WO2015076475A1 - Composition containing hgf protein or gene thereof for preventing or treating erectile dysfunction and use thereof - Google Patents

Composition containing hgf protein or gene thereof for preventing or treating erectile dysfunction and use thereof Download PDF

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WO2015076475A1
WO2015076475A1 PCT/KR2014/006602 KR2014006602W WO2015076475A1 WO 2015076475 A1 WO2015076475 A1 WO 2015076475A1 KR 2014006602 W KR2014006602 W KR 2014006602W WO 2015076475 A1 WO2015076475 A1 WO 2015076475A1
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composition
hgf
protein
erectile dysfunction
endothelial cells
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PCT/KR2014/006602
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French (fr)
Korean (ko)
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서준규
류지간
두라이 다스난도
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인하대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/4753—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a composition for endothelial cell regeneration, a composition for preventing or treating erectile dysfunction, a method for regenerating endothelial cells of an individual, or a method for preventing or treating erectile dysfunction of an individual.
  • HGF is a growth factor found, isolated and cloned as a growth promoter for primary cultured hepatocytes. HGF is produced by a variety of mesenchymal cells, most of which target epithelial cells, neurons, vascular 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 a mediator of epithelial-mesenchymal interactions during development and is involved in the formation of internal organs such as liver, kidney, lung, placenta and skeletal system. It is expected that HGF functions as an organ regeneration factor that promotes regeneration of the liver, digestive tract, kidney, and lung in the adult. However, to date, there has been no teaching or description about the prevention or treatment of erectile dysfunction of HGF, and there is no research on this.
  • erectile dysfunction is a type of male sexual dysfunction, a phenomenon in which the male genitals are not erection or erection is not sustained, the sexual activity is not possible.
  • the causes of erectile dysfunction are largely divided into psychogenic causes and organic causes.
  • Psychogenic erectile dysfunction is due to excessive secretion of noradrenaline due to excessive effects of sympathetic nerve due to psychological and psychological effects, increased penile cavernous smooth muscle tension, and suppression of neurotransmitter secretion.
  • organic erectile dysfunction is classified into neurogenic, vascular, and endocrine dysfunction depending on the cause.
  • vascular erectile dysfunction is impaired due to hyperlipidemia, diabetes, hypertension, smoking, systemic cardiovascular diseases, etc., resulting in impaired release of loose substances such as nitric oxide (NO) from vascular endothelial cells. It is a disorder that occurs.
  • NO nitric oxide
  • One aspect of the present invention is to provide a composition for endothelial cell regeneration.
  • Another aspect of the present invention is to provide a composition for preventing or treating erectile dysfunction.
  • Another aspect of the invention is to provide a method for regenerating endothelial cells of a subject.
  • Another aspect of the invention is to provide a method for preventing or treating erectile dysfunction in a subject.
  • One aspect of the present invention provides a composition for endothelial cell regeneration comprising an HGF protein or a polynucleotide encoding the same as an active ingredient.
  • composition is preferably a pharmaceutical composition.
  • HGF protein means hepatocyte growth factor.
  • HGF is a heterodimeric protein consisting of a chain of 69 kDa and a chain of 34 kDa, and may have four Kringle structures in the chain.
  • C-met with tyrosine kinase activity may be a receptor for HGF.
  • the HGF protein includes a protein having an amino acid sequence represented by SEQ ID NO: 1 or a protein encoded by a nucleotide sequence represented by SEQ ID NO: 2, and a functional equivalent of the protein.
  • the term "functional equivalent” means at least 70%, preferably 80%, more preferably 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 1 as a result of the addition, substitution, or deletion of the amino acid.
  • it refers to a protein having a sequence homology of 95% or more, and having a substantially homogeneous physiological activity with a protein having an amino acid sequence represented by SEQ ID NO: 1.
  • the HGF proteins of the present invention include not only proteins having their natural amino acid sequence, but also variants thereof, are also included in the scope of the present invention.
  • the variant means a protein in which the amino acid sequence of the HGF protein and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.
  • Amino acid exchange in proteins and fragments that do not alter the activity of the molecule as a whole is known in the art (H. Neurode, R. L. Hill, The Proteins, Academic Press, New York, 1979).
  • HGF proteins or variants thereof can be extracted from nature or synthesized (Merrifleld, J. Amer. Chem. Soc. 85: 2149-2156, 1963) or by genetic recombination methods based on DNA sequences (Sambrook et al. , Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd Edition, 1989).
  • the HGF protein may be encoded by the nucleotide sequence of SEQ ID NO: 2, and a variant capable of functioning identically with the nucleotide is included within the scope of the present invention.
  • the composition may comprise not only all of the HGF protein, but also its biologically active fragments or fusion proteins thereof.
  • the biologically active fragment may be one comprising substantially the vascular endothelial and smooth muscle cell regeneration activity of the HGF protein or a gene encoding the same.
  • the biologically active fragment may comprise an active domain involved in vascular endothelial and smooth muscle cell activity of native HGF.
  • the fusion protein may include substantially the vascular endothelial and smooth muscle cell regeneration activity of the HGF protein or a gene encoding the same.
  • the fusion protein may be one in which the HGF protein or fragment thereof is linked to a fusion partner useful for isolation of the HGF protein or fragment thereof, a fusion partner useful for delivery to a target site, a fusion partner for increasing stability in the body, and the like.
  • the linkage may be linked via the N terminus or C terminus or side chain of the HGF protein or fragment thereof.
  • the linkage may be made by a covalent or non-covalent bond.
  • the fusion partner may be one comprising a polypeptide.
  • Useful fusion partners for separation may be His sequences, such as His6 sequences.
  • Fusion partners useful for delivery to a target site, or fusion partners for increasing stability in the body may be polymers that confer resistance to degradation in the body, such as antibody constant regions, eg, Fc regions or PEG.
  • the polynucleotide encoding the HGF may be in a fused form with other substances as well as itself.
  • the other substance may be such that the polynucleotide encoding the HGF can be delivered into a cell, expressed in a cell by a gene expression apparatus within the cell, and maintained stable within and outside the cell. It may be one or more selected from the group consisting of.
  • the fusion can be by covalent or non-covalent bonds.
  • the fusion includes the form that is collected in the vesicles.
  • the polynucleotide encoding the HGF may be operably linked with gene expression regulators such as promoters, operators, enhancers and / or transcription terminators.
  • the polynucleotide encoding the HGF may be, for example, inserted into a plasmid or viral genome to be expressed in a cell.
  • the polynucleotide encoding the HGF may be a structure linked to a regulator that is specifically expressed in endothelial cells and smooth muscle cells of the penis, for example, cavernous endothelial cells and smooth muscle cells or penile vascular endothelial cells and smooth muscle cells.
  • the polynucleotide encoding the HGF may be, for example, in the form of a structure linked to a regulator that is inserted into the adenovirus genome so as to be specifically expressed in corpus cavernothelium and smooth muscle cells or penile vascular endothelial cells and smooth muscle cells.
  • the structure may be collected in a virus particle.
  • the term "active ingredient” includes an agent which, when administered to a subject, has an activity that further promotes endothelial cell regeneration as compared to otherwise.
  • the subject may be one or more mammals, eg, humans, mice, hamsters, dogs, cats, horses, cattle, pigs and goats.
  • endothelial cells may be penile cavernous endothelial cells, penile vascular endothelial cells or smooth muscle cells, preferably penile cavernous endothelial cells or penile vascular endothelial cells.
  • the composition may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. When formulating the composition, it can be prepared using a commonly used diluent or excipient.
  • the composition may be a preparation for oral administration or a preparation for parenteral administration.
  • the term "administration" means providing a subject with any composition of the present invention in any suitable manner.
  • compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration and can be appropriately selected by those skilled in the art.
  • the composition of the present invention may be administered in an amount of 0.0001 mg / kg to 10000 mg / kg per day. Administration of the composition may be administered once a day, may be divided several times.
  • the pharmaceutical composition of the present invention can be administered to a subject by various routes.
  • it may be administered by oral, rectal or intravenous, intramuscular, subcutaneous, intracavernous injection.
  • the composition may be for preventing or treating erectile dysfunction of an individual.
  • compositions for preventing or treating erectile dysfunction comprising an HGF protein or a polynucleotide encoding the same as an active ingredient.
  • the composition is preferably a pharmaceutical composition or a food composition.
  • the HGF protein and polynucleotides encoding it are as described above.
  • active ingredient when administered to a subject, includes those having the activity of preventing or treating erectile dysfunction, as compared to otherwise.
  • the subject may be one or more mammals, eg, humans, mice, hamsters, dogs, cats, horses, cattle, pigs and goats.
  • prevention includes preventing the impotence from being reduced or the duration of the impotence in comparison with the composition not administered.
  • treatment includes the further recovery of weakened or reduced erection duration as compared to when the composition is not administered.
  • the HGF protein according to the present invention increases the intracavernosal pressure and improves erection by inducing regeneration of penile cavernous endothelial cells and smooth muscle cells or penile vascular endothelial cells and smooth muscle cells. It can have an excellent effect.
  • the composition may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
  • the composition may also be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions according to conventional methods.
  • the composition may be an injectable formulation.
  • the carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polycellulose Vinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil and the like.
  • compositions When formulating the composition can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents or surfactants commonly used.
  • Solid preparations 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.
  • 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.
  • Liquid preparations for oral administration include suspending agents, solvents, emulsions or syrups, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, 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 solvents, suspensions, emulsions, lyophilized formulations or suppositories.
  • non-aqueous solvent or suspending agent propylene glycol, polyethylene glycol, vegetable oils such as olive oil or injectable esters such as ethyl oleate may be used.
  • base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter or glycerogelatin may be used.
  • composition may further contain one or more known active ingredients having a prophylactic or therapeutic effect of erectile dysfunction together with HGF protein.
  • the composition may be to increase the intracavernosal pressure.
  • the composition may be to increase the level of endothelial cells, smooth muscle cells, perivascular specific proteins in the corpus cavernosum.
  • the endothelial cells, smooth muscle cells, and perivascular specific proteins may be platelet / endothelial cell adhesion molucule-1 (PECAM-1), smooth muscle actin (SMA), and platelet-derived growth factor beta (PDGF-beta), respectively. have.
  • the erectile dysfunction may be caused by damage to penile endothelial cells or smooth muscle cells.
  • the damage of the penile endothelial cells and smooth muscle cells may be caused by one or more of hyperlipidemia, diabetes, hypertension, and penile nerve damage.
  • composition of the present invention may 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 comprises the step of administering to the subject an endothelial cell regeneration composition
  • an endothelial cell regeneration composition comprising administering to the subject a composition for endothelial cell regeneration comprising an effective amount of HGF protein or a polynucleotide encoding the same as an effective ingredient for endothelial cell regeneration.
  • the endothelial cell regeneration composition is as described above.
  • the term "individual" means a subject in need of treatment of a disease.
  • the subject may be a subject in which endothelial cells are damaged.
  • the subject may be, for example, one or more selected from the group consisting of mammals, eg, human or non-human primates, mice, rats, dogs, cats, horses and cattle.
  • “amount effective for endothelial cell regeneration” includes an amount that, when administered to an individual, further promotes endothelial cell regeneration compared to otherwise. “Amounts effective for endothelial cell regeneration” include, for example, factors including the type and severity of the disease administered, the age and sex of the patient, the sensitivity to the drug, the time of administration, the route and rate of administration, the duration of treatment, and the drug used concurrently. And other factors well known in the medical field, and can be easily determined by those skilled in the art in such an amount that the maximum effect can be obtained without side effects in consideration of all the above factors.
  • the effective amount is 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, 0.001 mg / kg to 1000 mg / kg, 0.01 mg / kg to 1000 mg / kg, 0.1 mg / kg to 1000 mg / kg, 1 mg / kg to 1000 mg / kg, 10 mg / kg to 1000 mg / kg, or 100 mg / kg to May be 1000 mg / kg.
  • administration comprises administering the composition via any route that can reach the erectile tissue of the penis.
  • routes of administration include, for example, oral, rectal, intravenous, intramuscular, subcutaneous or penile corpus cavernosum.
  • the composition may be to increase the intracavernosal pressure.
  • the composition may be to increase the level of endothelial cells, smooth muscle cells, perivascular specific proteins in the corpus cavernosum.
  • the endothelial cells, smooth muscle cells, and perivascular specific proteins may be platelet / endothelial cell adhesion molucule-1 (PECAM-1), smooth muscle actin (SMA), and platelet-derived growth factor beta (PDGF-beta), respectively. have.
  • Another aspect of the invention comprises administering to a subject a pharmaceutical composition for preventing or treating erectile dysfunction comprising an effective amount of an HGF protein or a polynucleotide encoding the same as an active ingredient for treating erectile dysfunction. It provides a method for preventing or treating erectile dysfunction.
  • the pharmaceutical composition for preventing or treating erectile dysfunction is as described above.
  • the term "individual” means a subject in need of treatment of a disease.
  • the subject may be an individual who has or is likely to exhibit an erectile dysfunction symptom.
  • the subject may be, for example, one or more selected from the group consisting of mammals, eg, human or non-human primates, mice, rats, dogs, cats, horses and cattle.
  • “amount effective to treat erectile dysfunction” means that when the composition is administered to a subject, an improvement in erectile dysfunction, eg, an increase in erection and / or an increase in erection duration, is compared to otherwise. Contains amount to let. “Amounts effective for treating erectile dysfunction” include, for example, the type and severity of the disease being administered, the age and sex of the patient, the sensitivity to the drug, the time of administration, the route and rate of release, the duration of treatment, and the drug used concurrently.
  • the effective amount is 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, 0.001 mg / kg to 1000 mg / kg, 0.01 mg / kg to 1000 mg / kg, 0.1 mg / kg to 1000 mg / kg, 1 mg / kg to 1000 mg / kg, 10 mg / kg to 1000 mg / kg, or 100 mg / kg to May be 1000 mg / kg.
  • administration comprises administering the composition via any route that can reach the erectile tissue of the penis.
  • routes of administration include, for example, oral, rectal, intravenous, intramuscular, subcutaneous or penile corpus cavernosum.
  • the composition may be to increase the intracavernosal pressure.
  • the composition may be to increase the level of endothelial cells, smooth muscle cells, perivascular specific proteins in the corpus cavernosum.
  • the endothelial cells, smooth muscle cells, and perivascular specific proteins may be platelet / endothelial cell adhesion molucule-1 (PECAM-1), smooth muscle actin (SMA), and platelet-derived growth factor beta (PDGF-beta), respectively. have.
  • the erectile dysfunction may be caused by damage to penile endothelial cells or smooth muscle cells.
  • the damage of the penile endothelial cells and smooth muscle cells may be caused by one or more of hyperlipidemia, diabetes, hypertension, and penile nerve damage.
  • composition of the present invention may 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 food composition for preventing or improving erectile dysfunction comprising an HGF protein or a polynucleotide encoding the same.
  • the HGF protein and polynucleotides encoding it are as described above.
  • the HGF protein of the present invention may be added to a dietary supplement for the purpose of preventing or improving erectile dysfunction.
  • the "health functional food” refers to a food having a bioregulatory function, such as prevention and improvement of disease, biodefence, immunity, recovery of disease, inhibition of aging, and may be harmless to the human body when taken in the long term.
  • the HGF protein of the present invention When the HGF protein of the present invention is used as a food additive, the HGF protein may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method.
  • the mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment).
  • the HGF protein of the present invention is added in an amount of up to 15% by weight, preferably up to 10% by weight relative to the raw material.
  • the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.
  • foods to which the substance may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, dairy products including gum, ice cream, various soups, beverages, teas, drinks, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.
  • the health beverage composition of the present invention may include various flavors or natural carbohydrates, and the like as an additional ingredient, as in a general beverage.
