JP2009506981A - Wound healing composition and use thereof - Google Patents

Abstract

The present invention relates to a method for treating wounds, comprising administering to a patient in need of such treatment an effective amount of a polypeptide comprising an amino acid sequence having an EGF-like domain of thrombomodulin or a function-conserving variant thereof. The present invention also relates to a composition used to promote wound healing, the composition comprising a polypeptide comprising an amino acid sequence having an EGF-like domain of thrombomodulin or a function-conserving variant thereof.
[Selection] Figure 1

Description

  The present invention relates to methods and compositions used to accelerate wound healing.

Angiogenesis and thrombomodulin Several molecules are known in the art as therapeutic agents useful for the treatment of diseases and disorders that result in abnormal blood clotting in the major blood vessels, but abnormally high or abnormally low blood vessels There remains a need in the art for compositions and methods useful for the treatment of diseases and disorders that are symptomatic of neoplastic activity, as well as for the treatment of such diseases. These diseases and conditions include the following: cardiovascular diseases and symptoms such as hypotension, hypertension and atherosclerosis; thrombotic symptoms such as stroke, heart attack and post-angioplasty stent placement; angiogenic disorders; pulmonary fibrosis And respiratory diseases and symptoms such as asthma; diseases and disorders related to tumor cell wetting, angiogenesis and metastasis; wound healing and blood coagulation disorders, and reproductive disorders such as early onset of uterine contractions and sexual impotence .

  Recently, many biomolecules have been identified that induce or promote angiogenesis in tissues. The most prominent among these are vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGFs), transforming growth factor (TGFs). ) And other growth factors. Lack of thrombomodulin, a blood coagulation regulator, causes embryonic lethality in mice before the functional cardiovascular system develops (Proc. Natl. Acad. Sci. USA. 1995; 92: p850-854. JP Cooke et al., Circulation 105 (2002) p2133 (non-patent document 2)). Current approaches aimed at promoting angiogenesis in related fields can be summarized into three main categories. (I) delivery of angiogenic growth factors using synthetic and natural polymeric scaffolds, (ii) delivery of plasmids containing DNA encoding angiogenic proteins, and (iii) angiogenic molecules and endothelium Combined delivery of cell transplants.

  In a related field, another study suggests that human recombinant thrombomodulin molecules can be incorporated into delivery systems instead of growth factors (Shi CS et al., “Evidence for human thrombomodulin domains as novel angiogenic factors” 2005 April 5; 111 (13): p1627-36 (non-patent document 3)). It has been proposed to release the loaded human recombinant thrombomodulin under physiological conditions to promote local and rapid angiogenesis.

  Thrombomodulin is an anticoagulant endothelial cell membrane glycoprotein. Recombinant thrombomodulin proteins TMD2 (including 6 EGF-like structures) and TMD23 (including TMD2 and serine-threonine-rich domains) exhibit mitogenic activity.

  The aforementioned study (Shi CS et al., “Evidence of human thrombomodulin domain as a novel angiogenic factor” April 2005 5; 111 (13): p1627-36) is in vitro and biological model model. Was used to elucidate the novel angiogenic effect of the recombinant domain. It has been shown that TMD23 has higher activity than TMD2 in stimulating DNA synthesis in cultured human umbilical vein endothelial cells (HUVECs). In addition, TMD23 is an effect of phosphorylation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase and HUVECs through the phosphatidylinositol 3 kinase / Akt / endothelial nitric oxide synthase pathway. Stimulated chemotactic movement and the formation of capillary-like tubes. TMD23 also stimulated endothelial cell expression of matrix metalloproteinases and plasminogen activators and mediated extracellular proteolysis leading to endothelial cell wetting and migration during angiogenesis. Furthermore, TMD23-containing implants in the rat cornea induced neovascular ingrowth from the corneal margin. Using a mouse angiogenesis assay, it was found that TMD23 co-injected with Matrigel and heparin not only induced neovascularization, but also enhanced angiogenesis in Matrigel-containing melanoma A2058 cells in nude mice. That is, the recombinant thrombomodulin domain TMD23 enhances the angiogenic reaction in vitro and in vivo.

Wound healing There are three stages in the wound healing process. That is, the inflammatory phase, proliferative phase, and mature phase.

  The inflammatory phase is characterized by hemostasis and inflammation. Collagen exposed during wound formation activates the blood coagulation cascade (both intrinsic and extrinsic pathways) and the inflammatory phase begins. After damage to the tissue occurs, cell membranes damaged by wound formation release strong vasoconstrictors such as thromboxane A2 and prostaglandin 2-α. This initial reaction suppresses bleeding. Soon after, local histamine release causes capillary dilation, allowing inflammatory cells to migrate to the wound surface.

  Platelets, the first responding cells, release multiple chemokines including epidermal growth factor (EGF), fibronectin, fibrinogen, histamine, platelet-derived growth factor (PDGF), serotonin, and von Willebrand factor . These factors stabilize the wound through clot formation. These mediators control bleeding and limit the extent of damage. Platelet degranulation also activates the complement cascade, specifically C5a, a potent chemoattractant of neutrophils.

