Wound Debridement System

Abstract

The present invention provides compositions and methods for wound debridement and healing. The debridement composition preferably comprises a metalloprotease enzyme, such as an egg membrane degrading enzyme.

Description

Detailed Description of the Invention

[0001] FIELD OF THE INVENTION The present invention provides compositions and methods for wound debridement and healing.

[0002] BACKGROUND OF THE INVENTION Chronic wounds represent a significant burden to patients, healthcare professionals, and the US healthcare system, affecting 5.7 million patients and costing an estimated $20 billion annually.

[0003] Chronic wounds are rarely seen in otherwise healthy individuals. In fact, patients with chronic wounds often suffer from "high-brand" conditions such as diabetes and obesity. A chronic wound is a wound in which the repair process fails to proceed in an adequate and timely manner to create anatomical and functional integrity at the injury site. Often disguised as a comorbid condition, chronic wounds represent an asymptomatic epidemic that affects a large portion of the global population and poses a serious and significant threat to U.S. public health and the economy. In developed countries, it is estimated that 1-2% of the population will experience a chronic wound during their lifetime. In the United States alone, chronic wounds affect 6.5 million patients. In Scandinavian countries, associated costs account for 2-4% of total healthcare expenditures.

[0004] The burden of chronic wound care is growing rapidly due to rising healthcare costs, an aging population, and the sharp rise in the incidence of diabetes and obesity in the United States and beyond. It has been claimed that chronic wound care costs exceed $25 billion annually. Add to this the rapidly expanding wound care needs of our veterans and the need to prioritize wound care and research, and the need to prioritize wound care and research seems compelling. Currently, there are more than 1,000 outpatient wound centers operated by inpatient acute care hospitals, long-term care facilities, and nursing homes in the United States—and this does not include all the wound care provided by clinicians in their offices. According to a new report from Global Industry Analysts, the annual market for wound care products will reach $15.3 billion by 2010. The United States represents the largest and fastest-growing market in the world. The amount spent on wound care, the lost productivity, and the diminished quality of life for afflicted individuals and their caring families pose significant costs to our society.

[0005] What is needed in the art are new and effective treatments for chronic wounds.

[0006] Summary of the Invention The present invention provides compositions and methods for wound debridement and healing.

[0007] Thus, in some embodiments, the present invention provides a method of debridement of a wound comprising contacting the wound with an effective amount of a metalloprotease.

[0008] In some preferred embodiments, an effective amount of metalloprotease is effective in removing devitalized tissue in a wound. In some preferred embodiments, the metalloprotease is selected from the group consisting of low vitelline membrane degrading enzyme (LCE), high vitelline membrane degrading enzyme (HCE), and combinations thereof. In some preferred embodiments, the LCE is trout LCE. In some preferred embodiments, the LCE has at least 90% sequence identity with SEQ ID NO: 1 and has LCE activity. In some preferred embodiments, the LCE activity is LCE protease activity assayed using fluorescein isothiocyanate (FITC)-labeled casein as a substrate, where the LCE protease activity causes an increase in fluorescence due to dequenching of FITC as casein is digested into smaller peptides. In some preferred embodiments, the LCE is recombinant LCE. In some preferred embodiments, the recombinant LCE includes a his-tag. In some preferred embodiments, the metalloprotease is provided in a matrix, bandage, wound dressing, cream, gel, emulsion, ointment, spray, powder, or lotion.

[0009] In some preferred embodiments, the method further comprises applying a wound-healing agent to the wound after debridement. In some preferred embodiments, the wound-healing agent comprises an extract of differentiable cells. In some preferred embodiments, the extract of differentiable cells is an extract of fish roe. In some preferred embodiments, the fish roe extract comprises, in an aqueous solution, about 100-380 mg / mL of protein; about 0.1-10 mg / mL of RNA; about 0.1-5 mg / mL of DNA; and 0.1-10% by weight of lipids. In some preferred embodiments, the fish roe extract is heat-treated by heating the extract to above 80°C, above 90°C, above 95°C, or above 100°C. In some preferred embodiments, the heat treatment is carried out for about 1 minute to about 30 minutes. In some preferred embodiments, the wound-healing agent is provided as a matrix, bandage, wound dressing, cream, gel, emulsion, ointment, spray, powder, or lotion.

[0010] In some preferred embodiments, the wound being treated is a chronic wound, hi some preferred embodiments, the chronic wound is a diabetic ulcer.

[0011] In some preferred embodiments, the present invention provides a formulation comprising a metalloprotease in an amount effective for wound debridement, in a cream, gel, emulsion, ointment, spray, powder, or lotion. In some preferred embodiments, the metalloprotease in an amount effective for wound debridement is effective for removing devitalized tissue in the wound. In some preferred embodiments, the metalloprotease is selected from the group consisting of low vitelline membrane-degrading enzyme (LCE), high vitelline membrane-degrading enzyme (HCE), and combinations thereof. In some preferred embodiments, the LCE is trout LCE. In some preferred embodiments, the LCE has at least 90% sequence identity with SEQ ID NO: 1 and has LCE activity. In some preferred embodiments, the LCE activity is LCE protease activity assayed using fluorescein isothiocyanate (FITC)-labeled casein as a substrate, and the LCE protease activity causes an increase in fluorescence due to the release of FITC quenching as casein is digested into smaller peptides. In some preferred embodiments, the LCE is recombinant LCE. In some preferred embodiments, the recombinant LCE comprises a his-tag. In some preferred embodiments, the formulation comprises urea and / or zinc chloride (ZnCl2) in an aqueous solution. In some preferred embodiments, the urea is provided at a concentration of 1 to 10 M, more preferably 2 to 6 M, even more preferably 3 to 5 M, and most preferably about 4 M. In some preferred embodiments, the zinc chloride is provided at a concentration of 2 to 40 mM, more preferably 4 to 20 mM, even more preferably 6 to 18 mM, and most preferably 8 to 18 mM.

[0012] In some preferred embodiments, the present invention provides a kit or system for treating a wound, comprising: a first container containing a metalloprotease in an amount effective to remove devitalized tissue from the wound; and a second container containing a wound-healing agent. In some preferred embodiments, the metalloprotease is selected from the group consisting of low vitelline membrane-degrading enzyme (LCE), high vitelline membrane-degrading enzyme (HCE), and combinations thereof. In some preferred embodiments, the LCE is trout LCE. In some preferred embodiments, the LCE has at least 90% sequence identity with SEQ ID NO: 1 and has LCE activity. In some preferred embodiments, the LCE activity is LCE protease activity assayed using fluorescein isothiocyanate (FITC)-labeled casein as a substrate, where the LCE protease activity causes an increase in fluorescence due to the dequenching of FITC as casein is digested into smaller peptides. In some preferred embodiments, the LCE is recombinant LCE. In some preferred embodiments, the recombinant LCE contains a his-tag. In some preferred embodiments, the metalloprotease is provided in a matrix, bandage, wound dressing, cream, gel, emulsion, ointment, spray, powder, or lotion. In some preferred embodiments, the wound healing agent comprises an extract of differentiable cells. In some preferred embodiments, the extract of differentiable cells is an extract of fish roe. In some preferred embodiments, the fish roe extract comprises, in an aqueous solution, about 100-380 mg / mL of protein; about 0.1-10 mg / mL of RNA; about 0.1-5 mg / mL of DNA; and 0.1-10% by weight of lipids. In some preferred embodiments, the fish roe extract is heat-treated by heating the extract to above 80°C, above 90°C, above 95°C, or above 100°C. In some preferred embodiments, the heat treatment is carried out for about 1 minute to about 30 minutes. In some preferred embodiments, the wound healing agent is provided in a matrix, bandage, wound dressing, cream, gel, emulsion, ointment, spray, powder, or lotion.

