tested jellyfish collagen treatment groups in the db / db (BKS.CG-m Dock 7m+ / _+Leprdb / J) diabetic mouse model are shown as "% remaining wound area over time" data. These experiments were performed as described in Example 4. Results for all animals in the test group are shown as mean ± standard error (n=12 through day 35). [Figure 29]Wound closure profiles for thiolated powder- and Integra FM-treated groups in a db / db (BKS.CG-m Dock 7m+ / _+Leprdb / J) diabetic mouse model are shown as "% remaining wound area over time" data. These experiments were performed as described in Example 4. Results are shown as the mean ± standard error for all animals in the test group (n=12 through day 35). [Figure 30] Wound contraction profiles of treatment groups in the db / db (BKS.CG-m Dock 7m+ / _+Leprdb / J) diabetic mouse model are shown as "% Wound Contraction" data. These experiments were performed as described in Example 4, and the test groups were control (film dressing only), cross-linked sponge, 1.0% EDC cross-linked powder, thiolated powder, and Integra® Flowable Matrix. Results for all animals in the test groups are shown as mean ± standard error (n = 12 through day 35, n = 4 from days 42 to 63). [Figure 31] The wound contraction profiles of all tested jellyfish collagen treatment groups in the db / db (BKS.CG-m Dock 7m+ / _+Leprdb / J) diabetic mouse model are shown as "% Wound Contraction" data. These experiments were performed as described in Example 4. Results for all animals in the test group are shown as mean ± standard error (n=12 up to day 35). [Figure 32] The wound contraction profiles of the thiolated powder-treated and Integra FM-treated groups in the db / db (BKS.CG-m Dock 7m+ / _+Leprdb / J) diabetic mouse model are shown as "% Wound Contraction" data. These experiments were performed as described in Example 4. Results for all animals in the test group are shown as mean ± standard error (n=12 through day 35). [Figure 33]The wound re-epithelialization profile, first measurable on day 4 after wounding, among different treatment groups in the db / db (BKS.CG-m Dock 7m+ / _+Leprdb / J) diabetic mouse model is shown as "% wound re-epithelialization." These experiments were performed as described in Example 4, and the test groups were control (film dressing only), 1.0% EDC cross-linked powder, thiolated powder, and Integra® Flowable Matrix. Results for all animals in the test group are shown as mean ± standard error (n = 12 up to day 35, n = 4 from days 42 to 63). [Figure 34] The wound re-epithelialization profile of all tested jellyfish collagen treatment groups in the db / db (BKS.CG-m Dock 7m+ / _+Leprdb / J) diabetic mouse model, which was first measurable on day 4 after wounding, is shown as "% wound re-epithelialization" among the different treatment groups. These experiments were performed as described in Example 4. Results of all animals in the test group are shown as mean ± standard error (n=12 up to day 35). [Figure 35] The wound re-epithelialization profiles of the tested thiolated powder and Integra FM treatment groups in the db / db (BKS.CG-m Dock 7m+ / _+Leprdb / J) diabetic mouse model, which were first measurable on day 4 after wounding, are shown as "% wound re-epithelialization" between the different treatment groups. These experiments were performed as described in Example 4. Results for all animals in the test group are shown as the mean ± standard error (n=12 up to day 35). DETAILED DESCRIPTION OF THE INVENTION

[0012] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. Those skilled in the art will understand that embodiments of the present invention may be practiced without these specific details while remaining within the scope of the claims.

[0013] In a first aspect, the present invention provides a composition for use in the treatment of wounds, the composition comprising jellyfish collagen.

[0014] The phrase "wound treatment" or term "wound therapy" refers to any treatment that assists the complex process by which skin and any associated tissue repairs itself after injury. Examples of wounds that may benefit from the use of jellyfish collagen include, but are not limited to, pressure sores, transplant sites, surgical wounds, ulcers, burns (thermal, chemical, or electrical), lacerations, abrasions, puncture wounds, dehiscence wounds, seromas, and / or hematomas. In a preferred embodiment, the wound is not associated with epidermolysis bullosa.

[0015] In some embodiments, the jellyfish collagen is not in hydrolysate form, which includes collagen that has been broken down by heat or protease / collagenase activity to produce collagen fragments defined as collagen peptides and gelatin-like molecules.

[0016] Jellyfish collagen for use in wound treatment may be in its ateloform. "Ateloform" includes the meaning of a less immunogenic derivative of collagen obtained by removing the N- and C-terminal telopeptide components known to induce antigenicity in humans. The telopeptides are generally removed by treating the collagen with type I pepsin.

[0017] Jellyfish collagen for use in treating wounds may be in its teloform, which includes collagen extracted under acidic conditions to produce soluble collagen containing telopeptides.

[0018] Jellyfish collagen for use in treating wounds may be thiolated. The term "thiolated" is intended to refer to jellyfish collagen that has reacted with a thiol, resulting in the introduction of an -SH group or "thiol" group.

[0019] Jellyfish collagen for use in wound treatment may be cross-linked. In the context of the present invention, the term "cross-linking" refers to the bonding of two independent collagen molecules by a covalent bond. Preferably, the cross-linked collagen molecules are in the form of collagen fibrils, resulting in interfibrillar cross-links. To create cross-linked thiolated jellyfish collagen, a "cross-linking agent" or "cross-linker" may be used. The term "cross-linking agent" or "cross-linker" refers to an agent that can form a covalent bond between two independent molecules under certain conditions. In the context of the present invention, a cross-linking agent is used to covalently bond two independent collagen molecules. Preferably, the cross-linked collagen molecules are in the form of collagen fibrils. Preferably, interfibrillar cross-links occur. In some cases, the cross-linker is typically composed of two or more reactive functional groups linked together by a hydrocarbon chain. The two or more functional groups do not necessarily have to be the same. The length of the hydrocarbon chain can also be varied to control the distance between the functional groups. The exact length of the hydrocarbon chain in the context of the present invention is not intended to be limiting.

[0020] Jellyfish collagen for use in treating wounds may be non-crosslinked.

[0021] The jellyfish collagen source may be from the subphylum Scyphozoa. The jellyfish collagen source for use in treating wounds may be selected from the group consisting of the order Rhizostomeae (including, but not limited to, Rhizostomas pulmo), Rhopilema esculentum, Rhopilema nomadica, Stomolophus meleagris, Cassiopea species (upside-down jellyfish) (including, but not limited to, Cassiopea andromeda), the order Semaeostomeae (including Aurelia species), and other species, such as Nemopilema nomurai, Rhopilema esculentum, Rhopilema nomadica, Stomolophus meleagris, or any combination thereof. Preferably, the jellyfish collagen source is Rhizostomas pulmo. Thus, the collagen may comprise at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Rhizostomas pulmo collagen.

[0022] Jellyfish collagen for use in treating wounds may have a concentration of at least 1 mg / mL. It is envisioned that the maximum concentration of jellyfish collagen that can be used is 50 mg / mL. Thus, concentrations of jellyfish collagen may range from 1 mg / mL to 50 mg / mL, 2 mg / mL to 50 mg / mL, 3 mg / mL to 50 mg / mL, 4 mg / mL to 50 mg / mL, 5 mg / mL to 50 mg / mL, 6 mg / mL to 50 mg / mL, 7 mg / mL to 50 mg / mL, 8 mg / mL to 50 mg / mL, 9 mg / mL to 50 mg / mL, 10 mg / mL to 50 mg / mL, 11 mg / mL to 50 mg / mL, 12 mg / mL to 50 mg / mL, 13 mg / mL to 50 mg / mL, 14 mg / mL to 50 mg / mL, 15 mg / mL to 50 mg / mL, 16 mg / mL to 50 mg / mL, 17 mg / mL to 50 mg / mL, 18 mg / mL to 50 mg / mL, 19 mg / mL to 50 mg / mL, 20 mg / mL to 50 mg / mL, 21 mg / mL to 50 mg / mL, 22 mg / mL to 50 mg / mL, 23 mg / mL to 50 mg / mL, 24 mg / mL to 50 mg / mL, 25 mg / mL to 50 mg / mL, 26 mg / mL to 50 mg / mL, 27 mg / mL to 50 mg / mL, 28 mg / mL to 50 mg / mL, 29 mg / mL to 50 mg / mL, 30 mg / mL to 50 mg / mL, 31 mg / mL to 50 mg / mL, 32 mg / mL to 50 mg / mL, 33 mg / mL to 5 L~50mg / mL, 14mg / mL~50mg / mL, 15mg / mL~50mg / mL, 16mg / mL~50mg / mL, 17mg / mL~50mg / mL, 18mg / mL~50mg / mL, 19mg / mL~50mg / m L, 20 mg / mL to 50 mg / mL, 25 mg / mL to 50 mg / mL, 30 mg / mL to 50 mg / mL, 35 mg / mL to 50 mg / mL, 40 mg / mL to 50 mg / mL, or 45 mg / mL to 50 mg / mL.

[0023] Jellyfish collagen for use in treating wounds can be stable at temperatures up to at least 37°C. The term "stable" is intended to refer to the ability of jellyfish collagen to remain substantially denatured under given environmental conditions and maintain its desirable properties. This is an advantageous property given that the intended use of the invention involves physical contact between the product and the subject. While it is envisioned that the subject is a human, the invention can also be utilized in the veterinary industry for use in treating wounds in, for example, dogs, cats, horses, cows, goats, sheep, etc.

[0024] Jellyfish collagen for use in treating wounds may be in the form of a hydrogel, paste, powder, preferably a micronized powder, membrane, scaffold, solution, sponge matrix, nanofiber electrospun matrix, or in lyophilized form.