  The inflammatory phase persists and more immune response cells migrate to the wound. Neutrophils, the second reactive cells that migrate into the wound, clean up debris, complement-mediated bacterial opsonization, and destroy bacteria through reactive oxygen generation mechanisms (ie, superoxide and Responsible for the production of hydrogen oxide). Neutrophils kill bacteria and remove foreign bodies from the wound.

  The next cells present in the wound are white blood cells and macrophages (monocytes). Macrophages, called orchestrators, are essential for wound healing. Numerous enzymes and cytokines are secreted by macrophages and collagenase creates wounds; interleukins and tumor necrosis factor (TNF) that stimulate fibroblasts (produce collagen) and promote angiogenesis; and keratinocytes Transforming growth factor (TGF) is included. This stage represents the tissue reconstruction process, ie the transition to the growth phase.

  The second stage in wound healing is the proliferative phase. Epithelialization, angiogenesis, granulation tissue formation, and collagen deposition are the main processes in this anabolic part of wound healing. Epithelialization occurs early in wound repair. If the basement membrane remains intact, epithelial cells migrate upward along a normal pattern. This is equivalent to a single burn. Epithelial progenitor cells remain intact under the wound and the normal epidermal layer is repaired in a few days. If the basement membrane has been destroyed, as with a second or third burn, the wound is re-epithelialized from normal cells around it and, if intact, from the skin appendages (eg, hair follicles, sweat glands) .

  Angiogenesis stimulated by TNF-α is characterized by endothelial cell migration and capillary formation. The regenerated capillaries deliver nutrients into the wound and help maintain the granulation tissue layer. Capillary extension to the wound surface is important for proper wound healing. Granulation tissue formation and tissue deposition require the supply of nutrients by capillaries, and failure of nutrient supply leads to chronic wound healing failure. Mechanisms that alter angiogenesis have been studied and are likely to improve the healing process.

  The final phase of the growth phase is the formation of granulation tissue. Fibroblasts differentiate and produce matrix, then collagen. The matrix is deposited on the wound surface and collagen is then deposited as the wound progresses to the final phase of repair. A wide variety of cytokines are involved in the growth phase of wound repair. The stage of control and the exact mechanism are not yet elucidated. Some cytokines include PDGF, insulin-like growth factor, (IGF) and EGF. These are all necessary for the production of collagen.

  The final stage of wound healing is the mature stage. The wound contracts, eventually leading to a reduction in the amount of visible scar tissue. The whole process is a continuous activity with the overlap of each healing phase and continuous reconstruction. The wound reaches maximum strength in one year and the tensile strength is 30% of normal skin. Collagen deposition continues for a long period, but the net increase in collagen deposition levels off after 21 days.

  Currently, the role of cytokines in actual clinical practice is limited. The only commercial product currently available that has proven effective in a randomized, double-blind study is PDGF, available as recombinant human PDGF-BB. A number of studies have demonstrated that recombinant human PDGF-BB shortens the healing period and improves the wound healing rate in stage III and IV ulcers. Many other cytokines currently under study in vitro include TGF-β, EGF and IGF-1.

  Proper wound healing involves a complex interaction of cells and cytokines. In recent years, more chemical mediators essential to this process have been identified. Sequential steps and specific processes are still not fully identified. In investigating the wound healing process, the key steps must be identified and important transmitters must be known.

Burns and Wound Healing Burns are damage caused by exposure to heat, electricity, radiation (eg, sunburn or laser surgery), or corrosive chemicals.

  Generally, three stages of burns are recognized. With a single burn, the outer layer of the skin, called the epidermis, becomes red, sensitive to contact, and often swollen. Although no treatment is required, application of the ointment relieves pain. Twice burns are characterized by variable destruction of the epidermis and blister formation, and nerve endings may be exposed. In more severe cases it is necessary to see a doctor and take care to avoid infection. Topical treatment includes the application of chemicals such as silver nitrate to form a soft crust, reducing the risk of infection, and relieving pain. A third-degree burn involves the destruction of the entire skin thickness and the underlying connective tissue. In more severe cases, even the underlying bone is charred. The affected surface area is more important than the depth of the burn. Shock symptoms must be prevented or dealt with, and blood transfusions may be necessary to reverse fluid loss. Administration of antibiotics and other drugs must prevent or cure the invasion of various bacteria. Morphine may be used to relieve pain. Long-term treatment may involve natural or artificial skin grafts.

Diabetes Mellitus and Wound Healing Diabetes mellitus is a group of metabolic diseases characterized by high blood glucose (glucose) levels, resulting from abnormalities in insulin secretion or action, or both. Diabetes mellitus is commonly referred to as diabetes and means "sweet urine". The increase in blood glucose level (hyperglycemia) led to the outflow of glucose into the urine, and so was the term for sweet urine. Normally, blood glucose levels are tightly controlled by insulin, a hormone produced by the pancreas. Blood glucose levels are lowered by insulin. As blood glucose levels rise (eg, after food intake), insulin is released from the pancreas, returning glucose levels to normal levels. In patients with diabetes mellitus, insulin is not produced or not produced sufficiently, resulting in a hyperglycemic state. Diabetes mellitus is a chronic condition, meaning it can last a lifetime.