[0013] In some preferred embodiments, the present invention provides an effective amount of a metalloprotease for use in wound debridement. In some preferred embodiments, the effective amount of the metalloprotease is effective in removing devitalized tissue in the wound. In some preferred embodiments, the metalloprotease is selected from the group consisting of low vitelline membrane degrading enzyme (LCE), high vitelline membrane degrading enzyme (HCE), and combinations thereof. In some preferred embodiments, the LCE is trout LCE. In some preferred embodiments, the LCE has at least 90% sequence identity with SEQ ID NO: 1 and has LCE activity. In some preferred embodiments, the LCE activity is assayed using fluorescein isothiocyanate (FITC)-labeled casein as a substrate, and the LCE protease activity causes an increase in fluorescence due to the dequenching of FITC as casein is digested into smaller peptides. In some preferred embodiments, the LCE is recombinant LCE. In some preferred embodiments, the recombinant LCE includes a his-tag. In some preferred embodiments, the metalloprotease is provided in a matrix, bandage, wound dressing, cream, gel, emulsion, ointment, spray, powder, or lotion.

[0014] In further preferred embodiments, the present invention provides a pharmaceutical or cosmetic composition comprising a recombinant metalloprotease selected from the group consisting of low egg cell membrane degrading enzyme (LCE), high egg cell membrane degrading enzyme (HCE), and a combination thereof, wherein the recombinant metalloprotease exhibits metalloprotease activity in an in vitro assay. In some preferred embodiments, the LCE is trout LCE. In some preferred embodiments, the sequence of the trout LCE has at least 90% sequence identity with SEQ ID NO: 1 and has LCE activity. In some preferred embodiments, the LCE activity is assayed using fluorescein isothiocyanate (FITC)-labeled casein as a substrate, and the LCE protease activity causes an increase in fluorescence due to the dequenching of FITC as casein is digested into smaller peptides. In some preferred embodiments, the pharmaceutical or cosmetic composition comprises urea and / or zinc chloride (ZnCl) in an aqueous solution. In some preferred embodiments, the urea is provided at a concentration of 1 to 10 M, more preferably 2 to 6 M, even more preferably 3 to 5 M, and most preferably about 4 M. In some preferred embodiments, the zinc chloride is provided at a concentration of 2 to 40 mM (more preferably 4 to 20 mM, even more preferably 6 to 18 mM, and most preferably 8 to 18 mM).

[0015] In further preferred embodiments, the present invention provides pharmaceutical or cosmetic compositions comprising a recombinant metalloprotease selected from the group consisting of low egg cell membrane degrading enzyme (LCE), high egg cell membrane degrading enzyme (HCE), and combinations thereof; and one or more pharmaceutically or cosmetically acceptable excipients and / or diluents, wherein the composition is in the form of a gel, cream, ointment, lotion, foam, non-aqueous solution, spray, salve, stick, soap, powder, film, emulsion, suspension, or dispersion, and the recombinant metalloprotease exhibits metalloprotease activity in an in vitro assay. In some preferred embodiments, the LCE is trout LCE. In some preferred embodiments, the sequence of the trout LCE has at least 90% sequence identity with SEQ ID NO: 1 and has LCE activity. In some preferred embodiments, the pharmaceutical or cosmetic composition comprises urea and / or zinc chloride (ZnCl) in an aqueous solution. The urea is provided at a concentration of 1 to 10 M, more preferably 2 to 6 M, even more preferably 3 to 5 M, and most preferably about 4 M. In some preferred embodiments, the zinc chloride is provided at a concentration of 2 to 40 mM, more preferably 4 to 20 mM, even more preferably 6 to 18 mM, and most preferably 8 to 18 mM.

[0016] In some preferred embodiments, the pharmaceutical or cosmetic composition is provided for use in treating a skin disease or condition, in some preferred embodiments, the skin condition or disease is dry skin, hyperkeratosis, calluses, warts, melasma, age spots, stretch marks, sun spots, brown spots, acne, eczema, or psoriasis.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Gel showing the results of overexpressing LCE1 in E. coli.

[0018] [Figure 2] Protease activity of fractions derived from recombinant LCE1 overexpression.

[0019] [Figure 3] Soluble recombinant LCE activity upon addition of urea and zinc chloride.

[0020] [Definition] The term "natural product" refers to any of a variety of organic chemical moieties whose molecular arrangements are derived from enzymatic conversions in living organisms, excluding amino acids, proteins, polypeptides, nucleic acids and sequences, and saturated fatty acids. Examples include, but are not limited to, lipids (i.e., not saturated fatty acids), carbohydrates / sugars and polysaccharides, steroids and their derivatives, terpenes and their derivatives, vitamins, carotenoids, and natural pharmaceuticals such as taxol. The term "synthetic natural product" refers to a natural product that is not obtained from its natural source.

[0021] "Cell" refers to the smallest structural unit of living matter capable of autonomous function, consisting of one or more nuclei, cytoplasm, and various organelles, all surrounded by a semipermeable membrane. The term "cell" includes all somatic cells obtained from or derived from a living or dead animal at any stage of development, as well as germ cells, including sperm and eggs (animal gametes consisting of an egg or embryo along with nutritive and protective envelopes). This includes the general categories of cells, both prokaryotes and eukaryotes. Cells intended for use in the present invention include all types of cells from all kingdoms and all living organisms: plants, animals, protists, fungi, archaea, and eubacteria. Stem cells are cells capable of producing cells with many different levels of specialization through continuous division. For example, hematopoietic stem cells produce both red and white blood cells. Humans possess stem cells from conception until death, but their capacity for differentiation is reduced in adults.

[0022] As used herein, the term "differentiation" in reference to cells refers to the process by which cells become structurally and functionally specialized, which is the gradual restriction of developmental potential that occurs during embryonic development and results in increasing functional specialization that leads to the formation of specialized cells, tissues, and organs.

[0023] The term "dedifferentiation" in relation to cells refers to the reverse process of differentiation, in which a cell becomes less structurally and functionally specialized, increasing the developmental potential of the cell.

[0024] "Differentiable" refers to the ability of a cell to differentiate into a desired cell type. As used herein, the term "differentiate" refers to specialization (differentiation) or reversion to a more primitive cell type (dedifferentiation).

[0025] As used in the context of "cell extract" or "egg extract" in this invention, "extract" refers to a preparation of any type of cell, as defined above, obtained by chemical or mechanical action, such as by pressing, distillation, evaporation, etc. The extract may contain all of the cellular components, including concentrated preparations of active ingredients, or any single component or combination. Such components of the extract include, but are not limited to, RNA, DNA, microRNA, lipids, free amino acids, all amino acid-based structures, including peptides and proteins, carbohydrates, minerals, or combinations thereof. Extracts contemplated by this invention include, but are not limited to, extracts of fish eggs, sea urchin eggs, frog eggs, adult stem cells, plant seeds, and plant stem cells.

[0026] The term "manage," when used in connection with a disease or condition, means providing a beneficial effect to a subject receiving a prophylactic or therapeutic agent, but does not result in a cure of the disease. In certain embodiments, a subject is administered one or more prophylactic or therapeutic agents to manage the disease and prevent the progression or worsening of the disease.

[0027] As used herein, the terms "prevent" and "preventing" include prevention of recurrence, spread, or onset. It is not intended that the present invention be limited to complete prevention. In some embodiments, onset is delayed or the severity of the disease is reduced.

[0028] As used herein, the terms "treat" and "treating" are not limited to cases where a subject (e.g., a patient) is cured and the disease is eradicated. Rather, the present invention also contemplates treatments that merely reduce symptoms and / or slow the progression of the disease.

[0029] Detailed Description of the Invention The present invention provides compositions and methods for wound debridement and healing.

[0030] The two main layers of skin are the epidermis and dermis. The epidermis is composed of densely packed epithelial cells, while the dermis is made up of dense, irregular connective tissue that houses blood vessels, hair follicles, sweat glands, and other structures. The hypodermis lies below the dermis and is composed mostly of loose connective tissue and adipose tissue. Muscles, tendons, ligaments, bones, and cartilage are all found subcutaneously. The epidermis is composed of keratinized, stratified squamous epithelium. The dermis contains blood and lymphatic vessels, nerves, and other structures such as hair follicles and sweat glands.

