BRPI0613227A2 - a wound healing composition and use of this - Google Patents

Abstract

A COMPOSITION FOR WOUND HEALING AND USE OF THIS The invention relates to a method for treating wound healing, comprising administering, to a patient in need of this treatment, an effective amount of a polypeptide comprising a amino acid sequence or a conservative variant this having an EGF-like domain of thrombomodulin. The invention also relates to a composition for use in accelerating wound healing, comprising a polypeptide comprising an amino acid sequence or a conservative variant thereof having an EGF-like domain of thrombomodulin.

Description

"A Composition for Wound and Wound Healing".

BACKGROUND OF THE INVENTION

Field of the Invention

The invention relates to a method and composition for use in accelerating wound healing.

Angiogenesis and Thrombomodulin

Although various molecules are known in the art as useful therapeutic agents for the treatment of diseases and disorders having as a symptom of these abnormal coagulation in critical blood vessels, there is a natural need for compositions and methods that are useful for treating these diseases as well as for treating diseases. disorders that have abnormally high or abnormally low angiogenic activity as symptoms. These conditions and conditions include the following: cardiovascular diseases and conditions such as hypotension, hypertension, and atherosclerosis; thrombotic conditions such as stroke, atachecardiac and post angioplasty probes; angiogenic disorders; respiratory diseases and conditions such as pulmonary fibrosis and asthma; diseases and disorders related to tumor cell invasion, angiogenesis and metastasis; deferred healing and coagulation disorders, and reproductive disorders such as premature contraction or impotence.

Currently, a series of biomolecules, which induce or promote tissue angiogenesis, have been identified. Most prominent of these are: growth factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EFG), platelet-derived growth factors (PDGFs), transforming growth factors (TGFs) ). The absence of blood coagulation regulating thrombomodulin causes embryonic mortality in mice prior to the development of a functional cardiovascular system. (Proc. Natl. Acad. Sci. USA. 1995; 92: 850-854; and J. P. Cooke et al., Circulation 105 (2002) 2133). Current approaches directed at promoting related field angiogenesis can be summarized into three main categories: (i) provision of angiogenic growth factors using synthetic and natural polymeric platforms, (ii) provision of DNA-containing plasmids encoding angiogenic proteins; and (iii) combined provision of angiogenic molecules and endothelial cell transplantation.

According to the related techniques, another report (Shi CS et al., Evidence of Human Thrombomodulin domaines to Novel Angiogenic Factor. Circulation. April 5, 2005; 111 (13): 1627-36) proposed loading application systems. recombinant human thrombomodulin molecules rather than growth defactors. It is proposed that loaded human recombinant thrombomodulinar may be released under physiological conditions, thereby promoting rapid localized angiogenesis.

Thrombomodulin is an anticoagulant endothelial cell membrane glycoprotein. Thrombomodulinarecombinant proteins, TMD2 (containing six EGF-like structures) and TMD23 (containing TMD2 and a serine-threonine-rich domain), exhibit mitogenic activity. The previous study (Shi SC et al., Evidence of Human Thrombomodulin domain as a Novel Angiogenic Factor). April 5, 2005; 111 (13): 1627-36) revealed the new angiogenic effects of the recombinant domain using in vitro and in vivo models. TMD23 has been shown to have higher activity than TMD2 in stimulating DNA synthesis in cultured human umbilical vein endothelial cells (HUVECs). Additionally, TMD23 stimulated chemotactic motility and capillary tube deformation in HUVECs, a mediated effect through kinase phosphorylation. extracellular signal-regulated p38 mitogen-activated protein echinase and the phosphatidylinositol-3 kinase / Akt / endothelial nitric oxide synthase pathway. TMD23 also stimulated endothelial cell expression of metalloproteinases and plasminogen matrix activators, which mediated extracellular proteolysis leading to endothelial cell invasion and migration during angiogenesis. In addition, implants containing rat corneal TMD23 induced the growth of new limbal blood vessels. Using the murine angiogenesis assay, TMD23 not only induced co-injected neovascularization with Matrigel eheparin, but also increased angiogenesis in Matrigel-containing A2058 demelanoma cells in nude mice. In conclusion, recombinant thrombomodulin domain TMD23 increased angiogenic response in vitro and in vivo.

Wound healing

The wound healing process has 3 phases.

They are: the inflammatory phase, the proliferative phase, and the dematuration phase.

The inflammatory phase is characterized by hemostasis and inflammation. Collagen exposed during dalesion formation activates the coagulation cascade (both intrinsic and extrinsic pathways), initiating the inflammatory phase. Following tissue damage, cell membranes, damaged by lesion formation, release thromboxane A2 and propostaglandin 2-alpha, potent vasoconstrictors. This initial response helps to limit bleeding. After a short period, capillary dilation occurs secondary to local histamine release, and inflammation cells are able to migrate to the deferred bed.

