Pharmaceutical composition for treating wounds

Abstract

The present invention relates to a pharmaceutical composition for treating wounds, wherein the pharmaceutical composition contains silk protein and plasma components as active ingredients. According to the present invention, wounds can be healed more effectively by utilizing the synergistic effect of silk protein and plasma components.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for treating wounds, and more particularly to a pharmaceutical composition comprising silk protein and a plasma or serum component for treating wounds. Background Art

[0002] Substances known to date for the treatment of skin wounds include growth factors (such as epidermal growth factor (EGF), acidic and basic fibroblast growth factors (acidic and basic FGF), transforming growth factors (TGF-α and TGF-β) and insulin-like growth factors (IGF-I and II)), adhesion factors (such as fibronectin, laminin and vitronectin), retinoids and retinoid-like compounds, etc.

[0003] Cytokines have been identified as growth factors associated with wound treatment, and representative examples thereof include: basic fibroblast growth factor, which is produced in keratinocytes and fibroblasts and promotes the growth of epithelial cells; platelet-derived growth factor (PDGF), which is produced in platelet endothelial tissue and promotes the proliferation of epithelial cells together with epidermal growth factor (EGF); transforming growth factor-β (TGF-β), which is produced in fibroblasts and platelets and promotes the formation of connective tissue; epithelial growth factor, which is produced by salivary glands and promotes the proliferation of epithelial cells; fibroblast growth factor (FGF); and interleukin-1, which is produced in macrophages and epithelial cells and promotes the growth and movement of epithelial cells. Becaplermin is commercially available as a topical wound treatment agent by Johnson & Johnson under the trade name "Regranex", which is a genetically engineered PDGF. EP Patent No. 0575484 discloses a pharmaceutical composition comprising PDGF and dexamethasone for regeneration and repair of mammalian tissue. U.S. Patent No. 5,981,606 discloses a pharmaceutical composition comprising TGF-β for wound healing. WO 96 / 30038 discloses a pharmaceutical composition comprising TGF-β, fibric acid and an antioxidant for wound healing. U.S. Patent No. 5,183,805 discloses a pharmaceutical composition comprising EGF and thus having the effect of regenerating tissue. Japanese Patent No. 05070365 and U.S. Patent No. 6,165,978 disclose a wound therapeutic agent comprising FGF.

[0004] Formulations utilizing hyaluronic acid as an active ingredient have also been reported to be useful for treating skin wounds (U.S. Pat. No. 5,897,880). LAM Pharmaceutics markets IPN Wound Gel TM Preparations containing sodium hyaluronate are sold under the trade name HYALURONICA®.

[0005] Topically applied fibronectin, a glycoprotein found in plasma, has also been reported to improve healing rates in corneal wounds (Nishida, Larch Ophthalmology 101: 1046 (1983)) and leg wounds (Wysocki et al. Arch. Dermatol. 124: 175, 1988).

[0006] In addition, it was found that plasma normalizes abnormal cells and various cell active substances at a wound site to exhibit a wound healing effect, and based on this, a wound treatment agent containing plasma was developed (Korean Patent No. 0518152 and Korean Patent Publication No. 2007-0113876).

[0007] Plasma is a pale yellow liquid from which the tangible components in mammalian blood, i.e., cells and cell fragments, are separated. When an anticoagulant is added to the blood and then the mixture is centrifuged, precipitated, or allowed to stand at low temperature (about 0°C), the plasma as the upper layer is separated from the tangible components as the lower layer. In this case, the proportion of plasma is about 55%, but it is slightly different between men and women. Plasma is composed of about 90% water, 7% to 8% plasma protein, and the remainder of other lipids, sugars, inorganic salts, and non-protein nitrogen compounds (such as urea, amino acids, and uric acid). Plasma protein is mainly produced in the liver, contains albumin and globulin as main components, and also contains fibrinogen related to blood coagulation. Lipids include cholesterol, lecithin, etc. Inorganic salts contain sodium, chlorine, potassium, calcium, magnesium, etc., and their composition is similar to that of seawater, and they play a key role in maintaining normal osmotic pressure in the body. In addition, the total amount and composition of plasma vary greatly depending on the disease, so it is used to diagnose the disease or detect the state of the disease.

[0008] Plasma is used in unlimited applications due to various active ingredients as described above. In particular, plasma contains active substances (such as albumin and fibrinogen) with high medical utility, and is widely used as a source of these substances. However, plasma has the disadvantage of low economic efficiency because it should be separated from the blood of livestock and humans.