[0031] Debridement is indicated for the removal of devitalized tissue, such as necrotic tissue, scabs, bioburden, biofilm, and apoptotic cells. Debridement is recognized as a key component of wound management to prepare the wound bed for re-epithelialization. Devitalized tissue, especially necrotic tissue, generally serves as a nutrient source for bacteria. Devitalized tissue also acts as a physical barrier to re-epithelialization, preventing applied topical compounds from directly contacting the wound bed and providing their beneficial properties. Necrotic tissue also inhibits angiogenesis, granulation tissue formation, epidermal renewal, and normal extracellular matrix (ECM) formation. Finally, the presence of necrotic tissue can prevent clinicians from accurately assessing the extent and severity of the wound and even masking a potential underlying infection.

[0032] The present invention provides reagents and systems necessary for debridement and wound healing. In some embodiments, the present invention provides metalloproteases for use in wound debridement. Preferred metalloproteases include, but are not limited to, low vitelline membrane-degrading enzyme (LCE), high vitelline membrane-degrading enzyme (HCE), and combinations thereof. The enzyme may preferably be sourced from fish such as trout or salmon, although the present invention is not limited to these enzyme sources. In some preferred embodiments, the enzyme is recombinantly produced. In further embodiments, the present invention provides formulations and articles containing an effective amount of a metalloprotease enzyme. The enzyme may be used alone for wound debridement, or in more preferred embodiments, the enzyme may be used in conjunction with a wound healing agent. Generally, debridement is performed first, followed by application of the wound healing agent. Preferred wound healing agents include, but are not limited to, extracts of differentiable cells, as described in detail below. The compositions, formulations, articles, and methods of the present invention may be used to treat any type of wound. In some embodiments, the wound is a chronic wound, such as a diabetic ulcer.

[0033] [A. Debridement enzymes] In preferred embodiments, the present invention provides a metalloprotease for use in wound debridement. In some preferred embodiments, the metalloprotease is provided in an amount effective to remove devitalized tissue from a wound site. The devitalized tissue may include one or all of necrotic tissue, scab, bioburden, biofilm, and apoptotic cells found at a wound site.

[0034] The present invention is not limited to the use of a specific metalloprotease enzyme. Preferred metalloproteases include, but are not limited to, LCE, HCE, and combinations thereof. The LCE and HCE are preferably sourced from fish such as salmonids or other marine animals (e.g., Oncorhynchus mykiss and Salmo salar).

[0035] Suitable LCE and HCE enzymes are identified by the sequences listed in Table 1.

[0036] [Table 1]

[0037] In some embodiments, the egg membrane degrading enzyme has at least 50%, 60%, 70%, 809%, 90%, 95%, 99%, or 100% sequence identity to a sequence listed in Table 1, provided that the enzyme has egg membrane degrading activity. In some embodiments, the egg membrane degrading enzyme is isolated from a natural source. In some preferred embodiments, the egg membrane degrading enzyme is recombinant. In some embodiments, the recombinant enzyme preferably contains additional sequences, such as a his-tag, that facilitate purification of the enzyme.

[0038] In some preferred embodiments, the egg membrane degrading enzyme is the LCE of O. mykiss (rainbow trout). In some embodiments, the LCE of O. mykiss has the following amino acid sequence (SEQ ID NO: 1):

[0039] [ka] In some preferred embodiments, the LCE utilized in the present invention has at least 50%, 60%, 70%, 809%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 1, provided that the enzyme has egg membrane degrading activity. In some embodiments, the LCE is isolated from a natural source. In some preferred embodiments, the LCE is recombinant. In some embodiments, the recombinant enzyme preferably contains additional sequences, such as a his-tag, that facilitate purification of the enzyme.

[0040] In some preferred embodiments, soluble recombinant LCE activity (e.g., in an aqueous solution or formulation) is enhanced by a formulation containing urea and / or zinc chloride (ZnCl2). In some preferred implementations, the urea is provided at a concentration of 1-10 M, more preferably 2-6 M, even more preferably 3-5 M, and most preferably about 4 M. In some preferred embodiments, the zinc chloride is provided at a concentration of 2-40 mM, more preferably 4-20 mM, even more preferably 6-18 mM, and most preferably 8-18 mM.

[0041] [B. Wound healing agents] In some embodiments, the present invention provides methods, systems, and kits that provide a wound healing agent in addition to a debridement enzyme, which in some preferred embodiments is applied to the wound after enzymatic debridement.

[0042] The present invention is not limited to the use of any particular wound healing agent. In some preferred embodiments, the wound healing agent comprises an extract derived from differentiable cells such as fish eggs. In some embodiments, the egg cell extract comprises, in an aqueous solution, about 100-380 mg / mL of protein; about 0.1-10 mg / mL of RNA; about 0.1-5 mg / mL of DNA; and 0.1-10% by weight of lipids. In some preferred embodiments, the composition has an osmolality of about 330-440 mOsm, a pH of about 5.0-7.7, and a density of about 0.8-1.4 g / mL. In some embodiments, the egg cell extract is selected from the group consisting of an extract of activated fish egg cells and an extract of unactivated fish egg cells. In some embodiments, the fish egg cell extract is derived from fertilized eggs. In some embodiments, the cell extract is heat-treated by heating to above 80°C, above 90°C, above 95°C, or above 100°C. In some embodiments, the heat treatment is performed for about 1 minute to about 30 minutes. In some embodiments, the egg cell extract is provided in a cream, gel, emulsion, ointment, spray, powder, or lotion.

[0043] As mentioned above, the compositions of the present invention utilize extracts of cells, eggs, and embryos derived from vertebrates, and include those from the Superclass Gnathostomata (jawed vertebrates), Euteleostomi (bony vertebrates), Class Actinopterygii (ray-finned vertebrates), Class Sarcopterygii (lobe-finned vertebrates and terrestrial vertebrates), Tetrapoda (tetrapods), Amniota (amniotes), Synapsida (synapsids), Class Mammalia (mammals), Early Therapsida (early therapsids), Class Reptilia (reptiles), Anapsida (land and sea turtles), Order Testudines (land and sea turtles), Diapsida (birds, crocodiles, lizards, snakes, and related animals), Archosauria (birds and crocodiles), Order Acropia (birds and crocodiles), and the Order Acropia (birds and crocodiles). Examples of suitable eggs include, but are not limited to, Crocodilia (caimans, crocodiles, and related animals), Lepidosauria (water lizards, lizards, snakes, and tuatara), Order Rhynchocephalia (tuatara), Order Squamata (water lizards, lizards, and snakes), Class Amphibia (amphibians), Subclass Dipnoi (lungfish), Actinistia, Order Coelacanthiformes (coelacanths), Class Chondrichthyes (rays, sharks, and related animals), Placodermi (armored fish and placoderms), and Class Cephalaspidomorphi, and more preferably, eggs or embryos of fish, shrimp, sea urchin, or amphibians. In some embodiments, unfertilized but activated eggs of fish, shrimp, sea urchin, or amphibians are used. The present invention is not limited to the use of any particular type of egg. Indeed, a variety of eggs are contemplated for use, including, but not limited to, eggs from Xenopus, shrimp, sea urchin, salmon, trout, or zebrafish. In some embodiments, the eggs are collected from mature females and spontaneously activate upon contact with water. In further embodiments, the eggs are washed in Ringer's saline. In some embodiments, the eggs are not from avian species.

