KR101567954B1 - Composition for preventing or treating erectile dysfunction comprising HGF protein or gene therefor and use thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for endothelial cell regeneration comprising an HGF protein, a biologically active fragment thereof or a fusion protein thereof as an active ingredient, a pharmaceutical composition for preventing or treating erectile dysfunction, a method for regenerating endothelial cells of an individual, A method for preventing or treating erectile dysfunction, and a health functional food.

Description

The present invention relates to a composition for preventing or treating erectile dysfunction, comprising a HGF protein or a gene thereof, and a composition for preventing or treating erectile dysfunction,

The present invention relates to a pharmaceutical composition for endothelial cell regeneration, a pharmaceutical composition for preventing or treating erectile dysfunction, a method for regenerating endothelial cells of an individual, a method for preventing or treating erectile dysfunction of a subject, or a health functional food.

HGF is a proliferation promoting factor for primary cultured hepatocytes and is an isolated and replicated growth factor. HGF is produced by various mesenchymal cells and mostly targets epithelial cells, neurons, endothelial cells and some mesenchymal cells. HGF is involved in cell proliferation promoting activity and has cell motility promoting activity and epithelial morphogenesis inducing activity. HGF is involved in the formation of internal organs such as liver, kidney, lung, placenta, and skeletal system as mediators of epithelial-mesenchymal interactions during development. HGF is an agent for regenerating the liver, digestive tract, kidney, and lungs. However, to date, there has been no description or description of techniques for the prevention or treatment of erectile dysfunction of HGF, and no studies have been conducted on it.

On the other hand, erectile dysfunction is a type of male sexual dysfunction. It is a phenomenon that can not be done because male penis is not erected or erect state is not maintained. The causes of erectile dysfunction are largely classified into psychogenic causes and substrate causes. Cardiogenic erectile dysfunction is caused by excessive secretion of noradrenaline due to excessive action of sympathetic nerves due to psychological and psychological influences, increased smooth muscle tension of the penis cavernosum, and suppression of neurotransmitter secretion. In addition, organic erectile dysfunction is classified as neurogenic, vascular, and endocrine erectile dysfunction, depending on its cause.

The vascular erectile dysfunction is caused by damage to penile vascular endothelial cells due to hyperlipidemia, diabetes, hypertension, smoking, systemic cardiovascular disease, etc., and the secretion of nitric oxide (NO) It is a disorder that occurs.

Recent studies have focused more on the underlying causes and have been used worldwide as oral PDE-5 (phosphodiesterase-5) inhibitors, including Viagra (sildenafil). These oral drugs are known to increase the concentration of cGMP due to the inhibition of PDE-5, which is specifically distributed in the corpus cavernosum, thereby enhancing blood flow in the corpus cavernosum, thereby inducing erection and treating erectile dysfunction. However, not only are several side effects reported, such as headache, flushing, indigestion, and heart attack, as well as PDE-5 inhibitor drugs such as Viagra control transient protein expression and related factors at the molecular level, It can not be said. Furthermore, in the case of erectile dysfunction due to diabetes, the effect of such a therapeutic agent is not well manifested, and even if it is effective, its efficacy can not be sustained for a long time.

Therefore, there is a need for a therapeutic agent for erectile dysfunction which fundamentally treats the abnormal normalized blood vessel structure in the erectile dysfunction penis, and the effect is also prolonged for a long time.

One aspect of the present invention is to provide a pharmaceutical composition for endothelial cell regeneration.

Another aspect of the present invention is to provide a pharmaceutical composition for preventing or treating erectile dysfunction.

Another aspect of the present invention is to provide a method for regenerating endothelial cells of an individual.

Another aspect of the present invention is to provide a method for preventing or treating erectile dysfunction of an individual.

Another aspect of the present invention is to provide a health functional food containing HGF protein and the like.

One aspect of the present invention provides a pharmaceutical composition for endothelial cell regeneration comprising an HGF protein or a polynucleotide encoding the same as an active ingredient.

In the present specification, "HGF protein" means hepatocyte growth factor. HGF is a heteroduplex protein consisting of an α chain of 69 kDa and a β chain of 34 kDa, and may have four Kringle structures in the α- chain. C- met with tyrosine kinase activity may be a receptor for HGF.

The composition may comprise the entire HGF protein as well as its biologically active fragments or fusion proteins thereof. The biologically active fragment may substantially comprise the vascular endothelial cell and smooth muscle cell regeneration activity of the HGF protein or the gene encoding the same. The biologically active fragment may comprise an active domain involved in vascular endothelial cell and smooth muscle cell activity of native HGF.

The fusion protein may substantially comprise the vascular endothelial cell and smooth muscle cell regeneration activity of the HGF protein or the gene encoding the same. The fusion protein may be that the HGF protein or fragment thereof is a fusion partner useful for the isolation of the HGF protein or fragment thereof, a fusion partner useful for delivery to the target site, a fusion partner for enhancing body stability, and the like. The linkage may be through the N-terminus or the C-terminus or side chain of the HGF protein or fragment thereof. The connection may be made by a covalent or non-covalent bond. The fusion partner may be one comprising a polypeptide. A fusion partner useful for separation may be a His sequence, e. G. His ₆ sequence. A fusion partner useful for delivery to a target site, or a fusion partner for enhancing body stability, may be an antibody constant region, for example, a Fc region or a polymer that confers resistance to degradation in the body, such as PEG.