  Diabetes can lead to several complications including cardiovascular disease, blindness, renal failure, and diabetic foot ulcers.

  A diabetic person is at risk of injury to the foot due to nerve damage (diabetic neuropathy) and bluntness caused by decreased blood flow to the lower limbs. The most serious injury is diabetic foot ulcers. Diabetic foot ulcers have a very high risk of infection and may not heal. Unhealed foot ulcers often cause amputations in diabetics.

  The FDA has approved a gel product (becaplermin or Regranex gel) as a treatment for diabetic foot ulcers. This product contains a genetically engineered platelet-derived growth factor, one of the proteins that the human body produces to promote the growth of new tissues. Clinical studies of this product show that the use of becaprelmin increases the likelihood of complete closure of ulcers by 20 weeks after treatment.

Another product that helps wound closure of slowly healing ulcers in diabetic patients is a skin substitute called DERMAGRAFT®. It consists of human cells known as fibroblasts placed on a lytic mesh material. When the mesh material is placed on the ulcer, it is gradually absorbed and human cells grow and replace the damaged tissue in the ulcer.
US patent application Ser. No. 11/149378 Proc. Natl. Acad. Sci. USA. 1995; 92: p850-854 JP Cooke et al., Circulation 105 (2002) p2133 Shi CS et al., “Evidence for a human thrombomodulin domain as a novel angiogenic factor” April 2005; 111 (13): p1627-36

  The present invention relates to a method for treating wounds, comprising administering to a patient in need of such treatment an effective amount of a polypeptide comprising an amino acid sequence having an EGF-like domain of thrombomodulin or a function-conserving variant thereof. The present invention also relates to a composition used to promote wound healing, the composition comprising a polypeptide comprising an amino acid sequence having an EGF-like domain of thrombomodulin or a function-conserving variant thereof.

  Other objects and features of the present invention will become apparent from the following detailed description considered in conjunction with the accompanying drawings. Of course, the drawings are for illustration purposes only and do not define the limits of the invention, see the appended claims for the scope of the invention. It should also be understood that the drawings are not necessarily drawn to scale and are intended only to conceptually describe the structures and procedures described herein unless otherwise specified. .

  Thrombomodulin is an anticoagulant endothelial cell membrane glycoprotein. A recombinant thrombomodulin domain containing six epidermal growth factor-like structures and a serine-threonine rich domain exhibits mitogenic activity.

  Previous work (US Patent Application No. 11/149378) has demonstrated that TMD23 can induce endothelial cell (HUVEC) migration and proliferation.

  In the present invention, it became clear that TMD23 effectively enhances the migration of keratinocytes (HaCaT), which is the main cell type in the epidermis, and that the antibody against TMD23 can specifically inhibit the migration of keratinocytes. It was. Thus, it was demonstrated for the first time that TMD23 can enhance the wound healing process by stimulating the migration of keratinocytes.

  Furthermore, animal experiments have shown that TMD23 can accelerate wound closure by enhancing migration and expansion of the epidermis into the wound area. On the other hand, the water transpiration rate from the wound opening was also effectively reduced.

  In summary, TM can effectively increase the healing rate of wounds.

  The present invention relates to a method for treating wounds, comprising administering to a patient in need of such treatment an effective amount of a polypeptide comprising an amino acid sequence having an EGF-like domain of thrombomodulin or a function-conserving variant thereof. In a preferred embodiment, the polypeptide further comprises an operably linked polypeptide comprising an amino acid sequence or function-conserving variant of the serine threonine-rich domain of thrombomodulin.

  The method of the present invention can be applied to promote wound healing, where wounds are incisions, lacerations, abrasions, stab wounds, blisters, skin tears, donor or transplant sites, acne, contusions, hematomas, pressure wounds and skin Selected from the group consisting of wounds caused by peeling or laser peeling.

  The method of the present invention can also be used for diabetes mellitus patients. In a preferred embodiment, the patient suffers from a diabetic ulcer.

  The method of the present invention is applicable even if the wound is a burn resulting from fire, heat, radiation, electricity or skin surgery.

  The present invention is also applicable to reconstruction surgery.

  In a preferred embodiment, the method is applied to a skin-borne product selected from the group consisting of gels, creams, pastes, lotions, sprays, suspensions, aqueous solutions, dispersions, hydrogels, and ointment formulations. In a more preferred embodiment, the skin-borne product is administered to patients in need of such treatment by application to the skin, infusion, or electroporation.

  The present invention also relates to a method for treating wounds, and an effective amount of a plasmid comprising a DNA encoding a polypeptide containing an amino acid sequence having an EGF-like domain of thrombomodulin or a function-conserving variant thereof in a patient in need of such treatment Administration. In a more preferred embodiment, this administration is performed using a plasmid vaccine. In further preferred embodiments, administration is by intravenous (iv), subcutaneous (sc), abdominal (ip), or muscular (im) route.