[0044] The extracts of the present invention can be prepared from any of the sources described herein. In some embodiments, the extract is a cellular extract. The cellular extracts of the present invention are preferably composed of cells, such as disrupted eggs. The cells can be disrupted by various methods, including, but not limited to, mechanical shearing or blending, sonication, or osmotic lysis. In some embodiments, the cellular extract is preferably further processed to produce a composition that is substantially free of lipids naturally associated with the cells (such as cell membrane components). By substantially free of lipids, we mean that the cellular extract contains less than about 1%, preferably less than about 0.5%, and more preferably less than about 0.1% lipids that are naturally associated with the cells used to make the cellular extract. In some embodiments, the extract contains less than about 1%, preferably less than 0.1%, of cholesterol or ovalbumin. Thus, in some embodiments, the cellular extract contains carbohydrates, proteins, glycosylated or otherwise modified proteins, peptides, amino acids, RNA (mRNA, sRNA, miRNA, rRNA), DNA, water, etc., and combinations thereof. In some embodiments, cellular extracts may contain small amounts of lipids naturally associated with cells, as well as nuclear components such as chromosomes, nucleic acids, and nuclear proteins. In some embodiments, cellular extracts are preferably cytoplasmic extracts or fractions prepared by removing nuclei, cell membranes, and other water-insoluble materials naturally associated with cells. In some embodiments, these components are removed by centrifuging or fractionating disrupted cells. In some embodiments, cellular extracts are preferably aqueous extracts or fractions containing water-soluble cellular components (such as proteins, mRNA, and carbohydrates).

[0045] Various methods can be used to prepare extracts. For example, in some embodiments, eggs are "dried" in a 15 mL glass centrifuge tube and ground by sedimentation at 15,000 g for 15 minutes. This produces three layers: an upper lipid fraction (collected, aliquoted, and frozen); a middle cellular or cytoplasmic fraction (also collected, aliquoted, and frozen); and a pellet fraction (discarded). In some embodiments, the cellular fraction or extract contains primarily cytoplasmic contents. The cellular fraction is used as the extract. In some embodiments, the cellular fraction may be used in combination with the lipid fraction. The cytoplasmic fraction can be further clarified by sedimentation at 50,000, 100,000, or 200,000 g to produce additional cellular extracts, which are primarily aqueous extract fractions. Regardless of the fraction used, the extract can be diluted to approximately 300 mOsm with cell lysis buffer (see above), if necessary. Thus, in some preferred embodiments, a water-soluble extract prepared from eggs or embryos is utilized.

[0046] In another embodiment, eggs are suspended in 0.5 volumes of cell lysis buffer and sonicated on ice until all eggs are lysed. Particulate matter is sedimented at 15,000 g for 15 minutes at 4°C. The supernatant is the extract. As above, the osmolality can be adjusted to 300 mOsm, if necessary. The extract can be clarified as above.

[0047] In yet another embodiment, eggs are suspended in cell lysis buffer as in Method 2. Eggs are lysed by Dounce homogenization using a glass mortar and pestle (Kontes, Type A or Type B). The lysate is sedimented and processed as above.

[0048] In some preferred embodiments, the present invention provides compositions prepared from natural sources, such as those described above, or artificial source materials, or a combination thereof. In some embodiments, the extract is characterized as having an osmolality of about 330-440 mOsm, preferably about 350 mOsm. In some embodiments, the extract has a pH of about 5.0 to about 7.7, preferably about 6.5 to 7.0. In some embodiments, the extract has a protein content of about 100-250 mg / mL, preferably about 160-190 mg / mL, and most preferably about 120 mg / mL. In some embodiments, the composition has a water content of about 20-90% water by weight, preferably about 37-79% water by weight. In some embodiments, the extract has a density of about 0.8 to about 1.4 g / mL, preferably about 1.1 g / mL. In some embodiments, the composition contains trace elements, including, but not limited to, calcium, phosphorus, zinc, copper, and iron. In some embodiments, the composition contains vitamins, including, but not limited to, vitamins A, C, E, riboflavin, niacin, B6, calcium pantothenate, and B12. In some embodiments, the present invention provides a fresh egg (roe) composition, containing 2.7-3.4% protein present in fresh egg (roe); 3-5% carbohydrates; 1.0-1.7% lipid in the form of phospholipids; 0.01-0.05% minerals (the lipid content should be lower and the total protein content higher in the extract); and 37-79% water by weight. In some embodiments, the extract further contains a lipid fraction. In some embodiments, the lipid fraction contains about 60% to about 80% unsaturated fatty acids. In further embodiments, the composition contains phospholipids, including phosphatidylcholine (lecithin) or phosphatidylethanolamine (cephalin), and, to a lesser extent, inositol phosphatides, cerebrosides, and sphingomyelin.In some embodiments, the lipid fraction is about 0.1% to about 1%, 2%, 3%, 4% or 5% of the total composition, while in other embodiments, the composition is substantially free or free of lipids.

[0049] In some embodiments, the eggs or extracts are treated to prevent bacterial growth. Various methods are contemplated. In some embodiments, the following methods are combined. In some embodiments, unfertilized or fertilized eggs (e.g., fish or amphibian eggs) are treated with a bactericidal or bacteriostatic agent before homogenization. Preferred agents include, but are not limited to, iodine-containing agents such as betadine, buffodine, and povidone-iodine, as well as other agents such as novasan, sodium hypochlorite, bacitracin, polymyxin B sulfate, silver-containing compounds such as silver sulfadiazine and silver nitrate, mafenide acetate, nystatin, gentamicin, and neomycin. In other embodiments, the extract is treated after homogenization to prevent bacterial growth. In some embodiments, the extract, such as a cellular extract or cytoplasmic fraction, is treated by heating. In some embodiments, the extract is heated to about 37, 40, 50, 60, 70, 80, or 90° C. for about 30 seconds or for 1, 2, 5, 10, 20, 30, 60, or 120 minutes.

[0050] In some embodiments, the eggs or extract are preferably filtered through a 0.22 or 0.45 μm filter to remove bacteria. In some embodiments, before or after filtration, the extract is heated (to about 37, 40, 50, 56, 60, 70, 80, 90, or 95°C for about 30 seconds or for 1, 2, 5, 10, 20, 30, 60, or 120 minutes), followed by further centrifugation (15 minutes to 2 hours) to spin down any bacteria present. Surprisingly, it has been found that the biological activity of the extract is retained even after aggressive heat treatment. In some embodiments, the retained biological activity is the ability to promote mitochondrial activity and / or increase hyaluronic acid production in skin fibroblasts.

[0051] In some embodiments, eggs are washed with a sulfur-containing agent (e.g., calcium polysulfide or calcium thiosulfate (lime sulfur)) before preparation. In some embodiments, sulfur is added to the extract to remove bacteria. In other embodiments, benzoyl peroxide is added to the extract. In some embodiments, eggs are washed with 0.001% to about 0.2% by weight of a metal chlorite and sufficient acid to adjust the pH of the solution to about 2.2 to about 4.5 to remove bacteria. In further embodiments, the eggs and / or extract are placed in a vacuum drum and mixed with a neutral solution containing salt, vitamin C or citric acid, and water to remove bacteria. In some embodiments, the eggs and / or extract are stirred, vortexed, sonicated, swirled, or shaken with brine or a liquid buffer to remove bacteria, and the liquid is removed using a vacuum filter to remove bacteria. For quality control, it may be possible to check the bacterial content in the liquid and in the processed eggs. In some embodiments, electrophoresis of the eggs and / or extract is used to remove bacteria. Such methods are intended to exploit the effects of the electric double layer, electric field strength, electric density gradient, buffer pH, buffer ionic strength, bacterial growth stage, and anionic surfactants on the electrophoretic mobility of several species of bacteria.

[0052] In some embodiments, lipids are removed by treating the homogenate before centrifugation or the extract after centrifugation. Various methods are contemplated. In some embodiments, lipids are removed by filtering through lipid-absorbing paper or a filter by applying a vacuum suction system to a container with a filter at the bottom, where the extract is placed in the container and sucked through the filter. In some embodiments, lipids are removed by using an absorbent material and an outer containment vessel. The extract is pumped into a container filled with absorbent material and then collected by applying a vacuum. In some embodiments, lipids are removed using a hollow fiber contraction system and / or an extraction solvent to remove lipids from a viscous liquid, where lipids in the liquid are separated from the liquid or lipids in a lipid-containing organism are separated from the lipid-containing organism by contacting the liquid with an extraction solvent using at least one hollow fiber contactor.