The polynucleotide encoding HGF may be in its own form as well as fused with other substances. For example, the other substance may be selected from the group consisting of allowing the polynucleotide encoding the HGF to be delivered into the cell, allowing the gene to be expressed by a gene expression mechanism in the cell in the cell, And the like. The fusion may be by covalent or noncovalent bonding. The fusion includes a form that is captured in vesicles.

The polynucleotide encoding the HGF may be operably linked to a gene expression regulatory factor such as a promoter, operator, enhancer and / or transcription termination factor. The polynucleotide encoding HGF may be, for example, inserted into a plasmid or viral genome so that it can be expressed in a cell. The HGF-encoding polynucleotide may be a structure linked to a regulatory factor that specifically causes expression of endothelial cells and smooth muscle cells of the penis, for example, spongy endothelial cells and smooth muscle cells or penile vascular endothelial cells and smooth muscle cells. The polynucleotide encoding HGF may be, for example, in the form of a construct inserted into the adenovirus genome and linked to regulatory factors that are specifically expressed in cancellous endothelial cells and smooth muscle cells or penile vascular endothelial cells and smooth muscle cells. The structure may be collected on the virus particles.

As used herein, the term "active ingredient" includes those having an activity of further promoting endothelial cell regeneration, when the composition is administered to an individual. The subject may be at least one selected from the group consisting of mammals such as humans, mice, hamsters, dogs, cats, horses, cows, pigs and chlorine.

In the present specification, the "endothelial cell" may be a corpus cavernosum endothelial cell, a penile vascular endothelial cell, or a smooth muscle cell.

The composition may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. When the composition is formulated, it can be prepared using a diluent or excipient usually used. The composition may be a preparation for oral administration or a preparation for parenteral administration.

As used herein, the term "administering" means providing any of the compositions of the present invention to a subject in any suitable manner.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the drug form, the administration route and the period, and can be appropriately selected by those skilled in the art. For a desired effect, the composition of the present invention may be administered in an amount of 0.0001 mg / kg to 10000 mg / kg per day. The composition may be administered once a day, or divided into several doses.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. For example, by injection into the oral, rectal or vein, muscle, subcutaneous, intracavernosal (intracavernous).

The composition may be for preventing or treating erectile dysfunction of an individual.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating erectile dysfunction, comprising an HGF protein or a polynucleotide encoding the same as an active ingredient.

The HGF protein and the polynucleotide encoding it are as described above.

The term "active ingredient" includes those having an activity of preventing or treating erectile dysfunction, when the composition is administered to a subject, as compared to otherwise. The subject may be at least one selected from the group consisting of mammals such as humans, mice, hamsters, dogs, cats, horses, cows, pigs and chlorine.

The term "prevention" includes preventing the erectile potency is weakened or the erectile duration is reduced compared to when the composition is not administered. The term "treatment" includes further restoring weakened erectile dysfunction or reduced erectile duration compared to no administration of the composition. For example, the HGF protein according to the present invention induces regeneration of the corpus cavernosum endothelial cells and smooth muscle cells or penile vascular endothelial cells and smooth muscle cells in an animal model of diabetic erectile dysfunction, thereby improving the erectile potency by increasing the corpus cavernosum pressure There can be excellent effects.

The composition may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. The composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method. In particular, the composition may be in a syringe-type formulation. The carrier, excipient and diluent may be selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, Vinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.

When the composition is formulated, it may be prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrating agent or a surfactant, which are usually used. Solid formulations for oral administration may include tablets, pills, powders, granules, capsules, and the like. The solid preparation may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose or gelatin in the composition. In preparing the composition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients.

The composition may be a preparation for oral administration or a preparation for parenteral administration. As the liquid preparation for oral administration, suspensions, solutions, emulsions or syrups are used, and various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations or suppositories. As the non-aqueous solvent or suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil or injectable ester such as ethyl oleate may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao paper, laurin or glycerogelatin can be used.

The composition may contain, in addition to the HGF protein, at least one known active ingredient having an effect of preventing or treating erectile dysfunction.

The composition may be one that increases the corpus cavernosal pressure. The composition may be one that increases the level of endothelial cell specific protein in endothelial cells. The endothelial cell specific protein may include PECAM-1 (platelet / endothelial cell adhesion molecule-1), smooth muscle actin (SMA), platelet-derived growth factor beta (PDGF-beta), or a combination thereof.

The erectile dysfunction may be caused by damage to the penile endothelial cells or smooth muscle cells. The damage to the penile endothelial cells and smooth muscle cells may be caused by one or more of hyperlipidemia, diabetes, hypertension and penile nerve damage.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention or treatment of erectile dysfunction.

Another aspect of the present invention relates to a method for regenerating endothelial cells of an individual comprising administering to a subject a pharmaceutical composition for endothelial cell regeneration comprising an effective amount of HGF protein or a polynucleotide encoding the same as an effective component for endothelial cell regeneration Reproducing method.

The pharmaceutical compositions for endothelial cell regeneration are as described above.

In the above method, the term "individual" means a subject in need of treatment for a disease. The subject may be an individual with endothelial cell damage. The subject may be, for example, one or more selected from the group consisting of mammals, such as human or non-human primates, mice, rats, dogs, cats, horses and cows.