Platelets, the first response cell, release multiple chemokines, including epidermal growth factor (EGF), fibronectin, fibrinogen, histamine, platelet-derived growth factor (PDGF), serotonin, and von Willebrand factor. These factors help to stabilize the wound through of the decoagulum formation. These mediators act to control bleeding to eliminate the extent of injury. Platelet degranulation also activates the complement cascade, specifically C5a, which is a potent neutrophil chemoattractant.

The inflammatory phase continues, and more immune response cells migrate to the wound. The second response cell migrating to the wound, the neutrophil, is responsible for cleaning tissue fragments, complement-mediated bacterial opsonization, and bacterial destruction via oxidative eruption mechanisms (ie, formation of superoxide and hydrogen peroxide). Neutrophils kill bacteria and decontaminate gauged from foreign debris.

The next cells present in the wound are the leukocytes and the macrophages (monocytes). Macrophage, referred to as the harmonizer, is essential for wound healing. Numerous enzymes and cytokines are secreted by the macrophage. These include collagenases, which remove the remnants of the wound;

interleukins and tumor necrosis factor (TNF), which stimulate fibroblasts (produce collagen) and promote angiogenesis; and transforming growth factor (TGF), which stimulates keratinocytes. This step marks the transition to the detained reconstruction process, that is, the proliferative phase.

The second stage of wound healing is proliferative aphasia. Epithelialization, angiogenesis, detained granulation formation, and collagen deposition are the main steps in this anabolic portion of wound healing. Epithelialization occurs prematurely in lesion repair. If the base membrane remains intact, the epithelial cells migrate upwards in the normal pattern. This is equivalent to a first degree skin burn. The epithelial progenitor cells remain intact underneath the wound, and the normal deepiderm layers are recovered within 2-3 days. If the basement membrane has been destroyed, similar to a second or third degree burn, then the wound is reepithelialized from normal skin cells and skin appendages, if intact (eg hair follicles, sweat glands). .

TNF-alpha stimulated angiogenesis is marked by endothelial cell migration and capillary formation. The new capillaries provide wound nutrients and help maintain granulation tissue oil. Deferred bed capillary migration is critical for proper wound healing. The degranulation and tissue deposition phase requires nutrients supplied by the capillaries, and failure to do so results in an unhealed wound. Mechanisms to modify angiogenesis are under study and have significant potential to improve the healing process.

The final part of the proliferative phase is the formation of granulation tissue. Fibroblasts differentiate and produce background and then collagen. The background substance is deposited on the wound bed; Collagen is then deposited as the wound goes through the final stage of repair. Many different cytokines are involved in the proliferative phase of wound repair. The steps and exact control mechanism have not been elucidated. Some of the cytokines include PDGF, insulin-like growth factor (IGF), and EGF. All of these are necessary for collagen formation.

The final phase of wound healing is the maturation stage. The wound contracts, ultimately resulting in a smaller amount of apparent scar tissue. The entire process is a dynamic continuous mass with overlap of each phase and continuous remodeling. The wound reaches its maximum resistance in one year, with a tensile strength that is 30% of normal skin. Collagen deposition continues for a prolonged period, but the network increases in collagen deposition plateaus after 21 days.

Currently, cytokines play a limited role in clinical practice. The only currently available commercial product that has proved effective in randomized double-blind studies is PDGF, available as recombinant human PDGF-BB. In multiple studies, recombinant human PDGF-BB has been shown to reduce healing time and improve the incidence of complete wound healing. in stage III and IV ulcers. Many other cytokines currently under study in vitro include TGF-beta, EGF, and IGF-I.

Proper wound healing involves complex interaction of cells and cytokines working together. In recent years, more chemical mediators integral to this process have been identified. The sequential steps and specific processes were not fully differentiated. When examining the wound healing process, it is necessary to identify the main steps and to know the important mediators.

Burn and Wound Healing

Sunburn is an injury resulting from exposure to heat, electricity, radiation (eg, sunburn and laser surgery), or caustic chemicals.

Three degrees of burns are commonly recognized. In first degree burns, the outer layer of the skin, called the epidermis, becomes red, touch sensitive, and often swollen. Medical attention is not required, but applying an ointment can relieve pain. Second degree burns are characterized by variable destruction of the epidermis and blister formation; nerve endings may be exposed. The most serious cases should be observed by a doctor and care should be taken to avoid infection. Local therapy included applying a chemical such as silver nitrate to produce a soft crust, reduce the threat of infection, and alleviate pain. Third-degree burns involve the destruction of the total thickness of the skin and underlying connective tissue. In more severe cases, underlying bones are also partially burned. The surface area involved is more significant than the depth of the burn. Shock must be avoided or countered; Blood transfusion may be required to replace lost body fluids. Invasion of various bacteria should be prevented or cured by administering antibiotics and other medications. Morphine may be employed to relieve pain. Long-term treatment may include transplantation of natural or artificial skin grafts.