[0009] Meanwhile, sericin is a type of silk protein, also known as sericin, and cocoon fibers have a structure in which two strands of silk protein are covered with three layers of sericin. It was separated from cocoon fibers by E Kramer in 1865 and named "sericin" after the Latin "sercum" and the Greek "serikon" (each word means silk). The original silk thread is rough and hard due to the sericin attached thereto. The sericin is melted away during the refining process, resulting in a soft texture peculiar to the silk thread. Sericin has a remarkably high content of serine of about 37 mol% in its total amino acids, and dissolves in hot water and gels when cooled.

[0010] Among silk proteins, spider silk proteins include dragline proteins and / or flagellar proteins. Dragline is used by orb web spiders to build the framework and radial lines with their network, and is the lifeline that enables spiders to move backward continuously. For this purpose, high tensile strength and elasticity are required. The combination of such properties leads to higher tensile strength than other known materials. Dragline silk is usually composed of two main proteins, and the primary structure of these proteins has a common repeating architecture. The spiral capture of the round web composed in part of sticky silk (formed by flagella-like glands, called "flagellar-like silk") is elastic and can be stretched to three times its original length before breaking, but the tensile strength is half of that of dragline silk. Variations in a single repeating unit can contain up to 60 amino acids and are repeated several times to form the largest part of the spider silk sequence. These repeating units contain a limited set of different amino acid motifs. One motif found in all dragline repeating units is usually a large block of 6 to 9 alanine residues. In the silk thread, several polyalanine motifs form a stack of crystalline β sheets that give it tensile strength.

[0011] Silk protein is mainly used as a covering material to block wounds from the outside. A method of producing gel-like silk protein and using it as a wound covering material (Korean Patent No. 1,617,075) and a method of producing silk protein in the form of a layer (film) and using it as a wound covering material (Korean Patent No. 0,315,168) are known. However, the effect of directly treating wounds has not been known so far.

[0012] In the present invention, as a result of extensive efforts to develop a more economical and effective wound therapeutic agent, a wound therapeutic agent comprising a plasma component and silk protein was produced, and it was found that the therapeutic agent exhibits an excellent wound repair effect when applied to a wound. Based on this finding, the present invention has been completed. Summary of the invention

[0013] An object of the present invention is to provide an economical and effective pharmaceutical composition for treating wounds.

[0014] Another object of the present invention is to provide a method for treating wounds, the method comprising administering a plasma or serum fraction and silk protein to a subject.

[0015] Another object of the present invention is to provide use of plasma or serum fractions and silk protein for treating wounds.

[0016] Another object of the present invention is to provide use of a plasma or serum fraction and silk protein for preparing a wound therapeutic agent.

[0017] In order to achieve the above object, the present invention provides a pharmaceutical composition for treating wounds, wherein the pharmaceutical composition comprises a plasma or serum component and silk protein as active ingredients.

[0018] The present invention also provides a method for treating a wound, the method comprising administering a plasma or serum fraction and a silk protein to a subject.

[0019] The present invention also provides the use of plasma or serum fraction and silk protein for treating wounds.

[0020] The present invention also provides the use of plasma or serum components and silk protein for preparing a wound therapeutic agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shown are the results of testing the therapeutic effects of plasma fractions on scratch wounds formed on NIH3T3 cells as mouse fibroblasts.

[0022] Figure 2 Shown are the results of testing the therapeutic effects of plasma fractions and sericin on scratch wounds formed on NIH3T3 cells as mouse fibroblasts.

[0023] Figure 3 Shown are the results of testing the therapeutic effects of plasma fractions and recombinant spider silk proteins on scratch wounds formed on NIH3T3 cells as mouse fibroblasts. DETAILED DESCRIPTION

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which the invention belongs. Generally, the nomenclature used herein is well known and commonly used in the art.

[0025] In the present invention, it was found that when a composition comprising a plasma or serum component and silk protein was applied to a wound site, a better wound repair effect was obtained compared to when a plasma component or silk protein was applied to the wound site.

[0026] Therefore, in one aspect, the present invention relates to a pharmaceutical composition for treating wounds, the pharmaceutical composition comprising a plasma or serum fraction and silk protein as active ingredients.

[0027] In the present invention, the pH of the composition may be 3.5 to 6.5, and the plasma or serum may be derived from humans or livestock.