In this method, "an amount effective for endothelial cell regeneration " includes an amount such that, when the composition is administered to an individual, it further promotes endothelial cell regeneration, as compared to otherwise. "Effective amount for endothelial cell regeneration" includes, for example, factors including the type and severity of the disease to be administered, the age and sex of the patient, sensitivity to the drug, administration time, administration route and rate of release, And other factors well known in the medical arts, and can be readily determined by those skilled in the art, taking into account all of the above factors and in an amount sufficient to achieve the maximum effect without adverse side effects. The effective amount is in the range of 0.00001 mg / kg to 1000 mg / kg, for example 0.00001 mg / kg to 100 mg / kg, 0.00001 mg / kg to 10 mg / kg, 0.00001 mg / kg to 10 mg / kg, 0.0001 mg / kg to 1000 mg / kg, from 0.01 mg / kg to 1000 mg / kg, from 0.1 mg / kg to 1000 mg / kg, from 1 mg / kg to 1000 mg / 1000 mg / kg.

In this method, administration comprises administration via any route through which the composition can reach the erectile tissue of the penis. Such administration routes include, for example, oral, rectal, intravenous, intramuscular, subcutaneous or intracavernosal administration.

The composition may be one that increases the corpus cavernosal pressure. The composition may be one that increases the level of endothelial cell specific protein in endothelial cells. The endothelial cell specific protein may include PECAM-1 (platelet / endothelial cell adhesion molecule-1), smooth muscle actin (SMA), platelet-derived growth factor beta (PDGF-beta), or a combination thereof.

The erectile dysfunction may be caused by damage to the penile endothelial cells or smooth muscle cells. The damage to the penile endothelial cells and smooth muscle cells may be caused by one or more of hyperlipidemia, diabetes, hypertension and penile nerve damage.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention or treatment of erectile dysfunction.

Another aspect of the present invention provides a method for preventing or treating erectile dysfunction, comprising administering to a subject a pharmaceutical composition for preventing or treating erectile dysfunction, which comprises an effective amount of HGF protein or a polynucleotide encoding the same as an effective ingredient for treating erectile dysfunction. Of the erectile dysfunction of the present invention.

The pharmaceutical compositions for preventing or treating erectile dysfunction are as described above.

In the above method, the term "individual" means a subject in need of treatment for a disease. The subject may be an individual having erectile dysfunction or having the potential to exhibit erectile dysfunction. The subject may be, for example, one or more selected from the group consisting of mammals, such as human or non-human primates, mice, rats, dogs, cats, horses and cows.

In the above method, "an effective amount for treating erectile dysfunction" means an improvement in erectile dysfunction, for example, an increase in erectile potency and / or an increase in erection duration, . "An amount effective to treat erectile dysfunction" includes, for example, the type and severity of the disease being treated, the age and sex of the patient, the sensitivity to the drug, the time of administration, the route of administration and rate of release, And other factors that are well known in the medical arts, and can be readily determined by those skilled in the art, taking into account all of these factors and in such amounts as to obtain maximum effect without side effects. The effective amount is in the range of 0.00001 mg / kg to 1000 mg / kg, for example 0.00001 mg / kg to 100 mg / kg, 0.00001 mg / kg to 10 mg / kg, 0.00001 mg / kg to 10 mg / kg, 0.0001 mg / kg to 1000 mg / kg, from 0.01 mg / kg to 1000 mg / kg, from 0.1 mg / kg to 1000 mg / kg, from 1 mg / kg to 1000 mg / 1000 mg / kg.

In this method, administration comprises administration via any route through which the composition can reach the erectile tissue of the penis. Such administration routes include, for example, oral, rectal, intravenous, intramuscular, subcutaneous or intracavernosal administration.

The composition may be one that increases the corpus cavernosal pressure. The composition may be one that increases the level of endothelial cell specific protein in endothelial cells. The endothelial cell specific protein may include PECAM-1 (platelet / endothelial cell adhesion molecule-1), smooth muscle actin (SMA), platelet-derived growth factor beta (PDGF-beta), or a combination thereof.

The erectile dysfunction may be caused by damage to the penile endothelial cells or smooth muscle cells. The damage to the penile endothelial cells and smooth muscle cells may be caused by one or more of hyperlipidemia, diabetes, hypertension and penile nerve damage.

The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention or treatment of erectile dysfunction.

Another aspect of the present invention provides a health functional food comprising an HGF protein or a polynucleotide encoding the same.

The HGF protein and the polynucleotide encoding it are as described above.

The HGF protein of the present invention may be added to a health functional food for the purpose of preventing or improving erectile dysfunction. In the present invention, the term "health functional food" refers to a food having a biological control function such as prevention and improvement of diseases, defense of the body, immunity, restoration of aftergrowth and aging, and may be harmless to the human body when taken over a long period of time.

When the HGF protein of the present invention is used as a food additive, the HGF protein can be directly added or used together with other food or food ingredients, and can be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the HGF protein of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material in the production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the food. Examples of the foods to which the above substances can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and natural sweeteners such as dextrin and cyclodextrin, or synthetic sweeteners such as saccharin and aspartame. The ratio of the natural carbohydrate is generally about 0.01 to 10 g, preferably about 0.01 to 0.1 g per 100 ml of the composition of the present invention.