  The invention further relates to a composition for use in accelerating wound healing, comprising a polypeptide comprising an amino acid sequence having an EGF-like domain of thrombomodulin or a function-conserving variant thereof. In a more preferred embodiment, the polypeptide further comprises an operably linked polypeptide comprising an amino acid sequence or function-conserving variant of the serine threonine-rich domain of thrombomodulin.

  The composition of the present invention is applicable for use in accelerating wound healing, where wounds are incisions, lacerations, abrasions, stab wounds, blisters, skin tears, donors or transplant sites, acne, contusions, hematomas, compression Selected from the group consisting of wounds and wounds caused by skin peeling or laser peeling.

  The composition of the present invention can be used in patients with diabetes mellitus. In a preferred embodiment, the patient suffers from a diabetic ulcer.

  The composition of the present invention is applicable even if the wound is a burn resulting from fire, heat, radiation, electricity or skin surgery.

  In a more preferred embodiment, the composition is also applicable to reconstruction surgery.

  In a more preferred embodiment, the method is applied to a skin-borne product selected from the group consisting of gels, creams, pastes, lotions, sprays, suspensions, aqueous solutions, dispersions, hydrogels, and ointment formulations. In a more preferred embodiment, the skin-borne product is administered to patients in need of such treatment by application to the skin, infusion, or electroporation.

Definitions of Terms The following definitions are for purposes of explanation and are not intended to limit the invention, in order to aid understanding of the following discussion.

  The present invention utilizes conventional molecular biology, microbiology, and recombinant DNA technology within the knowledge of those skilled in the art. Such techniques are explained fully in the literature.

  “Wounds” are characterized by open and closed wounds. An open wound is an incision wound (caused by a smooth, sharp object such as a knife or razor), a laceration (a rough or irregular wound caused by a severe or tearing force), an exfoliation of the epidermis and an abrasion (the top layer of the skin The wound on the peeled surface, often caused by falling down against a rough surface, and categorized into various types, including puncture wounds (caused by objects that puncture skin such as nails, needles, etc.) Is possible. The classification of closed wounds is much less than this, but as dangerous as open wounds. Closed wounds are contusions or bruises (caused by blunt trauma that damages tissue under the skin), hematomas (caused by blood concentrating under damaged blood vessels), and (added over time) A pressure wound (caused by a large or extreme amount of force applied).

  There are three stages in the healing process of a “wound”. That is, the inflammatory phase, proliferative phase, and mature phase.

  The inflammatory phase is characterized by hemostasis and inflammation. Collagen exposed during wound formation activates the blood coagulation cascade (both intrinsic and extrinsic pathways) and the inflammatory phase begins. After damage to the tissue occurs, cell membranes damaged by wound formation release strong vasoconstrictors such as thromboxane A2 and prostaglandin 2-α. This initial reaction suppresses bleeding. Soon after, local histamine release causes capillary dilation, allowing inflammatory cells to migrate to the wound surface. The inflammatory phase persists and more immune response cells migrate to the wound. Neutrophils, the second reactive cells that migrate into the wound, clean up debris, complement-mediated bacterial opsonization, and destroy bacteria through reactive oxygen generation mechanisms (ie, superoxide and Responsible for the production of hydrogen oxide). Neutrophils kill bacteria and remove foreign bodies from the wound.

  The second stage in wound healing is the proliferative phase. Epithelialization, angiogenesis, granulation tissue formation, and collagen deposition are the main steps in this anabolic part of wound healing. Epithelialization occurs early in wound repair. If the basement membrane remains intact, epithelial cells migrate upward along a normal pattern. This is equivalent to a single burn. Epithelial progenitor cells remain intact under the wound and the normal epidermal layer is repaired in a few days. If the basement membrane has been destroyed, as with a second or third burn, the wound is re-epithelialized from normal cells around it and, if intact, from the skin appendages (eg, hair follicles, sweat glands) . The final phase of the growth phase is the formation of granulation tissue. Fibroblasts differentiate and produce matrix, then collagen. The matrix is deposited on the wound surface and collagen is then deposited as the wound progresses to the final phase of repair. A wide variety of cytokines are involved in the growth phase of wound repair. The stage of control and the exact mechanism are not yet elucidated. Some cytokines include PDGF, insulin-like growth factor, (IGF) and EGF. These are all necessary for the production of collagen.

  The final stage of wound healing is the mature stage. The wound contracts, eventually leading to a reduction in the amount of visible scar tissue. The whole process is a continuous activity with the overlap of each healing phase and continuous reconstruction. The wound reaches maximum strength in one year and the tensile strength is 30% of normal skin. Collagen deposition continues for a long period, but the net increase in collagen deposition levels off after 21 days.

  “Angiogenesis” is generally thought to be largely regulated by growth factors and other ligands. Angiogenesis and concurrent tissue development and regeneration depend on tightly controlled processes such as endothelial cell proliferation, migration, differentiation, and survival. During these processes, stimulant and inhibitor ligands appear to interact directly or indirectly with cellular receptors. Angiogenesis begins with erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. Endothelial cells covering the vessel wall then break the basement membrane. Angiogenic stimulants induce endothelial cell migration through the eroded basement membrane. The migrating cells then form “sprouting” from the parent vessel, and the endothelial cells begin to mitose and proliferate. Endothelial cell germination fuses together to form a capillary loop, and blood vessels are born.