Diabetes Mellitus and Wound Healing

Diabetes mellitus is a group of metabolic diseases characterized by high blood sugar (glucose), which results from defects in insulin secretion, or insulin intake, or both. Diabetes mellitus, commonly referred to as diabetes, means "sweet urine". Elevated blood glucose levels (hyperglycemia) lead to naurine glucose shedding, and thus the term sweet urine. Normally, blood glucose levels are strictly controlled by insulin, a hormone produced by the pancreas. Insulin lowers blood glucose level. When blood glucose rises (eg after food intake), insulin is released from the pancreas to normalize the glucose level. In patients with diabetes mellitus, the absence or insufficient production of insulin causes hyperglycemia. Diabetes mellitus is a chronic medical condition, meaning it can last a lifetime.

Diabetes can lead to a number of complications including heart and vascular disease, blindness, kidney failure and foot ulcers.

People with diabetes are at risk for foot injury due to numbness caused by nerve damage (diabetic neuropathy) and low blood flow to the legs and feet. The most serious injury is a foot ulcer. Diabetic foot ulcers are at high risk of becoming infected, and sometimes they cannot be healed. Uncurable foot ulcers are a frequent cause of amputation in people with diabetes.

The FDA has released a gel product (becaplermin, or Regranex Gel) that is used as a treatment for diabetic foot ulcers. This product contains genetically modified platelet derived growth factor, one of the proteins that the body produces to encourage new tissue growth.

Clinical studies of the product indicated that the probability of complete ulcer closure after up to 20 weeks of treatment was higher when becaplermin was used.

Another product that helps in closing deferred slow healing ulcers in patients with diabetes is a skin substitute called DERMAGRAFT®. This is composed of human cells known as fibroblasts that are placed in a dissolvable mesh material. When the mesh material is placed in the ulcer, it is gradually absorbed and the human cells grow and replace the damaged tissue in the ulcer.

SUMMARY OF THE INVENTION

The invention relates to a method for treating wound healing comprising administering to a patient in need of such treatment an effective amount of a polypeptide comprising an amino acid sequence or a conservative variant thereof having thrombomodulin EGF-like domain. The invention also relates to a composition for use in accelerating wound healing comprising a polypeptide comprising a amino acid sequence or a conservative variant thereof having thrombomodulin EGF-like domain.

Other objectives and features of the present invention will become apparent from the following detailed description taken in conjunction with the accompanying drawings. It should be understood, however, that the drawings are intended for illustration purposes only and not as a definition of the limits of the invention, to which reference should be made to the appended claims. It should further be understood that the drawings are not necessarily made to scale and that, unless otherwise indicated, they are intended merely to conceptually illustrate the structures and procedures described herein.

BRIEF DESCRIPTION OF DRAWINGS

In the drawings:

Figure 1 shows the effect of TMD23 on HaCaT epithelial cell migration.

Figure 2 is a time period for wound closure in DMAp or CGS-21680 treated CD-I mice (in solution form) by topical application. The test substance and vehicle were each administered topically once a day for 10 consecutive days. CGS-21680 (10μg / mouse) as a positive control was administered topically at the same time period. Wound closure (%) and half wound closure time (CT50) were determined and one-way ANOVA, followed by Dunnett's test, was applied for comparison between the treated groups and their corresponding vehicle groups on days 3, 5, 7 , 9 and 11.

Figure 3 is a deferred closure time period in DMAc or Regranex-treated CD-I mice (in gel form) by topical application. Test substances were each administered topically once daily for 10 consecutive days. Wound closure (%) and half wound closure time (CT50) were determined and one-way ANOVA, followed by Dunnett's test, were applied for comparison between treated groups and their corresponding vehicles on days 3, 5, 7, 9 and 11.

Figure 4 is a deferred closure time period in diabetic (Lepr db) mice treated with DMAp orCGS-21680 (in solution form) by topical application. Test substance and vehicle were each administered topically once daily for 14 consecutive days. CGS-21680 (10μg / mouse) as a positive control was administered topically over the same period. Wound closure (%) and half wound closure time (CT50) were determined and one-way ANVA followed by Dunnett's test were applied for comparison between treated groups and their corresponding vehicles on days 3, 5, 7, 9 , 11, 13 and 15.

Figure 5 is a deferred closure time period in diabetic (Lepr db) mice treated with DMAc or Regranex (gel form) by topical application. Detesting substances were each administered topically once a day for 14 consecutive days. Wound closure (%) and half wound closure time (CT50) were determined and one-way ANVA followed by Dunnett's test were applied for comparison between treated groups and their corresponding vehicles on days 3, 5, 7, 8 , 11, 13 and 15.

Figure 6 shows that TMD23 reduces the rate of evaporation of the wound opening.

DETAILED DESCRIPTION OF THE INVENTION

Thrombomodulin is an endothelial cell membrane glycoprotein anticoagulant. A recombinant detrombomodulin domain containing six epidermal factor-decreasing factor-like structures and serine-threonine-rich domain exhibits mitogenic reactivity.