The health functional food may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, And the like, and the like. The health functional food may include flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Brief Description of the Drawings Figure 1 is a graph showing measurement results of intracapsular pressure (erectile force) according to penile nerve electrical stimulation by administration of HGF protein in a diabetic mouse model.
FIG. 2 shows the expression levels of PECAM-1, a vascular endothelial cell-specific protein and SMA, a vascular smooth muscle cell-specific protein, in the penile tissues of a diabetic mouse model in combination with the expression amount of cMET, a HGF receptor, And the result of analysis by a focusing microscope.
FIG. 3 is a graph showing the result of analysis of hydroethidine staining with a confocal microscope to compare the formation patterns of peroxidized anions by administration of HGF protein in penile tissues of a diabetic mouse model.
FIG. 4 is a diagram showing the results of analysis of immunohistochemical staining of nitrotyrosine with a confocal microscope to compare the production patterns of peroxynitrite by administration of HGF protein in penile tissues of a diabetic mouse model.
FIG. 5 is a graph showing the results of analysis of immunohistochemical staining for PDGFR-b by confocal microscopy to compare the degree of peripheral vascular cell expression, which plays an important role in the stability of blood vessels by administration of HGF protein in penile tissues of a diabetic mouse model Fig.
FIG. 6 is a graph showing a result of confocal microscopy of the expression level of c-MET, an HGF receptor, by treatment with HGF protein after treatment of vascular endothelial cells obtained by primary culturing in mouse penis tissue under high glucose conditions .
FIG. 7 is a graph showing the results of analysis of the degree of expression of PECAM-1, a vascular endothelial cell-specific protein by administration of HGF protein, by confocal microscopy after treatment of vascular endothelial cells obtained by primary culturing in mouse penile tissue under hyperglycemic conditions .
FIG. 8 is a graph showing the results of analysis of the degree of tube formation by HGF protein administration by a phase contrast microscope after treatment of vascular endothelial cells obtained by primary culture in mouse penile tissues under hyperglycemic conditions.
FIG. 9 is a graph showing the degree of apoptosis by administration of HGF protein after treatment with vascular endothelial cells obtained by primary culturing in mouse penis tissues under hyperglycemic conditions by TUNEL staining.

Hereinafter, preferred embodiments, experimental examples, and production examples are provided to facilitate understanding of the present invention. However, the following Examples, Experimental Examples and Preparation Examples are provided only for the purpose of easier understanding of the present invention, and are not intended to limit the scope of the present invention.

Example  1. In a diabetic erectile dysfunction model HGF  Erectile dysfunction treatment effect of protein

(C57BL / 6J) mice were divided into four groups (N = 10 / group, group 1, normal mouse, group 2, normal mice + streptozotocin ), Diabetic induction (8 weeks after intraperitoneal injection at a dose of 50 mg / kg for 5 consecutive days) + PBS (Phophate buffered solution) [20 ㎕], group 3, diabetes induction with streptozotocin [50 mg / mice were injected with mouse + HGF protein 1 time [day 0; 4.2 占 퐂 / 20 占 퐇]; group 4, diabetes induction with streptozotocin [50 mg / kg concentration (Day 3, 0; 4.2 / / 20]) after 8 weeks of intraperitoneal administration for 5 consecutive days.

After anesthesia with intramuscular injection of ketamine (100 mg / kg) and xylazine (5 mg / kg), HGF protein was injected into the corpus cavernosum and the erectile force was measured after stimulation of the cavernous nerve at 2 weeks. Immunohistochemical staining of platelet-endothelial cell adhesion molecule-1 (PECAM-1) and smooth muscle cell-specific protein SMA and c-MET, an HGF receptor, (PDGFR) -beta (platelet-derived growth factor receptor-beta) were examined by immunohistochemical staining for pericyte. The production of superoxide anion was also confirmed by hydroethidine staining and the degree of peroxynitrite expression was evaluated by immunohistochemical staining for nitrotyrosine.

As a result, it was confirmed that the group of HGF protein administration group showed the highest improvement of erectile performance and the erectile power was restored to the normal level. The amount of cancellous endothelial cells (including vascular endothelial cells) and smooth muscle cells was significantly decreased in the penile tissues of the streptozotocin-induced diabetic animal model compared to the normal control, and the number of cancellous endothelial cells (including vascular endothelial cells) and smooth muscle cells Was recovered to a nearly normal level. In addition, HGF significantly increased peripheral vascular expression and decreased peroxide anion and peroxynitrite production. This will be described in more detail below.

(1) Analysis of erection power by electrical stimulation

The experimental method for measuring the erectile force was prepared such that the left epidermis of the lower abdomen of the prepared mouse was widened and the penile cavernosal nerve (penile nerve) located on the posterior lateral side of the prostate was visible. For electrical stimulation of the penis nerve, a platinum electrode was placed on the penile nerve and electrical stimulation was applied for 1-5 minutes at a frequency of 12 hertz. When an electrical stimulus is applied, the penis becomes erect. At this time, the pressure was measured through a pressure transducer (Biospac System, USA) connected to the computer to collect data about the pressure inside the penis (erosion pressure of the penis) during erection through a catheter inserted into the penis corpus cavernosum. Respectively. The measured pressure values reflect penile erection.

Brief Description of the Drawings Fig. 1 is a diagram showing penile sponge pressure (erectile force) according to penile nerve electrical stimulation in the HGF protein administration group and the control group in the diabetic mouse model. In Fig. 1A, ICP (Intracavernous Pressure) on the vertical axis refers to the pressure inside the penis (erosion pressure of the penis) during erection and is generally an index showing the erection power. The horizontal axis represents time after electric stimulation. In FIG. 1A, the electrical stimulation period for one minute is indicated by a black bar on the abscissa. FIG. 1A shows the results of measurement of erectile activity after nerve stimulation with 1 V or 5 V on the penile corpus cavernosum 2 weeks after the HGF protein was injected once or twice into the corpus cavernosum.

1B and 1C, Mean Systolic Blood Pressure (MSBP) on the vertical axis represents mean systolic blood pressure. In general, when expressing erectile function, the maximal ICP or the area of the penumbra pressure curve is divided by the mean systolic blood pressure (MSBP) This can affect the internal pressure. In Fig. 1, Control, DM + PBS, HGF once administration, and HGF twice administration are the individuals belonging to the fourth group in the above first group.