  As used herein, “burn” is damage caused by exposure to heat, electricity, radiation (eg, sunburn or laser surgery), or corrosive chemicals.

  Diabetes mellitus is a group of metabolic diseases characterized by high blood glucose (glucose) levels and is caused by abnormalities in insulin secretion or action, or both. Diabetes mellitus is commonly referred to as diabetes and means "sweet urine". The increase in blood glucose level (hyperglycemia) led to the outflow of glucose into the urine, and so was the term for sweet urine. Normally, blood glucose levels are tightly controlled by insulin, a hormone produced by the pancreas. Blood glucose levels are lowered by insulin. Insulin is released from the pancreas as blood glucose levels rise (eg, after food intake), returning glucose levels to normal levels. In patients with diabetes mellitus, insulin is not produced or not produced sufficiently, resulting in a hyperglycemic state. Diabetes mellitus is a chronic condition, meaning it can last a lifetime.

  Diabetes often accompanies peripheral neuropathy, especially when combined with vascular damage, can lead to foot ulcers and can progress to necrosis, infection, gangrene, and amputation of the foot. It may be necessary.

  "Nucleic acid" or "nucleotide sequence" is a ribonucleoside (adenosine, guanosine, uridine, siditin; RNA molecule) or deoxyribonucleoside (deoxyadenosine, deoxyguanosine, deoxythymidine) that is either single-stranded or double-helical , Or deoxycytidine; a DNA molecule). Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are contemplated. The term nucleic acid means only the primary and secondary structure of a molecule in a particular DNA or RNA molecule and is not limited to a particular tertiary or quaternary form. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (eg, restriction enzyme fragments), plasmids, and chromosomes. In discussing the structure of a particular double-stranded DNA molecule, in this application, the sequence is expressed only in the 5 'to 3' direction along the non-transcribed strand of DNA (ie, the strand having a sequence homologous to mRNA). It is described according to the usual practice. “Recombinant DNA” is a DNA molecule that has been biologically manipulated.

  When referring to a fragment of DNA, “operably linked” indicates that the fragments are arranged to cooperate, eg, the transcription step encounters a transcription terminator fragment when RNA polymerase binds to the promoter fragment. Then, the transcription is advanced through the coding fragment until the polymerase stops.

  As used herein, “nucleic acid fragment” is intended to mean any nucleic acid molecule originating from cDNA, genomic DNA, synthetic DNA or RNA. The term “fragment” is intended to mean a nucleic acid fragment that is single-stranded or double-stranded and is based on a complete or partial natural nucleotide sequence encoding the polypeptide of interest. Fragments optionally include other nucleic acid fragments.

  A nucleic acid fragment of the invention that encodes a polypeptide of the invention is suitably derived from a genome or cDNA, eg, by preparing a genome or cDNA library and performing hybridization with a synthetic oligonucleotide probe according to standard techniques. It can be obtained by screening a DNA sequence encoding all or part of a polypeptide.

  The polypeptide may be detected by methods known in the art that are specific for that polypeptide. These detection methods include the use of specific antibodies, production of enzyme products, or loss of enzyme substrates. For example, an enzyme assay is used to measure the activity of the polypeptide.

  The polypeptides of the present invention can be purified by a variety of procedures known in the art, including chromatography (eg, ion exchange, affinity, hydrophobicity, chromatographic fractionation, size exclusion), electrophoresis procedures (eg, preparative isoelectric). Point electrophoresis (IEF)), differential solubility (eg, ammonium sulfate precipitation) methods, or extraction, but are not limited thereto.

  As used herein, the term “plasmid vaccine” refers to a plasmid DNA designed to contain not only the gene integrated into the construct, but also the gene for the desired vaccine antigen, which can be produced in a suitable host system. As a purified preparation.

  The following examples are non-limiting and are merely representative of various aspects and features of the present invention.

Effect of TMD23 on HaCaT epithelial cell migration The effect of TMD23 on HaCaT cell migration was evaluated using a Boyden chamber of a 6.5 mm diameter polycarbonate filter (8 μm pore size). The lower surface of the filter is coated with type IV collagen. TMD23 (100 ng / ml) or TMD23 (100 ng / ml) + anti-TM antibody (1 μg / ml) was added to DMEM in the lower well. A cell suspension (50 μL) containing 1 × 10 ⁴ cells was loaded into each upper well. The number of cells that migrated through the membrane during 8 hours was counted under a microscope after fixation with methanol and staining with 10% GIEMSA. As shown in FIG. 1, TMD23 significantly induced chemotactic migration ability in HaCaT cells.