In the previous report (application N2: US 11 / 149,378) it was shown that TMD23 could induce endothelial cell migration and proliferation (HUVEC).

In the present invention, it has been found that TMD23 could effectively enhance keratinocyte migration (HaCaT), the main cell type in the epidermis and the TMD23 anti-counterpart could specifically inhibit keratinocyte migration. Therefore, it was demonstrated for the first time that TMD23 could increase the wound healing process by stimulating keratinocyte migration.

In addition, animal experiments have shown that TMD23 could increase migration and extension of the epidermis in the wound area, thereby accelerating wound closure. On the other hand, the rate of water evaporation from the wound opening has been effectively reduced as well. In conclusion, TM can effectively increase the rate of wound healing.

The present invention relates to a wound healing treatment method comprising administering to a patient in need of such treatment an effective amount of a polypeptide comprising an amino acid sequence or a conservative variant of this thrombomodulin-like EGF tendodomain. In the preferred embodiment, the opolipeptide further comprises a operably linked polypeptide comprising an amino acid sequence or a serine-threonine detrombomodulin rich domain conservative variant.

The method of the invention may be applied to accelerate wound healing, where the wound is selected from the group consisting of incisions, lacerations, abrasions, perforation wounds, blisters, skin tears, donation or graft sites, acnes, bruises, bruise, crush injuries. and injuries caused by dermal abrasion or laser removal.

The method of the invention may be used in patients with diabetes mellitus. In a preferred embodiment, the patient is suffering from diabetes ulcers.

The method of the invention may also be applied when the wound is a burn resulting from fire, heat, radiation, electricity or dermatological surgery.

The method can be applied to the reconstructive surgeon.

In a preferred embodiment, the method is applied to a product for application to the skin selected from the group consisting of gel, cream, paste, lotion, spray, suspension, solution, dispersion ointment, and hydrogungant. In a more preferred embodiment, the skin application product is administered to the patient in need of retreatment by application to the skin, by injection or electroporation.

The present invention also relates to a method of treating wound healing comprising administering to a patient in need of such treatment an effective amount of plasmid consisting of DNA encoding a polypeptide comprising an amino acid sequence or a conservative variant thereof having EGF-detrombomodulin-like domain . In a preferred embodiment, administration is by plasmid vaccine means. In a more preferred embodiment, administration is by intravenous (iv), subcutaneous (sc), intraperitoneal (ip), or intramuscular (im) route.

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

The composition of the invention may be applied in wound healing acceleration, where the wound is selected from the group consisting of incisions, lacerations, abrasions, puncture wounds, blisters, skin tears, donor or graft sites, acnes, bruises, bruise, bruising. crushing and injury caused by dermal abrasion or laser removal.

The composition of the invention may be used in patients with diabetes mellitus. In a preferred setting, the opacient suffers from diabetic ulcers.

The composition of the invention may also be applied when the wound is a burn resulting from fire, heat, radiation, electricity or dermatological surgery.

In a preferred embodiment, the composition may be applied to reconstructive surgery.

In a preferred embodiment, the method is applied to a product for application to the skin selected from the group consisting of gel, cream, paste, lotion, spray, suspension, solution, dispersion ointment, and hydrogungant. In a more preferred embodiment, the skin application product is administered to the patient in need of retreatment by skin application, injection or electroporation.

DEFINITION OF TERMS

The following definitions are offered for illustrative and non-limiting purposes to assist in understanding the following discussion.

In accordance with the present invention, conventional molecular biology, microbiology, and DNA recombinant techniques known to those skilled in the art may be employed. These techniques are fully explained in the literature.

"Wounds" can be characterized as open wounds and closed wounds. Open wounds can be classified into a number of different types, including incisions (caused by a clean, sharp-edged object such as a knife or blade), lacerations (coarse, irregular wounds caused by crushing or tearing forces), abrasions or abrasions (a wound). the superficial layers of the skin are scraped, often caused by a sliding fall on a rough surface, and puncture wounds (caused by a skin-piercing object, such as a nail or a needle). Closed wounds have far fewer categories but are as dangerous as open wounds. These are bruises or blows (caused by blunt force trauma that damage tissues under the skin), bruising (caused by damage to a blood vessel, which in turn causes blood to accumulate under the skin) and crushing injuries (caused by a large or extreme amount force applied over a stormy period).

The "wound healing" process has 3 stages. They are: the inflammatory phase, the proliferative phase, and the maturation phase.

The inflammatory phase is characterized by hemostasis and inflammation. Collagen exposed during deferred formation activates the coagulation cascade (both intrinsic and extrinsic pathways), initiating the inflammatory phase. After tissue healing occurs, the cell membranes damaged by wound formation release thromboxane A2 and prostaglandin 2-alpha, potent vasoconstrictors. This initial answer helps to limit bleeding. After a short period, capillary vasodilation occurs following local release of histamine, and inflammation cells are able to migrate to the wound bed. The inflammatory phase continues, and more immune response cells migrate to the wound. The second response cell to migrate to the calibrated neutrophil is responsible for cleaning debris, complement-mediated opsonization of bacteria, and destruction of bacteria via oxidative burning mechanisms (ie, deoxide and hydrogen peroxide formation). Neutrophils kill and decontaminate the wound from foreign debris.