  • the natural carbohydrate may be a monosaccharide such as glucose or fructose, a disaccharide such as maltose or sucrose, and a natural sweetener such as dextrin or cyclodextrin, or a synthetic sweetener such as saccharin or aspartame.
  • the proportion 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 dietary supplement includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonated beverages. And a carbonation agent used in the.
  • the health functional food may include a pulp for the production of natural fruit juice, fruit juice drinks and vegetable drinks.
  • the components can be used independently or in combination.
  • the proportion of such additives is not critical but is usually 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.
  • HGF protein induces the regeneration of vascular endothelial cells, smooth muscle cells, perivascular cells reduced by diabetes mellitus, PECAM-1, SMA, PDGF- specific proteins, vascular endothelial cells, smooth muscle cells, perivascular cells increase beta expression;
  • HGF protein has an effect of promoting tube production and apoptosis in vascular endothelial cells. Through the above mechanism, HGF protein has an excellent effect on improving erectile dysfunction by increasing intracavernosal pressure, and thus may be useful for preventing or treating erectile dysfunction.
  • FIG. 1 is a diagram showing the results of measurement of intracavernosal pressure (erectile power) according to penile nerve electrical stimulation by HGF protein administration 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, by HGF protein administration in the penile tissues of a diabetic mouse model. It is a figure which shows the result analyzed by confocal microscope together.
  • Figure 3 is a diagram showing the results of the analysis of the hydroethidin (hydroethidin) staining by confocal microscopy to compare the production of peroxide anion by HGF protein administration in the penile tissue of the diabetic mouse model.
  • FIG. 4 is a diagram showing the results of immunohistochemical staining for nitrotyrosine by confocal microscopy to compare the production pattern of peroxynitrite by HGF protein administration in the penile tissue of the diabetic mouse model.
  • FIG. 5 is a confocal microscope analysis of immunohistochemical staining for PDGFR-b in order to compare the expression level of perivascular cells that play an important role in the stability of blood vessels by HGF protein administration in the penile tissue of the diabetic mouse model. Is a diagram showing.
  • FIG. 6 is a diagram showing the results of analyzing the expression of HGF receptor c-MET by confocal microscopy by HGF protein administration after vascular endothelial cells obtained by primary culture in mouse penis tissues under high glucose conditions. .
  • FIG. 7 is a diagram showing the results of analyzing the expression level of PECAM-1, a vascular endothelial cell-specific protein by HGF protein administration after treatment of primary cultures in mouse penile tissues under hyperglycemic conditions. .
  • FIG. 8 is a diagram showing the results of analysis of the degree of tube formation by HGF protein administration by phase contrast microscopy after treatment of vascular endothelial cells obtained by primary culture in mouse penis tissue under hyperglycemic conditions.
  • FIG. 9 is a diagram illustrating the degree of apoptosis by HGF protein administration after treatment of vascular endothelial cells obtained by primary culture in mouse penis tissues under hyperglycemic conditions by TUNEL staining.
  • N 10 / group; group 1, normal mice; group 2, normal mice + streptozotocin
  • PBS Phosphate
  • mice were anesthetized by intramuscular injection of ketamine (100 mg / kg) and xylazine (5 mg / kg) and injected with HGF protein in the cavernous corpus cavernosum for 2 weeks after penile cavernous nerve stimulation.
  • PCAM-1 Platelet-endothelial adhesion molecule-1
  • SMA and c-MET smooth muscle cell-specific proteins SMA and c-MET, an HGF receptor
  • HGF protein showed the highest improvement in erection, and it was confirmed that the erection was restored to the normal level.
  • the amount of cavernous endothelial cells (including vascular endothelial cells) and smooth muscle cells in the penis of streptozotocin-induced diabetic animals was significantly reduced compared to the normal control group. The amount of was remarkably restored to almost normal level.
  • HGF significantly increased the expression of perivascular cells and decreased peroxide anion and peroxynitrite production. It will be described in more detail below.
  • the left epidermis was opened in the lower abdomen of the prepared mouse, and the penile cavernous nerve (the penis nerve) located on the posterior and posterior side of the prostate gland was prepared.
  • the penile cavernous nerve the penis nerve located on the posterior and posterior side of the prostate gland was prepared.
  • a platinum electrode was placed in the penis nerve, and then electrostimulated for about 1 minute at a strength of 1-5 volts 12 hertz.
  • the catheter inserted into the corpus cavernosum was measured through a pressure transmitter connected to a computer (BioPack system, USA) to collect data on the pressure inside the penis (intracavernosal pressure) during the erection. 1 is shown. The measured pressure values will reflect penile erection.
  • FIG. 1 is a diagram showing the corpus cavernosum (erectile power) according to penile nerve electrical stimulation in the HGF protein-administered group and the control group in the diabetic mouse model.
  • ICP intracavernous pressure
  • the horizontal axis represents time after electric stimulation.
  • the electrical stimulation period of 1 minute is indicated by a black bar on the horizontal axis.
  • Figure 1A is the result of measuring the erection after neurostimulation at 1V or 5V in the corpus cavernosum 2 weeks after the injection of HGF protein into the corpus cavernosum once or twice.
  • MSBP mean systolic blood pressure
  • the erection power is expressed by dividing the maximum ICP or area of the cavernosal pressure curve by the mean systolic blood pressure (MSBP), which is the blood pressure itself. This may affect the internal pressure.
  • the control (Control) DM + PBS, one HGF administration, two HGF administration are individuals corresponding to the fourth group in the first group, respectively.
  • HGF double HGF protein twice-administered group
  • DM + PBS PBS-administered group
  • PECAM-1 vascular endothelial cell-specific protein
  • SMA smooth muscle cell-specific protein
  • the penis is a type of special vascular tissue that normally requires cavernous sinusoids and normal blood vessel activity for normal penile erection.
  • the corpus cavernosum and blood vessels are the innermost monolayer cells, which consist of endothelial cells, which are the vascular wall cells that come into direct contact with blood, and the smooth muscle cells that are responsible for the contraction and relaxation of blood vessels. These vascular endothelial cells and smooth muscle cells play an important role in penile blood vessel relaxation and thereby penile erection.
  • the cavernous corpus cavernosum and the abnormal structure or activity of blood vessels cause erection.
  • the present inventors expressed the expression of PECAM-1, a protein specific for spongy bodies and endothelial cells of blood vessels, and the expression of SMA, a smooth muscle cell specific protein. I checked it.
  • the penile tissue was fixed at 4 ° C. in 4% paraformaldehyde for 24 hours, and then frozen in the freezing section using a freeze embedding agent to easily fix the tissue and cut it into 7 ⁇ m thickness to prepare a penile tissue section.
  • FIG. 2A is a confocal micrograph showing the expression level of HGF protein c-Met and the expression level of PECAM-1, a vascular endothelial cell specific protein, in the penile tissues of diabetic mice.
  • HGF protein c-Met the expression level of HGF protein c-Met and the expression level of PECAM-1, a vascular endothelial cell specific protein, in the penile tissues of diabetic mice.
  • FIG. 2A is a confocal micrograph showing the expression level of HGF protein c-Met and the expression level of PECAM-1, a vascular endothelial cell specific protein, in the penile tissues of diabetic mice.
  • FIG. 2A is a confocal micrograph showing the expression level of HGF protein c-Met and the expression level of PECAM-1, a vascular endothelial cell specific protein, in the penile tissues of diabetic mice.
  • FIG. 2A it can be seen that the expression of the receptor c-Met is increased in diabetic mice administered
  • FIG. 2B is a graph showing the statistical treatment of c-Met expression in the area of the corpus cavernosum.
  • Figure 2C is a graph showing the statistical processing of the expression of vascular endothelial cells in the area of the corpus cavernosum. This is the result of demonstrating that vascular endothelial cells reduced by diabetes are induced by HGF protein and that erection is restored accordingly.
  • 2D is a confocal micrograph showing the degree of expression of SMA, a smooth muscle cell specific protein, and the HGF receptor c-Met in the penile tissues of diabetic mice.
  • the control group is the result for penile tissue sections prepared in the same manner as the experimental group, except that the first group of streptozotocin was not injected.
  • 2D and 2E are results demonstrating that smooth muscle cells reduced by diabetes are induced to regenerate by HGF protein, thereby restoring erection.
  • Hydroetitidine is a redox-sensitive probe used to detect peroxide anions in cells. When superoxide reacts with hydroethidine, two-electron oxidized product called ethidium is formed, and ethidium adheres to DNA and absorbs wavelengths of 500-530 nm, thus 590-620 nm. Fluoresce.
  • Peroxide anion one of the active oxygen, is a highly reactive anion free radical that is made in large quantities by NADPH oxidase, which is harmful and can cause DNA damage.
  • the present inventors confirmed the degree of response through hydroetitidine to investigate whether peroxide anion is generated in the penile erectile tissue after administration of streptozotocin to normal mice.
  • Figure 3 is a confocal micrograph showing the degree of luminescence after treatment with hydroethidine reacting with the peroxide anion of the HGF protein-administered group and the control group in diabetic mouse penis tissue.
  • FIG. 3A it can be seen that the peroxide anion is significantly increased in the penis of the group in which diabetic mice are injected with PBS (DM + PBS) compared to normal mice (Control).
  • Control is the result for penile tissue sections prepared in the same manner as the experimental group, except that the first group of streptozotocin was not injected.
  • FIG. 3B is a graph showing the statistical treatment of the expression level of hydroethidine in the area of the corpus cavernosum. This is the result of demonstrating that the peroxide anion increased by diabetes is reduced in its production by HGF protein and therefore erection is restored.
  • Nitric oxide (NO) is an important cytotoxic mediator produced by nitric oxide synthase (NO synthase, NOS). Recently, peroxynitrite (ONOO-) produced by the reaction of NO and superoxide (ONOO-) is reported to cause more damage to cells than direct damage by NO.
  • Peroxynitrite nitrates tyrosine groups in proteins and oxidizes sulfahydryl groups to inhibit peroxidation of membrane lipids, cleavage of DNA, electrolyte migration in the mitochondria, and calcium leakage, which disrupts cell energy production. It is known to play a decisive role in cell damage such as causing necrosis. Since peroxynitrite changes to 3-nitrotyrosine by nitrifying the tyrosine group of the protein, immunostaining for nitrotyrosine may indirectly determine the presence and activity of peroxynitrite.
  • the present inventors stained nitrotyrosine to check whether or not peroxynitrite is produced in penile erectile tissue after administration of streptozotocin to normal mice.
  • Fluorescence immunostaining with anti-PECAM-1 antibody and anti-nitrotyrosine antibody and the second antibody (TRITC-labeled anti-rat antibody, FITC-labeled anti-rabbit antibody) were reacted in the manner specified above.
  • the degree of expression was analyzed by a confocal microscope that can identify the fluorescence microscope or fluorescent material, the results are shown in FIG.
  • FIG. 4A is a confocal micrograph showing the expression level of nitrotyrosine in the HGF protein-administered group and the control group in the penile tissue of diabetic mice.
  • the nitrotyrosine is significantly increased in the penis of the group (DM + PBS) injected diabetic mice compared to the normal mouse (Control).
  • the control group is the result for penile tissue sections prepared in the same manner as the experimental group, except that the first group of streptozotocin was not injected.
  • FIG. 4B is a graph showing the statistical treatment of nitrotyrosine expression in the area of the corpus cavernosum. This is the result demonstrating that peroxynitrite increased by diabetes is reduced in production by HGF protein and therefore erection is restored.
  • Angiogenesis refers to the generation of microvessels from existing blood vessels.
  • pericyte constituting the blood vessels (pericyte) enters to form the structure of endothelial vascular periphery cells, new blood vessels mature and stabilize.
  • PDGF-BB platelet-derived growth factor secreted from vascular endothelial cells is known to contribute to the recruitment and differentiation of perivascular cells.
  • the present inventors investigated the expression of PDGFR-beta by injecting HGF into the penile tissue. Therefore, the present inventors examined the expression level by staining PDGFR-beta in order to investigate the generation of perivascular cells in the penile erectile tissue after administration of streptozotocin to normal mice.
  • Fluorescence immunostaining with anti-PECAM-1 antibody and anti-PDGFR-beta antibody was carried out by the method specified above and a second antibody (TRITC-labeled anti-rat antibody, FITC-labeled anti-rabbit antibody) was reacted.
  • the degree of expression was analyzed by a confocal microscope capable of identifying a fluorescence microscope or fluorescent substance, and the results are shown in FIG. 5.
  • FIG. 5A is a confocal micrograph showing the expression level of perivascular cells of the HGF protein-administered group and the control group in diabetic penis tissue.
  • the control group is the result for penile tissue sections prepared in the same manner as the experimental group, except that the first group of streptozotocin was not injected.
  • Figure 5B is a graph showing the amount of perivascular cells in the corpus cavernosum area statistically processed. This is a result of proving that the perivascular cells reduced by diabetes are increased in amount by HGF protein and thus the erection is restored.
  • Vascular endothelial cells obtained from primary cultures of mouse penis tissues were subjected to in vitro experiments (group 1, NG (Normal Glucose), and vascular endothelial cells obtained from primary cultures of mouse penile tissues +). Glucose 5mmol, 48 hours treatment; Group 2, NG + HGF, vascular endothelial cells obtained from primary culture of mouse penis tissue + Glucose 5mmol, HGF protein 200ng / ml after 48 hours treatment, 24 hours treatment; Group 3, HG (hyperglycemia) , Endothelial cells + Glucose 30mmol obtained by primary culture of mouse penis tissue, 48 hours treatment; Group 4, HG + HGF, 30mmol of vascular endothelial cells + Glucose obtained by primary culture of mouse penis tissue, 48 hours treatment HGF protein 200ng / ml, 24 hours treatment).
  • TUNEL TdT mediated dUTP biotin nick end labeling method
  • c-Met was expressed in vascular endothelial cells obtained by primary culture of mouse penis tissue, and c-Met was expressed more in the normal glucose group than in the hyperglycemic group.
  • the HGF protein treated group was found to have a higher expression level of c-Met than the other group.
  • PECAM-1 staining and tube formation analysis showed that the expression and tube formation of PECAM-1 in the hyperglycemic group was low, but it was recovered in the HGF protein treatment group.
  • TUNEL analysis showing the apoptosis, apoptosis occurred a lot in the hyperglycemic group, but the apoptosis was confirmed in the HGF protein treated group. It will be described in detail below.
  • the primary culture test method for obtaining endothelial cells from mouse penis tissue is as follows.
  • HBSS Hanks Balanced Salt Solution
  • Complement medium 199 including 20% Fetal bovine serum, 1% Penicillin / Streptomycin solution, 50 mg / ml heparin, and 5 ng / ml bFGF
  • Complement medium 199 including 20% Fetal bovine serum, 1% Penicillin / Streptomycin solution, 50 mg / ml heparin, and 5 ng / ml bFGF
  • the present inventors obtained endothelial cells from mouse penis tissue and carried out in vitro experiments.
  • Fluorescent staining of endothelial cells obtained from penile tissue is as follows.
  • FIG. 6 is a confocal micrograph showing the expression of HGF receptor c-Met in HGF protein treated group and control group after inducing hyperglycemia by treating glucose (30 mmol) in endothelial cells obtained from mouse penis tissue.
  • the expression of c-Met was decreased in the hyperglycemic group than the normal glucose, and the expression of the HGF receptor c-Met was increased when HGF protein was treated in the normal and hyperglycemic groups. This result shows that HGF protein is effective on penile endothelial cells when HGF protein is treated in endothelial cells obtained from mouse penis tissue.
  • the present inventors examined the expression level of PECAM-1, a protein specific for vascular endothelial cells, to investigate the changes in vascular endothelial cells after HGF protein administration to endothelial cells obtained from penile tissues. Fluorescence immunostaining with anti-PECAM-1 antibody by the method specified above, and after the final washing, a mounting solution containing DAPI containing a staining nucleus is added and covered with a cover slide. After that, the expression level was analyzed by a confocal microscope that can identify a fluorescence microscope or a fluorescent substance, and the results are shown in FIG. 7.