[0025] A "hydrogel" is a network of hydrophilic polymer chains, resulting in a highly absorbent material. The term "paste" is intended to refer to a semi-solid preparation, usually intended for external application to the skin. Typically, when used in a pharmaceutical context, they consist of a fatty base (e.g., petrolatum) and are at least 25% solid material (e.g., zinc oxide). Those skilled in the art will recognize that the form of jellyfish collagen selected may depend on the particular wound being treated. For example, burns may require a hydrogel or fine collagen mesh formulation.

[0026] The composition for use according to the present invention may contain additional pharmaceutically active ingredients. Examples of additional pharmaceutically active ingredients include growth factors, anti-inflammatory agents, and antibacterial agents. Examples of anti-inflammatory agents include nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin salsalate, diflunisal, ibuprofen, ketoprofen, nabumetone, piroxicam, naproxen, diclofenac, indomethacin, and sulindac. It is understood that the concentration of the selected anti-inflammatory agent will depend on the type and severity of the wound being treated. Examples of antibacterial agents that may be used include, but are not limited to, nanosilver, penicillin, ofloxacin, tetracycline, aminoglycosides, and erythromycin. Mixtures of two or more of the aforementioned pharmaceutically active ingredients, with or without the excipients and carriers listed above, are contemplated.

[0027] In some embodiments, the compositions for use according to the invention further comprise at least one growth factor. In preferred embodiments, the at least one growth factor is platelet-rich plasma (PRP), epidermal growth factor 38 (EGF), transforming growth factor-beta (TGF-B, TGF-B2, TGF-B3), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), granulocyte-monocyte colony-stimulating growth factor, platelet-derived growth factor, insulin-like growth factor 1 (IGF1), basic fibroblast growth factor (bFGF), and / or vascular endothelial growth factor 5 (VEGF), or any combination thereof.

[0028] Compositions for use according to the invention may further comprise at least one antibacterial compound. Preferably, the at least one antibacterial compound is nanocilliver, penicillin, ofloxacin, tetracycline, aminoglycosides and erythromycin, flucloxacillin, clarithromycin, doxycycline, gentamicin, metronidazole, co-amoxiclav, cotrimoxazole (in penicillin), ceftriaxone, piperacillin / tazobactam, clindamycin, ciprofloxacin, vancomycin, teicoplanin, linezolid, and / or standard of care antibacterial agents, or any combination thereof.

[0029] Compositions for use in treating wounds may be formulated for topical application to the wound. The term "topical application" in the context of the present invention is intended to refer to the application of jellyfish collagen to the specific site of the wound to be treated. The wound to be treated may be on the skin of a subject or on a mucous membrane of a subject, for example, inside the mouth. Compositions for use according to the present invention may also be formulated for administration by any other route known in the art. For example, compositions for use according to the present invention may be formulated for administration by negative pressure wound therapy (NPWT), which involves the controlled application of subatmospheric pressure to the local wound environment using an occlusive wound dressing connected to a vacuum pump (also known as vacuum-assisted closure (VAC)).

[0030] In some embodiments, compositions for use according to the present invention comprise collagen in a dose of 0.01 g / L to 200 g / L, preferably 1 g / L to 50 g / L per dose.

[0031] Compositions for use according to the invention may be formulated as creams, bigels, ointments, masks, serums, emulsions, lotions, pastes, foams, aerosols, sticks, shampoos, conditioners, patches, hydroalcoholic or oily aqueous solutions, oil-in-water or water-in-oil or multiple emulsions, aqueous or oily gels, liquids, pasty, or solid anhydrous products, electrospun collagen nanofiber matrices, membranes, and / or oil dispersions in an aqueous phase using microspheres, which microspheres are polymeric nanoparticles such as nanospheres and nanocapsules or lipid vesicles of ionic and / or non-ionic type, more preferably electrospun collagen nanofiber matrices and / or membranes.

[0032] Compositions comprising jellyfish collagen for use according to the present invention may further comprise pharmaceutically acceptable excipients and / or carriers and / or pharmaceutically active ingredients. The excipients and carriers may increase the stability of the pharmaceutically active ingredient or jellyfish collagen and / or improve its biopharmaceutical profile, and may have an active substance bound thereto or not. Examples of suitable pharmaceutically acceptable excipients and carriers may include sterile water, olive oil, ethyl oleate, monosaccharides such as fructose, glucose, and galactose, non-reducing disaccharides such as sucrose, lactose, trehalose, non-reducing oligosaccharides such as raffinose and melezitose, non-reduced starch-derived polysaccharide products such as maltodextrin, dextran, and cyclodextrin, and non-reducing alditols such as mannitol and xylitol. Further suitable excipients include cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, and / or polyvinylpyrrolidone.Mixtures of any two or more of the above excipients or carriers (or any other suitable equivalents) are also contemplated.It is understood that other substances with similar effects are also suitable.

[0033] In a preferred embodiment, the pharmaceutically active ingredient may be 1% lidocaine. Lidocaine, or lidocaine hydrochloride, is an anesthetic commonly used as a numbing agent. The lidocaine may be present in concentrations up to 5%, such as 0.1%-5% lidocaine, 0.5%-5% lidocaine, 1%-5% lidocaine, 1.5%-5% lidocaine, 2%-5% lidocaine, 2.5%-5% lidocaine, 3%-5% lidocaine, 3.5%-5% lidocaine, 4%-5% lidocaine, or 4.5%-5% lidocaine. Preferably, the lidocaine is present in a concentration of 0.1-2%. Examples of additional anesthetics suitable for the same purpose include, but are not limited to, benzocaine, butamben, dibucaine, lidocaine, oxybuprocaine, pramoxine, proparacaine, proxymetacaine, and tetracaine.

[0034] The composition for use according to the present invention may be used to treat a wound selected from the list consisting of a pressure sore, a transplant site, a surgical wound, an ulcer, preferably a diabetic ulcer, a thermal wound, a chemical burn, an electrical burn, a laceration, an abrasion, a puncture wound, an avulsion wound, a seroma, and / or a hematoma.

[0035] It is envisaged that the present invention may form at least a part of a wound dressing, with or without the further presence of the aforementioned medicinal agents. The wound dressing may include additional components such as alginates and cellulose derivatives, which may enhance absorbency, softness and comfort, and help to maintain an environment conducive to healing.

[0036] In one embodiment, a composition for use according to the invention may comprise jellyfish collagen in the form of a micronised powder, preferably having a particle size of between 1 μm and 1000 μm, more preferably between 200 μm and 500 μm.

[0037] In certain embodiments, compositions for use according to the invention may comprise jellyfish collagen in the form of a collagen three-dimensional sponge scaffold.

[0038] In some embodiments, the compositions for use according to the invention promote improved vascularization in treated wounds, preferably compared to untreated wounds and / or wounds treated with bovine collagen.

[0039] In a second aspect of the present invention, there is provided a method for producing the aforementioned jellyfish collagen, comprising at least: i) extracting acid-soluble collagen from a jellyfish collagen source; ii) purifying the jellyfish collagen to provide a solution of purified jellyfish collagen.

[0040] A "solution of purified jellyfish collagen" refers to a solution of isolated jellyfish collagen that is substantially monomeric or substantially free of collagen fibrils. In this context, "substantially free" refers to a solution of collagen in which less than 2% by weight of the collagen is composed of fibrils. To maintain the collagen solution under these conditions, the isolated collagen can be stored under conditions that disfavor collagen fibril formation. This can mean that the collagen is stored under acidic conditions, where acidic means any solution having a pH between pH 1 and pH 6.5, or it can mean that the collagen is stored under basic conditions, where basic means any solution having a pH between pH 8 and pH 14. As a non-limiting example, collagen can be stored in a 0.1 M weak acid solution. The weak acid can be acetic acid or hydrochloric acid. The concentration of collagen in the collagen solution can be in the range of 0.1 mg / ml to 30 mg / ml. Preferably, the concentration of the collagen solution is between 1 mg / ml and 10 mg / ml.

[0041] There are multiple methods for "isolating" or "purifying" jellyfish collagen from its anatomical environment. Many of these are well known and routine to those skilled in the art. For example, collagen can be purified from jellyfish by acid extraction, whereby various anatomical parts of the jellyfish are immersed in an acidic solution. "Soaking" or "soaked" refers to the process of incubating the jellyfish in an acidic solution for a sufficient time to liberate the collagen molecules. An alternative method of collagen purification is enzymatic extraction, whereby the jellyfish is incubated with at least one proteolytic enzyme for a sufficient time under conditions that favor the degradation of the anatomical environment to liberate the collagen molecules. The exact temperature, pH, and incubation time for the enzymatic extraction method will vary depending on the proteolytic enzyme used. The most suitable conditions are well known to those skilled in the art. As a non-limiting example, the enzyme pepsin can be incubated with the jellyfish under acidic conditions to liberate the collagen molecules. It is contemplated that any enzyme may be used in the enzymatic extraction method, and the above examples are not intended to be limiting in any way.

[0042] The collagen can then be further isolated or purified from undesirable contaminants from the acid or enzyme extraction process by many different means. For example, insoluble contaminants can be removed by centrifugation. If a purer source of collagen is required, the isolated collagen can be subjected to gel filtration or alternative chromatographic methods that allow for purification of collagen molecules from other soluble contaminants from the extraction process. The exact method of further purification is not particularly limited. Any method well known and routinely used by protein biochemists can be adapted for the purpose of obtaining purified or isolated jellyfish collagen. This step may allow for the transfer of jellyfish collagen to the desired storage buffer to obtain the desired solution of purified jellyfish collagen. This can be achieved by first equilibrating the chromatography equipment with the desired storage buffer prior to purification. There are many well-known alternative methods that can be used for this purpose. Preferably, the collagen solution used in the present invention is 70% to 99% pure, where pure refers to the weight percent of the solution attributable to collagen molecules. More preferably, the collagen solution is at least 95%, 96%, 97%, 98%, or 99% pure.