As shown in FIG. 1, HGF protein (HGF double) showed the highest improvement in erectile function compared with PBS (DM + PBS). Two parameters for penile erection, the highest penile corpus cavernosal pressure and the area of the penile pressure curve, showed a significant increase after administration of HGF protein.

(2) a vascular endothelial cell-specific protein PECAM -1 and Smooth muscle cell  SMA expression analysis, a specific protein

The penis is a special type of vascular tissue. For normal penile erection, normal activities of cavernous sinusoids and penis blood vessels are essential. Spongy body Oriental structure and blood vessels are the innermost monolayer cells, composed of endothelial cells, which are blood vessel walls that come into direct contact with blood, and smooth muscle cells, which constitute several cell layers of blood vessels and contracts and relaxes blood vessels. These vascular endothelial cells and smooth muscle cells play an important role in the relaxation of the penile blood vessels and the penile erection resulting therefrom. The abnormal structure and activity of the corpus cavernosum oriental structure and blood vessels cause the erectile power to decrease.

In order to investigate the structural changes of blood vessels in erectile tissues after administration of streptozotocin to normal mice, the present inventors examined the expression of PECAM-1, a specific protein in the endothelial cells of cavernosal and blood vessels, and the expression of SMA, I checked.

Penile tissues were fixed in 4% paraformaldehyde at 4 ° C for 24 hours. After freezing, the tissue was easily fixed with a freezing agent in a freezing machine and cut into 7 μm thickness to prepare a penile tissue section.

Next, prepared penile tissue sections were placed on a slide, and tissues of the slides prepared for PECAM-1 and SMA expression analysis were fixed in 4% paraformaldehyde for about 5 minutes. Fixed penile tissue sections are washed three times in wash buffer (2% FBS + 0.1% Sodium Azide in PBS) followed by blocking with non-specific protein blocking buffer (5% BSA in PBS) for 1 hour. The first antibody (anti-PECAM-1 rat antibody and FITC-labeled anti-SMA antibody, anti-c-Met rapto antibody) was reacted at a ratio of 1:50 for 16 hours at 4 ° C, (TRITC-labeled anti-rat antibody, FITC-, and TRITC-labeled anti-rat antibody) prepared so as to be able to identify the antibody specifically reacted with PECAM-1 by fluorescence - rabbit antibody) was reacted at a ratio of 1: 1000 at room temperature for 2 hours. After the reaction was completed, the cells were washed three times with washing buffer to remove the remaining antibody, and the degree of expression was analyzed by a fluorescence microscope or a confocal microscope capable of confirming the fluorescent material. The results are shown in FIG.

2A is a confocal microscope photograph showing the expression level of c-Met HGF receptor and the expression level of PECAM-1, a vascular endothelial cell-specific protein, in the HGF protein administration group and the control group in penile tissues of diabetic mice. As shown in Fig. 2A, the expression of the receptor c-Met is increased in diabetic mice to which HGF protein is administered. The expression of c-Met was increased in the group treated with HGF protein 1 than in the group treated with PBS alone, and c-Met was found to be the most expressed in the group treated with HGF protein 2 times.

The number of vascular endothelial cells was significantly reduced in the penis of the group (DM + PBS) injected with PBS in the diabetic mice compared to the control mice. Here, the control group is the result of a penile tissue section prepared in the same manner as the experimental group, except that the first group streptozotocin was not injected. HGF protein in the DM + HGF single and two times (DM + HGF double) groups compared with the other groups (DM + PBS: PBS instead of HGF protein) Expression was increased. FIG. 2B is a graph showing statistical processing of the amount of c-Met expressed in the corpus cavernosum area. 2C is a graph showing statistical treatment of vascular endothelial cell expression in the corpus cavernosum area. This is a result of proving that the reduced endothelial cell by diabetes is induced by its HGF protein to regenerate and thus the erectile power is restored.

FIG. 2D is a confocal microscopic photograph showing the degree of expression of SMA, a smooth muscle cell specific protein of the HGF protein-treated group and the control group, and c-Met HGF receptor, in penile tissues of diabetic mice. As shown in FIG. 2D, the number of smooth muscle cells of the vascular smooth muscle was significantly decreased in penis of the group (DM + PBS) injected with PBS in the diabetic mouse compared with the normal mouse. Here, the control group is the result of a penile tissue section prepared in the same manner as the experimental group, except that the first group streptozotocin was not injected. Expression of smooth muscle cell specific protein in HGF protein (DM + HGF single) and twice (DM + HGF double) compared to the other group (DM + PBS: PBS instead of HGF protein) . 2D and 2E are results that demonstrate that smooth muscle cells reduced by diabetes are induced by HGF protein to regenerate and thus erectile potency is restored.

(3) Production of peroxide anion Hydroethidine  Reaction analysis

Hydroethidine is a probe that is sensitive to redox reactions and is used to detect peroxide anions in cells. When superoxide and hydroethidine are reacted, a two-electron oxidized product called ethidium is generated. Ethidium absorbs the wavelength of 500-530 nm attached to the DNA to form a 590-620 nm Of fluorescence. Peroxide anions, one of the active oxygen species, are highly reactive anion free radicals and are produced in large quantities by NADPH oxidase, which is harmful and can cause DNA damage.

Therefore, the present inventors confirmed the degree of the reaction through hydroethidine in order to investigate the generation of peroxide anion in the erectile tissue of the penis after administration of streptozotocin to normal mice.