  • Angiogenesis is not only a simple proliferation of vascular endothelial cells, but also consists of several complex and sequential processes, each of which is essential for the construction of a vascular network.
  • the angiogenesis process can be divided into three types: proliferation, migration, and tube formation.
  • the tube formation step is a step in which endothelial cells migrated and proliferated during angiogenesis continue to divide and grow into hollow tube shapes, differentiate into final blood vessels, and blood enters to complete blood vessel formation.
  • the inventors conducted the following experiment to observe whether the HGF protein is involved 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, then divided into four groups as described above, treated with glucose and HGF proteins, respectively, and seeded on matrigel in a 48-well cell culture dish. Incubate in 5% CO 2 incubator. After 4, 8, 24 hours, the degree of tube formation was observed through a microscope.
  • the tube was clearly generated after 8 hours, and the tube formation was significantly reduced in the HG group, and then recovered to the control level in the HG + HGF group. This is the result demonstrating that the formation of tubes reduced by hyperglycemic conditions is restored by HGF protein.
  • TUNEL analysis is an experimental method to determine whether DNA fragmentation has occurred by attaching digoxigenin-labeled uridine triphosphate (UTP) to the end of a DNA fragment using an enzyme called terminal deoxynucleotidyl transferase (TdT).
  • UTP digoxigenin-labeled uridine triphosphate
  • TdT terminal deoxynucleotidyl transferase
  • apoptosis was increased in the HG group compared to NG and NG + HGF groups, and apoptosis was significantly reduced in the HG + HGF group. This is a result demonstrating that apoptosis increased by hyperglycemic conditions is reduced by HGF protein.
  • HGF protein according to the present invention induces the regeneration of vascular endothelial cells, smooth muscle cells, perivascular cells reduced by diabetes mellitus, PECAM- specific proteins, vascular endothelial cells, smooth muscle cells, perivascular cells 1, SMA, PDGF-beta was confirmed to increase the expression.
  • HGF protein has been shown to promote tube production and inhibit cell death in vascular endothelial cells. Through the above mechanism, HGF protein has been found to have an excellent effect on improving erectile dysfunction by increasing intracavernosal pressure, and thus it may be usefully used for the prevention or treatment of erectile dysfunction.
  • the above ingredients are mixed and filled in an airtight cloth to prepare a powder.
  • tablets are prepared by tableting according to a conventional method for preparing tablets.
  • the above ingredients are mixed and filled into gelatin capsules to prepare capsules.
  • the amount of the above ingredient is prepared per ampoule (2 ml).
  • Vitamin B6 0.5 mg
  • composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method.
  • the granules may be prepared and used for preparing a health food composition according to a conventional method.

Abstract

The present invention provides a composition for endothelial cell generation, containing HGF protein, a biologically active fragment thereof, or a fusion protein thereof, as an active ingredient; a composition for preventing or treating erectile dysfunction; a method for regenerating endothelial cells of an individual; and a method for preventing or treating erectile dysfunction of an individual.

Description

ＨＧＦ 단백질 또는 그 유전자를 포함하는 발기부전 예방 또는 치료용 조성물 및 그의 용도A composition for preventing or treating erectile dysfunction comprising a HWF protein or a gene thereof and use thereof
본 발명은 내피세포 재생용 조성물, 발기부전 예방 또는 치료용 조성물, 개체의 내피세포를 재생시키는 방법, 또는 개체의 발기부전을 예방 또는 치료하는 방법에 관한 것이다.The present invention relates to a composition for endothelial cell regeneration, a composition for preventing or treating erectile dysfunction, a method for regenerating endothelial cells of an individual, or a method for preventing or treating erectile dysfunction of an individual.
HGF는 초대배양 간세포에 대한 증식촉진인자로서 발견되고 분리 및 복제된 증식인자이다. HGF는 여러 가지 간엽계 세포에 의해 생산되며, 대부분 상피계 세포, 뉴런, 혈관내피세포, 일부의 간엽계세포를 표적으로 한다. HGF는 세포증식촉진활성에 관여하여 세포운동성 촉진활성, 상피형태형성 유도활성을 갖는다. HGF는 발생과정에서 상피-간엽 상호작용의 매개체로서 간, 신장, 폐 등의 내장기관, 태반, 골격계의 형성에 관여한다. HGF가 성체에서는 간을 비롯하여 소화관, 신장, 폐의 재생을 촉진하는 기관재생인자로 기능한다는 점에서 여러 질환의 치료약으로 기대를 모으고 있다. 그러나 현재까지 HGF의 발기부전 예방 또는 치료에 대한 기술내용이 교시되거나 기재된 바 없으며, 이에 대한 연구도 전무한 상태이다.HGF is a growth factor found, isolated and cloned as a growth promoter for primary cultured hepatocytes. HGF is produced by a variety of mesenchymal cells, most of which target epithelial cells, neurons, vascular 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 a mediator of epithelial-mesenchymal interactions during development and is involved in the formation of internal organs such as liver, kidney, lung, placenta and skeletal system. It is expected that HGF functions as an organ regeneration factor that promotes regeneration of the liver, digestive tract, kidney, and lung in the adult. However, to date, there has been no teaching or description about the prevention or treatment of erectile dysfunction of HGF, and there is no research on this.
한편, 발기부전은 남성 성기능 장애의 일종으로서, 남성의 성기가 발기되지 않거나 발기 상태가 지속되지 않아 성행위를 할 수 없는 현상이다. 발기부전의 원인은 크게 심인성 원인과 기질성 원인으로 구별된다. 심인성 발기부전은 심리적, 정신적 영향에 의한 교감신경의 과다한 작용으로 인한 노르아드레날린(noradrenaline)의 과도한 분비, 음경해면체 평활근 긴장도 증가, 신경 전달물질의 분비 억제 등에 기인한다. 또한, 기질성 발기 부전은 그 원인에 따라, 신경인성, 혈관성, 및 내분비성 발기부전으로 분류된다.On the other hand, erectile dysfunction is a type of male sexual dysfunction, a phenomenon in which the male genitals are not erection or erection is not sustained, the sexual activity is not possible. The causes of erectile dysfunction are largely divided into psychogenic causes and organic causes. Psychogenic erectile dysfunction is due to excessive secretion of noradrenaline due to excessive effects of sympathetic nerve due to psychological and psychological effects, increased penile cavernous smooth muscle tension, and suppression of neurotransmitter secretion. In addition, organic erectile dysfunction is classified into neurogenic, vascular, and endocrine dysfunction depending on the cause.
상기 혈관성 발기부전은 고지질혈증, 당뇨, 고혈압, 흡연, 전신 심혈관질환 등으로 인해서 음경 혈관내피세포가 손상되어 혈관내피세포에서 일산화질소(nitric oxide: NO) 등의 이완성 물질의 분비가 원활하지 않아 생기는 장애이다.The vascular erectile dysfunction is impaired due to hyperlipidemia, diabetes, hypertension, smoking, systemic cardiovascular diseases, etc., resulting in impaired release of loose substances such as nitric oxide (NO) from vascular endothelial cells. It is a disorder that occurs.
최근 연구는 기질성 원인에 더 비중을 두고 있고, 그의 치료로서 주로 비아그라(viagra, 성분명: 실데나필)를 포함한 경구용 PDE-5(phosphodiesterase-5) 저해제가 전세계적으로 사용되고 있다. 이러한 경구용 약물은 음경해면체에 특이적으로 분포하는 PDE-5의 저해에 의한 cGMP의 농도를 증가시킴으로써 음경해면체 내 혈류를 증대시켜 발기를 유도하여 발기부전의 치료효과가 있는 것으로 알려져 있다. 그러나 두통, 안면홍조, 소화불량, 및 심장마비 등 여러 가지 부작용이 보고되고 있을 뿐만 아니라, 비아그라와 같은 PDE-5 저해제 계열의 약물들은 분자적 수준에서 일시적인 단백질 발현 및 관련 인자들을 조절하는 것으로 근본적인 치료라 할 수 없다. 더욱이 당뇨에 의한 발기부전의 경우, 이와 같은 치료제의 효과가 잘 나타나지 않을 뿐만 아니라, 설령 효과가 있다 해도 그 효능이 장기간 지속되지 못한다는 단점이 있다.Recent studies have placed more emphasis on organic causes, and oral PDE-5 (phosphodiesterase-5) inhibitors, including viagra (component name sildenafil), have been used worldwide for its treatment. Such oral drugs are known to have an effect of treating erectile dysfunction by increasing the concentration of cGMP by inhibiting PDE-5, which is specifically distributed in the corpus cavernosum, thereby increasing the blood flow in the corpus cavernosum. However, not only side effects such as headache, hot flashes, indigestion, and heart attack have been reported, but drugs of the PDE-5 inhibitor family such as Viagra are fundamentally controlled by regulating transient protein expression and related factors at the molecular level. I can not say. Moreover, in case of erectile dysfunction due to diabetes, not only the effect of such a therapeutic agent does not appear well, but even if there is a disadvantage that its efficacy does not last long.
따라서, 발기부전 음경 내의 비정상화된 혈관 구조를 근본적으로 치료하고, 그 효능도 장시간 지속되는 발기부전 치료제의 필요성이 요구되고 있는 실정이다.Therefore, there is a need for a treatment for an erectile dysfunction which fundamentally treats an abnormal vascular structure in an erectile dysfunction penis, and whose efficacy also lasts for a long time.
본 발명의 일 양상은 내피세포 재생용 조성물을 제공하는 것이다.One aspect of the present invention is to provide a composition for endothelial cell regeneration.
본 발명의 다른 일 양상은 발기부전 예방 또는 치료용 조성물을 제공하는 것이다.Another aspect of the present invention is to provide a composition for preventing or treating erectile dysfunction.
본 발명의 다른 일 양상은 개체의 내피세포를 재생시키는 방법을 제공하는 것이다.Another aspect of the invention is to provide a method for regenerating endothelial cells of a subject.
본 발명의 다른 일 양상은 개체의 발기부전을 예방 또는 치료하는 방법을 제공하는 것이다.Another aspect of the invention is to provide a method for preventing or treating erectile dysfunction in a subject.
본 발명의 일 양상은 HGF 단백질 또는 그를 코딩하는 폴리뉴클레오티드를 유효성분으로 포함하는 내피세포 재생용 조성물을 제공한다.One aspect of the present invention provides a composition for endothelial cell regeneration comprising an HGF protein or a polynucleotide encoding the same as an active ingredient.
상기 조성물은 바람직하게는 약학적 조성물이다. The composition is preferably a pharmaceutical composition.
본 명세서에 있어서, "HGF 단백질"이란 간세포생장인자(hepatocyte growth factor)를 의미한다. HGF는 69kDa의 사슬과 34kDa의 사슬로 된 헤테로 2합체 단백질이고, 사슬에 4개의 크린글(Kringle) 구조가 있는 것일 수 있다. 티로신인산화효소 활성이 있는 c-met은 HGF의 수용체일 수 있다. In the present specification, "HGF protein" means hepatocyte growth factor. HGF is a heterodimeric protein consisting of a chain of 69 kDa and a chain of 34 kDa, and may have four Kringle structures in the chain. C-met with tyrosine kinase activity may be a receptor for HGF.
상기 HGF 단백질은 서열번호 1로 표시되는 아미노산 서열을 갖는 단백질 또는 서열번호 2로 표시되는 염기서열로 인코딩 되는 단백질, 및 상기 단백질의 기능적 동등물을 포함한다. 상기 "기능적 동등물"이란 아미노산의 부가, 치환 또는 결실의 결과, 상기 서열번호 1로 표시되는 아미노산 서열과 적어도 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 더 더욱 바람직하게는 95% 이상의 서열 상동성을 갖는 것으로, 서열번호 1로 표시되는 아미노산 서열을 갖는 단백질과 실질적으로 동질의 생리활성을 나타내는 단백질을 말한다. 또한, 본 발명의 HGF 단백질에는 이의 천연형 아미노산 서열을 갖는 단백질뿐만 아니라 이의 변이체가 또한 본 발명의 범위에 포함된다. 상기 변이체란 HGF 단백질의 아미노산 서열과 하나 이상의 아미노산 잔기가 결실, 삽입, 비보전적 또는 보전적 치환 또는 이들의 조합에 의하여 상이한 서열을 가지는 단백질을 의미한다. 분자의 활성을 전체적으로 변경시키지 않는 단백질 및 단편에서의 아미노산 교환은 당해 분야에 공지되어 있다 (H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). HGF 단백질 또는 이의 변이체는 천연에서 추출하거나 합성 (Merrifleld, J. Amer. chem. Soc. 85:2149-2156, 1963) 또는 DNA 서열을 기본으로 하는 유전자 재조합 방법에 의해 제조될 수 있다 (Sambrook et al, Molecular Cloning, Cold Spring Harbour Laboratory Press, New York, USA, 2판, 1989). The HGF protein includes a protein having an amino acid sequence represented by SEQ ID NO: 1 or a protein encoded by a nucleotide sequence represented by SEQ ID NO: 2, and a functional equivalent of the protein. The term "functional equivalent" means at least 70%, preferably 80%, more preferably 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 1 as a result of the addition, substitution, or deletion of the amino acid. Preferably, it refers to a protein having a sequence homology of 95% or more, and having a substantially homogeneous physiological activity with a protein having an amino acid sequence represented by SEQ ID NO: 1. In addition, the HGF proteins of the present invention include not only proteins having their natural amino acid sequence, but also variants thereof, are also included in the scope of the present invention. The variant means a protein in which the amino acid sequence of the HGF protein and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchange in proteins and fragments that do not alter the activity of the molecule as a whole is known in the art (H. Neurode, R. L. Hill, The Proteins, Academic Press, New York, 1979). HGF proteins or variants thereof can be extracted from nature or synthesized (Merrifleld, J. Amer. Chem. Soc. 85: 2149-2156, 1963) or by genetic recombination methods based on DNA sequences (Sambrook et al. , Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd Edition, 1989).
상기 HGF 단백질은 서열번호 2의 염기서열로 암호화될 수 있으며, 상기 뉴클레오티드와 기능적으로 동일한 작용을 할 수 있는 변이체가 본 발명의 범위 내에 포함된다. The HGF protein may be encoded by the nucleotide sequence of SEQ ID NO: 2, and a variant capable of functioning identically with the nucleotide is included within the scope of the present invention.
상기 조성물은 상기 HGF 단백질 전체뿐만 아니라 그의 생물학적 활성 단편 또는 그의 융합 단백질을 포함할 수 있다. 상기 생물학적 활성 단편은 HGF 단백질 또는 그를 코딩하는 유전자의 혈관내피세포 및 평활근세포 재생 활성을 실질적으로 포함하는 것일 수 있다. 상기 생물학적 활성 단편은 천연 HGF의 혈관내피세포 및 평활근세포 활성에 관여하는 활성 도메인을 포함하는 것일 수 있다.The composition may comprise not only all of the HGF protein, but also its biologically active fragments or fusion proteins thereof. The biologically active fragment may be one comprising substantially the vascular endothelial and smooth muscle cell regeneration activity of the HGF protein or a gene encoding the same. The biologically active fragment may comprise an active domain involved in vascular endothelial and smooth muscle cell activity of native HGF.