[0028] In the present invention, the plasma component can be obtained by separating albumin, fibrinogen and thrombin, and can be obtained by further removing at least one selected from heparin, antithrombin III, coagulation factor 2, coagulation factor 8, coagulation factor 9 and IgG.

[0029] In the present invention, the plasma or serum component may be a lyophilized preparation, and the plasma or serum component may be present in the composition in an amount of 0.001% to 99.999%.

[0030] In the present invention, the silk protein may be present in the composition in an amount of 0.001% to 99.999%.

[0031] In the present invention, the composition preferably contains 0.01% to 10% of the plasma component and 0.001% to 10% of the silk protein, more preferably 0.1% to 2% of the plasma component and 0.01% to 1% of the silk protein, and even more preferably 0.2% to 1% of the plasma component and 0.02% to 0.1% of the silk protein.

[0032] When the composition contains the plasma in an amount of 10% or more, it is not expected that the wound healing effect will be improved in proportion to the amount of the added plasma, and when the composition contains the plasma in an amount of less than 0.01%, no wound healing effect is expected. When the composition contains the silk protein in an amount of 10% or more, it is not expected that the wound healing effect will be improved in proportion to the amount of the added silk protein, and when the composition contains the silk protein in an amount of less than 0.001%, no wound healing effect is expected.

[0033] In the present invention, the silk protein may be of natural origin or may be recombinant silk protein, and the silk protein may be a repeating peptide unit constituting a protein selected from dragline silk, elastin, fibroin, foot silk, sericin, flagellar silk protein and collagen.

[0034] In the present invention, the silk protein may have a structure in which a repeating silk peptide unit having a glycine or serine content of 1% or more is repeated 1 to 160 times.

[0035] In the present invention, the silk protein may have a molecular weight of 5 to 300 kDa.

[0036] In the present invention, the recombinant silk protein may be a recombinant spider protein.

[0037] In the present invention, the recombinant spider silk protein may be prepared by the method disclosed in Korean Patent No. 10-1317420, and may have a high molecular weight and a structure in which a peptide having the sequence of the following SEQ ID NO: 1 or SEQ ID NO: 2 is repeated 1 to 96 times.

[0038] SED ID NO 1:

[0039] NH2-SGRGGLGGQGAGMAAAAAMGGAGQGGYGGLGSQGT-COOH

[0040] SED ID NO 2:

[0041] NH2-SSTGSSSNTDSNSNSVGSSTSGGSSTYGYSSNSRDGSV-COOH

[0042] In the present invention, the wound is selected from unhealed traumatic wounds, tissue destruction caused by irradiation, abrasions, bone gangrene, lacerations, avulsions, penetrating wounds, gunshot wounds, wounds, burns, frostbite, skin ulcers, dry skin, skin keratosis, cracks, ruptures, dermatitis, pain caused by dermatophytes, surgical or vascular disease wounds, contusions, corneal wounds, pressure sores, bedsores, conditions related to poor circulation and diabetes (such as diabetic skin erosions), chronic ulcers, sutures after plastic surgery, spinal cord injury wounds, gynecological wounds, chemical wounds and acne.

[0043] In the present invention, the formulation of the therapeutic composition may be selected from creams, ointments, gels, solutions, powders and patches.

[0044] In another aspect, the invention relates to a method for treating a wound, the method comprising administering to a subject a plasma or serum fraction and a silk protein.

[0045] In another aspect, the present invention relates to use of the plasma or serum fraction and silk protein for treating wounds.

[0046] In another aspect, the present invention relates to use of the plasma or serum fraction and silk protein for preparing a wound therapeutic agent.

[0047] As used herein, the term "trauma" refers to a state of bodily injury and encompasses pathological conditions in which tissue forming an internal or external surface of the body (such as skin, muscle, nervous tissue, bone, soft tissue, internal organs, or vascular tissue) is ruptured or destroyed.

[0048] In the present invention, examples of the wound include, but are not limited to, unhealed traumatic wounds, tissue destruction caused by irradiation, abrasions, bone gangrene, lacerations, avulsions, penetrating wounds, gunshot wounds, wounds, burns, frostbite, contusions (bruises), skin ulcers, dry skin, skin keratinization, cracks, ruptures, dermatitis, pain caused by dermatophytes, surgical trauma or vascular disease trauma, corneal trauma, pressure sores, bedsores, conditions related to poor circulation and diabetes (such as diabetic skin erosions), chronic ulcers, sutures after plastic surgery, spinal cord injury trauma, gynecological trauma, chemical trauma and acne, and cover injuries at any part of the subject. In this regard, the formulation according to the present invention may be very useful for repairing, replacing, improving, accelerating, promoting or healing these damaged tissues.