The second stage of wound healing is proliferative aphasia. Epithelialization, angiogenesis, detained granulation formation, and collagen deposition are the main steps in this anabolic portion of wound healing. Epithelialization occurs early in wound repair. If the thinning membrane remains intact, the epithelial cells migrate upwards in the normal pattern. This is equivalent to a first burn. Epithelial progenitor cells remain intact below the wound, and normal epidermal layers are recovered within 2-3 days. If the basement membrane has been destroyed, similar to a second or third degree burn, then the wound is reepithelialized from normal peripheral cells and from skin appendages, if intact (eg hair follicles, sweat glands). . The final part of the proliferative phase is the formation of granulation tissue. Fibroblasts differentiate and produce background substance and then collagen. Background substance is deposited on the wound bed; Collagen is then deposited as the wound goes through the final repair phase. Many different cytokines are involved in the proliferative phase of wound repair. The steps and exact control mechanism have not been elucidated. Some of the cytokines include PDGF, insulin-like growth factor (IGF), and EGF. All are required for collagen formation.

The final phase of wound healing is the maturation stage. The wound contracts, ultimately resulting in a smaller amount of apparent scar tissue. The entire process is a dynamic continuous process with overlap of each phase and continuous remodeling. The wound reaches maximum resistance within one year, with a tensile strength that is 30% of normal skin. Collagen deposition continues for a prolonged period, but the network increases on collagen deposition plateaus after 21 days.

"Angiogenesis" is generally considered to be largely regulated by growth factors and other ligands. Angiogenesis, and the detained simultaneous development and regeneration, depend on the strictly controlled processes of endothelial cell proliferation, migration, differentiation, and survival. Both stimulating and inhibiting ligands appear to interact, directly or indirectly, with cell receptors during these processes. Angiogenesis begins with the erosion of the base membrane by enzymes released by endothelial cells elucocytes. Endothelial cells, which line the lumen of vasosanguines, then protrude through the base membrane. Angiogenic stimulators induce endothelial cells to migrate through the eroded base membrane. Migrant cells then form a "bud" from the mother blood vessel, where the endothelial cells undergo mitosis and proliferate. Endothelial shoots split together to form capillary loops, creating the new blood vessel. As used herein, the term "burn" is injury resulting from exposure to heat, electricity, radiation (eg, sunburn and laser surgery), or products caustic chemicals.

"Diabetes mellitus" is a group of metabolic diseases characterized by high blood sugar (glucose), which results from defects in secretion or action of insulin, or both. Diabetes mellitus, commonly referred to as diabetes, means "sweet urine". High blood glucose levels (hyperglycemia) lead to naurine glucose shedding, thus the term sweet urine. Normally, blood glucose levels are tightly controlled by insulin, a hormone produced by the pancreas. Insulin lowers blood glucose level. When blood glucose rises (eg after food intake), insulin is released from the pancreas to normalize the glucose level. In patients with diabetes mellitus, the absence or insufficient production of insulin causes hyperglycemia. Diabetes mellitus is a chronic medical condition, meaning that it can extend for a lifetime.

Diabetes can sometimes lead to peripheral aneuropathy which, specifically when combined with damaged blood vessels, can lead to foot ulcers, possibly progressing to necrosis, infection and gangrene, sometimes requiring limb amputation.

A "nucleic acid" or "nucleotide sequence" refers to the polymeric form of deribonucleoside phosphate ester (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleoside (deoxyadenosine, deoxyguanosine, deoxythymidine, or "deoxycytidine; deDNA ") both in the form of a single ribbon and a single-stranded propeller. DNA-DNA, DNA-RNA, and double-stranded RNA-RNA helices are possible. The term nucleic acid, and in particular DNA or RNA molecules, refers only to the primary and secondary structures of the molecule, and is not limited to any specific tertiary or quaternary forms. Thus, this term includes double-stranded DNA found, among others, in linear or circular DNA molecules (eg, restriction fragments), plasmids, and chromosomes. In the discussion of the structure of specific double-stranded DNA molecules, sequences may be described herein according to the normal convention of providing only the 5 'to 3' 'direction sequence along the non-transcribed strand of DNA (ie, the strand having one sequence). homologous to mRNA). A "Recombinant DNA" is a DNA molecule that has undergone molecular biological manipulation.

"Operably linked", when referring to DNA segments, indicates that the segments are arranged so that they function together, for example, the transcriptional process occurs by binding RNA polymerase to the promoter segment and proceeding with transcription through the DNA segments. coding until the polymerase stops when it encounters a transcription termination segment.