상기 융합 단백질은 HGF 단백질 또는 그를 코딩하는 유전자의 혈관내피세포 및 평활근세포 재생 활성을 실질적으로 포함하는 것일 수 있다. 상기 융합 단백질은 HGF 단백질 또는 그의 단편이 HGF 단백질 또는 그의 단편의 분리에 유용한 융합 파트너, 표적부위로의 전달에 유용한 융합 파트너, 체내 안정성을 증가시키기 위한 융합 파트너 등에 연결된 것일 수 있다. 상기 연결은 HGF 단백질 또는 그의 단편의 N 말단 또는 C 말단 또는 측쇄를 통하여 연결된 것일 수 있다. 상기 연결은 공유 또는 비공유 결합에 의하여 이루어진 것일 수 있다. 상기 융합 파트너는 폴리펩티드를 포함하는 것일 수 있다. 분리에 유용한 융합 파트너는 His 서열, 예를 들면, His6 서열일 수 있다. 표적부위로의 전달에 유용한 융합 파트너, 또는 체내 안정성을 증가시키기 위한 융합 파트너는 항체 불변 영역, 예를 들면, Fc 영역 또는 PEG와 같은 체내 분해에 저항을 부여하는 중합체일 수 있다.The fusion protein may include substantially the vascular endothelial and smooth muscle cell regeneration activity of the HGF protein or a gene encoding the same. The fusion protein may be one in which the HGF protein or fragment thereof is linked to a fusion partner useful for isolation of the HGF protein or fragment thereof, a fusion partner useful for delivery to a target site, a fusion partner for increasing stability in the body, and the like. The linkage may be linked via the N terminus or C terminus or side chain of the HGF protein or fragment thereof. The linkage may be made by a covalent or non-covalent bond. The fusion partner may be one comprising a polypeptide. Useful fusion partners for separation may be His sequences, such as His6 sequences. Fusion partners useful for delivery to a target site, or fusion partners for increasing stability in the body, may be polymers that confer resistance to degradation in the body, such as antibody constant regions, eg, Fc regions or PEG.
상기 HGF를 코딩하는 폴리뉴클레오티드는 그 자체뿐만 아니라, 다른 물질과 융합된 형태의 것일 수 있다. 예를 들면, 상기 다른 물질은 상기 HGF를 코딩하는 폴리뉴클레오티드를 세포 내로 전달될 수 있도록 하는 것, 세포 내에서 세포 내의 유전자 발현기구에 의하여 발현될 수 있도록 하는 것, 및 세포 내외에서 안정하게 유지되도록 하는 것으로 이루어진 군으로부터 선택된 하나 이상일 수 있다. 상기 융합은 공유 또는 비공유 결합에 의하여 이루어질 수 있다. 상기 융합은 소포 중에 포집되는 형태를 포함한다.The polynucleotide encoding the HGF may be in a fused form with other substances as well as itself. For example, the other substance may be such that the polynucleotide encoding the HGF can be delivered into a cell, expressed in a cell by a gene expression apparatus within the cell, and maintained stable within and outside the cell. It may be one or more selected from the group consisting of. The fusion can be by covalent or non-covalent bonds. The fusion includes the form that is collected in the vesicles.
상기 HGF를 코딩하는 폴리뉴클레오티드는 프로모터, 오퍼레이터, 인핸서 및/또는 전사종결인자와 같은 유전자 발현 조절인자와 작동가능하게 연결된 것일 수 있다. 상기 HGF를 코딩하는 폴리뉴클레오티드는 예를 들면, 플라스미드 또는 바이러스 게놈에 삽입되어 세포 내에서 발현될 수 있도록 된 것일 수 있다. 상기 HGF를 코딩하는 폴리뉴클레오티드는 음경의 내피세포 및 평활근세포, 예를 들면, 해면체 내피세포 및 평활근세포 또는 음경 혈관 내피세포 및 평활근세포에 특이적으로 발현되도록 하는 조절인자와 연결된 구조체일 수 있다. 상기 HGF를 코딩하는 폴리뉴클레오티드는 예를 들면, 아데노바이러스 게놈에 삽입되어 해면체 내피세포 및 평활근세포 또는 음경 혈관 내피세포 및 평활근세포에 특이적으로 발현되도록 하는 조절인자와 연결된 구조체의 형태일 수 있다. 상기 구조체는 바이러스 입자에 포집되어 있는 것일 수 있다.The polynucleotide encoding the HGF may be operably linked with gene expression regulators such as promoters, operators, enhancers and / or transcription terminators. The polynucleotide encoding the HGF may be, for example, inserted into a plasmid or viral genome to be expressed in a cell. The polynucleotide encoding the HGF may be a structure linked to a regulator that is specifically expressed in endothelial cells and smooth muscle cells of the penis, for example, cavernous endothelial cells and smooth muscle cells or penile vascular endothelial cells and smooth muscle cells. The polynucleotide encoding the HGF may be, for example, in the form of a structure linked to a regulator that is inserted into the adenovirus genome so as to be specifically expressed in corpus cavernothelium and smooth muscle cells or penile vascular endothelial cells and smooth muscle cells. The structure may be collected in a virus particle.
본 명세서에 있어서, "유효성분"이란 상기 조성물이 개체에 투여되는 경우, 그렇지 않은 경우에 비하여 내피세포 재생을 더 촉진하는 활성을 갖는 것을 포함한다. 상기 개체는 포유동물, 예를 들면, 인간, 마우스, 햄스터, 개, 고양이, 말, 소, 돼지 및 염소로 이루어진 군으로부터 선택된 하나 이상일 수 있다.As used herein, the term "active ingredient" includes an agent which, when administered to a subject, has an activity that further promotes endothelial cell regeneration as compared to otherwise. The subject may be one or more mammals, eg, humans, mice, hamsters, dogs, cats, horses, cattle, pigs and goats.
본 명세서에 있어서, 상기 "내피세포"는 음경 해면체 내피세포, 음경 혈관내피세포 또는 평활근 세포일 수 있으며, 바람직하게는 음경 해면체 내피세포 또는 음경혈관내피세포이다. In the present specification, the "endothelial cells" may be penile cavernous endothelial cells, penile vascular endothelial cells or smooth muscle cells, preferably penile cavernous endothelial cells or penile vascular endothelial cells.
상기 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. 상기 조성물을 제제화할 경우에는 보통 사용하는 희석제 또는 부형제를 사용하여 조제될 수 있다. 상기 조성물은 경구투여용 제제 또는 비경구 투여용 제제일 수 있다.The composition may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. When formulating the composition, it can be prepared using a commonly used diluent or excipient. The composition may be a preparation for oral administration or a preparation for parenteral administration.
본 명세서에 있어서, 용어 "투여"는 임의의 적절한 방법으로 개체에게 소정의 본 발명의 조성물을 제공하는 것을 의미한다.As used herein, the term "administration" means providing a subject with any composition of the present invention in any suitable manner.
본 발명의 약학적 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르며, 당업자에 의해 적절하게 선택될 수 있다. 바람직한 효과를 위해서, 본 발명의 조성물은 1일 0.0001 mg/kg 내지 10000 mg/kg의 양으로 투여할 수 있다. 상기 조성물의 투여는 하루에 한번 투여할 수도 있고, 수 회 나누어 투여할 수도 있다.Preferred dosages of the pharmaceutical compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration and can be appropriately selected by those skilled in the art. For the 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. Administration of the composition may be administered once a day, may be divided several times.
본 발명의 약학적 조성물은 개체에게 다양한 경로로 투여될 수 있다. 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 음경 해면체 내(intracavernous)에 주사에 의해 투여될 수 있다.The pharmaceutical composition of the present invention can be administered to a subject by various routes. For example, it may be administered by oral, rectal or intravenous, intramuscular, subcutaneous, intracavernous injection.
상기 조성물은 개체의 발기부전을 예방 또는 치료하기 위한 것일 수 있다.The composition may be for preventing or treating erectile dysfunction of an individual.
본 발명의 다른 양상은 HGF 단백질 또는 그를 코딩하는 폴리뉴클레오티드를 유효성분으로 포함하는 발기부전 예방 또는 치료용 조성물을 제공한다.Another aspect of the present invention provides a composition for preventing or treating erectile dysfunction comprising an HGF protein or a polynucleotide encoding the same as an active ingredient.
상기 조성물은 바람직하게는 약학적 조성물 또는 식품 조성물이다. The composition is preferably a pharmaceutical composition or a food composition.
HGF 단백질 및 그를 코딩하는 폴리뉴클레오티드는 상기에서 기술한 바와 같다.The HGF protein and polynucleotides encoding it are as described above.
용어 "유효성분"이란 상기 조성물이 개체에 투여되는 경우, 그렇지 않은 경우에 비하여 발기부전의 예방 또는 치료하는 활성을 갖는 것을 포함한다. 상기 개체는 포유동물, 예를 들면, 인간, 마우스, 햄스터, 개, 고양이, 말, 소, 돼지 및 염소로 이루어진 군으로부터 선택된 하나 이상일 수 있다.The term "active ingredient" when administered to a subject, includes those having the activity of preventing or treating erectile dysfunction, as compared to otherwise. The subject may be one or more mammals, eg, humans, mice, hamsters, dogs, cats, horses, cattle, pigs and goats.
용어 "예방"이란, 상기 조성물이 투여되지 않은 것에 비하여 발기력이 약화되거나 발기력 지속시간이 감소되는 것을 방지하는 것을 포함한다. 용어 "치료"란 상기 조성물이 투여되지 않은 것에 비하여, 약화된 발기력 또는 감소된 발기 지속시간을 더 회복시키는 것을 포함한다. 예를 들면, 본 발명에 따른 HGF 단백질은 당뇨성 발기부전 동물모델에 있어서, 음경 해면체 내피세포 및 평활근 세포 또는 음경 혈관 내피세포 및 평활근 세포의 재생을 유도함으로써 음경해면체 내압을 상승시켜 발기력을 개선시키는 우수한 효과가 있을 수 있다.The term "prevention" includes preventing the impotence from being reduced or the duration of the impotence in comparison with the composition not administered. The term “treatment” includes the further recovery of weakened or reduced erection duration as compared to when the composition is not administered. For example, in the diabetic erectile dysfunction animal model, the HGF protein according to the present invention increases the intracavernosal pressure and improves erection by inducing regeneration of penile cavernous endothelial cells and smooth muscle cells or penile vascular endothelial cells and smooth muscle cells. It can have an excellent effect.
상기 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. 또한 상기 조성물은 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 특히, 상기 조성물은 주사형 제형일 수 있다. 상기 담체, 부형제 및 희석제는 락토오스, 덱스트로오스, 수크로오스, 소르비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시 벤조에이트, 프로필히드록시 벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 포함할 수 있다.The composition may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The composition may also be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions according to conventional methods. In particular, the composition may be an injectable formulation. The carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polycellulose Vinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil and the like.
상기 조성물을 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제 또는 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함될 수 있다. 상기 고형제제는 상기 조성물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로오스, 락토오스 또는 젤라틴 등을 섞어 조제될 수 있다. 또한 상기 조성물의 조제시, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다.When formulating the composition can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents or surfactants commonly used. Solid preparations 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 addition, when preparing the composition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients.
상기 조성물은 경구투여용 제제 또는 비경구 투여용 제제일 수 있다. 경구투여를 위한 액상 제제로는 현탁제, 내용액제, 유제 또는 시럽제 등이 사용되고, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제 또는 좌제가 포함될 수 있다. 또한, 비수성용제 또는 현탁제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름 또는 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지 또는 글리세로제라틴 등이 사용될 수 있다.The composition may be a preparation for oral administration or a preparation for parenteral administration. Liquid preparations for oral administration include suspending agents, solvents, emulsions or syrups, and may include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, 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 solvents, suspensions, emulsions, lyophilized formulations or suppositories. In addition, as the non-aqueous solvent or suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil or injectable esters such as ethyl oleate may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter or glycerogelatin may be used.
상기 조성물은 HGF 단백질과 함께 발기부전의 예방 또는 치료 효과를 갖는 공지의 유효성분을 1종 이상 더 함유할 수 있다.The composition may further contain one or more known active ingredients having a prophylactic or therapeutic effect of erectile dysfunction together with HGF protein.
상기 조성물은 음경해면체 내압을 증가시키는 것일 수 있다.The composition may be to increase the intracavernosal pressure.
상기 조성물은 음경해면체에서 내피세포, 평활근세포, 혈관주위세포 특이적인 단백질의 수준을 증가시키는 것일 수 있다. 상기 내피세포, 평활근세포, 혈관주위세포 특이적인 단백질은 각각 PECAM-1(platelet/endothelial cell adhesion molucule-1), SMA(smooth muscle actin), PDGF-beta(platelet-derived growth factor beta)이 될 수 있다.The composition may be to increase the level of endothelial cells, smooth muscle cells, perivascular specific proteins in the corpus cavernosum. The endothelial cells, smooth muscle cells, and perivascular specific proteins may be platelet / endothelial cell adhesion molucule-1 (PECAM-1), smooth muscle actin (SMA), and platelet-derived growth factor beta (PDGF-beta), respectively. have.
상기 발기부전은 음경 내피세포 또는 평활근 세포의 손상에 의하여 야기된 것일 수 있다. 상기 음경 내피세포 및 평활근 세포의 손상은 고지질혈증, 당뇨병, 고혈압 및 음경신경손상 중 하나 이상에 의하여 야기된 것일 수 있다.The erectile dysfunction may be caused by damage to penile endothelial cells or smooth muscle cells. The damage of the penile endothelial cells and smooth muscle cells may be caused by one or more of hyperlipidemia, diabetes, hypertension, and penile nerve damage.
본 발명의 조성물은 발기부전의 예방 또는 치료를 위하여 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The composition of the present invention may 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.
본 발명의 다른 양상은 내피세포 재생에 유효한 양의 HGF 단백질 또는 그를 코딩하는 폴리뉴클레오티드를 유효성분으로 포함하는, 내피세포 재생용 조성물을 개체에 투여하는 단계를 포함하는, 개체의 내피세포를 재생시키는 방법을 제공한다.Another aspect of the present invention comprises the step of administering to the subject an endothelial cell regeneration composition comprising administering to the subject a composition for endothelial cell regeneration comprising an effective amount of HGF protein or a polynucleotide encoding the same as an effective ingredient for endothelial cell regeneration. Provide a method.
상기 내피세포 재생용 조성물은 상기에서 기술한 바와 같다.The endothelial cell regeneration composition is as described above.
상기 방법에 있어서, 용어 "개체"란 질병의 치료를 필요로 하는 대상을 의미한다. 상기 개체는 내피세포가 손상되어 있는 개체일 수 있다. 상기 개체는 예를 들면, 포유동물, 예를 들면, 인간 또는 비-인간인 영장류, 생쥐, 쥐, 개, 고양이, 말 및 소로 이루어진 군으로부터 선택된 하나 이상일 수 있다.In the method, the term "individual" means a subject in need of treatment of a disease. The subject may be a subject in which endothelial cells are damaged. The subject may be, for example, one or more selected from the group consisting of mammals, eg, human or non-human primates, mice, rats, dogs, cats, horses and cattle.
상기 방법에 있어서, "내피세포 재생에 유효한 양"은 상기 조성물이 개체에 투여되는 경우, 그렇지 않은 경우에 비하여 내피세포 재생을 더 촉진하도록 하는 양을 포함한다. "내피세포 재생에 유효한 양"은 예를 들면, 투여되는 질환 종류 및 중증도, 환자의 연령 및 성별, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정되며 상기 요소를 모두 고려하여 부작용 없이 최대 효과를 얻을 수 있는 양으로, 당업자에 의해 용이하게 결정될 수 있다. 상기 유효한 양은 0.00001mg/kg 내지 1000mg/kg, 예를 들면, 0.00001mg/kg 내지 100mg/kg, 0.00001mg/kg 내지 10mg/kg, 0.00001mg/kg 내지 10mg/kg, 0.0001mg/kg 내지 1000mg/kg, 0.001mg/kg 내지 1000mg/kg, 0.01mg/kg 내지 1000mg/kg, 0.1mg/kg 내지 1000mg/kg, 1mg/kg 내지 1000mg/kg, 10mg/kg 내지 1000mg/kg, 또는 100mg/kg 내지 1000mg/kg일 수 있다.In the method, “amount effective for endothelial cell regeneration” includes an amount that, when administered to an individual, further promotes endothelial cell regeneration compared to otherwise. “Amounts effective for endothelial cell regeneration” include, for example, factors including the type and severity of the disease administered, the age and sex of the patient, the sensitivity to the drug, the time of administration, the route and rate of administration, the duration of treatment, and the drug used concurrently. And other factors well known in the medical field, and can be easily determined by those skilled in the art in such an amount that the maximum effect can be obtained without side effects in consideration of all the above factors. The effective amount is 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, 0.001 mg / kg to 1000 mg / kg, 0.01 mg / kg to 1000 mg / kg, 0.1 mg / kg to 1000 mg / kg, 1 mg / kg to 1000 mg / kg, 10 mg / kg to 1000 mg / kg, or 100 mg / kg to May be 1000 mg / kg.