Material DMAp was 100 μg of recombinant TMD23 purified protein (SEQ ID NO. 1) in 20 μl of 0.5% CMC / PBS solution. 0.5% CMC (carboxymethyl cellulose) / PBS (phosphate buffered saline) at pH 7.4 was used as a solution form vehicle for DMAp and CGS-21680. CGS-21680 hydrochloride (2-p- [2-carboxyethyl] phenethylamino-5′-N-ethylcarboxamidoadenosine) is a G protein activator and was purchased from Sigma-Aldrich.

  The DMAc gel was 100 μg recombinant TMD23 purified protein (SEQ ID NO. 1) in a 20 mg base gel. The base gel was used as a negative control for DMAc gel and Regranex gel. Base gel was sodium carboxymethylcellulose, sodium chloride, sodium acetate trihydrate, glacial acetic acid, methylparaben, propylparaben, m-cresol, lysine hydrochloride, benzyl alcohol, methylchloroisothiazoline, methylisothiazolinin, acryloyldimethyltaurate ammonium / It consists of a VP copolymer and water for injection. Regranex Gel was purchased from Johnson & Johnson.

  For the chemicals used in the examples, 10% neutral buffered formalin solution (Shiyak Kogyo, Japan), carboxymethylcellulose (Sigma-Aldrich, USA), GCS-21680 hydrochloride (Tocris, USA), hexo Barbital (Sigma-Aldrich, USA), phosphate buffered saline (PBS, pH 7.4, Sigma-Aldrich, USA) and sodium chloride (Wako, Japan) were used.

  The instrument used in the examples includes an animal cage (Allentown, USA), Image Pro Plus (Media Cybernetics, version 4.5.29), Pipetman (Gilson, Germany) and a sharp drilling instrument with an inner diameter of 12 mm (Germany). Sinter, R.O.C) was used.

Animals Male CD-1 (Crl) -derived mice weighing 24 ± 2 g were obtained from BioLasco Taiwan (under Charles River Laboratories Technology licensee). The assigned space for 10 mice was 29x18x13 cm. All mice are housed in a controlled temperature (22 ° C-24 ° C) and humidity (50% -60%) environment for at least one week with a light / dark cycle of 12 hours prior to the experiment at the MDS Pharma Services-Taiwan Laboratory. It was done. Standard laboratory mouse feed [MF-18 (Oriental Yeast, Japan)] and RO water were ad libitum. All aspects of this experiment, including breeding environment, experimentation and mouse sacrifice, are generally described in the Guide for the Care and Use of Laboratry Animals: National Academy Press, Washington, DC, 1996. Went along.

  Insulin-independent diabetes mellitus (NIDDM) male mice (C57BLKS / J-m + / + Lepr db) weighing 50 ± 5 g (9 weeks old) were obtained from the Institute for Animal Breeding (IAR, Japan). These mice show hyperinsulinemia, hyperglycemia, and islet atrophy. During the experiment, mice were housed under specific pathogen free (SPF) conditions in individually ventilated cage racks (IVC rack, 36 miniisolator system). Each APEC_cage (length 26.7 x width 20.7 x 14.0 height, cm notation) was sterilized with an autoclave to accommodate 7 mice, which were then controlled at a controlled temperature (22 ° C-24 ° C). C.) and humidity (50% to 60%), and kept in a hygienic environment under a 12 hour light / dark cycle. Mice were fed ad libitum with feed for sterilized experimental mice and sterilized distilled water. All aspects of this experiment, including breeding environment, experimentation and mouse sacrifice, are generally described in the Guide for the Care and Use of Laboratry Animals: National Academy Press, Washington, DC, 1996. Went along.

Promotion of wound closure in mice by TMD23 A group of 5 male CD-1 (Crl) -induced mice weighing 24 ± 2 g each was used. During the test period, mice were housed individually in individual cages. Under hexobarbital (90 mg / kg, IP) anesthesia, the hair on the shoulder and back area of each mouse was shaved. A sharp piercing device (12 mm inner diameter) was applied to remove the skin containing the muscle layer and associated tissue. Vehicle (0.5% CMC / PBS, pH 7.4, 20 μl / mouse) and 10 μ / mouse CGS-21680 as well as 20 mg / mouse DMAc, Legranex and base gel and 100 μg / mouse DMAp as positive controls, Immediately following skin injury, each was administered topically (TOP) once a day for 10 consecutive days. The area of the wound traced on a transparent plastic sheet was measured after 1, 3, 5, 7, 9, 11 days using Image Pro Plus (Media Cybernetics, version 4.5.29). Wound closure rate (%) was calculated and wound semi-closure time (CT50) was analyzed using linear regression using Graph Prism (Graph Software, USA). A one-way analysis of variance followed by Dunnett's trial was applied to compare the treatment group with the corresponding vehicle group at each measurement time point. Differences were considered statistically significant when P <0.05. CGS-21680 (10 μg / mouse × 10) as a positive standard showed a significant increase in wound closure (P <0.05; days 3, 5, 7, 9 and 11) and CT50 was the corresponding vehicle control Decreased relative to value. The results are summarized in FIG. 2 (solution phase), FIG. 3 (gel phase), and Table 1 below.