As used herein, the term "nucleic acid fragment" is intended to indicate any cDNA nucleic acid molecule, genomic DNA, synthetic DNA or RNA origin. The term "fragment" is intended to indicate a nucleic acid segment that may be single or double stranded, and which may be based on a complete or partial naturally occurring nucleotide sequence encoding a polypeptide of interest. The fragment may optionally contain other nucleic acid segments.

The nucleic acid fragment of the invention encoding the polypeptide of the invention may suitably be genomic or cDNA origin, for example obtained by preparing a genomic library or cDNA and exploiting DNA sequences encoding all or part of the polypeptide by hybridization using synthetic oligonucleotide probes according to the invention. with standard techniques.

Polypeptides can be detected using methods known in the art that are specific for polypeptides. These detection methods may include the use of antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme analysis may be used to determine polypeptide activity.

The polypeptides of the present invention may be purified by a variety of known procedures in the art, including but not limited to chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocalization, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focus ( IEF)), differential solubility (eg, ammonium sulfate precipitation), or extraction.

As used herein, the term "plasmid vaccines" is defined as purified plasmid DNA preparations designed to contain a gene or genes for the intended vaccine antigen, as well as genes incorporated into the construct to allow production in a suitable host system.

EXAMPLES

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

Example 1: Effect of TMD23 on HaCaT Epithelial Cell Migration

The effect of TMD23 on HaCaT cell migration was evaluated using Boyden's chamber with 6.5mm diameter (8-μπι pore size) depolycarbonate filters. The bottom surface of the filter was coated with collagen type IV.TMD23 (100 ng / ml) or TMD23 (100 ng / ml) plus anti-TM antibody (1 µg / ml) was added in DMEM in the lower wells. The cell suspension (50 μΐϋ) containing 1 χ 10 4 cells was loaded into each upper well. The number of cells that migrated across the membrane within hours was counted under a microscope after being fixed with methanol and stained with 10% GIEMSA. As shown in Figure 1, TMD23 significantly induced chemotactic migration in HaCaT cells.

Example 2: Materials

DMAp was 100 μg of recombinant purified protein TMD23 (SEQ ID NO: 1) in 20 μΐ of 0.5% CMC / PBS solution. CMC (carboxymethylcellulose) / PBS (phosphate buffered saline) at a pH of 7.4 was used as the carrier in the dissolution form for DMAp and CGS-21680. (2-p- [2-Carboxyethyl] phenethylamino-5'-N-ethylcarboxamidoadenosine) hydrochloride CGS-21680era was a G protein activator and was purchased from sigma-Aldrich.Gel DMAc was 100 μ9 of the recombinant purified TMD23 protein (SEQ ID N2 1) in 20 mg of base gel. The base gel was used as a negative control of DMAc gel and Regranex gel. The gelbase was composed of sodium carboxymethylcellulose, sodium chloride, sodium acetate trihydrate, glacial acetic acid, methylparaben, propyl paraben, m_Cresol 1-lysine hydrochloride, benzyl alcohol, methylchloroisothiazoline, methylisothiazolinine, acryloyl dimethyltaurate copolymer / ammonium water for injection and . GelRegranex was purchased from Johnson & Johnson.

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

For equipment used in the examples, animal cage (Allentown, USA), Image - ProPlus (MediaCybernetics, Version 4.5.29), pipette (Gilson, Germany) and sharp drill, 12 mm inner diameter (Sinter, R. 0. C .)was used.

Example 3: Animals

Male (Cri.) CD-I derived mice weighing 24 ± 2 g were provided by BioLasco Taiwan (under License Charles River Laboratories Technology). The space allocation for 10 animals was 29 χ 18 χ 13 cm. All animals were kept in an environment of temperature (22 ° to 24 ° C) and humidity (50% - 60%) controlled with 12 hour light / dark cycles for at least one week at MDS Pharma Services - Taiwan Laboratory use. Free access to standard laboratory feed for mice [MF-18 (Oriental Yeast Co., Ltd., Japan)] and RO water was provided. All aspects of this work including animal housing, experimentation and disposal were performed in accordance with the Guide to Laboratory Animal Care and Use (National Academy Press, Washington, D.C., 1996).

Male mice (NIDDM) with non-insulin dependent diabetes mellitus (C57BLKS / J-m + / + Lepr db), weighing 50 ± 5 g (9 weeks old), provided by the Institute for Animal Reproduction (IAR, Japan), were used. These animals exhibit hyperinsulinemia, hyperglycemia and islet atrophy. The animals were housed on shelves with Individually Ventilated Cages (IVC Racks, 36 Mini Isolator systems) under Specific Pathogen Free (SPF) condition throughout the experiment. Each APEC cage (in cm, length 26.7 χ 20.7 width χ 14.0 height) was autoclaved and contained 7 mice, and the animals were then kept in a hygienic environment at temperature (22 ° to 24 ° C). ° C) and humidity (50% -60%) controlled with 12 hour light / dark cycles. The animals were allowed free access to sterile laboratory feed and sterile distilled water adlibitum. All aspects of this work, that is, housing, experimentation, and animal disposal, are performed according to the Guide for Laboratory Animal Care and Use (National Academy Press, Washington, D.C., 1996).