상기 방법에 있어서, 투여는 상기 조성물을 음경의 발기조직에 도달할 수 있는 임의의 경로를 통한 투여를 포함한다. 상기 투여 경로는 예를 들면, 경구, 직장, 정맥, 근육, 피하 또는 음경 해면체 내에 투여 등이 포함된다.In this method, administration comprises administering the composition via any route that can reach the erectile tissue of the penis. Such routes of administration include, for example, oral, rectal, intravenous, intramuscular, subcutaneous or penile corpus cavernosum.
상기 조성물은 음경해면체 내압을 증가시키는 것일 수 있다. The composition may be to increase the intracavernosal pressure.
상기 조성물은 음경해면체에서 내피세포, 평활근세포, 혈관주위세포 특이적인 단백질의 수준을 증가시키는 것일 수 있다. 상기 내피세포, 평활근세포, 혈관주위세포 특이적인 단백질은 각각 PECAM-1(platelet/endothelial cell adhesion molucule-1), SMA(smooth muscle actin), PDGF-beta(platelet-derived growth factor beta)이 될 수 있다.The composition may be to increase the level of endothelial cells, smooth muscle cells, perivascular specific proteins in the corpus cavernosum. The endothelial cells, smooth muscle cells, and perivascular specific proteins may be platelet / endothelial cell adhesion molucule-1 (PECAM-1), smooth muscle actin (SMA), and platelet-derived growth factor beta (PDGF-beta), respectively. have.
본 발명의 다른 양상은 발기부전을 치료하기에 유효한 양의 HGF 단백질 또는 그를 코딩하는 폴리뉴클레오티드를 유효성분으로 포함하는, 발기부전 예방 또는 치료용 약학적 조성물을 개체에 투여하는 단계를 포함하는, 개체의 발기부전을 예방 또는 치료하는 방법을 제공한다.Another aspect of the invention comprises administering to a subject a pharmaceutical composition for preventing or treating erectile dysfunction comprising an effective amount of an HGF protein or a polynucleotide encoding the same as an active ingredient for treating erectile dysfunction. It provides a method for preventing or treating erectile dysfunction.
상기 발기부전 예방 또는 치료용 약학적 조성물은 상기에서 기술한 바와 같다.The pharmaceutical composition for preventing or treating erectile dysfunction is as described above.
상기 방법에 있어서, 용어 "개체"란 질병의 치료를 필요로 하는 대상을 의미한다. 상기 개체는 발기부전 증상을 가지고 있거나 발기부전 증상을 나타낼 가능성이 있는 개체일 수 있다. 상기 개체는 예를 들면, 포유동물, 예를 들면, 인간 또는 비-인간인 영장류, 생쥐, 쥐, 개, 고양이, 말 및 소로 이루어진 군으로부터 선택된 하나 이상일 수 있다.In the method, the term "individual" means a subject in need of treatment of a disease. The subject may be an individual who has or is likely to exhibit an erectile dysfunction symptom. The subject may be, for example, one or more selected from the group consisting of mammals, eg, human or non-human primates, mice, rats, dogs, cats, horses and cattle.
상기 방법에 있어서, "발기부전을 치료하기에 유효한 양"은 상기 조성물이 개체에 투여되는 경우, 그렇지 않은 경우에 비하여 발기부전의 개선, 예를 들면, 발기력의 강화 및/또는 발기 지속 시간을 증가시키는 양을 포함한다. "발기부전을 치료하기에 유효한 양"은 예를 들면, 투여되는 질환 종류 및 중증도, 환자의 연령 및 성별, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정되며 상기 요소를 모두 고려하여 부작용 없이 최대 효과를 얻을 수 있는 양으로, 당업자에 의해 용이하게 결정될 수 있다. 상기 유효한 양은 0.00001mg/kg 내지 1000mg/kg, 예를 들면, 0.00001mg/kg 내지 100mg/kg, 0.00001mg/kg 내지 10mg/kg, 0.00001mg/kg 내지 10mg/kg, 0.0001mg/kg 내지 1000mg/kg, 0.001mg/kg 내지 1000mg/kg, 0.01mg/kg 내지 1000mg/kg, 0.1mg/kg 내지 1000mg/kg, 1mg/kg 내지 1000mg/kg, 10mg/kg 내지 1000mg/kg, 또는 100mg/kg 내지 1000mg/kg일 수 있다.In the method, “amount effective to treat erectile dysfunction” means that when the composition is administered to a subject, an improvement in erectile dysfunction, eg, an increase in erection and / or an increase in erection duration, is compared to otherwise. Contains amount to let. “Amounts effective for treating erectile dysfunction” include, for example, the type and severity of the disease being administered, the age and sex of the patient, the sensitivity to the drug, the time of administration, the route and rate of release, the duration of treatment, and the drug used concurrently. It is determined according to the elements included and other well-known factors in the medical field, and can be easily determined by those skilled in the art in such an amount that the maximum effect can be obtained without side effects in consideration of all the above factors. The effective amount is 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, 0.001 mg / kg to 1000 mg / kg, 0.01 mg / kg to 1000 mg / kg, 0.1 mg / kg to 1000 mg / kg, 1 mg / kg to 1000 mg / kg, 10 mg / kg to 1000 mg / kg, or 100 mg / kg to May be 1000 mg / kg.
상기 방법에 있어서, 투여는 상기 조성물을 음경의 발기조직에 도달할 수 있는 임의의 경로를 통한 투여를 포함한다. 상기 투여 경로는 예를 들면, 경구, 직장, 정맥, 근육, 피하 또는 음경 해면체 내에 투여 등이 포함된다.In this method, administration comprises administering the composition via any route that can reach the erectile tissue of the penis. Such routes of administration include, for example, oral, rectal, intravenous, intramuscular, subcutaneous or penile corpus cavernosum.
상기 조성물은 음경해면체 내압을 증가시키는 것일 수 있다. The composition may be to increase the intracavernosal pressure.
상기 조성물은 음경해면체에서 내피세포, 평활근세포, 혈관주위세포 특이적인 단백질의 수준을 증가시키는 것일 수 있다. 상기 내피세포, 평활근세포, 혈관주위세포 특이적인 단백질은 각각 PECAM-1(platelet/endothelial cell adhesion molucule-1), SMA(smooth muscle actin), PDGF-beta(platelet-derived growth factor beta)이 될 수 있다.The composition may be to increase the level of endothelial cells, smooth muscle cells, perivascular specific proteins in the corpus cavernosum. The endothelial cells, smooth muscle cells, and perivascular specific proteins may be platelet / endothelial cell adhesion molucule-1 (PECAM-1), smooth muscle actin (SMA), and platelet-derived growth factor beta (PDGF-beta), respectively. have.
상기 발기부전은 음경 내피세포 또는 평활근 세포의 손상에 의하여 야기된 것일 수 있다. 상기 음경 내피세포 및 평활근 세포의 손상은 고지질혈증, 당뇨병, 고혈압 및 음경신경손상 중 하나 이상에 의하여 야기된 것일 수 있다.The erectile dysfunction may be caused by damage to penile endothelial cells or smooth muscle cells. The damage of the penile endothelial cells and smooth muscle cells may be caused by one or more of hyperlipidemia, diabetes, hypertension, and penile nerve damage.
본 발명의 조성물은 발기부전의 예방 또는 치료를 위하여 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The composition of the present invention may 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.
본 발명의 다른 양상은 HGF 단백질 또는 그를 코딩하는 폴리뉴클레오티드를 포함하는 발기부전 예방 또는 개선용 식품 조성물을 제공한다. Another aspect of the present invention provides a food composition for preventing or improving erectile dysfunction comprising an HGF protein or a polynucleotide encoding the same.
HGF 단백질 및 그를 코딩하는 폴리뉴클레오티드는 상기에서 기술한 바와 같다.The HGF protein and polynucleotides encoding it are as described above.
본 발명의 HGF 단백질은 발기부전의 예방 또는 개선을 목적으로 건강기능식품에 첨가될 수 있다. 본 발명에서, '건강기능식품'이란 질병의 예방 및 개선, 생체방어, 면역, 병후의 회복, 노화 억제 등 생체조절기능을 가지는 식품을 말하는 것으로, 장기적으로 복용하였을 때 인체에 무해한 것일 수 있다.The HGF protein of the present invention may be added to a dietary supplement for the purpose of preventing or improving erectile dysfunction. In the present invention, the "health functional food" refers to a food having a bioregulatory function, such as prevention and improvement of disease, biodefence, immunity, recovery of disease, inhibition of aging, and may be harmless to the human body when taken in the long term.
본 발명의 HGF 단백질을 식품 첨가물로 사용할 경우, 상기 HGF 단백질을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조 시에 본 발명의 HGF 단백질은 원료에 대하여 15중량 % 이하, 바람직하게는 10 중량 % 이하의 양으로 첨가된다. 그러나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.When the HGF protein of the present invention is used as a food additive, the HGF protein may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). Generally, in the manufacture of food or beverages, the HGF protein of the present invention is added in an amount of up to 15% by weight, preferably up to 10% by weight relative to the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, it may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.
상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소시지, 빵, 초콜릿, 캔디류, 스낵류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 수프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There is no particular limitation on the kind of food. Examples of foods to which the substance may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, dairy products including gum, ice cream, various soups, beverages, teas, drinks, Alcoholic beverages and vitamin complexes, and the like and include all of the health foods in the conventional sense.
본 발명의 건강음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 포함할 수 있다. 상기 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토오스, 수크로오스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 천연 감미제, 또는 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 ml 당 일반적으로 약 0.01 내지 10 g, 바람직하게는 약 0.01 내지 0.1 g 이다.The health beverage composition of the present invention may include various flavors or natural carbohydrates, and the like as an additional ingredient, as in a general beverage. The natural carbohydrate may be a monosaccharide such as glucose or fructose, a disaccharide such as maltose or sucrose, and a natural sweetener such as dextrin or cyclodextrin, or a synthetic sweetener such as saccharin or aspartame. The proportion 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.
상기 건강기능식품은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 사용되는 탄산화제 등을 포함할 수 있다. 상기 건강기능식품은 천연 과일주스, 과일주스 음료 및 야채 음료의 제조를 위한 과육을 포함할 수 있다. 상기 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0.01 내지 0.1 중량부의 범위에서 선택되는 것이 일반적이다.The dietary supplement includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonated beverages. And a carbonation agent used in the. The health functional food may include a pulp for the production of natural fruit juice, fruit juice drinks and vegetable drinks. The components can be used independently or in combination. The proportion of such additives is not critical but is usually 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.
본 발명에 따른 HGF 단백질은 당뇨에 의해 감소된 혈관내피세포, 평활근세포, 혈관주위세포의 재생을 유도하고, 혈관내피세포, 평활근세포, 혈관주위세포 특이적 단백질인 PECAM-1, SMA, PDGF-beta의 발현을 증가시킨다. 또한 HGF 단백질은 혈관내피세포에서 튜브생성 촉진 및 세포사멸사 억제효과를 보인다. 상기 기전을 통해서 HGF 단백질은 음경해면체 내압을 상승시킴으로써 발기부전에 대하여 우수한 발기력 개선 효과가 있어, 발기부전의 예방 또는 치료에 유용하게 이용될 수 있다.HGF protein according to the present invention induces the regeneration of vascular endothelial cells, smooth muscle cells, perivascular cells reduced by diabetes mellitus, PECAM-1, SMA, PDGF- specific proteins, vascular endothelial cells, smooth muscle cells, perivascular cells increase beta expression; In addition, HGF protein has an effect of promoting tube production and apoptosis in vascular endothelial cells. Through the above mechanism, HGF protein has an excellent effect on improving erectile dysfunction by increasing intracavernosal pressure, and thus may be useful for preventing or treating erectile dysfunction.
도 1은 당뇨 마우스 모델에서 HGF 단백질 투여에 의한 음경신경 전기자극에 따른 음경해면체 내압(발기력) 측정 결과를 나타낸 도이다.1 is a diagram showing the results of measurement of intracavernosal pressure (erectile power) according to penile nerve electrical stimulation by HGF protein administration in a diabetic mouse model.
도 2는 당뇨 마우스 모델의 음경 조직에서 HGF 단백질 투여에 의한 혈관내피세포 특이적 단백질인 PECAM-1 및 혈관평활근세포 특이적 단백질인 SMA의 발현 정도를 HGF 수용체(receptor)인 c-MET의 발현량과 함께 공초점 현미경으로 분석한 결과를 나타낸 도이다.FIG. 2 shows the expression levels of PECAM-1, a vascular endothelial cell-specific protein, and SMA, a vascular smooth muscle cell-specific protein, by HGF protein administration in the penile tissues of a diabetic mouse model. It is a figure which shows the result analyzed by confocal microscope together.
도 3은 당뇨 마우스 모델의 음경 조직에서 HGF 단백질 투여에 의한 과산화물 음이온의 생성 양상을 비교하기 위해 히드로에티딘(hydroethidin) 염색을 공초점 현미경으로 분석한 결과를 나타낸 도이다.Figure 3 is a diagram showing the results of the analysis of the hydroethidin (hydroethidin) staining by confocal microscopy to compare the production of peroxide anion by HGF protein administration in the penile tissue of the diabetic mouse model.
도 4는 당뇨 마우스 모델의 음경 조직에서 HGF 단백질 투여에 의한 퍼옥시나이트라이트의 생성양상을 비교하기 위해 니트로티로신에 대한 면역조직화학염색을 공초점 현미경으로 분석한 결과를 나타낸 도이다.4 is a diagram showing the results of immunohistochemical staining for nitrotyrosine by confocal microscopy to compare the production pattern of peroxynitrite by HGF protein administration in the penile tissue of the diabetic mouse model.
도 5는 당뇨 마우스 모델의 음경 조직에서 HGF 단백질 투여에 의한 혈관의 안정성에 중요한 역할을 하는 혈관주위세포의 발현정도를 비교하기 위해 PDGFR-b에 대한 면역조직화학염색을 공초점 현미경으로 분석한 결과를 나타낸 도이다.5 is a confocal microscope analysis of immunohistochemical staining for PDGFR-b in order to compare the expression level of perivascular cells that play an important role in the stability of blood vessels by HGF protein administration in the penile tissue of the diabetic mouse model. Is a diagram showing.
도 6은 마우스 음경 조직에서 일차배양해서 얻은 혈관내피세포를 고혈당(high glucose) 조건 하에서 처리 후 HGF 단백질 투여에 의한 HGF 수용체인 c-MET의 발현 정도를 공초점 현미경으로 분석한 결과를 나타낸 도이다.6 is a diagram showing the results of analyzing the expression of HGF receptor c-MET by confocal microscopy by HGF protein administration after vascular endothelial cells obtained by primary culture in mouse penis tissues under high glucose conditions. .
도 7은 마우스 음경 조직에서 일차배양해서 얻은 혈관내피세포를 고혈당 조건 하에서 처리 후 HGF 단백질 투여에 의한 혈관내피세포 특이적 단백질인 PECAM-1의 발현 정도를 공초점 현미경으로 분석한 결과를 나타낸 도이다.7 is a diagram showing the results of analyzing the expression level of PECAM-1, a vascular endothelial cell-specific protein by HGF protein administration after treatment of primary cultures in mouse penile tissues under hyperglycemic conditions. .