創傷閉鎖率（％）及び創傷半閉鎖時間（ＣＴ_５０）を求め、一元配置分散分析とそれに続くダネット試験を用いて治療グループと、それに対応するビヒクルグループとを比較した。＊Ｐ＜０．０５対ビヒクルコントロール Effect of test items on wound closure rate and semi-closure time in CD-1 mice

Wound closure rate (%) and wound half-closure time (CT ₅₀ ) were determined and treatment groups were compared to corresponding vehicle groups using a one-way analysis of variance followed by Dunnett's test. * P <0.05 vs. vehicle control

  Throughout the experiment, DMAp (100 μ / mouse) showed the most lasting effect on wound healing and CT50 values were significantly shortened. DMAc and regranex showed a tendency to promote wound healing, but did not significantly affect CT50. The base gel had no effect in the skin wound mouse model.

Promotion of wound closure in diabetic mice by TMD23 A group of 5 male mice of C57BLKS / J-m + / + Lepr db weighing 50 ± 5 g was used. During the experiment, mice were individually housed in individually ventilated cage racks (IVC rack, 36 miniisolator system). Under hexobarbital (90 mg / kg, IP) anesthesia, the hair on the shoulder and back area of each mouse was shaved. A sharp piercing device (12 mm inner diameter) was applied to remove the skin containing the muscle layer and associated tissue. The wound area traced on a transparent plastic sheet on the first, third, fifth, seventh, ninth, eleventh, thirteenth and fifteenth days was measured using an image analyzer (ProPlus, Media Cybernetics, version 4.5.29). did. Solution or gel-like test substance and vehicle, positive control CGS-21680 or Regranex were administered topically once daily for 14 consecutive days immediately after wounding. Test substances DMAp (100 μg / mouse), vehicle (0.5%, CMC / PBS, pH 7.4) and CGS-21680 (10 μg / mouse) were in solution and a dose of 20 μl / mouse was used. On the other hand, DMAc, Regranex and base gel (as vehicle control group) were in gel form and were administered at 20 mg / mouse. Wound semi-closure time (CT50) was determined by linear regression using Graph Pad Prism (Graph Pad Software, USA), and one-way analysis of variance followed by Dunnett's test at treatment time and vehicle group at each time point. Applied for comparison. Differences were considered statistically significant at the P <0.05 level. CGS-21680 (10 μg / mouse × 14) and Regranex (20 mg / mouse × 14) as positive control substances showed a significant increase (P <0.05) in wound closure rate (3, 5, 7, 9, On days 11, 13, 15), CT50 was significantly reduced compared to the corresponding vehicle control value. The results are summarized in FIG. 4 (solution phase), FIG. 5 (gel phase), and Table 2 below.

創傷閉鎖率（％）及び創傷半閉鎖時間（ＣＴ_５０）を求め、一元配置分散分析とそれに続くダネット試験を用いて治療グループと、それに対応するビヒクルグループとを比較した。＊Ｐ＜０．０５対ビヒクルコントロール Effect of test items on wound closure rate and semi-closure time in Lepr db mice

Wound closure rate (%) and wound half-closure time (CT ₅₀ ) were determined and treatment groups were compared to corresponding vehicle groups using a one-way analysis of variance followed by Dunnett's test. * P <0.05 vs. vehicle control

  Throughout the experiment, DMAp (100 μg / mouse), DMAC, Regranex (20 mg / mouse each) and CGS-21680 (10 μg / mouse) showed a sustained enhancement effect on wound healing and CT50 values were significantly shortened . In the initial phase, the base gel vehicle appears to show some delay in wound healing compared to 0.5% CMC / PBS, pH 7.4.

Decrease in transpiration rate from wound opening by TMD23 In order to measure epithelial formation rate, the transpiration rate from wound area was measured using TM210 skin moisture transpiration measuring device Tevameter (Courage + KHAZAKA electronic Co., Cologne, Germany). It was measured. A lower water transpiration rate represents a higher level of epithelialization or keratinization. The results (FIG. 6) show that TMD23 effectively increases the rate of wound epithelialization.

  It will be readily appreciated by those skilled in the art that the present invention is highly adapted not only to the original, but also to achieve the objectives and obtain the results and advantages described. The cell lines, animals and treatments and methods for producing them are representative of preferred embodiments, are exemplary, and are not intended to limit the scope of the invention. Modifications and other uses in the present invention are within the scope of those skilled in the art. These modifications are within the spirit of the invention and are defined by the scope of the claims.

  It will be readily apparent to those skilled in the art that various alternatives and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

  All patents and publications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All patents and publications are hereby incorporated by reference to the same extent as each publication is specifically and individually intended to be incorporated by reference.

  The present invention described with reference to examples in the present application can be appropriately implemented regardless of lack of any element or limitation, and is not particularly disclosed in the present application. The terms and expressions used are used as descriptive terms and are not limiting, and these terms and expressions are not intended to exclude the illustrated and described configurations and some equivalents thereof. Rather, it will be appreciated that various modifications are possible within the scope of the claimed invention. Accordingly, although the present invention has been specifically disclosed by preferred embodiments and arbitrary configurations, modifications and variations of the concept disclosed herein may be made by those skilled in the art, and such modifications and variations are accompanied. It is considered to be within the scope of the invention as defined by the claims.