[0043] In some embodiments, the method of manufacturing comprises: iii) adding a cross-linking agent to form cross-linked jellyfish collagen.

[0044] In some embodiments, the crosslinking agent is EDC, genipin, or polyethylene glycol (PEG). Preferably, the crosslinking agent is EDC. EDC can be present at a concentration of 0.01% to 5%, 0.05% to 5%, 0.1% to 5%, 0.2% to 5%, 0.3% to 5%, 0.4% to 5%, 0.5% to 5%, 0.6% to 5%, 0.7% to 5%, 0.8% to 5%, 0.9% to 5%, 1% to 5%, 1.5% to 5%, 2% to 5%, 3% to 5%, 3.5% to 5%, 4% to 5%, or 4.5% to 5%. Preferably, the concentration of EDC is 0.5% to 1%.

[0045] In some embodiments, the method of producing jellyfish collagen further comprises digesting the extracted jellyfish collagen with a peptidase to provide Ateromedusa collagen. Preferably, in the step of digesting the collagen with a peptidase, the peptidase is pepsin. The pepsin can be of mammalian origin, non-mammalian origin (e.g., papain), or microbial origin. In some embodiments, the step of digesting the collagen with a peptidase occurs after the extraction step and before the purification step. The method for producing jellyfish collagen described above may further comprise the steps of: i) providing jellyfish collagen containing S-S bonds; and ii) introducing -SH groups into the jellyfish collagen containing S-S bonds by reducing the S-S bonds to provide a collagen thiol containing -SH groups.

[0046] For jellyfish collagen to contain -SH groups, it must first contain S-S bonds or "disulfide" groups. Naturally occurring collagen typically does not contain S-S bonds. Disulfide groups can be incorporated into collagen by several routes. Jellyfish collagen containing S-S bonds can be obtained from jellyfish collagen containing one or both of lysine and hydroxylysine residues. Collagen takes the form of a triple helix of polypeptide chains, one or more of which generally contain one or both of lysine and hydroxylysine residues. Lysine and hydroxylysine are α-amino acids with an ε-amino group. As used herein, the term "residue" refers to the portion of a compound that remains after incorporation into another substance, for example, by chemical reaction and bond formation. Thus, an amino acid "residue" refers to the polymerized form of an amino acid monomer present in a polypeptide. Hydroxylysine residues, although four to five times less abundant in collagen than lysine residues, are also present in sufficient amounts for use in the methods disclosed herein. Thus, collagen containing lysine or hydroxylysine residues may contain a residue of formula (XI) prior to the treatment described herein. [ka] In the formula, R 5 is H or OH. 5 When R is H, a lysine residue is present. 5 is OH, a hydroxylysine residue is present. X is selected from an OH group and a chemical bond, and Y is selected from H and a chemical bond, provided that one or both of X and Y are chemical bonds that form peptide bonds within the collagen. The peptide chains that form part of the modified collagen are shown in formula (XI) enclosed in brackets "[ ]". The residue can be in a terminal position of the peptide, for example, when one or the other of X and Y is OH and H, respectively. When both X and Y are peptide bonds, the lysine residue is non-terminal within the peptide chain that forms part of the collagen.

[0047] The jellyfish collagen containing one or both of lysine and hydroxylysine residues is preferably solubilized jellyfish collagen containing one or both of lysine and hydroxylysine. Solubilization can be achieved by pepsin digestion or acid digestion to provide pepsin-solubilized jellyfish collagen containing one or both of lysine and hydroxylysine, or by acid digestion to provide acid-solubilized jellyfish collagen containing one or both of lysine and hydroxylysine.

[0048] Jellyfish collagen, such as pepsin-solubilized or acid-solubilized jellyfish collagen containing one or both of lysine and hydroxylysine residues, can be reacted with an activated dicarboxylic acid derivative containing a disulfide (i.e., S-S) group to provide collagen containing S-S bonds, in which the carbonyl group of the activated dicarboxylic acid derivative can react with the e-amino group of a lysine or hydroxylysine residue present in the jellyfish collagen to form an amide bond.

[0049] The activated dicarboxylic acid derivative is preferably a compound of the formula: ZN-C(O)-R 3-C(O)-NH-R 1 -SSR 2 -NH-C(O)-R 3 -C(O)-NZ (I) In the formula, R 1 , R 2 , and R 3 are independently a divalent linking group, preferably a divalent organic linking group, more preferably a divalent hydrocarbon linking group, for example, an alkanediyl group having 1 to 6 carbon atoms or an alkenediyl or alkynediyl group having 2 to 6 carbon atoms. Even more preferably, R 1 , R 2 , and R 3 R independently represents an ethanediyl group or a propanediyl group, and most preferably represents ethanediyl, i.e., -CHCH-. 1 Group, R 2 groups, and R 3 The groups may be optionally substituted, independently, by replacing 1 to 4 hydrogen atoms with hydroxyl groups or halogens, such as F or Cl. In a preferred embodiment, R 1 and R 2 are identical, and ZN together represent a nitrogen-containing heterocyclic group, preferably a nitrogen-containing heterocyclic group having 5 to 6 atoms in the heterocycle, where the N atom is directly attached to the carbonyl group of the compound of formula (I) such that Z represents a divalent linking group with two valencies attached to the nitrogen. The heterocyclic group can be saturated or unsaturated, such that Z can represent an alkanediyl, alkenediyl, or alkynediyl, particularly having 2 to 4 carbon atoms. Z can optionally contain one or two heteroatoms selected from O, S, and N.

[0050] More preferably, ZN is a nitrogen-containing heteroaryl group having 5-6 atoms in the aryl ring, 1-3 of which are heteroatoms selected from O, N, and S, at least one of which is N directly bonded to the carbonyl group of the compound of formula (I). Even more preferably, the heteroaryl group has 5-6 atoms in the aryl ring, 2 of which are N. Alternatively, ZN is a nitrogen-containing heterocyclic group having 5-6 atoms in the heterocycle with one N atom directly bonded to the carbonyl group of the compound of formula (I), and Z is α,ω-organodionediyl. For example, α,ω-organodionediyl is —C(O)R 4 C(O)—, where R 4 is an alkanediyl or alkenediyl having 2 or 3 carbon atoms.

[0051] The ZN group may be optionally substituted by replacing one to four hydrogen atoms with hydroxyl groups or halogens, such as F, Br, or Cl, or by replacing two hydrogen atoms attached to the same carbon with oxygen atoms to form a carbonyl group, the latter substitution may occur once or twice.

[0052] Most preferably, the ZN group is 1-imidazole, i.e., [ka] or 1-pyrrolidine-2,5-dione, i.e. [ka] is. Preferred activated dicarboxylic acid derivatives are [ka] may be selected from:

[0053] The activated dicarboxylic acid derivative (I) can be synthesized in two steps: First, a diaminodisulfide of formula (IV) can be reacted with at least two molar equivalents of a dicarboxylic acid anhydride of formula (V) to give a dicarboxylic acid diamide of formula (VI). H2N-R 1 -SSR 2 -NH2(IV)+ [ka] HO-C(O)-R 3 -C(O)-NH-R 1 -SSR 2 -NH-C(O)-R 3 -C(O)-OH (VI) In the formula, R 1 , R 2 , and R 3 is as defined above. R 1 and R 2 are identical, it will be apparent that the diaminodisulfide of formula (IV) is a symmetrical molecule, which leads to the symmetrical activated dicarboxylic acid derivative (I).

[0054] The first reaction step can be carried out by dissolving the diaminodisulfide of formula (IV) in a solvent such as water and adding the dicarboxylic acid anhydride of formula (V). It is preferable to carry out the reaction under basic conditions so that a base can be added before the addition of the acid anhydride. For example, aqueous sodium hydroxide can be added to adjust the pH to 10. The pH may decrease after the addition of the dicarboxylic acid anhydride, so it is preferable to maintain the pH within the range of 7 to 10 during the reaction by adding additional base. This reaction can be carried out at room temperature with stirring and can be completed within 30 minutes to 2 hours. The dicarboxylic acid diamide of formula (VI) The product can be precipitated by adding an acid, such as aqueous hydrochloric acid, to lower the pH, for example, to pH 1. The precipitated dicarboxylic acid diamide (VI) can be isolated by filtration, washed with water, and then dried under reduced pressure.

[0055] In the second step of the synthesis, the dicarboxylic acid diamide (VI) is activated by the addition of a nitrogen-containing heterocyclic compound to provide the activated dicarboxylic acid derivative (I). HO-C(O)-R 3 -C(O)-NH-R 1 -SSR 2 -NH-C(O)-R 3 -C(O)-OH (VI)→ ZN-C(O)-R 3 -C(O)-NH-R 1 -SSR 2 -NH-C(O)-R 3 -C(O)-NZ (I) In the formula, R 1 , R 2 , R 3 , and NZ are as defined above.

[0056] In one embodiment, the nitrogen-containing heterocyclic compound can be a carbodiimide, such as a compound of formula (VII): [ka] wherein Z is as defined above. Preferably, Z and N taken together are a nitrogen-containing heteroaryl group having 5 to 6 atoms in the aryl ring, 1 to 3 of which are heteroatoms selected from O, N, and S, at least one of which is N. More preferably, carbodiimide (VII) is 1,1'-carbonyl-diimidazole or the like.

[0057] At least 2 molar equivalents of carbodiimide should be used per mole of dicarboxylic acid diamide (VI). Theoretically, the reaction produces 2 molar equivalents of carbon dioxide and 2 molar equivalents of imidazole per mole of dicarboxylic acid diamide (VI). The evolution of carbon dioxide gas indicates that the reaction is proceeding. The reaction can be carried out under reduced pressure.