도 8은 마우스 음경 조직에서 일차배양해서 얻은 혈관내피세포를 고혈당 조건 하에서 처리 후 HGF 단백질 투여에 의한 튜브 형성 정도를 위상차현미경으로 분석한 결과를 나타낸 도이다.8 is a diagram showing the results of analysis of the degree of tube formation by HGF protein administration by phase contrast microscopy after treatment of vascular endothelial cells obtained by primary culture in mouse penis tissue under hyperglycemic conditions.
도 9는 마우스 음경 조직에서 일차배양해서 얻은 혈관내피세포를 고혈당 조건 하에서 처리 후 HGF 단백질 투여에 의한 세포사멸사(apoptosis) 정도를 TUNEL 염색으로 분석한 도이다.9 is a diagram illustrating the degree of apoptosis by HGF protein administration after treatment of vascular endothelial cells obtained by primary culture in mouse penis tissues under hyperglycemic conditions by TUNEL staining.
이하 본 발명의 이해를 돕기 위하여 바람직한 실시예, 실험예 및 제조예를 제시한다. 그러나 하기의 실시예, 실험예 및 제조예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 본 발명의 내용을 한정하는 것이 아니다.Hereinafter, preferred examples, experimental examples, and preparation examples are provided to help understanding of the present invention. However, the following Examples, Experimental Examples and Preparation Examples are provided only to more easily understand the present invention, and do not limit the content of the present invention.
실시예 1. 당뇨성 발기부전 모델에서 HGF 단백질의 발기부전 치료 효과Example 1 Effect of HGF Protein on Erectile Dysfunction in a Diabetic Erectile Dysfunction Model
생후 2개월 된 수컷 마우스(C57BL/6J)를 대상으로 하였고, 총 4 개의 군으로 나누어 실험을 진행하였다 (N = 10/군; 1군, 정상 마우스; 2군, 정상 마우스 + 스트렙토조토신(streptozotocin)을 이용한 당뇨 유도[50 mg/kg 농도로 5일 연속으로 복강 내 투여 후 8주째 된 마우스] + PBS(Phophate buffered solution) 투여 [20 ㎕]; 3군, 스트렙토조토신을 이용한 당뇨 유도[50 mg/kg 농도로 5일 연속으로 복강 내 투여 8주 후] + HGF 단백질을 1번 주사[day 0; 4.2 μg/20 ㎕]한 마우스; 4군, 스트렙토조토신을 이용한 당뇨 유도[50 mg/kg 농도로 5일 연속으로 복강 내 투여 8주 후] + HGF 단백질을 2번 주사[day -3, 0; 4.2 μg/20 ㎕]한 마우스). 상기 마우스를 케타민(ketamine)(100 mg/kg)과 자일라진(xylazine)(5 mg/kg) 근육 주사로 마취한 후 HGF 단백질을 음경해면체 내에 주사한 뒤 2주째에 음경해면체 신경자극 후 발기력을 측정하였다. 음경해면체 조직에서 혈소판-내피세포 부착 분자-1(PECAM-1) 및 평활근 세포 특이적 단백질인 SMA과 HGF 수용체인 c-MET을 같이 면역조직화학염색을 시행하여 혈관내피세포의 발현 변화와 혈관 주위의 수축을 조절하고 형태를 유지하는 혈관주위세포(pericyte)의 발현 변화를 PDGFR-beta(plateletderived growth factor receptor-beta)에 대한 면역조직화학염색으로 살펴보았다. 또한 과산화물 음이온(superoxide anion)의 생성을 히드로에티딘 염색으로 확인하였고, 퍼옥시나이트라이트(peroxinitrite) 발현정도를 니트로티로신(nitrotyrosine)에 대한 면역조직화학염색으로 평가하였다.Two-month-old male mice (C57BL / 6J) were studied and divided into four groups (N = 10 / group; group 1, normal mice; group 2, normal mice + streptozotocin) Induction of diabetes mellitus [mouse 8 weeks after intraperitoneal administration at 50 mg / kg concentration] + administration of Phosphate buffered solution (PBS) [20 μl]; group 3, induction of diabetes mellitus with streptozotocin [50 mg 8 weeks after intraperitoneal administration at / kg concentration] + mice injected with HGF protein once [day 0; 4.2 μg / 20 μl]; group 4, induction of diabetes with streptozotocin [50 mg / kg concentration] 8 weeks after intraperitoneal administration] + 2 injections of HGF protein [day −3, 0; 4.2 μg / 20 μl] mice). The mice were anesthetized by intramuscular injection of ketamine (100 mg / kg) and xylazine (5 mg / kg) and injected with HGF protein in the cavernous corpus cavernosum for 2 weeks after penile cavernous nerve stimulation. Measured. Platelet-endothelial adhesion molecule-1 (PECAM-1) and smooth muscle cell-specific proteins SMA and c-MET, an HGF receptor, were used in the corpus cavernosum tissues to change the expression of vascular endothelial cells and perivascular Changes in the expression of pericyte, which regulates contraction and maintains morphology, were examined by immunohistochemical staining for plateletderived growth factor receptor-beta (PDGFR-beta). In addition, the production of superoxide anion was confirmed by hydroetitidine staining, and the expression level of peroxinitrite was evaluated by immunohistochemical staining for nitrotyrosine.
그 결과, HGF 단백질 투여군 중에서 2번 주사한 군에서 가장 높은 발기력 개선효과를 보였고, 발기력이 정상 수준으로 회복되었음을 확인할 수 있었다. 스트렙토조토신 당뇨 유도 동물모델의 음경에서 해면체내피세포(혈관내피세포 포함) 및 평활근세포의 양이 정상 대조군에 비해서 현저하게 감소되었고, HGF 단백질 투여 후 해면체내피세포(혈관내피세포 포함) 및 평활근세포의 양이 거의 정상 수준으로 현저하게 회복되었다. 또한 HGF는 혈관주위세포의 발현도 현저하게 증가시켰고, 과산화물 음이온 및 퍼옥시나이트라이트 생성은 감소시켰다. 이하에서 보다 자세하게 설명하고자 한다.As a result, the group of two injections of HGF protein showed the highest improvement in erection, and it was confirmed that the erection was restored to the normal level. The amount of cavernous endothelial cells (including vascular endothelial cells) and smooth muscle cells in the penis of streptozotocin-induced diabetic animals was significantly reduced compared to the normal control group. The amount of was remarkably restored to almost normal level. In addition, HGF significantly increased the expression of perivascular cells and decreased peroxide anion and peroxynitrite production. It will be described in more detail below.
(1) 전기 자극에 따른 발기력 분석(1) Analysis of erection power according to electrical stimulation
발기력 측정에 관한 실험방법은, 준비된 마우스의 하복부에서 좌측 표피부분을 개복하고, 전립선의 후외측에 위치한 음경해면체신경(음경신경)이 잘 보이도록 준비하였다. 음경신경의 전기자극을 위해서 백금전극을 음경신경에 위치시킨 후, 1-5 볼트 12 헤르츠 강도로 약 1분 동안 전기자극을 가하였다. 전기자극을 가하게 되면, 음경은 발기를 하게 된다. 이때 음경 해면체에 삽입된 카테터를 통해서 발기 시 음경내부의 압력(음경해면체 내압)에 대한 데이터 수집을 위하여 컴퓨터와 연결되어 있는 압력 전달기(바이오스팩 시스템, 미국)를 통해서 측정하였고, 그 결과를 도 1에 나타내었다. 측정된 압력 수치는 음경 발기력을 반영하게 된다.In the experimental method for measuring the erection power, the left epidermis was opened in the lower abdomen of the prepared mouse, and the penile cavernous nerve (the penis nerve) located on the posterior and posterior side of the prostate gland was prepared. For electrical stimulation of the penis nerve, a platinum electrode was placed in the penis nerve, and then electrostimulated for about 1 minute at a strength of 1-5 volts 12 hertz. When electrical stimulation is applied, the penis will have an erection. At this time, the catheter inserted into the corpus cavernosum was measured through a pressure transmitter connected to a computer (BioPack system, USA) to collect data on the pressure inside the penis (intracavernosal pressure) during the erection. 1 is shown. The measured pressure values will reflect penile erection.
도 1은 당뇨 마우스 모델에서 HGF 단백질 투여군과 대조군에서 음경 신경 전기자극에 따른 음경해면체 내압(발기력)을 나타낸 도면이다. 도 1A에서, 세로축의 ICP(Intracavernous Pressure: 음경해면체내 압력)는 발기 시 음경내부의 압력(음경해면체내압)을 말하며 일반적으로 발기력을 나타내는 지표이다. 가로축은 전기자극 후의 시간을 나타낸다. 도 1A에서 1분간의 전기자극 기간은 가로축에 검정색 막대로 표시하였다. 도 1A는 HGF 단백질을 음경해면체 내에 한번 또는 두번 주사 후 2주째에 음경해면체에 1V 또는 5V로 신경자극 후 발기력을 측정한 결과이다. 또한, 도 1B, 1C에서 세로축의 MSBP(Mean Systolic Blood Pressure)는 평균 수축기 혈압을 나타낸다. 일반적으로 발기력을 표현할 때는 최고 음경해면체내압(Maximal ICP) 또는 음경해면체내압 곡선의 면적(Total ICP [area under the curve])을 평균 수축기 혈압(MSBP)으로 나눈 값으로 나타내게 되는데 이는 혈압자체가 음경해면체내압에 영향을 줄 수 있기 때문이다. 도 1에서, 대조군(Control), DM+PBS, HGF 1회 투여, HGF 2회 투여는 각각 상기한 제1 군에서 제4 군에 해당하는 개체이다.1 is a diagram showing the corpus cavernosum (erectile power) according to penile nerve electrical stimulation in the HGF protein-administered group and the control group in the diabetic mouse model. In FIG. 1A, the intracavernous pressure (ICP) of the vertical axis refers to the pressure inside the penis (penis cavernous pressure) during an erection and is generally an indicator of erectile power. The horizontal axis represents time after electric stimulation. In FIG. 1A, the electrical stimulation period of 1 minute is indicated by a black bar on the horizontal axis. Figure 1A is the result of measuring the erection after neurostimulation at 1V or 5V in the corpus cavernosum 2 weeks after the injection of HGF protein into the corpus cavernosum once or twice. 1B and 1C, the mean systolic blood pressure (MSBP) on the vertical axis represents mean systolic blood pressure. In general, the erection power is expressed by dividing the maximum ICP or area of the cavernosal pressure curve by the mean systolic blood pressure (MSBP), which is the blood pressure itself. This may affect the internal pressure. In Figure 1, the control (Control), DM + PBS, one HGF administration, two HGF administration are individuals corresponding to the fourth group in the first group, respectively.
도 1에 나타난 바와 같이, HGF 단백질 2회 투여군(HGF double)에서 PBS 투여군(DM+PBS)에 비해서 가장 높은 발기력 개선효과를 보였다. 음경발기에 대한 두 가지 변수, 즉 최고 음경해면체 내과 음경압력곡선의 면적이 HGF 단백질 투여 후 현저한 상승을 보였다.As shown in FIG. 1, the HGF protein twice-administered group (HGF double) showed the highest improvement in erection compared to the PBS-administered group (DM + PBS). Two variables for penile erection, namely, the area of maximal corpus cavernosum and the penile pressure curve, were markedly increased after HGF protein administration.
(2) 혈관내피세포 특이적 단백질인 PECAM-1 및 평활근세포 특이적 단백질인 SMA 발현분석(2) Analysis of expression of PECAM-1, a vascular endothelial cell-specific protein, and SMA, a smooth muscle cell-specific protein
음경은 일종의 특수한 혈관조직으로서, 정상적인 음경 발기를 위해서는 해면체 동양구조(cavernous sinusoids)와 음경혈관의 정상적인 활동이 필수적이다. 해면체 동양구조와 혈관은 가장 내벽을 이루는 단층 세포로서 혈액과 직접 접촉하게 되는 혈관벽 세포인 내피세포와 혈관 중막의 여러 개의 세포층을 구성하고 혈관의 수축, 이완을 담당하는 평활근세포로 이루어져 있다. 이러한 혈관내피세포와 평활근세포는 음경혈관의 이완 및 이로 인한 음경 발기에 중요한 역할을 한다. 음경해면체 동양구조와 혈관의 비정상적인 구조나 활동력은 발기력을 감소시키는 원인이 된다.The penis is a type of special vascular tissue that normally requires cavernous sinusoids and normal blood vessel activity for normal penile erection. The corpus cavernosum and blood vessels are the innermost monolayer cells, which consist of endothelial cells, which are the vascular wall cells that come into direct contact with blood, and the smooth muscle cells that are responsible for the contraction and relaxation of blood vessels. These vascular endothelial cells and smooth muscle cells play an important role in penile blood vessel relaxation and thereby penile erection. The cavernous corpus cavernosum and the abnormal structure or activity of blood vessels cause erection.
이에 본 발명자들은 정상 마우스에 스트렙토조토신 투여 후 음경 발기조직 내 혈관의 구조적 변화를 조사하고자 해면체 및 혈관의 내피세포에 특이적 단백질인 PECAM-1의 발현과 평활근세포 특이적 단백질인 SMA의 발현을 확인해 보았다.In order to investigate the structural changes of blood vessels in the penile erectile tissues after administration of streptozotocin to normal mice, the present inventors expressed the expression of PECAM-1, a protein specific for spongy bodies and endothelial cells of blood vessels, and the expression of SMA, a smooth muscle cell specific protein. I checked it.
음경조직을 4℃에서 4% 파라포름알데히드에서 24 시간 동안 고정한 후 동결 절편기에서 동결용 포매제를 이용하여 조직을 절편 만들기 쉽게 고정시킨 후 7 ㎛두께로 잘라서 음경조직 절편을 준비하였다.The penile tissue was fixed at 4 ° C. in 4% paraformaldehyde for 24 hours, and then frozen in the freezing section using a freeze embedding agent to easily fix the tissue and cut it into 7 μm thickness to prepare a penile tissue section.
다음으로, 준비된 음경조직 절편을 슬라이드 상에 올리고, PECAM-1 및 SMA 발현 분석을 위해 준비된 슬라이드 상의 조직을 4% 파라포름알데히드에서 약 5분간 고정시켰다. 고정된 음경조직 절편들을 세척 버퍼(2% FBS + 0.1% Sodium Azide in PBS)에 3회 세척한 후에 비특이적 단백질 차단 버퍼(5% BSA in PBS)로 1시간 블로킹(blocking)한다. 첫 번째 항체(항-PECAM-1 랫 항체 및 FITC-표지된 항-SMA 항체, 항-c-Met 가토 항체)를 1:50 비율로 4 ℃에서 16시간 동안 반응시킨 후 남아 있는 항체를 제거하기 위해서 세척 버퍼로 다시 3회 세척한 다음, PECAM-1에 특이적으로 반응한 항체를 형광으로 확인할 수 있도록 제작된 두 번째 항체(TRITC-표지된 항-랫 항체, FITC- 및 TRITC-표지된 항-가토 항체)를 1:1000 비율로 상온에서 2시간 동안 반응시켰다. 반응이 끝난 후, 남아있는 항체를 제거하기 위해 세척 버퍼로 다시 3회 세척한 후에 형광현미경이나 형광물질을 확인할 수 있는 공초점 현미경으로 발현 정도를 분석하였고, 그 결과를 도 2에 나타내었다.Next, the prepared penile tissue sections were placed on the slides and the tissues on the slides prepared for PECAM-1 and SMA expression analysis were fixed in 4% paraformaldehyde for about 5 minutes. Fixed penile tissue sections were washed three times in wash buffer (2% FBS + 0.1% Sodium Azide in PBS) and then blocked for 1 hour with non-specific protein blocking buffer (5% BSA in PBS). Removing the remaining antibody after reacting the first antibody (anti-PECAM-1 rat antibody and FITC-labeled anti-SMA antibody, anti-c-Met rabbit antibody) at 1: 50 ratio for 16 hours at 4 ° C Second wash (TRITC-labeled anti-rat antibody, FITC- and TRITC-labeled anti-antibodies), which were then washed three times with wash buffer again, followed by fluorescence to identify antibodies that specifically reacted to PECAM-1. Rabbit antibody) was reacted at room temperature in a ratio of 1: 1000 for 2 hours. After the reaction was completed, and washed again three times with a wash buffer to remove the remaining antibody, the expression level was analyzed by confocal microscopy to confirm the fluorescence microscope or fluorescent material, the results are shown in FIG.