[0049] The plasma used in the present invention includes plasma separated from the blood of any mammalian species (including humans), for example, the blood of livestock (including sheep, goats, pigs, horses, dogs, cattle), other primates and rodents. In the present invention, plasma can be easily separated from blood by well-known conventional methods such as centrifugation, sedimentation (precipitation) or filtration. The centrifugation can be carried out under conditions suitable for sedimentation of blood cells from plasma. For example, the blood is centrifuged at about 1,400g for about 10 minutes, which is sufficient to precipitate blood cells and white blood cells and almost all cell debris (including platelets). The supernatant containing plasma can be easily separated from the settled cells by standard techniques.

[0050] The filtration can be performed by passing the blood through a filter suitable for separating blood cells from plasma, and the filter is preferably a microporous membrane that allows proteins to easily penetrate therein. In addition to fresh liquid plasma or liquid preparations obtained by centrifugation or sedimentation after blood collection, methods for preserving plasma in various states before use are also known. Examples of such preparations include fresh frozen preparations, frozen precipitation preparations, freeze-dried preparations, concentrated preparations, etc. In the present invention, any type of plasma described above can be used.

[0051] The composition according to the invention can be applied directly to the wound in liquid or powder form, i.e., can be spread on the wound. When the composition is provided as a sheet, the wound area to which the composition is applied can be appropriately bandaged to protect the wound and prevent reduction of the therapeutic effect of the active ingredient. The dressing can be any commercially available or generally known dressing. Examples of commercially available dressings include Comfeel, Duoderm, Tegaderm and Opsite.

[0052] The composition according to the present invention containing a pharmaceutically effective amount of plasma components as an active ingredient can be formulated in various forms with a pharmaceutically acceptable carrier. The formulation can be carried out according to methods known in the art. The formulation includes, but is not limited to, conventional external formulations, such as liquid coatings, sprays, lotions, creams, gels, pastes, liniments, ointments, aerosols, powders and transdermal absorbers. These formulations are disclosed in the literature [Remington's Pharmaceutical Science, 15th edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042 (Chapter 87: Blaug, Seymour)], which is a commonly known prescription in all pharmaceutical and chemical fields.

[0053] In the external preparation of the present invention, the pharmaceutically acceptable carrier may vary depending on its formulation, but may include: hydrocarbons, such as vaseline, liquid paraffin and gelled hydrocarbons (also known as a composite ointment base (plastibase) of liquid paraffin and polyethylene); animal and vegetable oils, such as medium-chain fatty acid triglycerides, pork fat, hard fat and cocoa butter; higher fatty alcohols and their esters, such as cetyl alcohol, stearyl alcohol, stearic acid and isopropyl palmitate; water-soluble bases, such as macrogol / polyethylene glycol, 1,3-butylene glycol, glycerol, gelatin, sucrose and sugar alcohols; emulsifiers, such as glycerol fatty acid esters, polyethylene glycol stearate and polyoxyethylene hardened castor oil; adhesives, such as acrylates and sodium alginate; propellants, such as liquefied petroleum gas and carbon dioxide; and preservatives, such as parabens, and the external preparation of the present invention can be prepared using the pharmaceutically acceptable carrier according to conventional methods. Furthermore, stabilizers, flavors, colorants, pH adjusters, diluents, surfactants, preservatives, antioxidants, etc. may be mixed therein as necessary. The external preparation of the present invention can be applied to a local wound by a conventional method.

[0054] In addition, the topical formulation according to the present invention can be used in a state of being adhered to a solid support (such as the wound peeling cover of a conventional band-aid). Adhesion is achieved by saturating the solid support with a plasma fraction from which tangible (commercial) components have been removed and then dehydrated. In an embodiment of the present invention, the solid support is first coated with an adhesive layer to improve the adhesion of the plasma fraction from which tangible (commercial) components have been removed to the solid support. Examples of the adhesive include polyacrylates and cyanoacrylates.