[0058] In another embodiment, the nitrogen-containing heterocyclic compound can be an N-hydroxy heterocyclic compound of formula (VIII). [ka] where Z and N together are a nitrogen-containing heterocyclic group having 5 to 6 atoms in the heterocyclic ring, one of which is N, and Z is α,ω-organodionediyl. For example, α,ω-organodionediyl is —C(O)R 4 C(O)—, where R 4 is an alkanediyl or alkenediyl having 2 or 3 carbon atoms. The N-hydroxyheterocyclic compound (VIII) is most preferably N-hydroxysuccinimide.

[0059] The second reaction step can be carried out by dissolving the dicarboxylic acid diamide (VI) in a solvent such as anhydrous dimethylformamide, followed by the addition of the nitrogen-containing heterocyclic compound (VII) or (VIII). The activated dicarboxylic acid derivative (I) precipitates from the solution. The product can be collected by filtration, washed with anhydrous ethyl acetate, and dried under reduced pressure.

[0060] Returning to the first step of the method of the present invention, a source collagen containing one or both of lysine and hydroxylysine residues can be reacted with an activated dicarboxylic acid derivative containing a disulfide group, such as the activated dicarboxylic acid derivative of formula (I), to provide a collagen containing an S-S bond, e.g., one or more collagens of types I, II, III, IV, V, VI, IX, X, and XI containing an S-S bond. In this reaction, the carbonyl group of the activated dicarboxylic acid derivative reacts with the e-amino group of a lysine or hydroxylysine residue present in the collagen to form an amide bond, thereby incorporating a disulfide group. The reaction can be represented as follows: ZN-C(O)-R 3 -C(O)-NH-R 1 -SSR 2 -NH-C(O)-R 3 -C(O)-NZ (I) + collagen-NH2 → ZN-C(O)-R 3 -C(O)-NH-R 1-SSR 2 -NH-C(O)-R 3 -C(O)-NH-collagen + HNZ This reaction can continue to provide cross-linked collagen when both activated carboxyl groups of the activated dicarboxylic acid derivative react with collagen, particularly different collagen triple helices or fibrils. ZN-C(O)-R 3 -C(O)-NH-R 1 -SSR 2 -NH-C(O)-R 3 -C(O)-NH-collagen + collagen-NH2 → Collagen-HN-C(O)-R 3 -C(O)-NH-R 1 -SSR 2 -NH-C(O)-R 3 -C(O)-NH-collagen + HNZ The reaction can be carried out by dissolving the source collagen in a solvent. Dissolving the source collagen can be carried out in a two-step process. In the first step, the source collagen can be mixed with methanol. In the second step, a polar aprotic solvent is added. For example, the source collagen can be added to a mixture of methanol and dimethyl sulfoxide to allow it to swell. Additional dimethyl sulfoxide can be added with stirring until dissolution of the source collagen is complete. The methanol can then be removed from the solution by evaporation under reduced pressure. This solubilization process can be used for both atelocollagen and telocollagen.

[0061] The activated dicarboxylic acid derivative can then be dissolved in a solvent, particularly an anhydrous polar aprotic solvent such as dimethyl sulfoxide. Because the activated dicarboxylic acid derivative of formula (I) is sensitive to water, the reaction with collagen is preferably carried out in an anhydrous polar aprotic solvent such as dimethyl sulfoxide. The activated dicarboxylic acid derivative dissolved in the solvent is then added to the source collagen solution. The carbonyl group of the activated dicarboxylic acid derivative can react with the e-amino group of a lysine or hydroxylysine residue present in the source collagen to form an amide bond.

[0062] The mixture can be stirred at room temperature, e.g., 22°C, until a gel forms. The gel-containing mixture can then be allowed to stand, e.g., for 12 to 18 hours. Dimethyl sulfoxide can then be extracted from the gel by blending with excess acetone, collecting the collagen gel by decantation, and then reblending with additional acetone. The mixture can then be stirred, e.g., for 0.5 to 1 hour, followed by isolating the collagen by filtration, washing with acetone, then washing with water-ethanol (30:70 v / v), and dehydrating with ethanol. This provides jellyfish collagen containing S-S bonds.

[0063] In another embodiment, jellyfish collagen containing S-S bonds can be provided by modifying a collagen-binding protein to include a photoreactive crosslinker containing disulfide groups, combining it with jellyfish collagen to provide a complex, and irradiating the complex to crosslink the photoreactive crosslinker and incorporate disulfide groups into the collagen.

[0064] This route creates protein-binding sites in jellyfish collagen by using a site-specific photocrosslinking strategy that allows for the creation of thiol groups in collagen. This involves introducing cysteine ​​residues into collagen-binding proteins by site-directed mutagenesis. A photoreactive crosslinker, preferably APDP, can be introduced to the protein's cysteine ​​-SH groups. The APDP-modified protein-collagen complex can be crosslinked by ultraviolet (UV) irradiation. The disulfide bridges are then cleaved by reduction, generating -SH groups in jellyfish collagen.

[0065] In the first step, a collagen-binding protein containing a cysteine ​​residue is provided. The cysteine ​​residue is necessary because it contains a sulfhydryl group necessary for reaction with a crosslinker. The collagen-binding protein can be, for example, pigment epithelium-derived factor (PEDF). PEDF is a known antiangiogenic / neurotrophic factor with a collagen-binding site identified by Yasui et al. as disclosed in Biochemistry, 2003, 42 (pp. 3160-3167).

[0066] If necessary, cysteines can be incorporated into the collagen-binding protein if they are not already present or if the collagen-binding protein does not contain enough cysteines. Cysteine ​​substitutions can be made by site-directed mutagenesis, for example, at the site where the collagen-binding site is located (F383) and on the opposite surface of the site (Y211). Methods for performing such site-directed mutagenesis can be found in JDJ Biol. Chem. 2002, 277, 4223-4231 and RRBiochemistry 1992, 31, 9526-9532.

[0067] The sulfhydryl groups introduced as a result of the cysteine ​​substitution can then be reacted with a photoreactive crosslinker. The photoreactive crosslinker must be bifunctional. Specifically, the photoreactive crosslinker must contain a functional group capable of reacting with the sulfhydryl groups to form disulfide bonds. One such suitable functional group is a pyridyl-dithio group, i.e., C5NH5-SS-, particularly 2-pyridyldithio. The photoreactive crosslinker must also contain a functional group capable of crosslinking with collagen under light irradiation. One such suitable functional group is an azide group, particularly an aryl azide group, such as phenyl azide, particularly para-C6H4-N3.

[0068] N-[4-(p-azidosalicylamido)butyl]-3'-(2'pyridyldithio)propionamide (APDP) is a preferred photoreactive crosslinker. The disulfide group of APDP reacts with the sulfhydryl group of cysteine ​​to form a disulfide bond between the cysteine ​​and the photoreactive crosslinker, thereby providing a collagen-binding protein modified with a photoreactive crosslinker containing an S-S group. 2-Pyridylthione is liberated as a leaving group as part of this reaction.

[0069] The photoreactive crosslinker-modified collagen-binding protein can then be combined with jellyfish collagen to provide a complex of the photoreactive crosslinker-modified collagen-binding protein and jellyfish collagen. In this step, the photoreactive crosslinker-modified collagen-binding protein binds to the protein-binding site of the jellyfish collagen, forming a complex. The photoreactive crosslinker-modified collagen-binding protein and collagen complex can then be irradiated, for example, with ultraviolet (UV) light. Upon irradiation, the crosslinkable functional groups present on the photoreactive crosslinker form covalent bonds with adjacent collagens in the complex. For example, if the crosslinkable functional group is a phenyl azide, irradiation with a wavelength in the range of 250-280 nm generates a nitrene, which can then attack nucleophilic or active hydrogen groups, such as C-H or C-NH, on the collagen, generating crosslinks by insertion across C-H or N-H bonds. In this way, the photoreactive crosslinker-modified collagen-binding protein containing S-S groups is incorporated into collagen, providing collagen containing S-S bonds. It should be apparent that this route provides disulfide groups near the collagen protein-binding site.

[0070] Sulfhydryl groups can then be introduced into the collagen containing S-S bonds, which can be provided by either of the methods described above, i.e., using either the activated dicarboxylic acid derivative or photoreactive crosslinker methods. The jellyfish collagen containing S-S bonds can then be reacted with a suitable reducing agent. The reducing agent reduces the disulfide bond to two sulfhydryl groups, thereby cleaving the activated dicarboxylic acid derivative residue or photoreactive crosslinker residue on which the disulfide group is located. This reduction proceeds via two sequential thiol-disulfide exchange reactions, which reduce the disulfide groups and generate jellyfish collagen containing sulfhydryl (-SH) groups.

[0071] Suitable reducing agents include, for example, dithiothreitol (DTT), (2S)-2-amino-1,4-dimercaptobutane (DTBA), and tris(2-carboxyethyl)phosphine HCl (TCEP hydrochloride). Dithiothreitol is a preferred reducing agent.

[0072] The reduction step can be carried out by adding jellyfish collagen containing S--S bonds to a buffer solution such as a glycine / sodium hydroxide buffer at a pH in the range of 7.5 to 9.5, more preferably about 8 to 9.5, preferably 8.0. Jellyfish collagen containing S--S bonds may be acidic in nature, and if so, neutralization with a base such as sodium hydroxide may be required.

[0073] Preferably, at least 2 molar equivalents of DTT reducing agent per mole of disulfide group are added to the same buffer, and the reaction is allowed to proceed for 2-6 hours at 30°C. After the reaction is complete, the pH of the solution can be lowered to 2, for example, using HCl. The mixture is then dialyzed against a dilute HCl solution, centrifuged, and lyophilized to obtain jellyfish collagen thiols with -SH groups.

[0074] Reduction can cause slight degradation of the collagen chains, and as a result, shorter reaction times, lower pH, and lower temperatures can all be used to minimize any degradation.