도 2A는 당뇨 마우스의 음경조직에서 HGF 단백질 투여군과 대조군의 HGF 수용체인 c-Met의 발현 정도 및 혈관내피세포 특이적 단백질인 PECAM-1의 발현 정도를 나타낸 공초점 현미경 사진이다. 도 2A에 나타난 바와 같이, HGF 단백질을 투여한 당뇨 마우스에서 그 수용체인 c-Met의 발현이 증가함을 알 수 있다. HGF 단백질을 1번 투여한 군에서 PBS만 투여한 군보다 c-Met의 발현이 증가하였고, HGF 단백질을 2번 투여한 군에서 c-Met이 가장 많이 발현됨을 알 수 있었다.FIG. 2A is a confocal micrograph showing the expression level of HGF protein c-Met and the expression level of PECAM-1, a vascular endothelial cell specific protein, in the penile tissues of diabetic mice. As shown in Figure 2A, it can be seen that the expression of the receptor c-Met is increased in diabetic mice administered with HGF protein. In the group administered with HGF protein once, the expression of c-Met was increased compared with the group administered with only PBS, and the expression of c-Met was most expressed in the group administered with HGF protein twice.
당뇨 마우스에 PBS를 주사한 군(DM+PBS)의 음경에서 정상 마우스(Control)에 비해 혈관내피세포의 수가 현저하게 감소되어 있음을 알 수 있다. 여기서, 대조군은 제1 군 스트렙토조토신을 주사하지 않은 것을 제외하고는, 실험군과 동일하게 준비된 음경조직 절편에 대한 결과이다. HGF 단백질을 1회(DM+HGF single) 및 2회 투여한 군(DM+HGF double)에서 그렇지 않은 군(DM+PBS: HGF 단백질 대신 PBS를 투여한 군)에 비하여 혈관내피세포 특이적 단백질의 발현이 증가되었다. 도 2B는 음경해면체 면적 중에 c-Met의 발현량을 통계 처리하여 그래프로 나타낸 그림이다. 도 2C는 음경해면체 면적 중에 혈관내피세포의 발현량을 통계 처리하여 나타낸 그래프이다. 이는 당뇨에 의하여 감소된 혈관내피세포가 HGF 단백질에 의하여 그의 재생이 유도되고, 그에 따라 발기력이 회복된다는 것을 증명하는 결과이다.It can be seen that the number of vascular endothelial cells in the penis of the group injected with PBS to diabetic mice (DM + PBS) was significantly reduced compared to the control mice. Here, the control group is the result for penile tissue sections prepared in the same manner as the experimental group, except that the first group of streptozotocin was not injected. Compared with the group treated with HGF protein (DM + HGF single) and twice (DM + HGF double), the group of vascular endothelial cell-specific proteins was compared with the group not treated with DM + PBS (PBS instead of HGF protein). Expression was increased. Figure 2B is a graph showing the statistical treatment of c-Met expression in the area of the corpus cavernosum. Figure 2C is a graph showing the statistical processing of the expression of vascular endothelial cells in the area of the corpus cavernosum. This is the result of demonstrating that vascular endothelial cells reduced by diabetes are induced by HGF protein and that erection is restored accordingly.
도 2D는 당뇨 마우스의 음경조직에서 HGF 단백질 투여군과 대조군의 평활근세포 특이적 단백질인 SMA의 발현 정도 및 HGF 수용체인 c-Met을 나타낸 공초점 현미경 사진이다. 도 2D에 나타난 바와 같이, 당뇨 마우스에 PBS를 주사한 군(DM+PBS)의 음경에서 정상 마우스에 비해 혈관 평활근세포의 수가 현저하게 감소되어 있음을 알 수 있다. 여기서, 대조군은 제1 군 스트렙토조토신을 주사하지 않은 것을 제외하고는, 실험군과 동일하게 준비된 음경조직 절편에 대한 결과이다. HGF 단백질을 1회(DM+HGF single) 및 2회 투여한 군(DM+HGF double)에서 그렇지 않은 군(DM+PBS: HGF 단백질 대신 PBS를 투여한 군)에 비하여 평활근세포 특이적 단백질의 발현이 증가되었다. 도 2D와 2E는 당뇨에 의하여 감소된 평활근세포가 HGF 단백질에 의하여 그의 재생이 유도되고, 그에 따라 발기력이 회복된다는 것을 증명하는 결과이다.2D is a confocal micrograph showing the degree of expression of SMA, a smooth muscle cell specific protein, and the HGF receptor c-Met in the penile tissues of diabetic mice. As shown in Figure 2D, it can be seen that the number of vascular smooth muscle cells in the penis of the group (DM + PBS) injected diabetic mice PDM significantly reduced compared to normal mice. Here, the control group is the result for penile tissue sections prepared in the same manner as the experimental group, except that the first group of streptozotocin was not injected. The expression of smooth muscle cell-specific protein in the group treated with HGF protein once (DM + HGF single) and twice (DM + HGF double) compared to the group not treated with DM + PBS (PBS instead of HGF protein). This has been increased. 2D and 2E are results demonstrating that smooth muscle cells reduced by diabetes are induced to regenerate by HGF protein, thereby restoring erection.
(3) 과산화물 음이온의 생성을 알 수 있는 히드로에티딘 반응 분석(3) Analysis of hydroetitidine reactions to know the formation of peroxide anions
히드로에티딘은 산화환원에 민감한 프로브(probe)로 세포 속의 과산화물 음이온을 탐지하는데 사용한다. 과산소(Superoxide)과 히드로에티딘이 반응하면 에티디움(ethidium)이라는 2개 전자 산화 생성물(two-electron oxidized product)이 생성되고 에티디움은 DNA에 붙어 500-530nm의 파장을 흡수하여 590-620nm의 형광을 낸다. 활성 산소 중 하나인 과산화물 음이온은 반응성이 강한 음이온 유리기로 NADPH 산화 효소에 의해 다량으로 만들어져 유해성을 띄며 DNA 손상까지 일으킬 수 있는 물질이다.Hydroetitidine is a redox-sensitive probe used to detect peroxide anions in cells. When superoxide reacts with hydroethidine, two-electron oxidized product called ethidium is formed, and ethidium adheres to DNA and absorbs wavelengths of 500-530 nm, thus 590-620 nm. Fluoresce. Peroxide anion, one of the active oxygen, is a highly reactive anion free radical that is made in large quantities by NADPH oxidase, which is harmful and can cause DNA damage.
이에 본 발명자들은 정상 마우스에 스트렙토조토신 투여 후 음경 발기조직 내 과산화물 음이온의 생성여부를 조사하고자 히드로에티딘을 통한 반응 정도를 확인하였다.Therefore, the present inventors confirmed the degree of response through hydroetitidine to investigate whether peroxide anion is generated in the penile erectile tissue after administration of streptozotocin to normal mice.
상기에 명시된 방법으로 항-PECAM-1 항체로 형광면역염색을 하고 마지막 세척을 마친 후 히드로에티딘[1mM in PBS]을 30분 처리한 후 형광현미경이나 형광물질을 확인할 수 있는 컨포칼 현미경으로 발현 정도를 분석하였고, 그 결과를 도 3에 나타내었다.Fluorescence immunostaining with anti-PECAM-1 antibody by the method described above, and after final washing, treated with hydroetitidine [1 mM in PBS] for 30 minutes, followed by expression with a confocal microscope to identify fluorescence microscope or fluorescent substance. The degree was analyzed and the results are shown in FIG. 3.
도 3은 당뇨 마우스의 음경조직에서 HGF 단백질 투여군과 대조군의 과산화물 음이온과 반응하는 히드로에티딘을 처리한 후 발광 정도를 나타낸 공초점 현미경 사진이다. 도 3A에 나타난 바와 같이, 당뇨 마우스에 PBS를 주사한 군(DM+PBS)의 음경에서 정상 마우스(Control)에 비해 과산화물 음이온이 현저하게 증가되어 있음을 알 수 있다. 여기서, Control은 제1 군 스트렙토조토신을 주사하지 않은 것을 제외하고는, 실험군과 동일하게 준비된 음경조직 절편에 대한 결과이다. HGF 단백질을 2회 투여한 군(DM+HGF double)에서 그렇지 않은 군 (DM+PBS: HGF 단백질 대신 PBS를 투여한 군, DM+HGF single: HGF 단백질을 1회 투여한 군)에 비하여 과산화물 음이온의 생성이 감소되었다. 도 3B는 음경해면체 면적 중에 히드로에티딘의 발현량을 통계 처리하여 나타낸 그래프이다. 이는 당뇨에 의하여 증가된 과산화물 음이온이 HGF 단백질에 의하여 그의 생성이 감소되고, 그에 따라 발기력이 회복된다는 것을 증명하는 결과이다.Figure 3 is a confocal micrograph showing the degree of luminescence after treatment with hydroethidine reacting with the peroxide anion of the HGF protein-administered group and the control group in diabetic mouse penis tissue. As shown in FIG. 3A, it can be seen that the peroxide anion is significantly increased in the penis of the group in which diabetic mice are injected with PBS (DM + PBS) compared to normal mice (Control). Here, Control is the result for penile tissue sections prepared in the same manner as the experimental group, except that the first group of streptozotocin was not injected. Peroxide anion compared to the group administered with HGF protein twice (DM + HGF double) compared with the group not treated with DM + PBS: PBS instead of HGF protein, DM + HGF single: HGF protein once The production of is reduced. Figure 3B is a graph showing the statistical treatment of the expression level of hydroethidine in the area of the corpus cavernosum. This is the result of demonstrating that the peroxide anion increased by diabetes is reduced in its production by HGF protein and therefore erection is restored.
(4) 퍼옥시나이트라이트의 생성을 알 수 있는 니트로티로신 반응의 여부 분석(4) Analysis of the nitrotyrosine reaction which can know the production of peroxynitrite
산화질소(Nitric oxide, NO)는 중요한 세포독성 매개체로 산화질소 합성효소 (NO synthase, NOS)에 의해 생산된다. 최근에는 NO에 의한 직접적인 손상보다는 NO와 과산소(superoxide)의 반응으로 생성되는 퍼옥시나이트라이트(ONOO-)가 세포에 더 큰 손상을 입히는 것으로 보고되고 있다. 퍼옥시나이트라이트는 단백질의 티로신기를 질소화하고 설파히드릴(sulfahydryl)기를 산화하여 세포막 지질의 과산화, DNA의 분열, 미토콘드리아에서 전해질 이동을 저해시키고 칼슘을 유출시켜 세포에서의 에너지 생산을 방해하여 세포 괴사를 초래하는 등 세포손상에 결정적인 역할을 한다고 알려져 있다. 퍼옥시나이트라이트는 단백질의 티로신기를 질화하여 3-니트로티로신으로 변화시키기 때문에 니트로티로신에 대한 면역염색을 시행하면 퍼옥시나이트라이트의 존재 및 활성도를 간접적으로 알 수 있다.Nitric oxide (NO) is an important cytotoxic mediator produced by nitric oxide synthase (NO synthase, NOS). Recently, peroxynitrite (ONOO-) produced by the reaction of NO and superoxide (ONOO-) is reported to cause more damage to cells than direct damage by NO. Peroxynitrite nitrates tyrosine groups in proteins and oxidizes sulfahydryl groups to inhibit peroxidation of membrane lipids, cleavage of DNA, electrolyte migration in the mitochondria, and calcium leakage, which disrupts cell energy production. It is known to play a decisive role in cell damage such as causing necrosis. Since peroxynitrite changes to 3-nitrotyrosine by nitrifying the tyrosine group of the protein, immunostaining for nitrotyrosine may indirectly determine the presence and activity of peroxynitrite.
이에 본 발명자들은 정상 마우스에 스트렙토조토신 투여 후 음경 발기조직 내 퍼옥시나이트라이트의 생성여부를 조사하고자 니트로티로신을 염색해 발현 정도를 확인해 보았다.Thus, the present inventors stained nitrotyrosine to check whether or not peroxynitrite is produced in penile erectile tissue after administration of streptozotocin to normal mice.
상기에 명시된 방법으로 항-PECAM-1 항체와 항-니트로티로신 항체로 형광면역염색을 하고 두 번째 항체(TRITC-표지된 항-랫 항체, FITC-표지된 항-가토 항체)를 반응시켰다. 형광현미경이나 형광물질을 확인할 수 있는 공초점 현미경으로 발현 정도를 분석하였고, 그 결과를 도 4에 나타내었다.Fluorescence immunostaining with anti-PECAM-1 antibody and anti-nitrotyrosine antibody and the second antibody (TRITC-labeled anti-rat antibody, FITC-labeled anti-rabbit antibody) were reacted in the manner specified above. The degree of expression was analyzed by a confocal microscope that can identify the fluorescence microscope or fluorescent material, the results are shown in FIG.
도 4A는 당뇨 마우스의 음경조직에서 HGF 단백질 투여군과 대조군의 니트로티로신의 발현 정도를 나타낸 공초점 현미경 사진이다. 도 4A에 나타난 바와 같이, 당뇨 마우스에 PBS를 주사한 군(DM+PBS)의 음경에서 정상 마우스(Control)에 비해 니트로티로신이 현저하게 증가되어 있음을 알 수 있다. 여기서, 대조군은 제1군 스트렙토조토신을 주사하지 않은 것을 제외하고는, 실험군과 동일하게 준비된 음경조직 절편에 대한 결과이다. HGF 단백질을 2회 투여한 군(DM+HGF double)에서 그렇지 않은 군(DM+PBS: HGF 단백질 대신 PBS를 투여한 군, DM+HGF single: HGF 단백질을 1번 투여한 군)에 비하여 니트로티로신의 생성이 감소되었다. 도 4B는 음경 해면체 면적 중에 니트로티로신의 발현량을 통계 처리하여 나타낸 그래프이다. 이는 당뇨에 의하여 증가된 퍼옥시나이트라이트가 HGF 단백질에 의하여 그의 생성이 감소되고, 그에 따라 발기력이 회복된다는 것을 증명하는 결과이다.4A is a confocal micrograph showing the expression level of nitrotyrosine in the HGF protein-administered group and the control group in the penile tissue of diabetic mice. As shown in Figure 4A, it can be seen that the nitrotyrosine is significantly increased in the penis of the group (DM + PBS) injected diabetic mice compared to the normal mouse (Control). Here, the control group is the result for penile tissue sections prepared in the same manner as the experimental group, except that the first group of streptozotocin was not injected. Nitrotyrosine compared to the group treated with HGF protein twice (DM + HGF double) compared to the group not treated with DM + PBS: PBS instead of HGF protein, and DM + HGF single: HGF protein once. The production of is reduced. Figure 4B is a graph showing the statistical treatment of nitrotyrosine expression in the area of the corpus cavernosum. This is the result demonstrating that peroxynitrite increased by diabetes is reduced in production by HGF protein and therefore erection is restored.
(5) 혈관주위세포(pericyte) 특이적 단백질인 PDGFR-beta 발현분석(5) Analysis of PDGFR-beta expression, a pericyte-specific protein
혈관 신생이란 이미 존재하는 혈관으로부터 미세혈관이 생성되는 것을 의미한다. Angiogenesis refers to the generation of microvessels from existing blood vessels.