[0055] Various formulations of this type are commercially available, and examples include Band-Aids with non-adhesive wound peel-off coverings in the form of perforated plastic films (Smith & Nephew Ltd), BAND-AID* from Johnson & Johnson in the form of tapes, patches, spots or plastic strips; Curity CURAD (Ouchless) Band-Aids from Colgate-Palmolive Co (Kendall); and STIK-TITE* elastic tape from American White Cross Laboratories, Inc.

[0056] In one embodiment, the pharmaceutical composition according to the present invention can be formulated into the form of a liquid coating agent by mixing the plasma powder with physiological saline at a predetermined volume ratio and adjusting the pH to 3.5 to 6.5. In another embodiment, the pharmaceutical composition according to the present invention can be formulated into the form of an ointment by mixing the plasma powder of the present invention with a water-soluble ointment base and then adding physiological saline thereto. In a preferred embodiment, the pharmaceutical composition according to the present invention can be formulated by adjusting the pH of the ointment to 3.5 to 6.5.

[0057] In the present invention, drug carriers (such as gels or microspheres) can be used to accelerate wound healing. US Patent No. 5,264,207, WO 2000 / 24378, WO 96 / 13164 and WO 94 / 13333 disclose one or more polymer microspheres used as carriers for active pharmaceutical or cosmetic substances.

[0058] The pharmaceutical composition of the present invention can be used to treat various trauma symptoms in mammals. The pharmaceutical composition effectively treats infection, malignant tumors, lack of large blood vessel arteries (stenosis), lack of small blood vessel arteries, deep vein obstruction or lack, superficial vein lack, lymphatic disorders, endogenous poor circulation, blood abnormalities, collagen vascular abnormalities, radiation dermatitis, and most malignant ulcers caused by nutritional reasons, etc.

[0059] The pharmaceutically effective amount of the plasma component used in the composition of the present invention refers to the amount of active ingredients that exhibit a therapeutic effect on the wound by normalizing abnormal cells and various cell active substances in the wound site. The effective amount may vary depending on the patient's wound type, application site, number of applications, application time, formulation, patient's condition, type of adjuvant, etc. The number of applications may be twice a day to once a week. In one embodiment, when the powder is applied to a wound that has lost all skin, the effective daily amount of the pharmaceutical composition of the present invention is 0.01 to 0.1 g / cm 2 , preferably 0.02 to 0.09 g / cm2 and more preferably 0.02 to 0.07 g / cm 2 .

[0060] Hereinafter, the present invention will be described in more detail with reference to Examples. However, it will be apparent to those skilled in the art that these Examples are provided only to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0061] Example 1. Preparation of plasma fraction

[0062] Fresh blood from patients determined to be negative for possible pathogens including HIV, HCV, and HBV (The Korean Society for Blood Transfusion) was thawed in a 30°C water bath, then centrifuged at 3,000 rpm for 10 minutes, and the pale yellow plasma as the upper layer excluding precipitates such as red blood cells and white blood cells was separated.

[0063] The separated plasma was collected in a glass tube to which an anticoagulant (such as EDTA or heparin) was not added, the plasma was allowed to stand overnight at 4°C, and the precipitated fibrinogen clot was removed using a Pasteur pipette. The blood from which the clot was removed was centrifuged at 4°C and 4,000 rpm for 20 minutes to obtain a plasma fraction from which fibrinogen was removed.

[0064] The thrombin present in the plasma fraction without fibrinogen was separated under acidic conditions. In order to precipitate the prothrombin present in the plasma fraction from which fibrinogen was removed, the pH of the plasma fraction was reduced to 5.1 to 5.3 using an acetic acid solution. The solution with the reduced pH was allowed to stand at 20°C for 1 hour to completely precipitate the prothrombin, followed by centrifugation at 20°C and 4,200 rpm for 20 minutes to remove the precipitated thrombin, and a plasma fraction from which fibrinogen and thrombin were removed was obtained.

[0065] Albumin contained in the plasma fraction free of fibrinogen and thrombin was removed using SwellGel Blue Albumin Removal Kit (Pierce Biotechnology, USA) according to the manufacturer's method. By the above-mentioned albumin removal method, a plasma fraction from which commercial components were removed was obtained.

[0066] Example 2. Virus Inactivation of Plasma Fractions

[0067] The commercial component-free plasma fraction obtained in Example 1 was continuously subjected to the following three methods to inactivate viruses that may be present in the plasma fraction.