[0075] If jellyfish collagen containing S-S bonds is prepared using a collagen-binding protein modified with a photoreactive crosslinker, it will be clear that the modified collagen-binding protein is still bound to the collagen via the photoreactive crosslinker prior to the step of forming collagen thiols. Reduction of the S-S bond will cleave the S-S bond of the photoreactive crosslinker.

[0076] When the jellyfish collagen contains one or both of lysine and hydroxylysine residues, there is provided a jellyfish collagen thiol containing a lysine or hydroxylysine residue of formula (X). [ka] In the formula, R 1 , R 3 , and R 5 is as defined above. For example, R 1 and R 3 are independently selected from divalent linking groups, preferably divalent organic linking groups, more preferably divalent hydrocarbon linking groups, such as alkanediyl groups having 1 to 6 carbon atoms or alkenediyl or alkynediyl groups having 2 to 6 carbon atoms. Even more preferably, R 1 and R 3 independently represent an ethanediyl group or a propanediyl group, and most preferably represent ethanediyl (i.e., -CH2CH2-). When the amino acid residue is a lysine residue, R 5 is H. If the amino acid residue is a hydroxylysine residue, R 5 is OH, X is selected from an OH group and a chemical bond, and Y is selected from H and a chemical bond, provided that one or both of X and Y are chemical bonds that form peptide bonds within the collagen. Peptide chains that form part of the modified collagen are shown in formula (X) enclosed in brackets "[ ]". The residue can be in a terminal position of the peptide, for example, when one or the other of X and Y is OH and H, respectively. When both X and Y are peptide bonds, the lysine residue is non-terminal within the peptide chain that forms part of the collagen.

[0077] The present invention further provides a method of producing the jellyfish collagen disclosed herein, which may or may not be thiolated, further comprising the steps of: i) mixing a solution of purified jellyfish collagen or collagen thiol with an aqueous neutralization buffer; and ii) incubating the mixture for a time sufficient for collagen fibrils to form, wherein a cross-linking agent is added in either step i) or step ii) to provide cross-linked collagen.

[0078] The term "neutralization buffer" refers to any buffer capable of diluting a solution of purified jellyfish collagen to increase or decrease its pH between pH 4 and pH 9. The composition of the neutralization buffer is not particularly limited, provided that the neutralization buffer only increases or decreases the pH of the solution of purified jellyfish collagen so that collagen fibril formation can proceed. Furthermore, the buffer must be substantially free of ions, compounds, or molecules that may interfere with any crosslinking process. Thus, a buffer that is substantially free of unreacted amines is particularly desirable. As a non-limiting example only, the neutralization buffer may be 1x to 10x phosphate-buffered saline (PBS), where 1x or 10x refers to the concentration of PBS. The composition of 1x PBS is well known to those skilled in the art. The exact concentration of PBS (i.e., 1x, or, for example, 10x) depends entirely on the dilution factor required upon mixing with the solution of purified jellyfish collagen so that the solution is substantially neutralized and collagen fibril formation can proceed. In some embodiments, the neutralization buffer is sodium hydroxide.

[0079] The terms "fibril formation" or "fibrillogenesis" refer to the process by which collagen molecules undergo controlled aggregation to form higher-order, well-structured macromolecular assemblies. In vivo, collagens are primarily extracellular proteins that aggregate into fibrillar structures to provide structural support to surrounding tissues and / or components of the extracellular matrix. Collagen aggregation, particularly of mammalian collagen, is a well-known phenomenon. Different isoforms of mammalian and marine collagen preferentially aggregate to form distinct macromolecular structures. The unique macromolecular structures formed by each collagen isoform are determined by the physicochemical properties of the collagen polypeptides and the conditions promoting fibril formation. Higher-order collagen structures, i.e., collagen fibrils obtained from mammals or fish, have been utilized in vivo to generate mammalian and / or fish collagen hydrogels. Therefore, to form hydrogels from jellyfish collagen, it is preferred that jellyfish collagen assemble to form higher-order structures. Preferably, the higher-order structures are fibrils.

[0080] Any crosslinking agent known to crosslink under conditions that result in collagen fibril formation is considered suitable for use in the present invention. In certain applications, it may be desirable to use a non-cytotoxic crosslinking agent. In certain embodiments, the crosslinking agent may be selected from genipin, 1,4-BDDGE, or mucochloric acid. Preferably, the crosslinking agent is either genipin or 1,4-BDDGE.

[0081] The invention will now be further described with reference to the following examples and studies. [Example]

[0082] Example 1 - Study to determine the effects of jellyfish collagen sponge, cross-linked jellyfish collagen sponge, and chemically modified (thiolated) jellyfish collagen paste in a mouse model of skin wound healing.

[0083] the purpose To compare the performance of jellyfish collagen sponge, cross-linked jellyfish collagen sponge, and chemically modified (thiolated) jellyfish collagen paste with the commercially available wound product Puracol® in an excision skin wound healing model in C57BL / 6J mice.

[0084] procedure Eighty male C57BL / 6J mice, 5-6 weeks old, were provided by Epistem and divided into two cohorts of 40 mice each. Each cohort of mice was allowed to acclimate for 2 weeks before being randomly assigned, with eight mice per cohort, to one of five treatment groups. On day 0 (wounding day), all animals were anesthetized, shaved, and two 6-mm diameter excision wounds were created at the same relative positions on either side of the dorsal midline of each mouse. Depending on the assigned treatment group, one of the following dressings was applied to the wound cavity of each mouse: preformed jellyfish collagen sponge (either standard or cross-linked), chemically modified (thiolated) collagen gel, precut Puracol® dressing, or no treatment. After wound treatment, a Tegaderm film dressing was applied to cover each wound. After treatment, mice were placed in a heated cabinet and allowed to recover from anesthesia before being returned to the holding room. All mice were individually housed from the time of wounding. Wounds were monitored at least daily for any signs of infection and removal of the Tegaderm film dressing. Normal mouse activity caused the Tegaderm to fold, preventing accurate visual assessment of wound condition and measurement of wound width during the in-life phase of the study.

[0085] From each cohort, four mice per treatment group were euthanized 3 days after wounding and four mice were euthanized 7 days after wounding. Mice were humanely killed by cervical dislocation, and a strip of dorsal skin containing both wounds was excised. Wound width was determined using a digital caliper, and a subjective visual score (1-5) was also assigned to the wound according to the degree of macroscopic healing. For histological processing and preparation of hematoxylin and eosin (H&E)-stained wound cross sections, each wound was bisected, one half was snap-frozen in liquid nitrogen, and the other half was fixed in 10% neutral-buffered formalin. For each wound section, wound width and the degree of re-epithelialization were determined using computer-assisted morphometry (Zeiss Axiohome system). In addition, a subjective score of granulation tissue maturity was assigned to each wound, and the area of ​​granulation tissue was determined.

[0086] result Each treatment group demonstrated healing of excision wounds from days 3 to 7, as evidenced by a decrease in wound width (Figures 1 and 2), an increase in re-epithelialization rate (Figure 3), and an increase in granulation tissue (Figure 4) (both granulation tissue maturity and granulation tissue area). In the Tegaderm-only control group, mean wound width decreased from (4.57±0.93) mm on day 3 to (3.71±0.92) mm on day 7 (values ​​quoted are mean±standard deviation).

[0087] The cross-linked jellyfish collagen sponge group showed the greatest wound closure, with a mean wound width of (5.11 ± 1.46) mm on day 3 and (3.16 ± 0.80) mm on day 7 (Figure 2). The least wound closure was observed in the Puracol® group, with a mean wound width of (3.96 ± 1.03) mm on day 7.

[0088] The re-epithelialization rate in the Tegaderm-only group increased from (17.2±10.5)% on day 3 to (59.7±37.9)% on day 7. The Puracol® group showed the highest re-epithelialization rate at (73.2±27.1)% on day 7, while the cross-linked jellyfish collagen sponge group showed a similar level of re-epithelialization rate at (70.7±30.3)% (Figure 3).

[0089] Granulation tissue score and granulation tissue area (as a percentage of total wound area) increased from day 3 to day 7 in all groups (Figure 4). In the Tegaderm-only group, the mean granulation tissue score on day 3 was (1.46±0.52), with granulation tissue occupying (33±24)% of the wound cavity. By day 7, this group's mean granulation tissue score had increased to (2.08±0.51), with (61±31)% of the wound cavity being granulation tissue. The Puracol® group had the lowest granulation tissue score on day 3 (1.17±0.39), with only (5±6)% of the wound cavity being granulation tissue, but by day 7, this group had the highest granulation tissue score of (2.63±0.52), with (81±20)% of the wound cavity being granulation tissue. While the jellyfish collagen sponge and chemically modified (thiolated) collagen paste groups showed similar amounts of granulation tissue as the Tegaderm-only group on day 3, treatment with the cross-linked jellyfish collagen sponge was associated with approximately half the granulation tissue in the wound cavity on day 3, at only 15 ± 21%. The lowest mean granulation tissue score on day 7 (1.67 ± 0.50) was observed in the chemically modified (thiolated) collagen paste group, with only 48 ± 41% of the wound cavity containing granulation tissue. Despite differences in the percentage of the wound cavity showing granulation tissue on day 3, both types of jellyfish collagen sponge were associated with similar levels of granulation tissue on day 7, with the standard sponge scoring 2.30 ± 0.82 and the cross-linked sponge scoring 2.18 ± 0.75.