[0053] In some embodiments, the homogenate and extract may be stabilized by adding one or more stabilizers, such as lipid stabilizers, or by packaging in a package designed to prevent oxidation. In some embodiments, an antioxidant, such as vitamin E, is added to the extract to reduce the rate of lipid oxidation. In some embodiments, the extract is packaged in a container under an inert atmosphere. In some embodiments, the extract is packaged in an air-free container, such as an aluminum-coated bag (less than 10 kg per bag for efficient oxygen removal), or in a container filled with nitrogen to remove oxygen, to reduce the rate of lipid oxidation. In other embodiments, the extract is packaged in a vacuum-packed container using a pump delivery system.

[0054] In some embodiments utilizing fish roe, the fish roe is treated as described above to prevent bacterial growth. The fish roe is then homogenized by subjecting it to pressure treatment. In some embodiments, the roe is subjected to a pressure of about 1 ton to about 100 tons, preferably about 5 to about 50 tons, more preferably about 10 to about 30 tons, and most preferably about 20 tons. In some embodiments, the pressure is applied via a hydropress. Suitable hydropresses are available from Speidel. In some embodiments, the components of the homogenate are separated. In some preferred embodiments, an aqueous cytoplasmic fraction is obtained, which contains proteins, DNA, RNA, and other components as described in more detail elsewhere herein. In some embodiments, the extract contains lipid components in addition to water-soluble components. In some embodiments, the extract is separated from the homogenate by centrifugation. In some embodiments, the centrifugation is a continuous feed process facilitated by a separator. Suitable separators are available, for example, from GEA Westfalia.

[0055] In some embodiments, the cell extract, most preferably the intermediate fraction, is further fractionated.Various methods can be used, including but not limited to FICOL gradient, gradient centrifugation, protein precipitation, lyophilization, column chromatography (such as size exclusion chromatography and affinity chromatography), gel separation, high-pressure liquid chromatography, ChIP, and immunoprecipitation.It will be appreciated that these fractionation steps will produce corresponding fractions, such as lyophilized fractions, affinity chromatography fractions, precipitated fractions, etc.

[0056] Thus, in some embodiments, the present invention provides powders prepared from extracts of the cells described above. In some embodiments, the extract of cells used to produce the powder is prepared from salmonid eggs. In some embodiments, the extract of cells used to produce the powder is prepared from salmon or trout eggs. In some embodiments, the powder is biologically active. In some preferred embodiments, the powder is lyophilized. In some embodiments, the powder contains less than about 10% moisture, and most preferably less than about 5% moisture; protein at a concentration of about 500 to about 800 mg / g powder, preferably about 600 to about 700 mg / g powder, and most preferably about 640 mg / g powder; DNA at a concentration of about 1 to about 50 μl / mg powder, preferably about 5 to about 25 μl / mg powder, and most preferably about 16 μl / mg powder; total RNA (e.g., mRNA, rRNA, and microRNA) at a concentration of about 1 to about 50 μl / mg powder, preferably about 5 to about 20 μl / mg powder, and most preferably about 12 μl / mg powder; and lipids at a concentration of about 100 to about 200 mg / g powder, most preferably about 150 mg / g powder. Powders can preferably be used as a substitute for non-powdered cell extracts to produce the formulations described herein.

[0057] In some embodiments, the fractions are then combined or resolubilized with appropriate ingredients to prepare compositions for topical administration, as described in more detail below.

[0058] As mentioned above, the present invention is not limited to the use of any particular wound healing agent.Other suitable wound healing agents include, but are not limited to, antibacterial polypeptides (such as alpha-defensin, LL37, beta-defensin, etc.); antibacterial drugs, such as pharmaceutically acceptable salts of beta-lactam drugs, quinolone drugs, ciprofloxacin, norfloxacin, tetracycline, erythromycin, amikacin, triclosan, doxycycline, capreomycin, chlorhexidine, chlortetracycline, oxytetracycline, clindamycin, ethambutol, hexamidine isothionate, metronidazole; pentamidine, gentamicin, kanamycin, lineomycin, methacycline, methenamine, minocycline, neomycin, netilmicin, paromomycin, streptomycin, tobramycin, miconazole, and amanfadine; antibacterial silver compositions, etc.

[0059] In yet other preferred embodiments, the recombinant enzymes described herein, including recombinant LCEs and HCEs, can be used to treat skin conditions and diseases. For example, the compositions can be used in cosmetics as exfoliants, moisturizers, or to improve abnormal skin pigmentation, such as melasma, age spots, sun spots, or brown spots. In some preferred embodiments, the recombinant enzymes can be provided in suitable cosmetic or pharmaceutical formulations, such as those described below. Diseases and conditions that can be treated using the recombinant enzymes and formulations described herein include dry skin, hyperkeratosis, calluses, warts, melasma, age spots, stretch marks, sun spots, brown spots, acne, eczema, and psoriasis.

[0060] C. Formulations and Devices In some embodiments, the active agent (i.e., a debridement enzyme such as LCE or HCE and / or a wound-healing agent) is formulated for delivery by various methods. In some embodiments, the extract is formulated for delivery to the skin, gastrointestinal tract, fat deposits, cartilage, bone, connective tissue, muscle, or internal organs. In some embodiments, the active agent is formulated for topical delivery. Typical formulations for topical delivery are described in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing, pp. 1288-1300 (1990). In accordance with the compositions and methods of the present invention, the active agent of the present invention can be administered in the form of a pharmaceutical composition additionally comprising a pharmaceutically acceptable carrier. Those skilled in the art will understand that suitable methods of administering the extract composition to animals, such as mammals, are available, and that while more than one method may be used to administer a particular composition, certain methods and doses may provide a more rapid and effective response than others. Pharmaceutically acceptable carriers are also well known to those skilled in the art. The choice of carrier will be determined, in part, by both the particular composition and the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical composition of the present invention.

[0061] In some preferred embodiments, the formulations of the present invention are designed for topical administration. Typical of such formulations are sprays, ointments, creams, and gels.

[0062] Thus, in some preferred embodiments, the recombinant LCE or HCE enzymes described herein are provided in pharmaceutical or cosmetic formulations. In some preferred embodiments, the formulations include one or more pharmaceutically or cosmetically acceptable excipients and / or diluents. In some preferred embodiments, the formulations are gels, creams, ointments, lotions, foams, non-aqueous solutions, sprays, salves, sticks, soaps, powders, films, emulsions, suspensions, or dispersions.

[0063] Ointments are generally prepared using either (1) an oleaginous base (i.e., composed of fixed oils or hydrocarbons), such as white petrolatum or mineral oil, or (2) an absorbent base (i.e., composed of anhydrous substances or substances capable of absorbing water), such as anhydrous lanolin. Usually, after forming the base, whether oleaginous or absorbent, the active ingredient (e.g., salmon egg extract) is added in an amount to give the desired concentration.

[0064] Creams are oil / water emulsions. They typically consist of an oil phase (internal phase) containing fixed oils, hydrocarbons (waxes, petrolatum, mineral oil, etc.), and an aqueous phase (continuous phase) containing water and water-soluble substances (added salts, etc.). The two phases are stabilized by the use of emulsifiers, e.g., surfactants such as sodium lauryl sulfate; hydrophilic colloids such as acacia colloidal clay, begum, etc. Once the emulsion is formed, active ingredients (e.g., salmon roe extract) are usually added in amounts to achieve the desired concentration.

[0065] The gel comprises a base selected from the above-mentioned oil base, water, or emulsion-suspension base. A gel additive that forms a matrix in the base is added to the base to increase its viscosity. Examples of gelling agents include hydroxypropyl cellulose, acrylic acid polymers, etc. Typically, the active ingredient (IGF-II) is added to the formulation at a desired concentration before adding the gelling agent.