Fluorescent staining with anti-PECAM-1 antibody was performed by the method described above. After the final washing, hydrotidine [1 mM in PBS] was treated for 30 minutes, and then expressed by a fluorescence microscope or a confocal microscope And the results are shown in FIG.

FIG. 3 is a photograph of confocal microscope showing the degree of luminescence after treatment of hydroethidine which reacts with peroxide anions of the HGF protein-treated group and the control group in penile tissues of diabetic mice. As shown in FIG. 3A, it can be seen that the peroxide anion was significantly increased in the penis of the group (DM + PBS) injected with PBS in the diabetic mouse compared to the control mice. Here, Control is the result for penile tissue sections prepared in the same manner as the experimental group, except that the first group, streptozotocin, was not injected. Compared with the other groups (DM + PBS: PBS instead of HGF protein and DM + HGF single: HGF protein), the HGF protein (DM + HGF double) . FIG. 3B is a graph showing statistical treatment of the amount of hydroethidine expressed in the cavernous surface area of the penis. This is a result of demonstrating that the peroxide anion increased by diabetes is reduced by the HGF protein and thus the erectile potency is restored.

(4) Analysis of the presence of nitrotoxin reaction, which indicates the formation of peroxynitrite

Nitric oxide (NO) is an important cytotoxic mediator produced by nitric oxide synthase (NOS). Recently, it has been reported that peroxynitrite (ONOO-) produced by the reaction of NO and superoxide rather than direct damage by NO causes more damage to the cells. Peroxynitrite nitrifies the tyrosine group of proteins and oxidizes the sulfahydryl group to inhibit cell membrane lipid peroxidation, DNA fragmentation, electrolyte migration in mitochondria, and calcium leaching to inhibit energy production in the cell, It is known that it plays a crucial role in cell damage such as necrosis. Since peroxynitrite nitrates the tyrosine group of protein and converts it to 3-nitrotyrosine, the presence and activity of peroxynitrite can be indirectly detected by immunostaining for nitrotyrosine.

Accordingly, the present inventors examined the degree of expression of nitrosyltrophin by staining nitrotoxin in order to investigate the generation of peroxynitrite in penile erectile tissue after administration of streptozotocin to normal mice.

The cells were subjected to fluorescent immunostaining with an anti-PECAM-1 antibody and an anti-nitrotoxin antibody and reacted with a second antibody (TRITC-labeled anti-rat antibody, FITC-labeled anti-raptor antibody) by the method described above. The degree of expression was analyzed using a fluorescence microscope or a confocal microscope capable of confirming the fluorescent material, and the results are shown in FIG.

FIG. 4A is a confocal microscopic photograph showing the degree of expression of nitrotoxin in the HGF protein-treated group and the control group in penile tissues of diabetic mice. As shown in FIG. 4A, it can be seen that nitrotyrosine was significantly increased in the penis of the group (DM + PBS) injected with PBS in the diabetic mouse compared to the control mice. Here, the control group is the result of a penile tissue section prepared in the same manner as the experimental group, except that the first group streptozotocin was not injected. Compared to the non-HGF protein (DM + HGF double) group (DM + PBS: PBS group instead of HGF protein group, DM + HGF single: HGF protein group 1) . 4B is a graph showing statistical treatment of the expression amount of nitrotoxin in the cavernous surface area of the penis. This is the result of demonstrating that increased production of peroxynitrite by diabetes decreases its production by the HGF protein, thereby restoring erectile power.

(5) pericytes ( pericyte ) Specific protein PDGFR - beta  Expression analysis

Angiogenesis refers to the production of microvessels from already existing blood vessels. As the final stage of this angiogenesis, the pericytes that constitute the blood vessels enter and form the structure of the endothelial cells - the pericytes, so that the new blood vessels mature and stabilize. At this time, platelet-derived growth factor (PDGF-BB) secreted from vascular endothelial cells is known to contribute to recruitment and differentiation of pericytes.

Therefore, the present inventors examined the expression of PDGFR-beta by injecting HGF into penile tissues. Therefore, the present inventors confirmed the expression level of PDGFR-beta by staining with PDGFR-beta in order to investigate the formation of pericytes in the erectile tissue after administration of streptozotocin in normal mice.

The cells were subjected to fluorescent immunostaining with an anti-PECAM-1 antibody and an anti-PDGFR-beta antibody in the manner described above, and a second antibody (TRITC-labeled anti-rat antibody, FITC-labeled anti-raptor antibody) was reacted. The degree of expression was analyzed using a fluorescence microscope or a confocal microscope capable of confirming the fluorescent material, and the results are shown in FIG.

FIG. 5A is a confocal microscope photograph showing the degree of peri-vascularity of HGF protein-treated group and control group in penile tissues of diabetic mice. As shown in FIG. 5A, it can be seen that the amount of pericytes in the penis of the group (DM + PBS) injected with PBS in the diabetic mouse was markedly reduced compared with that in the normal mouse. Here, the control group is the result of a penile tissue section prepared in the same manner as the experimental group, except that the first group streptozotocin was not injected. Compared with the group that did not receive HGF protein (DM + HGF double) (DM + PBS: group that received PBS instead of HGF protein, group that received DM + HGF single: The amount of cells was increased. FIG. 5B is a graph showing the amount of peripheral blood cells in the corpus cavernosum area by statistical processing. This is a result of demonstrating that the amount of pericytes reduced by diabetes is increased by the HGF protein and thus the erectile power is restored.