[0068] (1) γ irradiation

[0069] Using a cobalt source at 15°C (60 Co) The plasma fraction free of fibrinogen / thrombin / albumin and containing silk peptides was irradiated with a total of 25 kGy of gamma rays at an intensity of 1.8 kGy / hr.

[0070] (2) Methylene blue light treatment

[0071] The plasma fraction containing no fibrinogen / thrombin / albumin and containing silk peptides that had been irradiated with gamma rays was added with methylene blue at a final concentration of 1 μM and irradiated with white light at 60,000 lux for 1 hour. The remaining methylene blue was removed by filtration, frozen at -80°C for 8 hours, dried at -48°C for 7 days, and lyophilized.

[0072] (3) Steam treatment

[0073] The plasma fraction containing no fibrinogen / thrombin / albumin and containing silk peptides was sieved, crushed and homogenized, and then steam was slowly injected into the fraction in a stainless steel tank to adjust the water content to 8% (w / w). The steam-treated plasma was transferred to a stainless steel cylinder filled with dry nitrogen, from which oxygen was removed, and then the plasma was heated at 60° C. for 10 hours to obtain a plasma fraction in which the virus was inactivated.

[0074] Example 3. Preparation of plasma fraction and recombinant spider silk protein

[0075] The plasma fraction in which the virus was inactivated obtained in Example 2 was added to 1N HCl (hydrochloric acid) or 1N NaOH (sodium hydroxide) while stirring. The pH was measured with a pH meter (Orion) and adjusted to 5.5, followed by lyophilization to obtain a plasma fraction.

[0076] Freeze drying was slowly performed using a common freeze dryer at -30°C to -40°C at 40.9 to 77.5 Torr for 12 hours, and a 0.2 μm sterilizing filter was installed to obtain a dried plasma component, which was used to detect the wound healing effect.

[0077] Recombinant spider silk protein was prepared using the following method.

[0078] The recombinant spider silk protein is prepared by a method for producing recombinant silk protein described in a prior art patent (Korean Patent No. 10-1317420).

[0079] A high molecular weight recombinant silk protein or silk-like protein having a structure in which a peptide having a sequence of SEQ ID NO: 1 is repeated 48 times is used.

[0080] SEQ ID NO: 1:

[0081] NH2-SGRGGLGGQGAGMAAAAAMGGAGQGGYGGLGSQGT-COOH

[0082] Example 4. Confirmation of wound healing effect of plasma fraction

[0083] In order to confirm the wound healing effect of the freeze-dried plasma fraction prepared in Example 3, an experiment for measuring wound healing was performed using mouse fibroblast NIH3T3 cell line (KCTC 4612). NIH3T3 cells were seeded on a cell culture plate at a concentration of 500,000 / plate and grown to confluence in a DMEM medium (DMEM, Gibco) for cell growth supplemented with 10% fetal bovine serum (FBS, Thermo Fisher) and 1% penicillin-streptomycin (Pen-strep, Sigma-Aldrich) for 1 week.

[0084] Then, the cells were starved for one day using serum-free medium without FBS. Then, in order to measure the wound healing effect, the cells grown on the plates were regularly scratched and treated with serum-free medium supplemented with 0.1% (w / v), 0.5% (w / v), 1% (w / v) and 2% (w / v) concentrations of plasma fraction.

[0085] Only serum-free medium was added as a negative control for the experiment.

[0086] The wound healing effect of the cells was observed to last for 12 hours. Figure 1 It can be seen that the growth of fibroblasts was observed in the scratch area of ​​the experimental group to which the wound therapeutic agent was added, and the most effective wound healing could be obtained when 0.5% of the plasma fraction was added.

[0087] Example 5. Confirmation of wound healing effect of wound treatment formulation containing plasma fraction and sericin

[0088] In order to confirm the wound healing effect of the wound treatment preparation containing the freeze-dried plasma fraction obtained in Example 3 and sericin (Sigma-Aldrich), an experiment for measuring wound healing was performed using the NIH3T3 cell line in the same manner as in Example 4. In the same manner as in Example 4, the cells were grown to confluence in a cell growth medium containing 10% FBS and then starved for one day using a serum-free medium. Then, in order to confirm the wound healing effect, the cells grown on the plate were scratched, the plasma component was added to the serum-free medium at a concentration of 0.5%, and sericin was added at concentrations of 0.05% (w / v) and 0.5% (w / v). As a control group, the group treated with only the serum-free medium and the group containing only the plasma component were experimented. The experimental group was cultured for 12 hours, and then it was determined whether the scratched wound was treated.