[0066] Serums can be watery or thicker liquids and are often (but not always) clear in color. Serums are aqueous and have a light consistency. They are easily and quickly absorbed by the skin and provide an excellent method for delivering topical ingredients, including vitamin C, peptides, alpha hydroxy acids, and retinol. Serums may be layered under other serums, as well as creams or lotions, making them a very emollient product for incorporating into skin treatment regimens. Serums are well tolerated by all skin types, unless the individual is sensitive to any of the ingredients. Serums may also contain glycerol or glycerin. The amount of extract incorporated into the formulations of the present invention is not critical; the concentration should be sufficient to allow easy application of an amount of the formulation to the wound area that delivers the desired amount of extract.

[0067] Sprays preferably include both aerosol and pump spray formulations.

[0068] The present invention can be formulated, if necessary, with additives commonly used in pharmacy, such as surfactants, oils and fats, polyhydric alcohols, lower alcohols, thickeners, ultraviolet absorbers, light scattering agents, preservatives, antioxidants, antibiotics, chelating agents, pH adjusters, flavoring agents, pigments, and water.

[0069] Examples of the surfactant include polyoxyethylene (hereinafter abbreviated as POE)-chain alkyl ethers such as POE-octyldodecyl alcohol and POE-2-decyltetradecyl alcohol, POE-alkyl ethers such as POE-oleyl alcohol ether and POE-cetyl alcohol ether, sorbitan esters such as sorbitan monooleate, sorbitan monoisostearate and sorbitan monolaurate, sorbitan monooleate, POE-sorbitan monoisostearate and POE-sorbitan monolaurate, and the like. POE-sorbitan esters such as sorbitan, fatty acid esters of glycerol such as glyceryl monooleate, glyceryl monostearate and glyceryl monomyristate, POE-fatty acid esters of glycerol such as POE-glyceryl monooleate, POE-glyceryl monostearate and POE-glyceryl monomyristate, POE-dihydrocholesterol ester, POE-hydrogenated castor oil fatty acid esters such as POE-hydrogenated castor oil and POE-isostearate, POE-octylphenol ether, etc. E-Alkyl aryl ethers, glycerol esters such as glycerol monoisostearate and glycerol monomyristate, POE-glycerol esters such as POE-glycerol monoisostearate and POE-glycerol monomyristate, polyglycerol fatty acid esters such as diglyceryl monostearate, decaglyceryl decaglyceryl decaisostearate and diglyceryl diisostearate, and other nonionic surfactants; potassium salts, sodium salts, diethanolamine salts, triglycerides, etc. These include triethanolamine salts, amino acid salts and salts of other higher fatty acids (e.g., myristic acid, stearic acid, palmitic acid, behenic acid, isostearic acid and oleic acid), alkali salts of the above-mentioned ether carboxylic acids, salts of N-acylamino acids, N-acylsarconates, higher alkyl sulfonates and other anionic surfactants; alkylamine salts, polyamines, aminoalcohol fatty acids, organic silicone resins, alkyl quaternary ammonium salts and other cationic surfactants; and lecithin, betaine derivatives and other amphoteric surfactants.

[0070] Examples of fats and oils include vegetable fats and oils such as castor oil, olive oil, cocoa oil, camellia oil, coconut oil, wood wax, jojoba oil, grapeseed oil, and avocado oil; animal fats and oils such as mink oil and egg yolk oil; waxes such as beeswax, spermaceti, lanolin, carnauba wax, and candelilla wax; hydrocarbons such as liquid paraffin, squalene, microcrystalline wax, ceresin wax, paraffin wax, and petrolatum; natural or synthetic fatty acids such as lauric acid, myristic acid, stearic acid, oleic acid, isostearic acid, and behenic acid; natural or higher alcohols such as cetyl alcohol, stearyl alcohol, hexyldecanol, octyldecanol, and lauryl alcohol; and esters such as isopropyl myristate, isopropyl palmitate, octyldodecyl myristate, octyldodecyl oleate, and cholesterol oleate.

[0071] Examples of polyhydric alcohols include ethylene glycol, polyethylene glycol, propylene glycol, 1,3-butylene glycol, 1,4-butylene glycol, dipropylene glycol, glycerol, diglycerol, triglycerol, tetraglycerol and other polyglycerols, glucose, maltose, maltose, sucrose, fructose, xylitose, sorbitol, maltotriose, threitol and erythritol.

[0072] Examples of thickening agents include naturally occurring polymeric substances such as sodium alginate, xanthan gum, aluminum silicate, quince seed extract, tragacanth gum, starch, collagen, and sodium hyaluronate; semi-synthetic polymeric substances such as methylcellulose, hydroxyethylcellulose, carboxymethylcellulose, soluble starch, and cationized cellulose; and synthetic polymeric substances such as carboxyvinyl polymers and polyvinyl alcohol.

[0073] Examples of ultraviolet absorbers include p-aminobenzoic acid, 2-ethoxyethyl p-methoxycinnamate, isopropyl p-methoxycinnamate, butyl methoxybenzoylmethane, glyceryl-mono-2-ethylhexanoyl-di-p-methoxybenzophenone, digalloyl trioleate, 2,2'-dihydroxy-4-methoxybenzophenone, ethyl-4-bishydroxypropylaminobenzoate, 2-ethylhexyl-2-cyano-3,3'-diphenylacrylate, ethylhexyl p-methoxycinnamate, ethylhexyl 2-salicylate, glyceryl p-aminobenzoate, homomethyl salicylate, methyl o-aminobenzoate, 2-hydroxy-4-methoxybenzophenone, amyl p-dimethylaminobenzoate, 2-phenylbenzimidazole-5-sulfonic acid, and 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid.

[0074] Examples of preservatives include benzoate, salicylate, sorbate, dehydroacetate, p-oxybenzoate, 2,4,4'-trichloro-2'-hydroxydiphenyl ether, 3,4,4'-trichlorocarbanilide, benzalkonium chloride, hinokitiol, resorcinol, and ethanol.

[0075] Examples of antioxidants include tocopherol, ascorbic acid, butylhydroxyanisole, dibutylhydroxytoluene, nordihydroguaiaretic acid, and propyl gallate.

[0076] Examples of chelating agents include sodium edetate and sodium citrate.

[0077] Examples of antibiotics include penicillin, neomycin, cephalothin, potassium permanganate, selenium sulfide, erythromycin, bacitracin, thetacycline, chloramphenicol, vancomycin, nitrofurantoin, acrisorcin, chlorodontoin, and flucytosine.

[0078] Some of these additives function to enhance the effectiveness of the composition by increasing the stability or transdermal absorption of the essential ingredients of the present invention.

[0079] Also, any dosage form is acceptable, whether it is a solution, emulsion, powder dispersion, or other. The applicability is broad and includes basic dosage forms such as lotions, emulsions, creams, and gels.

[0080] In addition to those mentioned above, suitable vehicles, carriers and adjuvants include water, petrolatum, petrolatum, mineral oil, vegetable oil, animal oil, organic and inorganic waxes, polymers such as xanthan, gelatin, cellulose, collagen, starch, kaolin, carrageenan, gum arabic, synthetic polymers, alcohols, polyols, etc. Carriers also include sustained release carriers such as liposomes, microsponges, microspheres, or microcapsules, aqueous-based ointments, water-in-oil or oil-in-water emulsions, gels, etc.

[0081] In addition to being included in topical delivery formulations, the active agents described above can be included in devices such as wound dressings and dressings, adhesive bandages, wound pads, skin patches, and similar devices. For example, the active agent can be included in one or more layers of such devices, allowing the active agent to be released from the device to the wound bed. Thus, in some embodiments, the active agent can be incorporated into a wound dressing or biological wound dressing that is suitable for functionalization. Examples of commercially available wound dressings that can be modified by adding the active agents of the present invention include, but are not limited to, Biobrane™, gauze, adhesive tape, bandages such as Band-Aids™, and other commercially available wound dressings (including, but not limited to, COMPEEL™, DUODERM™, TAGADERM™, and OPSITE™).