[0090] Observation of H&E-stained wound cross sections showed that the distinct structures of both the Puracol® and cross-linked jellyfish collagen sponge were visible over the wounds on day 3. With the cross-linked jellyfish collagen sponge, migration of healing epithelium underneath the sponge was readily apparent. Representative images of wounds from each group are shown in Figure 5 (Tegaderm only), Figure 6 (jellyfish collagen sponge), Figure 7 (chemically modified (thiolated) jellyfish collagen paste), Figure 8 (cross-linked jellyfish collagen sponge), and Figure 9 (Puracol®). These show the wound margins with unwounded skin (often with a hyperplastic epithelial layer) adjacent to the wound. The Tegaderm dressing can be seen as a wavy line on the surface of the skin in the images.

[0091] Collagen I immunoreactivity was observed in all wound sections, and representative images are shown in Figure 10, which are from wounds also shown in H&E images. Prominent collagen I immunoreactivity can be observed at the wound edges and throughout the wound cavity, even in 7-day wounds.

[0092] conclusion The animals tolerated the various test items well with no adverse effects. Over the seven days following injury, the wounds healed well, with most animals showing evidence of re-epithelialization, and both the amount and maturity of granulation tissue increased over time. Both types of jellyfish collagen sponges were superior to Tegaderm dressing alone in terms of the extent of granulation tissue formation in the wound cavity by day 7, with the cross-linked jellyfish collagen sponge demonstrating a higher rate of re-epithelialization. In contrast, the performance of the jellyfish sponge and the reference product, Puracol® dressing, was similar.

[0093] This study demonstrates that jellyfish collagen is a suitable alternative to known wound care products on the market with at least comparable efficacy. These known products are typically mammalian in origin and therefore suffer from several drawbacks. It would therefore be highly advantageous to provide an alternative source of collagen suitable for wound care.

[0094] Example 2 - Immunohistochemical labeling using anti-mouse CD31 antibody on mouse wound healing formalin-fixed paraffin-embedded (FFPE) samples.

[0095] the purpose The purpose of this study was to section 152 FFPE mouse wounds from Study 18 / 099 (Study to Determine the Effect of Test Items in a Mouse Model of Skin Wound Healing) and immunohistochemically label them with anti-mouse CD31, an endothelial cell marker commonly used to determine and measure angiogenesis.

[0096] procedure One hundred fifty-two FFPE mouse wounds obtained during Study 18 / 099 (a study to determine the effects of test items in a mouse model of skin wound healing) at 3 and 7 days were sectioned at 3 μm thickness to provide two non-contiguous sections per slide. Sections were mounted on charged slides and dried overnight at 37°C.

[0097] One hundred fifty-two FFPE sections were dewaxed, rehydrated, and then subjected to antigen retrieval using protein digestion with Proteinase K (Dako S3020) for 5 minutes at room temperature. Endogenous peroxidase was blocked with 0.3% H2O2 in TBST for 15 minutes, followed by blocking of nonspecific binding with 2.5% goat serum for 30 minutes. Sections were incubated with rat monoclonal anti-mouse CD31 antibody, clone MEC13.3 (BD ​​Pharmingen 550274), at 0.3125 μg / ml for 1 hour at room temperature. A corresponding rat monoclonal IgG2a isotype control was included in one sample at a concentration matching the primary antibody. A sample of untreated mouse skin was included as a positive tissue control. Sections were washed with TBST and then incubated with anti-rat mouse adsorbent polymer (Vector ImmunPress MP-7444). All labeling was visualized using DAB (Vector ImmPact SK-4105), and sections were counterstained with hematoxylin before permanent mounting. Each labeled slide was checked against its corresponding block to ensure agreement and microscopic examination was performed as part of a quality control procedure.

[0098] Images were captured using an AxioVision Imaging System consisting of a Zeiss Axioscope A1 microscope, an AxioCam MRc camera, and AxioVision software installed on a computer workstation, with 30 representative images taken. Images were taken using a 10x objective.

[0099] Representative images of each of the wound dressing treatment groups (Tegaderm only, jellyfish collagen sponge, chemically modified (thiolated) jellyfish collagen paste, cross-linked jellyfish collagen sponge, and Puracol) were taken on day 3 (Figures 11, 13, 15, 17, and 19) and day 7 (Figures 12, 14, 16, 18, and 20). For orientation, each wound was imaged with the epidermis at the top, i.e., uppermost, of the image.

[0100] Results and Conclusions Wound samples taken from study 18 / 099 were successfully labeled with the mouse-specific endothelial cell marker CD31, indicating that angiogenesis occurred in these samples treated with jellyfish collagen.

[0101] Similar to the in vivo data, the above in vitro data demonstrate that jellyfish collagen is a suitable alternative to bovine collagen for use in wound treatment without the disadvantages associated with the use of mammalian collagen, such as increased risk of disease / prion transmission, increased risk of contamination, and the significant costs associated with obtaining collagen from mammalian sources. This finding is surprising given the significant differences in the physicochemical and amino acid profiles of jellyfish collagen and mammalian collagen.

[0102] Example 3 - db / db (BKS.CG-m Dock 7 m + / _+Lepr db / J) Investigation of the effect of a jellyfish-derived collagen preparation on the healing of full-thickness excision wounds in diabetic mice.

[0103] the purpose To investigate the effects of three jellyfish-derived collagen preparations on wound healing in BKS.Cg-m Dock7m+ / +Leprdb / J (db / db) diabetic mice (a model of delayed skin wound healing).

[0104] procedure Test materials: - Form a 96-well plate (approximately 6 mm diameter) with Jellagen non-crosslinked sponge (sterilized with -CHCl3). - Micronized 0.5% EDC* Cross-linked Jellagen powder (CHCl3 sterilized) (4g). - Micronized 1% EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) cross-linked Jellagen powder (sterilized in CHCl3) (4 g). -Promogran™ (Protease Modulating Matrix, Acelity (USA)). Lot 1912V001, expiration date February 28, 2021. BKS.Cg-m Dock7m+ / +Leprdb / J diabetic mouse model The methods followed are briefly outlined below: The live phase of the study was conducted between September 19, 2019 and October 9, 2019. Diabetic mice (BKS.Cg-m Dock7m+ / +Leprdb / J, Jackson Labs, Bar Harbor, ME, USA) were brought to the UK at approximately 9–10 weeks of age and allowed to acclimate for one week before the start of the study. Animals were maintained in accordance with UK Home Office regulations and specific requirements for diabetic animals. Animals were randomly assigned to one of five treatment groups (Table 1). [Table 1]

[0105] Briefly, according to the protocol, mice were anesthetized using isoflurane and air, and the dorsal flank skin of the mice was excised and cleaned. A single, standardized, full-thickness wound (10 mm x 10 mm) was created on the left dorsal flank approximately 5 mm from the spine. The wound was cleaned with a gauze swab soaked in sterile saline and dried with sterile gauze. 15 μL of sterile saline was then applied to the surface of each wound. The test material was then applied directly to the saline-moistened wound surface.

[0106] Group 2 wounds received a single, non-crosslinked Jellagen sponge approximately 6 mm in diameter (96-well format), Group 5 wounds received a 6 mm disc of the commercial control Promogran™ (Acelity, USA), and Groups 3 and 4 wounds received 5 mg of EDC-crosslinked Jellagen powder (0.5% and 1.0% EDC crosslinking, respectively). Immediately after product application, all wounds were covered with a semi-occlusive film dressing, Tegaderm™ Film (3M Deutschland GmbH, Germany). The film dressing was inspected daily for the duration of the study and replaced if dislodged.

[0107] On days 4, 8, 12, and 16 after wounding, all animals were re-anesthetized, the film dressing and any loose debris were removed, and the wounds (and surrounding skin) were gently cleansed (without disturbing any existing product within the wound) using gauze soaked in sterile saline. The wounds were then photographed (not shown), moistened with 15 μl of sterile saline, and the product was reapplied to the wounds of selected groups on selected days.

[0108] It was originally planned to reapply the product to the wounds of Group 2 (non-crosslinked Jellagen sponge) and Group 5 (Promogran™, Acelity (USA)) on day 4 after wounding. Given the observation that none of these products underwent significant degradation, it was decided (and the sponsor agreed) not to reapply at this time. However, the product was reapplied on days 8, 12, and 16 after wounding without prior removal of the existing undegraded product.

[0109] After reapplication of the product (where indicated), the wounds were re-covered with Tegaderm™ film dressing (as above) and the animals were allowed to recover in a warm environment (approximately 35° C.) All wounds were digitally photographed along with a calibration / identification plate immediately after wounding and then after cleaning on days 4, 8, 12, 16, and 20.

[0110] All animals were sacrificed 20 days after wounding. To facilitate the detection of cell proliferation in tissue sections, all animals received an intraperitoneal injection of 5-bromo-2'-deoxyuridine (Sigma B5002) in saline (30 μg / g) 1 hour before sacrifice. Wound and surrounding tissues were then harvested from all wounds. Tissues were fixed (neutral buffered formalin, Sigma) and embedded in paraffin wax to facilitate histological examination.

[0111] overview This study investigated the effects of topical application of three jellyfish-derived preparations (non-crosslinked Jellagen sponge, 0.5% EDC-crosslinked Jellagen powder, and 1.0% EDC-crosslinked Jellagen powder) on the repair of full-thickness excisional skin wounds in poorly healing db / db diabetic mice.

[0112] The healing of wounds treated with these formulations / products was compared with each other, with the healing of wounds treated with a commercial control (Promogran™ Protease Modulating Matrix, Acelity, USA) and with the healing of wounds treated with a control (film dressing only).

[0113] Wound healing was assessed over 20 days in terms of (i) the onset of a neodermal repair response and (ii) wound closure. The onset of neodermal tissue formation was expressed as the number of responding wounds in each group at each time point. Wound closure was examined both overall and in terms of its components, wound contraction and wound re-epithelialization. Wound closure (contraction and re-epithelialization) was determined from digital photographs taken at 0, 4, 8, 12, 16, and 20 days after wounding. At the histological level, H&E-stained sections of wound tissue (taken at 20 days) were briefly examined and compared in terms of granulation tissue formation and re-epithelialization, and (in the case of the two EDC-crosslinked powder formulations) their potential use as structural scaffolds to support tissue regeneration.