[0089] As a result, if Figure 2 It can be seen that the group (BP 0.5%, Ser 0.05%) to which the plasma fraction (0.5%) and sericin (0.05%) were added had a better therapeutic effect than the group containing only the plasma fraction. This indicates that the optimal concentration of mixed sericin is 0.05%.

[0090] Example 6. Confirmation of wound healing effects of fractions containing plasma fractions and recombinant spider silk proteins

[0091] In order to confirm the wound healing effect of the preparation containing the freeze-dried plasma fraction and recombinant silk protein obtained in Example 3, an experiment for measuring wound healing was performed in the same manner as in Example 4 using the NIH3T3 cell line.

[0092] In the same manner as in Example 4, cells were grown to confluence in a cell growth medium containing 10% FBS and starved for one day using a serum-free medium, and the cells grown on the plate were scratched to confirm a wound healing effect.

[0093] The prepared plasma fraction was added to a serum-free medium at a concentration of 0.5% (w / v), and the recombinant silk protein was added thereto at concentrations of 0.05% (w / v), 0.1% (w / v), and 0.5% (w / v). As a control group, a group treated with only a serum-free medium and a group containing only the plasma fraction were experimented. The experimental groups were cultured for 12 hours, and then it was determined whether the scratch wound was treated.

[0094] As a result, if Figure 3It can be seen that the group to which both the plasma fraction and the recombinant spider silk protein were added was more effectively filled with new cells, thus exhibiting a better therapeutic effect than the group treated with the plasma fraction alone. The best wound healing ability was observed when 0.05% of the recombinant spider silk protein was added. This suggests that the optimal concentration of the recombinant spider silk protein mixture is 0.05%.

[0095] Industrial Applicability

[0096] According to the present invention, wounds can be treated more effectively through the synergistic effect of silk protein and plasma components.

[0097] Although the specific configuration of the present invention has been described in detail, it should be understood by those skilled in the art that this description is provided for the purpose of describing the preferred embodiment for illustrative purposes and should not be understood as limiting the scope of the present invention. Therefore, the essential scope of the present invention is defined by the appended claims and their equivalents.

[0098] Sequence Listing Free Text

[0099] Electronic files are attached. <110> Medex Biotech Co., Ltd. KAIST <120> Pharmaceutical composition for treating wounds <130> PP-B2310 <150> KR 10-2018-0146287 <151> 2018-11-23 <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> Spider silk peptide <400> 1 Ser Gly Arg Gly Gly Leu Gly Gly Gln Gly Ala Gly Met Ala Ala Ala 1 5 10 15 Ala Ala Met Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser 20 25 30 Gln Gly Thr 35 <210> 2 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> sericin silk peptide <400> 2 Ser Ser Thr Gly Ser Ser Ser Asn Thr Asp Ser Asn Ser Asn Ser Val 1 5 10 15 Gly Ser Ser Thr Ser Gly Gly Ser Ser Thr Tyr Gly Tyr Ser Ser Asn 20 25 30 Ser Arg Asp Gly Ser Val 35

Claims

1. Use of a composition comprising a platelet-free plasma or serum fraction and silk protein in the preparation of a therapeutic agent for treating wounds, wherein the silk protein is sericin or spider silk protein, wherein the wound is selected from unhealed traumatic wounds, tissue destruction caused by irradiation, abrasions, lacerations, avulsions, penetrating wounds, gunshot wounds, cuts, burns, frostbite, skin ulcers, cracks, ruptures, surgical or vascular disease wounds, contusions, corneal wounds, pressure sores, bedsores, diabetic skin erosions, chronic ulcers, sutures after plastic surgery, gynecological wounds, chemical wounds and acne.

2. The method according to claim 1, wherein the plasma or serum is derived from humans or livestock.

3. The use according to claim 1, wherein the silk protein is of natural origin or derived from recombinant silk protein.

4. The use according to claim 1, wherein the silk protein has a structure in which a peptide having a glycine or serine content of 1% or more is repeated 1 to 160 times.

5. The use according to claim 1, wherein the composition is provided as a formulation selected from the group consisting of creams, ointments, gels, solutions, powders and patches.

6. The use according to claim 1, wherein the pH of the composition is 3.5 to 6.

5.

7. The use according to claim 1, wherein the plasma fraction is a lyophilized preparation.

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