Example 4: TMD23 speeds wound closure in mice

Groups of 5 male CD-I-derived (Cri.) Mice each weighing 24 ± 2 g were used. During the test period, the animals were used alone in individual cages. Under hexobarbital anesthesia (90 mg / kg, PI), each animal's shoulder and back region were shaved. A sharp puncture (12 mm ID) was applied to remove the skin including papanniculus carnosus and adherent tissues. DMAc, Regranex and 20 mg / mouse deBAse gel and 100 μg / mouse DMAp, as well as the vehicle (0.5% CMC / PBS pH 7.4, 20 μΐ / mouse) and 10 µg / mouse CGS-21680 as the Positive controls were each administered topically (TOP) immediately after the cutaneous wound once a day for 10 consecutive days. The deferred area drawn on transparent plastic sheets was measured by an Image - ProPlus (Media Cybernetics, Version 4.5.29) on days 1, 3, 5, 7, 9ell. Wound closure percentage (%) was calculated, and half wound closure time (CT50) was analyzed by linear regression using Graph-Prism (GraphSoftware USA). One-way ANOVA followed by Dunnet's test were applied for comparison between treated groups and their corresponding vehicle groups at each measured timepoint. Differences are considered statistically significant at P <0.05. CGS-21680 (10 μg / χ 10 mouse) as positive standard exhibited significant increase (P <0.05) in wound closure (on days 3, 5, 7, 9, and 11) with decreased CT50 relative to vehicle control values. corresponding. The results are summarized in Figure 2 (in the solution phase), Figure 3 (in the gel phase) and Table 1 below: <table> table see original document page 27 </column> </row> <table>

Wound closure (%) and half wound closure time (CT50) were determined and one-way ANOVA by Dunnett's test was used for comparison between the treated group and its corresponding vehicle groups.

* P <0.05, vs. vehicle control.

Table 1: The effect of test articles on wound closure and closure time of half of the wound in the CD-1 mouse.

It is concluded that DMAp (100 μ9 / σβπηΗΗΐοη9θ) demonstrated a more persistent effect on wound healing throughout the study with a significant reduction in the CT50 value, DMAc and Regranex tended to promote wound healing but no significant effect on the CT50; The base gel had no effect on the mouse skin wound model.

Example 5: TMD23 accelerates wound closure in mice with diabetes.

Groups of 5 male C57BLKS / J-M + / + Leprdb mice weighing 50 ± 5 g were used. During the test period, the animals were housed alone on shelves of Individually Ventilated Cages (IVC Racks, 36 Mini Insulator Systems). Under hexobarbital anesthesia (90 mg / kg, PI), each animal's shoulder and back were shaved. A sharp puncture (12 mm ID) was applied to remove the skin including panniculus carnosus and adherent tissues. The deferred area, drawn on transparent plastic sheets, was measured on days 1, 3, 5, 7, 9 and 11, 13 and 15 using a imaging analyzer (ProPlus, Media Cybernetics, Version 4.5.29). Test substances in the form of solution or gel and vehicle, GCS-21680 or Regranex positive control were each applied topically starting on day 1 immediately after wound formation once a day for a total of 14 consecutive days. Test substances DMAp (100 μg / mouse), vehicle (0.5% CMC / PBS pH 7.4) and CGS-21680 (10 μg / mouse) were used as a solution and dosing volume at 20 μΐ / mouse. Meanwhile, DMAc, Regranex and Base gel (as vehicle control group) are in gel form and were administered at 20 mg / mouse. Half-wound closure time (CT50) was determined by linear regression using Graph-Pad Prism (Graph Pad Software USA) and one-way ANOVA followed by Dunnet's test were applied for comparison between treated groups and vehicle groups in assessed point measurement. . Differences are considered statistically significant at P <0.05. CGS-21680 (10μg / χ 14 mouse) and Regranex (20 mg / χ 14 mouse) as the positive pattern substance showed a significant increase (P <0.05) in the wound closure percentage (on days 3, 5, 7, 9, 11, 13 and 15) and significant decrease in CT50 relative to the corresponding vehicle control values. The results are summarized in Figure 4 (in the solution phase), Figure 5 (in the defrost phase) and Table 2 below:

Table 2: The effect of the test articles on wound closure and wound closure time in Mice Lepr db

<table> table see original document page 29 </column> </row> <table> <table> table see original document page 30 </column> </row> <table>

Wound closure (%) and half wound closure time (TCso) were determined and one-way ANOVA followed by Dunnett's test were used for comparison between the treated group and their corresponding vehicle groups.

* P <0.05, vs. vehicle control.