[0114] Summary of Results - Wound Closure Each wound was digitally photographed along with the identification / calibration plate immediately after wounding, and then at days 4, 8, 12, 16, and 20. For a given wound at a given time point, wound closure was expressed as the percentage of remaining wound area relative to the initial wound area immediately after injury (i.e., day 0). The mean percentage remaining wound area data for all treatment groups is set forth in Table 2 below and shown in Figure 21.

[0115] [Table 2]

[0116] When examining wound closure (in terms of change in "% open wound area" over time), the following was observed: -All three Jellagen products evaluated were found to significantly promote closure compared with a control 'film dressing only' treatment from day 12 onwards.

[0117] -When comparing the three Jellagen products, it was found that application of the non-crosslinked sponge resulted in a level of closure similar to that of the 1.0% EDC crosslinked powder, and both were slightly greater than those responding to the 1.0% EDC crosslinked powder. No statistically significant differences in closure were observed between the three Jellagen product treatment groups.

[0118] Summary of Results - Wound Contraction Contraction is the centripetal movement of the wound edges due to compression of granulation tissue within the "body" of the wound. The "compression" forces that drive this process are thought to reside in cells of the fibroblast lineage. In this study, contraction rate was calculated as follows: [Table 3] While wounds in nondiabetic mice close primarily by contraction, the ability of wounds in diabetic mice (such as those used in this study) to contract is greatly reduced (presumably due to insufficient granulation tissue formation). As a result, untreated wounds in diabetic animals tend to close by re-epithelialization to an extent greater than wounds in nondiabetic animals. The observation of enhanced contraction suggests improved granulation tissue function, which in turn may be explained by an increased amount of granulation tissue formed, an increased rate at which it forms, or an increased ability of the tissue to contract. The mean wound contraction rate data for all treatment groups are listed in Table 3 (below) and shown in Figure 22. [Table 4]

[0119] When examining the effect of treatment on wound contraction, the following was observed: All treatments tested were found to significantly accelerate wound contraction from day 8 onwards compared to the control treatment (film dressing only).

[0120] When comparing the three Jellagen products, shrinkage tended to be more extensive in response to the 1.0% EDC cross-linked powder, which tended to be more extensive than that observed in response to the 0.5%, which in turn tended to be more extensive than that observed in response to the non-cross-linked sponge. Statistical analysis showed that application of the 1.0% EDC cross-linked powder significantly enhanced shrinkage compared to treatment with the non-cross-linked sponge.

[0121] Summary of Results - Wound Re-epithelialization For a given wound at a given time point, the area of ​​re-epithelialization was expressed as a percentage of the initial area of ​​that wound immediately after injury. Mean wound re-epithelialization data for all treatment groups are listed in Table 4 (below) and shown in Figure 23. [Table 5]

[0122] When examining the effect of treatment on wound re-epithelialization, the following was observed: -When the Jellagen treatment group was examined, application of a non-crosslinked sponge was found to be more effective than application of EDC crosslinked powder.

[0123] Re-epithelialization in response to the non-crosslinked sponge was found to exceed that observed in the Promogran™-treated group on days 8, 12, and 16, but was lower on day 20.

[0124] - Re-epithelialization in response to EDC cross-linked powder tended to be lower than that in response to treatment with the "film dressing only" control in the majority of studies, although similar levels of re-epithelialization were achieved by the study conclusion at day 20.

[0125] Summary of Results - Initiation of new skin tissue generation All wounds in the study were visually evaluated daily through day 8, and then on days 10, 12, 14, 16, and 20 to establish a "healed" status. Each wound was scored for whether it exhibited "neodermal tissue generation activity" within the central wound area. Scoring was performed independently by two independent observers, and the mean % of wounds exhibiting "neodermal tissue generation activity" at each evaluation time point was compared between treatment groups. The number of wounds that responded in each treatment group on each day is presented in Table 5. The mean time to response for each group is shown in Table 6. Note that 10 of the 10 wounds in the "film dressing only" treatment group did not respond during the study period, so the mean "time to response" for this group is an unknown value greater than 20. [Table 6] [Table 7]

[0126] Compared to the "film dressing only" control treatment, application of all three Jellagen products accelerated the initiation of "neodermal tissue formation" within the central wound area. Of the three Jellagen treatments investigated, 1.0% EDC cross-linked powder provided the most rapid "initiation," followed by 0.5% EDC cross-linked powder and finally the non-cross-linked sponge. All three products were equally, if not more, effective than Promogran™.

[0127] Summary of Results - Wound Histology Wound tissue, along with the surrounding wound skin, was harvested from each animal at the end of the study, 20 days after wounding. Tissue samples were fixed, processed, and embedded in paraffin wax. Sections (approximately 6 μm) were taken from the center of each wound in a craniocaudal direction. These sections were stained with hematoxylin and eosin (H&E) and digitally scanned. Representative examples of the appearance of wounds in each experimental group are presented in Figures 24 and 25. The typical level of granulation tissue deposition and the degree of wound re-epithelialization for each group of wounds are described below.

[0128] Control-treated wounds exhibited limited granulation tissue formation and limited re-epithelialization (both limited to the wound edges).

[0129] Application of the non-crosslinked sponge resulted in increased granulation tissue formation and re-epithelialization compared to the control. Granulation tissue formed throughout the wound base, but was found to be of variable "quality." No product residue was evident in the majority of the wounds.

[0130] Application of both EDC cross-linked powders resulted in increased granulation tissue formation in the majority of wounds with minimal re-epithelialization. These products appear to function as a scaffold for granulation tissue deposition and, once fully cellularized, may support re-epithelialization. Based on overall wound imaging and histological appearance, these products do not appear to have experienced significant degradation over the 20-day study period.

[0131] EDC cross-linked jellyfish collagen powder appeared to promote the growth of new blood vessels from the wound edge. This was most pronounced for the 1.0% powder and was visible to the naked eye as redness advancing from the outer edge of the hydrated mass on the wound surface and was also evident on histological sections (Figure 26).

[0132] Research results All three Jellagen products evaluated in this study were found to have a positive impact on wound healing in the poorly healing db / db diabetic mouse model.

[0133] A non-crosslinked collagen sponge was found to promote wound closure by promoting both contraction and re-epithelialization, and to promote granulation tissue formation. The sponge material underwent extensive compression and limited degradation and did not appear to be incorporated into the wound tissue.

[0134] Crosslinking powders promote wound closure primarily by accelerating the wound contraction process, which may be explained by the ability of these products to act as a scaffold for granulation tissue formation.

[0135] Example 4 - db / db(BKS.Cg-m Dock7 m + / +Lepr db / J) Investigation of the effect of a jellyfish-derived collagen preparation on the healing of full-thickness excision wounds in diabetic mice. the purpose To investigate the effects of three jellyfish-derived collagen preparations on wound healing in BKS.Cg- m Dock7m+ / +Leprdb / J (db / db) diabetic mice (a model of delayed skin wound healing).

[0136] procedure Test materials: - Cross-linked jellyfish collagen sponge (Jellagen). Fabricated in a 24-well plate using 500 μL of AMY2 (4.5 mg / mL). Cross-linked with 1% EDC (sterilized with CHCl3). Date of manufacture: October 2020. Cut the sponge to the size of the wound. Micronized 1% EDC* cross-linked jellyfish collagen powder (Jellagen) (sterilized in CHCl3) (individual wound aliquots of approximately 2.5 mg). Micronized thiolated jellyfish collagen powder (Jellagen) (sterilized with CHCl3) (individual wound aliquots of approximately 2.5 mg). -Comparator: Integra® Flowable Wound Matrix (Integra Lifesciences Inc., Princeton, USA). Product code: FDR 301, lot: 4679547, expiration date: September 30, 2021.

[0137] BKS.Cg-m Dock7m+ / +Leprdb / J diabetic mouse model The methodology followed is briefly outlined below. Diabetic mice (BKS.Cg-m Dock7m+ / +Leprdb / J, Jackson Labs, Bar Harbor, ME, USA) were brought to the UK at approximately 9-10 weeks of age and allowed to acclimate for one week before the start of the study. Animals were maintained in accordance with UK Home Office regulations and specific requirements for diabetic animals. Animals were randomly assigned to one of five treatment groups (Table 7). [Table 8]

[0138] Briefly, according to the protocol, mice were anesthetized using isoflurane and air, and the dorsal flank skin of the mice was excised and cleaned. A single, standardized, full-thickness wound (10 mm x 10 mm) was created on the left dorsal flank approximately 5 mm from the spine. The wound was cleaned with a gauze swab soaked in sterile saline and dried with sterile gauze. 15 μL of sterile saline was then applied to the surface of each wound. The test material was then applied directly to the saline-moistened wound surface.

[0139] On the day of reapplication (see Table 7 above), the animals were re-anesthetized, the film dressing and any loose debris were removed, and the wounds (and surrounding skin) were gently cleansed (without disturbing any existing product within the wound) using gauze soaked in sterile saline. The wounds were then photographed (not shown), moistened with 15 μl of sterile saline, and the product was reapplied to the wounds of the selected group on the selected day. After product reapplication (if indicated), the wounds were re-covered with Tegaderm™ film dressing, and the animals were allowed to recover in a warm environment (approximately 35°C). Immediately after wounding, and on days 4, 8, 12, 16, 20, 24, 28, 35, 42, 49, 56, and 63 after wounding, the wounds were digitally photographed along with the calibration / identification plate.

[0140] Eight animals from each group were sacrificed on day 35 post-wounding, and all remaining animals were culled on day 63 post-wounding.