Example  2. Mouse penis tissue Primary culture  After induction of hyperglycemia with the obtained endothelial cells HGF  Assessing the Effectiveness of Proteins

Endothelial cells obtained by primary culture of mouse penile tissues were divided into four groups (1 group, NG (Normal Glucose), vascular endothelial cells obtained by primary culture of mouse penis tissue, Glucose 5 mmol, 48 hours, Group 2, NG + HGF, HGF protein (200ng / ml) for 24 hours, Group 3, HG (hyperglycemia) after treatment with 5mmol of endothelial cells + , HGF + HGF, HGF + HGF protein treated with 30 mmol of glucose endothelial + Glucose for 48 hours, primary cultured mouse penile tissues, 200ng / ml, 24 hours treatment).

Immunohistochemical staining of the HGF receptor c-Met and platelet-endothelial cell adhesion molecule-1 (PECAM-1) after glucose and HGF treatment was performed and the degree of tube formation was determined by tube formation assay , And TUNEL (TdT mediated dUTP biotin nick end labeling method) analysis was performed to investigate the degree of apoptosis.

As a result, c-Met was expressed in vascular endothelial cells obtained by primary culture of mouse penile tissue, and c-Met was more expressed in normal glucose group than in hyperglycemic group. The expression of c-Met was higher in the HGF protein-treated group than in the non-treated group. Analysis of PECAM-1 staining and tube formation revealed that PECAM-1 expression and tube formation were less in the hyperglycemic group but recovered in the HGF protein treated group. The results of TUNEL analysis revealed that apoptosis was observed in hyperglycemia group but decreased in HGF protein treated group. Hereinafter, it will be described in detail.

(One) HGF Lt; RTI ID = 0.0 > c- MET Analysis of Expression Patterns

The primary culture method for obtaining endothelial cells from mouse penile tissues is as follows. Cut the penile tissues of 8-week-old C57BL / 6J mice, remove the urethra and stomach vein, and put them in Hanks Balanced Salt Solution (HBSS). After washing on a clean bench with HBSS and PBS, the tissues were cut and seeded in a 60-mm cell culture dish and matrigel (5 ng) containing bFGF (recombinant mouse FGF basic) / ml bFGF: matrigel = 1: 50). Incubate for 5 minutes at 37 ° C in this state. When Matrigel is hardened, add 3 ml of Complement medium 199 (20% Fetal bovine serum, 1% Penicillin / Streptomycin solution, 50 mg / ml heparin and 5 ng / ml bFGF) and incubate at 37 ℃ in 5% CO2 incubator. After 3 weeks, the cells from the tissues are transferred into a 0.2% gelatin-coated culture dish.

The present inventors obtained endothelial cells from mouse penile tissues and conducted in vitro experiments.

The method of fluorescence staining of endothelial cells obtained from penile tissues is as follows. Prepare a 12-well cell culture dish, place a cover slide, and apply 0.2% gelatin coating. Cells are subcultured and placed in wells and incubated at 37 ° C in a 5% CO2 incubator for 1-2 days. The slides with the cells were removed from the wells, washed three times in washing buffer (2% FBS + 0.1% sodium azide in PBS) and fixed in 4% paraformaldehyde for about 10 minutes at room temperature. Wash three times with wash buffer, increase permeability by treatment with cold methanol for 1-2 minutes, wash with wash buffer, and block with non-specific protein blocking buffer (5% BSA in PBS) for 1 hour. The first antibody (anti-c-Met antibody) was reacted at 4: 1 in a ratio of 1: 100 for 16 hours, washed again with washing buffer three times to remove the remaining antibody, The second antibody (FITC-labeled anti-rabbit antibody), which was designed to detect the reacted antibody by fluorescence, was reacted at a ratio of 1: 200 at room temperature for 2 hours. After the reaction is completed, wash the wells three times with a washing buffer to remove any residual antibody, then insert a mounting solution containing DAPI that can stain the nuclei and cover with a cover slide. Thereafter, the degree of expression was analyzed with a fluorescence microscope or a confocal microscope capable of confirming the fluorescent material, and the results are shown in Fig.

FIG. 6 is a photomicrograph showing the expression of the HGF receptor c-Met in the HGF protein-treated group and the control group after treatment of glucose (30 mmol) on endothelial cells obtained from mouse penile tissue to induce hyperglycemia. As shown in FIG. 6, the expression of c-Met was decreased in the hyperglycemic group than in the normal glucose group, and the expression of the HGF receptor c-Met was increased in the normal group and the hyperglycemic group when the HGF protein was treated. This is the result of demonstrating that HGF protein is effective on penile endothelial cells by increasing HGF protein in endothelial cells obtained from mouse penis tissue.

(2) a vascular endothelial cell-specific protein PECAM -1 expression analysis

The present inventors examined the expression of PECAM-1, a specific protein in vascular endothelial cells, in order to investigate changes in vascular endothelial cells after administration of HGF protein to endothelial cells obtained from penile tissues. Fluorescent immunostaining with anti-PECAM-1 antibody is performed by the method described above. After the final wash, the mounting solution containing DAPI, which can stain the nucleus, is inserted and covered with a cover slide. Then, the degree of expression was analyzed by a fluorescence microscope or a confocal microscope capable of confirming a fluorescent substance, and the results are shown in FIG. As shown in Fig. 7, there was no difference in expression of PECAM-1 between the NG group and the NG + HGF group. PECAM-1 expression was significantly reduced in the HG group compared with the NG group, and the HG + HGF group was restored to the control (NG group) level. This is the result of demonstrating that the amount of endothelial cells reduced by hyperglycemic conditions is restored by the HGF protein.