It was concluded that DMAp (100 μg / mouse), DMAC eRegranex (each 20 mg / mouse), and CGS-21680 (10μg / mouse) demonstrated persistent increasing wound healing effect throughout the study with a significant reduction in CT50 value. In the initial phase, Gel Vehicle appeared to have a short delay in wound healing compared to 0.5% CMC / PBS pH 7.4 vehicle.

Example 6: TMD23 Reduces Wound Opening Evaporation Rate

To measure epithelialization rate, wound area water evaporation rate was measured using the TM210Tewameter (COURAGE + KHAZAKA Electronic Co., Cologne, Germany). Lower evaporation rate level is representative of higher level of epithelialization or keratinization. The results (Figure 6) showed that TMD23 could effectively increase the deepithelialization rate of the wound.

One skilled in the art readily observes that the present invention is well adapted to accomplish the objectives and obtain the results and advantages mentioned as well as those inherent in them. The cell lines, animals, processes and methods for producing these are representative of preferred embodiments, are exemplary, and are not intended to limit the scope of the invention. Modifications to this and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

It will be immediately apparent to a person skilled in the art that various substitutions and modifications may be made to the invention presented herein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification are indicative of the levels of those ordinary expertise in the art to which the invention belongs. All patents and publications are incorporated herein by reference to the same extent as if each individual publication had been specifically and individually indicated as incorporated by reference.

The invention illustratively described herein may suitably be practiced in the absence of any element or element, limitation or limitation, which is not specifically disclosed herein. The terms and expressions that have been used are used as terms of description and not limitation, and there is no intention that in the use of these terms and expression any equivalents of the characteristics shown and described or portions thereof are acknowledged, but it is recognized that various modifications are possible within scope of the claimed invention. Thus, it should be understood that while the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts disclosed herein may be used by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. .

Other configurations are presented within the following claims.

Claims (21)

A method for treating deferred healing, characterized in that it comprises administering to a patient in need of such treatment an effective amount of a polypeptide comprising a amino acid sequence or a conservative variant thereof having thrombomodulin EGF-like domain. A method according to claim 1, characterized in that the polypeptide further comprises an operably linked polypeptide comprising an amino acid sequence or a thrombomodulin rich serine-threonine domain conservative variant. Method according to claim 1, characterized in that the wound is selected from the group consisting of incisions, lacerations, abrasions, puncture wounds, blisters, skin tears, donation or graft sites, acnes, bruises, bruise, crush injuries and injuries caused by dermal abrasion or laser removal. Method according to claim 1, characterized in that it is used in patients with diabetes mellitus. Method according to claim 4, characterized in that the patient suffers from dediabetic ulcers. Method according to claim 1, characterized in that the wound is a burn resulting from fire, heat, radiation, electricity or dermatological surgery. Method according to claim 1, characterized in that it can be applied to the reconstructive surgeon. Method according to claim 1, characterized in that it is applied to a skin application product selected from the group consisting of gel formulation, cream, paste, lotion, spray, suspension, solution, dispersion ointment, hydrogel and ointment. Method according to claim 8, characterized in that the skin application product is administered to the patient in need of such treatment by skin application, injection or electroporation. A method for treating deferred healing, comprising administering to a patient in need of such treatment an effective amount of plasmid consisting of DNA encoding a polypeptide comprising an amino acid sequence or a conservative variant thereof, having an EGF-like domain detrombomodulin. Method according to claim 10, characterized in that the administration is by means of plasmid devacin. Method according to claim 10, characterized in that the administration is by intravenous (iv), subcutaneous (sc), intraperitoneal (ip), or intramuscular (im) administration. 13. Composition for use in deferred healing, characterized in that it comprises a polypeptide comprising an amino acid sequence or a conservative variant thereof having thrombomodulin EGF-like domain. Composition according to Claim 13, characterized in that the polypeptide further comprises an operably linked polypeptide comprising an amino acid sequence or a conservative variant of thrombomodulin serine-threonine rich domain. Composition according to Claim 13, characterized in that the wound is selected from the group consisting of incisions, lacerations, abrasions, perforation wounds, blisters, skin tears, donation or graft sites, acnes, bruises, bruise, crush injuries and injuries caused by dermal abrasion or laser removal. Composition according to Claim 13, characterized in that it is used in patients with diabetes mellitus. Composition according to Claim 16, characterized in that the patient suffers from dediabetic ulcers. Composition according to Claim 13, characterized in that the wound is a burn resulting from fire, heat, radiation, electricity or dermatological surgery. Composition according to Claim 13, characterized in that it can be applied to the reconstructive surgeon. Composition according to Claim 13, characterized in that it is applied to a skin application product selected from the group consisting of a formulation such as defrost, cream, paste, lotion, spray, suspension, solution, dispersion, hydrogel and ointment. . Composition according to Claim 20, characterized in that the skin application product is administered to the patient in need of such treatment by skin application, injection or electroporation.