[0082] [Example] Example 1 Generation of recombinant LCEs from trout The ORF encoding trout coriolisin (LCE1) was cloned into pMAL-c4x. In this design, an amino-terminal maltose nucleotide sequence, which binds the protein to an 8x histidine tag, was fused to a TEV protease site and cloned into the amino terminus of LCE1. The expression construct was transformed into Rosetta(DE3)pLysS under ampicillin selection. The resulting strain was designated Rosetta(DE3)pLysS / MBP_LCE1. A single colony of Rosetta(DE3)pLysS / MBP_LCE1 expression was grown overnight in 3 mL of LBAMP100 with vigorous shaking at 37°C. The overnight growth was subcultured at a 1 / 125 ratio into 25 mL of LBAMP100. When these cultures reached a density of 0.3-0.5 A600, an uninduced sample was taken, and the culture was induced with 0.5 mM IPTG. After 4 hours of shaking at 37°C, the induced sample was harvested. The uninduced and induced samples were resolved on 10% SDS-PAGE and stained with Coomassie Brilliant Blue. The gel was destained and imaged (Figure 1).

[0083] Example 2 Measurement of protease activity of recombinant LCE1 Samples collected as described in Example 1 were analyzed for the presence of activated proteases. Samples were aliquoted into 20 μL equivalent fractions, as determined by bacterial cell density. 20 μg of lysozyme was added to each aliquot and incubated at room temperature for at least 30 minutes. The protease activity of the expressed LCE was measured in an assay using fluorescein isothiocyanate (FITC)-labeled casein as a general protease substrate. The fluorescein label on FITC-casein is highly quenched. Upon digestion with LCE1 present in the sample, the FITC-casein substrate is cleaved into smaller peptides, which abolish quenching of the fluorescent label. Fluorescence of the FITC-labeled peptide fragments was measured at Ex / Em = 485 / 530 nm. Mass spectrometry grade (MSG), chemically stabilized trypsin was used as a general protease control. The results are shown in Figures 2 and 3. As shown, the activity of soluble LCE was improved by the addition of urea and zinc chloride (Figure 3).

[0084] Example 3 Preparation of fish roe extract Fresh, unfertilized salmon (Salmo salar) eggs collected from reproductive females (late fall) are kept on ice, and extracts are preferably prepared immediately. Dried eggs can be frozen in a cryoprotectant (e.g., 1.5 M 1,2-propanediol and 0.2 M sucrose) without disrupting the egg membrane. Freezing should be performed gradually (-1°C / min) to -80°C. Eggs must be thawed and kept on ice throughout the extract preparation process.

[0085] Eggs are washed twice in HBSS or seawater with protease inhibitors (10 μg / mL). The wash solution is removed, and the eggs are lysed and homogenized in a pre-chilled Dounce-glass homogenizer. While avoiding the transfer of eggshells, the lysate is transferred to a Beckman Ultra Clear polyallomer centrifuge tube (5 mL) and centrifuged at 15,000 g for 15 minutes at 4°C in a Beckman ultracentrifuge using an SW55T1 rotor. This yields three fractions: an upper lipid fraction, a middle cytoplasmic fraction, and a lower fraction containing eggshell and nuclear debris. The middle cytoplasmic fraction is the collected extract. This extract is expected to contain most cytosolic organelles, including mitochondria, lysosomes, and peroxisomes, and should be clear, viscous, and orange in color. Protease inhibitors (10 μg / mL stock) are added, and the extract is stored at -80°C.

[0086] Further fractionation of the cytoplasmic extract is possible. Centrifugation at 100,000 g for 60 minutes at 4°C yields two to three fractions, where the upper / middle cytoplasmic fraction contains the cytosol with endoplasmic reticulum, SVs, and microsomes. The pH of the extract is measured with litmus paper, the protein concentration by Bradford assay, and the osmolality by osmometer.

[0087] Mid-blastula stage zebrafish embryos are collected, the liquid removed, and frozen to -20°C. To prepare the extract, the embryos are thawed on ice, lysed, and homogenized using a Dounce-glass homogenizer in a small volume of HBSS or seawater (preferably less than 50% liquid by volume). The lysate is filtered through sterile linen cloth and centrifuged at 5,000 g for 20 minutes at 4°C in an SX4250 rotor using a Beckman X-22R centrifuge. The cytoplasmic extract (supernatant) is collected and protease inhibitors (10 μg / mL) are added. The extract can be Millipore filtered (0.22 μm MilliQ sterile filter). The extract is stored at -80°C. The pH of the extract is measured using litmus paper, the protein concentration is measured by Bradford assay, and the osmolality is measured using an osmometer.

[0088] This general process is useful for preparing extracts from sea urchin, shrimp, fish eggs / roe, or frog eggs. Briefly, eggs (roe) are collected from gravid female fish or gravid frogs immediately after egg release during a spawning program (hCG hormone injection (1 mL / kg) 6–8 h before egg release, usually at dawn (2–4 AM)). The eggs / roe are either freeze-dried, frozen at −20°C, or used fresh. Roe are collected from different species of fish. For sea urchins, 0.5 M KCl is injected around the mouth to induce egg release. Extracts are prepared from eggs / roe by disruption (cell cracker or dounce homogenization) or centrifugation at different speeds (with total contents, with / without eggshell (zona pellucida), with / without nucleus / cytoplasm, with / without organelles, with / without lipids, to separate cytoplasm). Further fractionation may be performed to isolate one or more of mRNA, proteins, small peptides, carbohydrates, and lipids. The main components of fatty acids in eggs / roe are oleic acid, linoleic acid, and omega-3 fatty acids.

[0089] Applying the above protocol to salmon egg extracts, the salmon egg extracts had surprisingly high protein concentrations ranging from 100 to 380 mg / mL, pHs ranging from 6.4 to 6.8, and osmolality of approximately 350 mOsm. The extracts were clear and viscous and could not be filtered (through a 0.45 μm MilliQ filter). Protein in the extracts readily precipitated upon the addition of water or aqueous solutions with low buffering capacity due to the high protein content and low pH. The extracts could be neutralized to pH 7.0 by adding alkaline (1 to 3 μL of 1 M NaOH / mL extract), thereby allowing dilution in water and aqueous solutions. Zebrafish extracts had protein concentrations ranging from 23 to 26 mg / mL, pHs ranging from 6.4 to 6.8, and osmolality ranging from 80 to 150 mOsm. The extracts were clear, non-viscous, filterable, and readily diluted in water at all dilutions.

[0090] The physical properties of the extracts of various fish eggs produced by these methods, including the contents of RNA, DNA and protein, were further evaluated using a Qube-iT fluorometer from InVitrogen.

[0091] Salmon egg homogenates (uncentrifuged) contain 3-4 mg / mL of RNA. After centrifugation at 9-15,000 g, the RNA content decreased to 2-3 mg / mL. This is likely due to the RNA being centrifuged or degraded. Interestingly, trout egg homogenates (uncentrifuged) contain 2-3 mg / mL of RNA, but after centrifugation at 9-15,000 g, the RNAsis concentration increased to 3-5 mg / mL. Extracts made from trout eggs are less viscous than extracts made from salmon eggs, allowing the RNA to better remain in aqueous suspension during centrifugation.

[0092] Salmon egg homogenates (uncentrifuged) contain 60–200 μg / mL of DNA. After centrifugation at 9–15,000 g, the DNA content decreased to 40–51 μg / mL, likely due to the DNA being centrifuged. Interestingly, trout egg homogenates (uncentrifuged) contain more DNA than salmon egg extracts: 130–530 μg / mL of DNA. After centrifugation at 9–15,000 g, the DNA content decreased to 70–125 μg / mL, but still higher than comparable salmon egg extracts. Extracts made from trout eggs are less viscous than extracts made from salmon eggs, allowing the DNA to better remain suspended in the aqueous phase during centrifugation.

[0093] Salmon egg homogenates (uncentrifuged) contain 180–260 mg / mL of protein. After centrifugation at 9–15,000 g, the protein content either remained unchanged or increased slightly to 200–260 mg / mL. Trout egg homogenates (uncentrifuged) contain 250–300 mg / mL of protein, and after centrifugation at 9–15,000 g, the protein content remains approximately the same (250–270 mg / mL). The protein fraction of the egg cytoplasm is not expected to be spun down at the applied g-force and can be expected to be similar to the unprocessed protein content of the egg cytoplasm.