  Other embodiments are set forth in the following claims.

The effect of TMD23 on HaCaT epithelial cell migration is shown. Time course of wound closure in CD-1 mice treated with topical application of DMAp or CGS-21680 (in solution). The test substance and vehicle were administered topically once a day for 10 consecutive days. CGS-21680 (10 μg / mouse) as a positive control was locally administered at the same timing. Wound closure rate (%) and wound semi-closed time (CT50) were measured, one-way analysis of variance and subsequent Dunnett tests were performed on days 3, 5, 7, 9, 11 and treatment groups and their corresponding vehicle groups And compared. Time course of wound closure in CD-1 mice treated with topical application of DMAc or Regranex (gelled). The test substance was administered topically once a day for 10 consecutive days. Wound closure rate (%) and wound semi-closed time (CT50) were measured, one-way analysis of variance and subsequent Dunnett tests were performed on days 3, 5, 7, 9, 11 and treatment groups and their corresponding vehicle groups And compared. Time course of wound closure in diabetic mice (Lepr db) treated with topical application of DMAp or CGS-21680 (in solution). Test substance and vehicle were administered topically once a day for 14 consecutive days. CGS-21680 (10 μg / mouse) as a positive control was locally administered at the same timing. Wound closure rate (%) and wound half-closed time (CT50) were measured, one-way analysis of variance and subsequent Dunnett tests were performed on days 3, 5, 7, 9, 11, 13, 15 and treatment groups and their Comparison was made with the corresponding vehicle group. Time course of wound closure in diabetic mice (Lepr db) treated with topical application of DMAc or Regranex (gelled). The test substance was administered topically once a day for 14 consecutive days. Wound closure rate (%) and wound half-closed time (CT50) were measured, one-way analysis of variance and subsequent Dunnett tests were performed on days 3, 5, 7, 9, 11, 13, 15 and treatment groups and their Comparison was made with the corresponding vehicle group. TMD23 shows reduced transpiration rate from wound opening.

Claims (21)

  A method for treating a wound, comprising administering to a patient in need of such treatment an effective amount of a polypeptide comprising an amino acid sequence having an EGF-like domain of thrombomodulin or a function-conserving variant thereof.   2. The method of claim 1, wherein said polypeptide further comprises an operably linked polypeptide comprising an amino acid sequence of a serine threonine-rich domain of thrombomodulin or a function-conserving variant.   The wound is selected from the group consisting of incision wounds, lacerations, abrasions, stab wounds, blisters, skin tears, donor or transplant sites, acne, contusions, hematomas, pressure wounds and wounds caused by skin peeling or laser peeling. The method of claim 1.   The method according to claim 1, which is used for patients with diabetes mellitus.   The method of claim 4, wherein the patient suffers from a diabetic ulcer.   The method of claim 1, wherein the wound is a burn resulting from fire, heat, radiation, electricity or skin surgery.   The method according to claim 1, which is applicable to reconstruction surgery.   2. The method of claim 1 applied to a skin-borne product selected from the group consisting of gels, creams, pastes, lotions, sprays, suspensions, aqueous solutions, dispersions, hydrogels, and ointment formulations.   9. The method of claim 8, wherein the skin-borne product is administered to the patient in need of such treatment by application to the skin, infusion, or electroporation.   A method for treating wounds, comprising administering an effective amount of a plasmid comprising a DNA encoding a polypeptide containing an amino acid sequence having an EGF-like domain of thrombomodulin or a function-conserving variant thereof to a patient in need of such treatment. A method of treating a wound, comprising:   The method according to claim 10, wherein the administration is performed using a plasmid vaccine.   11. The method of claim 10, wherein the administration is by intravenous (iv), subcutaneous (sc), abdominal cavity (ip), or muscular (im) route.   A composition for use in wound healing comprising a polypeptide comprising an amino acid sequence having an EGF-like domain of thrombomodulin or a function-conserving variant thereof.   14. The composition of claim 13, wherein the polypeptide further comprises an operably linked polypeptide comprising an amino acid sequence of a serine threonine-rich domain of thrombomodulin or a function-conserving variant.   The wound is selected from the group consisting of incision wounds, lacerations, abrasions, stab wounds, blisters, skin tears, donor or transplant sites, acne, contusions, hematomas, pressure wounds and wounds caused by skin peeling or laser peeling. The composition according to claim 13.   The composition according to claim 13, which is used for patients with diabetes mellitus.   The composition of claim 16, wherein the patient suffers from a diabetic ulcer.   14. A composition according to claim 13, wherein the wound is a burn resulting from fire, heat, radiation, electricity or skin surgery.   The composition according to claim 13, which is applicable to reconstruction surgery.   14. A composition according to claim 13, applied to a skin-borne product selected from the group consisting of gels, creams, pastes, lotions, sprays, suspensions, aqueous solutions, dispersions, hydrogels, and ointment formulations.   21. The composition of claim 20, wherein the skin-borne product is administered to the patient in need of such treatment by application to the skin, infusion, or electroporation.

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