[0141] Summary of Results - Wound Closure Each wound was digitally photographed along with the identification / calibration plate immediately after wounding, and then at days 4, 8, 12, 16, 20, 24, 28, 35, 42, 49, 56, and 63. For a given wound at a given time point, wound closure was expressed as the percentage of remaining wound area relative to the initial wound area immediately after injury (i.e., day 0). The mean percentage remaining wound area data for all treatment groups is shown in Figure 27. Due to the presence of the sponge, it was not always possible to clearly visualize the wound edges for the cross-linked sponge-treated group, and therefore values ​​for that test group are not included in Figure 27.

[0142] When examining wound closure (in terms of change in "% open wound area" over time), the following was observed: All treatments tested were found to increase wound closure compared with the "film dressing only" control for at least 35 days after wound healing. Some regression of wound healing was observed between days 35 and 63 in all groups, except for the thiolated powder and control treatment groups.

[0143] Each of the evaluated Jellagen products was found to significantly accelerate wound closure compared to the "film dressing only" control treatment from at least Day 12 onwards (Day 4 for the 1.0% EDC crosslinked powder and thiolated powder) (Figure 27).

[0144] -When comparing the two Jellagen products, wounds with thiolated powder applied showed the greatest response by day 35 (n=12), followed by the 1% EDC crosslinking powder (Figure 28). No significant differences were observed at later time points (day 35 and beyond) (n=4).

[0145] -When comparing the thiolated powder to the Integra product, a very similar closure profile was observed up to day 35 (n=12) (Figure 29), with slightly increased closure observed with the thiolated powder from day 42 to day 63 (n=4) (Figure 27).

[0146] Summary of Results - Wound Contraction Contraction is the centripetal movement of the wound edges due to compression of granulation tissue within the "body" of the wound. The "compression" forces that drive this process are thought to reside in cells of the fibroblast lineage. In this study, contraction rate was calculated as follows: [Table 9]

[0147] While wounds in nondiabetic mice close primarily by contraction, the ability of wounds in diabetic mice (such as those used in this study) to contract is greatly reduced (presumably due to insufficient granulation tissue formation). As a result, untreated wounds in diabetic animals tend to close by re-epithelialization to an extent greater than wounds in nondiabetic animals. The observation of enhanced contraction suggests improved granulation tissue function, which in turn may be explained by an increased amount of granulation tissue formed, an increased rate at which it forms, or an increased ability of the tissue to contract. Average wound contraction rate data for all treatment groups is shown in Figure 30.

[0148] When examining the effect of treatment on wound contraction, the following was observed: All treatments tested were found to significantly increase wound contraction compared with a control treatment of 'film dressing only' for at least 35 days after wounding.

[0149] Each of the three Jellagen products evaluated was found to significantly enhance wound contraction compared to the "film dressing only" control treatment from at least Day 12 onwards (near significance at Day 4 for the cross-linked sponge, Day 8 for the 1% EDC cross-linked powder, and Day 8 for the thiolated powder) (Figure 30).

[0150] When comparing the three Jellagen products (Figure 31), wounds with thiolated powder applied showed the greatest contraction by day 35 (n=12), while the cross-linked sponge and 1% EDC cross-linked powder promoted slightly lower contraction, comparable to the Integra product (Figure 30).

[0151] When comparing the thiolated powder to the Integra product (Figure 32), increased shrinkage was observed with the thiolated powder, reaching statistical significance at 20 and 28-42 days (p≦0.029) and approaching significance at 16 and 24 days (p=0.060 and 0.068, respectively).

[0152] Summary of Results - Wound Re-epithelialization For a given wound at a given time point, the area of ​​re-epithelialization was expressed as a percentage of the initial area of ​​that wound immediately after injury. Mean wound re-epithelialization data for all treatment groups are listed in Table 4 (below) and shown in Figure 33. Because it was not possible to accurately measure re-epithelialization of wounds to which cross-linked sponges were applied while the sponges were still in place, the results of that treatment are not included in Figure 33.

[0153] When examining the effect of treatment on wound re-epithelialization, the following was observed: -All treatments tested were found to increase wound re-epithelialization up to at least 35 days after wounding compared with a control treatment of 'film dressing only'.

[0154] Both the Jellagen 1% EDC crosslinked powder product and the Jellagen thiolated powder product were found to significantly promote wound contraction compared to the "film dressing only" control, at least from Day 16 onwards. Specifically, wounds receiving the thiolated powder showed significantly increased wound re-epithelialization compared to control wounds from Days 12 through 35 (p≦0.007), and wounds receiving the 1% EDC crosslinked powder product showed significantly increased wound re-epithelialization compared to control wounds from Days 16 through 35 (p≦0.028), reaching significance at Day 12 (p=0.052).

[0155] When the 1% EDC cross-linked powder was compared to the thiolated powder (Figure 34), wounds receiving the thiolated powder showed the greatest re-epithelialization by day 35 (n=12), while the 1% EDC cross-linked powder promoted slightly less re-epithelialization.

[0156] When comparing the thiolated powder to the Integra product (Figure 34), the Integra product generally demonstrated slightly increased levels of re-epithelialization up to 35 days post-wounding, reaching significance at days 16, 28, and 35 (p<0.039). However, the thiolated powder demonstrated a significant increase in re-epithelialization at day 8 post-wounding (p=0.010).

Claims

1. 1. A composition for use in the treatment of wounds, comprising jellyfish collagen, said jellyfish collagen being in the form of a micronized powder.

2. 10. The composition of claim 1, wherein the jellyfish collagen is crosslinked.

3. The composition of claim 1 , wherein the jellyfish collagen is not cross-linked.

4. The composition according to any one of claims 1 to 3, wherein the jellyfish collagen is in its ateloform.

5. The composition according to any one of claims 1 to 3, wherein the jellyfish collagen is in its telomorphic form.

6. The composition of any one of claims 1 to 5, wherein the jellyfish collagen is thiolated.

7. The composition of any one of claims 1 to 6, wherein the source of the jellyfish collagen is from the subphylum Scyphozoa.

8. 8. The composition of claim 7, wherein the source of jellyfish collagen is selected from the group consisting of Rhizostomas pulmo, Rhopilema esculentum, Rhopilema nomadica, Stomolophus meleagris, Aurelia species, Cassiopea andromeda, Nemopilema nomurai, or any combination thereof.

9. The composition according to any one of claims 1 to 8, wherein the composition further comprises at least one growth factor.

10. 10. The composition of claim 9, wherein the at least one growth factor is platelet-rich plasma (PRP), epidermal growth factor 38 (EGF), transforming growth factor-beta (TGF-B, TGF-B2, TGF-B3), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), granulocyte-monocyte colony-stimulating growth factor, platelet-derived growth factor, insulin-like growth factor 1 (IGF1), basic fibroblast growth factor (bFGF), and / or vascular endothelial growth factor 5 (VEGF), or any combination thereof.

11. The composition of any one of claims 1 to 10, wherein the composition further comprises at least one antibacterial compound.

12. 12. The composition of claim 11, wherein the at least one antibacterial compound is nanosilver, penicillin, ofloxacin, tetracycline, aminoglycoside, erythromycin, gentamicin, flucloxacillin, clarithromycin, doxycycline, metronidazole, co-amoxiclav, cotrimoxazole (in penicillin), ceftriaxone, piperacillin / tazobactam, clindamycin, ciprofloxacin, vancomycin, teicoplanin, linezolid, and / or standard of care antibacterial agents, or any combination thereof.

13. 13. The composition of any one of claims 1 to 12, wherein the jellyfish collagen is administered at a dose of 0.01 g / L to 200 g / L per administration.

14. The composition according to any one of claims 1 to 13, wherein the composition further comprises a pharmaceutically acceptable excipient and / or carrier, and / or a pharmaceutically active ingredient.

15. 15. The composition of claim 14, wherein the pharmaceutically active ingredient is lidocaine.

16. 16. The composition of any one of claims 1 to 15, wherein the wound to be treated is a pressure sore, a transplant site, a surgical wound, an ulcer, a diabetic ulcer, a thermal injury, a chemical burn, an electrical burn, a laceration, an abrasion, a puncture wound, an avulsion wound, a seroma, and / or a hematoma.

17. The composition of any one of claims 1 to 16, wherein the micronized powder has a particle size of from 1 μm to 1000 μm.

18. The composition of any one of claims 1 to 17, wherein the composition promotes improved vascularization in the treated wound.

19. 19. The composition of claim 18, wherein the improved vascularization is compared to untreated wounds and / or wounds treated with bovine collagen.

20. 2. A method for producing jellyfish collagen according to claim 1, comprising at least: i) extracting acid-soluble collagen from a jellyfish collagen source; ii) purifying the jellyfish collagen to provide a solution of purified jellyfish collagen.

21. The method comprises:

21. The method of producing jellyfish collagen of claim 20, further comprising the step of: iii) adding a cross-linking agent to form cross-linked jellyfish collagen.

22. 22. The method for producing jellyfish collagen of claim 21, wherein the cross-linking agent is EDC, genipin, or polyethylene glycol (PEG).

23. 23. The method for producing jellyfish collagen according to claim 22, wherein the cross-linking agent is EDC.

24. 24. The method for producing jellyfish collagen according to claim 23, wherein the EDC is at a concentration of 0.01% to 5%.

25. 25. A method for producing jellyfish collagen according to any one of claims 20 to 24, wherein the method further comprises digesting the extracted jellyfish collagen with a peptidase to provide Ateromedusa collagen.

26. 26. The method for producing jellyfish collagen of claim 25, wherein the peptidase is pepsin.

27. 27. A method for producing jellyfish collagen according to claim 25 or 26, wherein the step of digesting the collagen with a peptidase occurs after the extraction step and before the purification step.

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