[0094] Previous measurements of protein content in extracts using the Nano-drop spectrophotometer showed a range of 150-250 mg / mL. This may be due to the Nano-drop's upper detection limit of approximately 250 mg / mL. The slightly higher fluorometer readings shown here are considered more accurate.

[0095] [Table 2]

[0096] The lipid content of the extracts was measured by ALS (Germany) and found to be within a narrow range of 3.7-4.5 g / 100 g for all extracts derived from salmon or trout roe (roe) prepared by centrifugation between 1,700 g and 15,000 g. Centrifugation at lower g-forces likely requires spinning at room temperature, thereby yielding lipid fractions equivalent to higher g-forces at 4°C. At centrifugal forces lower than 1,700 g, which are also applicable for extract production, the lipid content can be higher (4-7%). The extracts may contain the following lipids (right column), and the lipid fraction removed from the extract during production may contain the following lipids (left column):

[0097] [Table 3] JPEG0007776142000005.jpg76169

[0098] Example 4. Extract Production As described in Example 1, the lipid content of the extract surprisingly does not change with centrifugation speeds ranging from 1,700 g to 15,000 g (see above), while other parameters such as RNA, DNA and protein content change with increasing g-force during centrifugation. Extracts can also be produced at lower centrifugal forces, up to 400 g, which can result in higher contents, especially of lipids.

[0099] An additional step of washing the eggs with buffodine (1:100 in 0.9% NaCl) for 10 minutes before preparing the homogenate is beneficial. This washing step appears to significantly reduce bacterial content. For safety reasons, all LEX batches packaged in final containers are gently pasteurized (incubated) by heating to 56°C for 20 minutes. This pasteurization completely sterilizes the extract, and zero bacteria are found in extracts plated on bacterial plates incubated at room temperature, 4°C, or 30°C for 3 days. A maximum of one colony per 100 μL of LEX is observed on agar plates incubated at room temperature. This is 100-fold lower than the safety limit for drinking water (100 bacteria / mL). The rarely observed single colonies likely arise from the air during LEX plating and are comparable to bacterial growth in the negative control (plate only). LEX stability and collagen secretion effects are maintained even after heating LEX to 56°C for 20 minutes. When applied to human fibroblasts in vitro at a concentration of 0.5% in cell culture medium for 8 days (medium was changed daily), the effect on collagen secretion (measured as collagen efflux from cells into cell culture medium and compared to untreated control cells) was comparable to cells treated with unheated extract kept at -80°C after preparation. Increases of 200-400% compared to the control were observed for both heated and unheated LEX.

[0100] Heat-treated LEX (HEX) was produced from salmon egg extract (LEX) prepared as described above. For HTX, salmon egg extract was diluted 1:10 in sterile 1x PBS buffer; 0.5 mL aliquots were prepared. All samples were heated to 95°C for 5 minutes, cooled, and then centrifuged at 12,100 x g for 2 minutes. The supernatant was collected, and the samples were labeled and stored at -80°C until use. [Brief explanation of the drawings]

[0101] [Figure 1] Gel showing the results of overexpressing LCE1 in E. coli. [Figure 2]Protease activity of fractions derived from recombinant LCE1 overexpression. [Figure 3] Soluble recombinant LCE activity upon addition of urea and zinc chloride.

Claims

1. 10. Use of a metalloprotease in the manufacture of a medicament for a method of debridement of a wound, wherein the metalloprotease is low membrane-degrading enzyme (LCE).

2. 2. The use of claim 1, wherein the effective amount of the metalloprotease is effective to remove devitalized tissue in a wound.

3. The use according to claim 1, wherein the LCE is an LCE of Oncorhynchus mykiss.

4. The use according to any one of claims 1 to 3, wherein the LCE has at least 90% sequence identity with SEQ ID NO: 1 and has LCE activity.

5. The use according to any one of claims 1 to 4, wherein the LCE is a recombinant LCE.

6. The use according to claim 5, wherein the recombinant LCE comprises a his-tag.

7. The use according to any one of claims 1 to 6, wherein the metalloprotease is provided in a matrix, bandage, wound dressing, cream, gel, emulsion, ointment, spray, powder or lotion.

8. The use according to any one of claims 1 to 7, wherein the method further comprises the step of applying a wound healing agent to the wound after debridement.

9. The use according to claim 8, wherein the wound healing agent comprises an extract of differentiable cells.

10. 10. The use according to claim 9, wherein the extract of differentiable cells is an extract of fish eggs.

11. The use according to claim 10, wherein the extract of fish roe comprises: 100-380 mg / mL protein in aqueous solution; 0.1-10 mg / mL RNA; 0.1 to 5 mg / mL of DNA; and 0.1 to 10% by weight lipids.

12. 12. The use according to claim 11, wherein the extract of fish roe is heat treated by heating the extract to above 80°C, above 90°C, above 95°C or above 100°C.

13. The use according to claim 12, wherein the heat treatment is carried out for 1 minute to 30 minutes.

14. The use according to any one of claims 10 to 13, wherein the fish roe extract is a salmon roe extract.

15. The use according to any one of claims 8 to 14, wherein the wound healing agent is provided in a matrix, bandage, wound dressing, cream, gel, emulsion, ointment, spray, powder or lotion.

16. The use according to any one of claims 1 to 15, wherein the wound is a chronic wound.

17. 17. The use according to claim 16, wherein the chronic wound is a diabetic ulcer.

18. The use according to any one of claims 1 to 17, wherein the LCE is a formulation comprising urea and / or zinc chloride in an aqueous solution.

19. 19. The use of claim 18, wherein the urea is provided at a concentration of 1 to 10 M.

20. 19. The use of claim 18, wherein the zinc chloride is provided at a concentration of 2 to 40 mM.

21. 1. A kit or system for treating a wound, comprising: a first container containing a metalloprotease in an amount effective to remove devitalized tissue from the wound; and a second container containing a wound healing agent; Equipped with The kit or system, wherein the metalloprotease is low egg membrane enzyme (LCE).

22. 22. The kit or system of claim 21, wherein the LCE is an Oncorhynchus mykiss LCE.

23. 23. The kit or system of claim 21 or 22, wherein the LCE has at least 90% sequence identity with SEQ ID NO: 1 and has LCE activity.

24. The kit or system according to any one of claims 21 to 23, wherein the LCE is a recombinant LCE.

25. The kit or system of claim 24, wherein the recombinant LCE comprises a his-tag.

26. 26. The kit or system of any one of claims 21 to 25, wherein the metalloprotease is provided in a matrix, bandage, wound dressing, cream, gel, emulsion, ointment, spray, powder, or lotion.

27. 27. The kit or system of claim 26, wherein the wound healing agent comprises an extract of differentiable cells.

28. 28. The kit or system of claim 27, wherein the extract of differentiable cells is an extract of fish eggs.

29. 29. The kit or system of claim 28, wherein the fish roe extract comprises: 100-380 mg / mL protein in aqueous solution; 0.1-10 mg / mL RNA; 0.1 to 5 mg / mL of DNA; and 0.1 to 10% by weight lipids.

30. 30. The kit or system of claim 29, wherein the fish roe extract is heat treated by heating the extract to above 80°C, above 90°C, above 95°C or above 100°C.

31. 31. The kit or system of claim 30, wherein the heat treatment is performed for 1 minute to 30 minutes.

32. 32. The kit or system of any one of claims 26 to 31, wherein the wound healing agent is provided in a matrix, bandage, wound dressing, cream, gel, emulsion, ointment, spray, powder or lotion.

33. 33. The kit or system of any one of claims 21 to 32, wherein the LCE is a formulation comprising urea and / or zinc chloride in an aqueous solution.

34. 34. The kit or system of claim 33, wherein the urea is provided at a concentration of 1 to 10 M.

35. 35. The kit or system of claim 34, wherein the zinc chloride is provided at a concentration of 2 to 40 mM.

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