(3) Assess the degree of tube formation during neovascularization

Angiogenesis consists of multiple, complex and sequential processes, not just simple proliferation of vascular endothelial cells, each of which is essential for the establishment of a vascular network. The angiogenesis process can be roughly divided into proliferation, migration, and tube formation. In the tube formation step, the endothelial cells migrating and proliferating during the angiogenesis process continue to divide and grow into a hollow tube shape and differentiate into a final blood vessel, into which blood enters and the blood vessel formation is completed. The present inventors conducted the following experiments to observe whether HGF protein participates in tube formation during angiogenesis through tube formation analysis.

Endothelial cells obtained by primary culture of penile tissues were subjected to serum starvation for 24 hours and then divided into four groups as described above. Glucose and HGF proteins were treated respectively and seeded on a 48-well cell culture dish matrigel. Gt; C, < / RTI > 5% CO2 incubator.

FIG. 8 shows the result of observing the degree of tube formation after 4, 8, and 24 hours through a microscope. Tubes were clearly produced after 8 hours, and tube formation was significantly reduced in the HG group and recovered to the control level in the HG + HGF group. This is the result of demonstrating that the formation of the reduced tube by hyperglycemic conditions is restored by the HGF protein.

(4) Cell Scheduled History  That knows the extent of TUNEL  analysis

TUNEL analysis is an experimental method to determine whether DNA fragmentation has occurred by attaching UDP (uridine triphosphate) labeled with digoxigenin using an enzyme called TdT (terminal deoxynucleotidyl transferase) to the DNA fragment end. When DNA fragmentation occurs in the course of DNA degradation in the cell nucleus, a breakdown occurs in the double helix structure and the 3'-OH group at the N-terminus is exposed, and the TUNEL is bound to the N terminus. The results of the TUNEL analysis are shown in FIG. At this time, DAPI (4 ', 6-diamidino-2-phenylindole) was used to stain nuclei of cells. As shown in FIG. 9, in the HG group, apoptosis was increased compared to the NG group and the NG + HGF group, and the apoptosis was significantly decreased in the HG + HGF group. This is the result of demonstrating that increased apoptosis by high glucose conditions is reduced by HGF protein.

The HGF protein of the present invention induces the regeneration of endothelial cells, smooth muscle cells and pericytes, which are reduced by diabetes, and the endothelial cells, smooth muscle cells, pericyte-specific protein PECAM- 1, SMA, and PDGF-beta, respectively. In addition, HGF protein showed the effect of promoting tube formation and apoptosis in vascular endothelial cells. Through the above mechanism, the HGF protein has an excellent erectile function improving effect against erectile dysfunction by raising the internal pressure of the corpus cavernosum, and thus it can be effectively used for preventing or treating erectile dysfunction.

Hereinafter, a pharmaceutical composition containing the composition of the present invention and a preparation example of a health functional food will be described, but the present invention is not intended to be limited thereto.

Formulation example  1. Preparation of Pharmaceutical Compositions

1-1. Sanje  Produce

20 mg of HGF protein

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

1-2. Manufacture of tablets

HGF protein 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

1-3. Preparation of capsules

HGF protein 10 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

1-4. Injection preparation

HGF protein 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ H ₂ O 26 mg

(2 ml) per 1 ampoule in accordance with the usual injection preparation method.

1-5. Liquid  Produce

20 mg of HGF protein

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Formulation example  2. Formulation of food composition

2-1. Manufacture of health food

100 mg of HGF protein

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

0.2 [mu] g vitamin B12

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

2-2. Health drink  Produce

100 mg of HGF protein

Vitamin C 15 g

Vitamin E (powder) 100 g

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3 g

Water quantification

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The resulting solution was filtered to obtain a sterilized container, which was sealed and sterilized, Used in the manufacture of health beverage compositions.

Although the composition ratio of the ingredients suitable for the beverage is comparatively low, it is also possible to arbitrarily modify the blending ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Claims (18)

delete delete A pharmaceutical composition for preventing or treating erectile dysfunction comprising hepatocyte growth factor (HGF) protein as an active ingredient. 4. The composition of claim 3, further comprising a pharmaceutically acceptable carrier, excipient or diluent. 4. The composition according to claim 3, wherein the composition increases the internal pressure of the penile cavernosum. 4. The composition of claim 3, wherein the composition increases the level of endothelial cell specific protein in endothelial cells, wherein the endothelial cell specific protein is platelet / endothelial cell adhesion molecule-1 : PECAM-1), smooth muscle actin (SMA), and platelet-derived growth factor beta (PDGF-beta). delete 4. The composition of claim 3, wherein the erectile dysfunction is caused by damage to the penile endothelial cells or smooth muscle cells. The composition according to claim 8, wherein the damage of the penile endothelial cells and smooth muscle cells is caused by at least one selected from the group consisting of hyperlipidemia, diabetes, hypertension and penile nerve damage. 4. The composition of claim 3, wherein the composition is in a form for injection. delete delete Comprising administering to a subject a pharmaceutical composition for the prevention or treatment of erectile dysfunction comprising an effective amount of a hepatocyte growth factor (HGF) protein effective to treat erectile dysfunction, Wherein the subject is a non-human mammal. 14. The method of claim 13, wherein said administration is administered in the oral, rectal, intravenous, muscular, subcutaneous or penile cavernosal. 14. The method of claim 13, wherein the erectile dysfunction is caused by damage to the penile endothelial cells or smooth muscle cells. 16. The method of claim 15, wherein the damage of the penile endothelial cells and smooth muscle cells is caused by at least one selected from the group consisting of hyperlipidemia, diabetes, hypertension and penile nerve damage. delete delete

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