이러한 혈관 신생의 단계 중 마지막 단계로, 혈관을 구성하는 혈관주위세포(pericyte)가 들어가게 되어 내피세포혈관주위세포의 구조를 형성함으로써, 새로운 혈관이 성숙되고 안정화된다. 이때, 혈관내피세포로부터 분비되는 혈소판 유래성장인자(platelet-derived growth factor, PDGF-BB)가 혈관주위세포의 모집과 차별화에 기여하는 것으로 알려져 있다.In the final stage of the angiogenesis, pericyte constituting the blood vessels (pericyte) enters to form the structure of endothelial vascular periphery cells, 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 the recruitment and differentiation of perivascular cells.
이에 본 발명자들은 음경 조직에 HGF를 주사하여 PDGFR-beta의 발현을 조사해 보았다. 이에 본 발명자들은 정상 마우스에 스트렙토조토신 투여 후 음경 발기조직 내 혈관주위세포 생성여부를 조사하고자 PDGFR-beta를 염색해 발현 정도를 확인해 보았다.The present inventors investigated the expression of PDGFR-beta by injecting HGF into the penile tissue. Therefore, the present inventors examined the expression level by staining PDGFR-beta in order to investigate the generation of perivascular cells in the penile erectile tissue after administration of streptozotocin to normal mice.
상기에 명시된 방법으로 항-PECAM-1 항체와 항-PDGFR-beta 항체로 형광면역염색을 하고 두 번째 항체(TRITC-표지된 항-랫 항체, FITC-표지된 항-가토 항체)를 반응시켰다. 형광현미경이나 형광물질을 확인할 수 있는 공초점 현미경으로 발현 정도를 분석하였고, 그 결과를 도 5에 나타내었다.Fluorescence immunostaining with anti-PECAM-1 antibody and anti-PDGFR-beta antibody was carried out by the method specified above and a second antibody (TRITC-labeled anti-rat antibody, FITC-labeled anti-rabbit antibody) was reacted. The degree of expression was analyzed by a confocal microscope capable of identifying a fluorescence microscope or fluorescent substance, and the results are shown in FIG. 5.
도 5A는 당뇨 마우스의 음경조직에서 HGF 단백질 투여군과 대조군의 혈관주위세포의 발현 정도를 나타낸 공초점 현미경 사진이다. 도 5A에 나타난 바와 같이, 당뇨 마우스에 PBS를 주사한 군(DM+PBS)의 음경에서 정상 마우스(Control)에 비해 혈관주위세포의 양이 현저하게 감소되어 있음을 알 수 있다. 여기서, 대조군은 제1 군 스트렙토조토신을 주사하지 않은 것을 제외하고는, 실험군과 동일하게 준비된 음경조직 절편에 대한 결과이다. HGF 단백질을 2번 투여한 군(DM+HGF double)에서 그렇지 않은 군(DM+PBS: HGF 단백질 대신 PBS를 투여한 군, DM+HGF single: HGF 단백질을 1번 투여한 군)에 비하여 혈관주위세포의 양이 증가되었다.5A is a confocal micrograph showing the expression level of perivascular cells of the HGF protein-administered group and the control group in diabetic penis tissue. As shown in Figure 5A, it can be seen that the amount of perivascular cells is significantly reduced in the penis of the group (DM + PBS) injected diabetic mice compared to the normal mouse (Control). Here, the control group is the result for penile tissue sections prepared in the same manner as the experimental group, except that the first group of streptozotocin was not injected. Vascular periphery compared to the group treated with HGF protein twice (DM + HGF double) compared to the group not treated with DM + PBS: PBS instead of HGF protein and DM + HGF single: HGF protein once The amount of cells increased.
도 5B는 음경해면체 면적 중에 혈관주위세포의 양을 통계 처리하여 나타낸 그래프이다. 이는 당뇨에 의하여 감소된 혈관주위세포가 HGF 단백질에 의하여 그의 양이 증가되고, 그에 따라 발기력이 회복된다는 것을 증명하는 결과이다.Figure 5B is a graph showing the amount of perivascular cells in the corpus cavernosum area statistically processed. This is a result of proving that the perivascular cells reduced by diabetes are increased in amount by HGF protein and thus the erection is restored.
실시예 2. 마우스 음경조직을 일차배양해서 얻은 내피세포로 고혈당 유도 후 HGF 단백질의 효과 평가Example 2 Evaluation of the Effect of HGF Protein after Induction of Hyperglycemia by Endothelial Cells Obtained by Primary Culture of Penile Tissue
마우스 음경 조직을 일차 배양하여 얻은 혈관내피세포를 대상으로 하였고, 총 4개의 군으로 나누어 생체외 실험을 진행하였다(1군, NG (Normal Glucose), 마우스 음경 조직을 일차 배양하여 얻은 혈관내피세포+Glucose 5mmol, 48시간 처리; 2군, NG+HGF, 마우스 음경 조직을 일차 배양하여 얻은 혈관내피세포+Glucose 5mmol, 48시간 처리 후 HGF 단백질 200ng/ml, 24시간 처리; 3군, HG (고혈당), 마우스 음경 조직을 일차 배양하여 얻은 혈관내피세포+Glucose 30mmol, 48시간 처리; 4군, HG+HGF, 마우스 음경 조직을 일차 배양하여 얻은 혈관내피세포+Glucose 30mmol, 48시간 처리 후 HGF 단백질 200ng/ml, 24시간 처리).Vascular endothelial cells obtained from primary cultures of mouse penis tissues were subjected to in vitro experiments (group 1, NG (Normal Glucose), and vascular endothelial cells obtained from primary cultures of mouse penile tissues +). Glucose 5mmol, 48 hours treatment; Group 2, NG + HGF, vascular endothelial cells obtained from primary culture of mouse penis tissue + Glucose 5mmol, HGF protein 200ng / ml after 48 hours treatment, 24 hours treatment; Group 3, HG (hyperglycemia) , Endothelial cells + Glucose 30mmol obtained by primary culture of mouse penis tissue, 48 hours treatment; Group 4, HG + HGF, 30mmol of vascular endothelial cells + Glucose obtained by primary culture of mouse penis tissue, 48 hours treatment HGF protein 200ng / ml, 24 hours treatment).
Glucose와 HGF 처리 후 HGF 수용체인 c-Met과 혈소판-내피세포 부착분자-1(PECAM-1)에 대한 면역조직화학염색을 시행하였고, 튜브 형성 분석(tube formation assay)을 통해 튜브 형성 정도를 알아보았으며, TUNEL(TdT mediated dUTP biotin nick end labeling method) 분석을 수행하여 세포사멸사(apoptosis) 정도를 조사하였다.After treatment with Glucose and HGF, immunohistochemical staining was performed on c-Met and platelet-endothelial adhesion molecule-1 (PECAM-1), and tube formation assay was used to determine the degree of tube formation. TUNEL (TdT mediated dUTP biotin nick end labeling method) analysis was performed to investigate the degree of apoptosis.
그 결과, 마우스 음경 조직을 일차 배양하여 얻은 혈관내피세포에서 c-Met이 발현됨을 알 수 있었고, 정상 글루코스(normal glucose) 군이 고혈당 군보다 c-Met이 더 발현되었다. HGF 단백질 처리군은 그렇지 않은 군에 비해 c-Met의 발현량이 높음을 알 수 있었다. PECAM-1 염색 및 튜브 형성 분석 결과 고혈당군에서 PECAM-1의 발현과 튜브 형성이 적었지만, HGF 단백질 처리군에서 회복됨을 알 수 있었다. 세포사멸사를 알 수 있는 TUNEL 분석 결과 고혈당군에서 세포사멸사가 많이 일어났지만 HGF 단백질 처리군에서 세포사멸사의 감소를 확인하였다. 이하에서 상술하고자 한다.As a result, it was found that c-Met was expressed in vascular endothelial cells obtained by primary culture of mouse penis tissue, and c-Met was expressed more in the normal glucose group than in the hyperglycemic group. The HGF protein treated group was found to have a higher expression level of c-Met than the other group. PECAM-1 staining and tube formation analysis showed that the expression and tube formation of PECAM-1 in the hyperglycemic group was low, but it was recovered in the HGF protein treatment group. As a result of the TUNEL analysis showing the apoptosis, apoptosis occurred a lot in the hyperglycemic group, but the apoptosis was confirmed in the HGF protein treated group. It will be described in detail below.
(1) HGF의 수용체인 c-MET의 발현 양상 분석(1) Analysis of expression patterns of c-MET, a receptor for HGF
마우스 음경 조직에서 내피세포를 얻는 일차배양 실험방법은 다음과 같다. 8주령 C57BL/6J 마우스의 음경 조직을 잘라 요도와 배정맥을 제거한 후 Hanks Balanced Salt Solution (HBSS)에 넣는다. 클린 벤치(clean bench)에서 HBSS와 PBS로 세척 후 조직을 잘라 60-mm 세포 배양 접시(cell culture dish)에 시딩(seeding) 하고 bFGF(recombinant mouse FGF basic)가 들어간 마트리겔(matrigel)(5 ng/ml bFGF:matrigel=1:50)로 조직을 덮는다. 이 상태로 37℃에서 5분 정도 인큐베이션한다. 마트리겔이 굳으면 Complement medium 199(20% Fetal bovine serum, 1% Penicillin/Streptomycin 용액, 50 mg/ml heparin 및 5 ng/ml bFGF 포함) 3ml을 넣고 37℃, 5% CO2 인큐베이터에서 배양한다. 3주정도 후 조직에서 나온 세포들이 배양 접시에 차면 0.2% 젤라틴 코팅한 배양 접시에 계대 배양한다.The primary culture test method for obtaining endothelial cells from mouse penis tissue is as follows. The penis tissues of the 8-week-old C57BL / 6J mice are cut to remove the urethra and the arteriovenous vein and then placed in Hanks Balanced Salt Solution (HBSS). After washing with HBSS and PBS on a clean bench, the tissue is cut, seeded into a 60-mm cell culture dish, and matrigel (5 ng) containing bFGF (recombinant mouse FGF basic). / ml bFGF: matrigel = 1: 50). Incubate at 37 ° C for about 5 minutes in this state. When the matrigel is hardened, 3 ml of Complement medium 199 (including 20% Fetal bovine serum, 1% Penicillin / Streptomycin solution, 50 mg / ml heparin, and 5 ng / ml bFGF) is added and incubated in a 37 ° C, 5% CO 2 incubator. After three weeks, the cells from the tissues are subcultured in a 0.2% gelatin coated petri dish.
본 발명자들은 마우스 음경 조직에서 내피세포를 얻어 생체 외 실험을 진행하였다.The present inventors obtained endothelial cells from mouse penis tissue and carried out in vitro experiments.
음경 조직에서 얻은 내피세포를 형광 염색하는 방법은 다음과 같다. Fluorescent staining of endothelial cells obtained from penile tissue is as follows.
12-웰 세포 배양 접시를 준비하여 커버 슬라이드(cover slide)를 넣고 0.2% 젤라틴 코팅을 한다. 세포를 계대 배양하여 웰에 넣고 37℃, 5% CO2 인큐베이터에서 1-2일 정도 배양한다. 세포가 붙어있는 슬라이드를 웰에서 꺼내어 세척 버퍼 (2% FBS + 0.1% Sodium Azide in PBS)에 3회 세척한 후에 4% 파라포름알데히드에서 약 10분간 실온에서 세포를 고정시켰다. 세척 버퍼로 3번 세척하고, 차가운 메탄올로 1-2분정도 처리하여 투과성을 높인 후, 세척 버퍼로 세척하고, 비특이적 단백질 차단 버퍼(5% BSA in PBS)로 1시간 블로킹한다. 첫 번째 항체(항-c-Met 항체)를 1:100 비율로 4℃에서 16시간 동안 반응시킨 후 남아 있는 항체를 제거하기 위해서 세척 버퍼로 다시 3회 세척한 다음, c-Met에 특이적으로 반응한 항체를 형광으로 확인할 수 있도록 제작된 두 번째 항체 (FITC-표지된 항-가토 항체)를 1:200 비율로 상온에서 2시간 동안 반응시켰다. 반응이 끝난 후, 남아있는 항체를 제거하기 위해 세척 버퍼로 다시 3회 세척한 후에 핵을 염색할 수 있는 DAPI가 들어간 마운팅 용액(mounting solution)을 넣고 커버 슬라이드로 덮는다. 그 후, 형광현미경이나 형광물질을 확인할 수 있는 공초점 현미경으로 발현 정도를 분석하였고, 그 결과를 도 6에 나타내었다.Prepare a 12-well cell culture dish, place a cover slide and apply 0.2% gelatin coating. Cells are passaged into the wells and incubated for 1-2 days in a 37 ° C., 5% CO 2 incubator. The cell-attached slides were removed from the wells, washed three times in wash buffer (2% FBS + 0.1% Sodium Azide in PBS), and the cells were fixed at room temperature for about 10 minutes in 4% paraformaldehyde. After washing three times with washing buffer, treatment with cold methanol for 1-2 minutes to increase permeability, washing with washing buffer, and blocking with non-specific protein blocking buffer (5% BSA in PBS) for 1 hour. The first antibody (anti-c-Met antibody) was reacted for 16 hours at 4 ° C. in a ratio of 1: 100, and then washed three times again with a washing buffer to remove the remaining antibody, followed by specific c-Met specificity. A second antibody (FITC-labeled anti-rabbit antibody), which was designed to identify the reacted antibody by fluorescence, was reacted for 2 hours at room temperature in a ratio of 1: 200. After completion of the reaction, wash again with washing buffer three times to remove the remaining antibody, and then put the mounting solution containing DAPI to stain the nucleus and cover with a cover slide. Then, the degree of expression was analyzed by a confocal microscope that can confirm the fluorescence microscope or fluorescent substance, the results are shown in FIG.
도 6은 마우스 음경 조직에서 얻은 내피세포에 글루코스를 처리(30mmol)하여 고혈당을 유도한 후, HGF 단백질 처리군과 대조군에서 HGF 수용체인 c-Met의 발현을 나타낸 컨포칼 현미경 사진이다. 도 6에 나타낸 바와 같이, 정상군(normal glucose)보다 고혈당군에서 c-Met의 발현이 감소하였고, 정상군 및 고혈당군에서 HGF 단백질을 처리한 경우 HGF 수용체인 c-Met의 발현이 증가하였다. 이는 마우스 음경 조직에서 얻은 내피세포에서 HGF 단백질을 처리했을 때 그 수용체가 증가함으로써 HGF 단백질이 음경 내피세포에 효과가 있음을 증명한 결과이다.6 is a confocal micrograph showing the expression of HGF receptor c-Met in HGF protein treated group and control group after inducing hyperglycemia by treating glucose (30 mmol) in endothelial cells obtained from mouse penis tissue. As shown in FIG. 6, the expression of c-Met was decreased in the hyperglycemic group than the normal glucose, and the expression of the HGF receptor c-Met was increased when HGF protein was treated in the normal and hyperglycemic groups. This result shows that HGF protein is effective on penile endothelial cells when HGF protein is treated in endothelial cells obtained from mouse penis tissue.
(2) 혈관내피세포 특이적 단백질인 PECAM-1 발현 분석(2) Analysis of PECAM-1 Expression, a Vascular Endothelial Cell Specific Protein
본 발명자들은 음경 조직에서 얻은 내피세포에 HGF 단백질 투여 후 혈관내피세포의 변화를 조사하고자 혈관내피세포에 특이적 단백질인 PECAM-1의 발현정도를 살펴보았다. 상기에 명시된 방법으로 항-PECAM-1 항체로 형광면역염색을 하고 마지막 세척을 마친 후 핵을 염색할 수 있는 DAPI가 들어간 마운팅 용액을 넣고 커버 슬라이드로 덮는다. 그 후 형광현미경이나 형광물질을 확인할 수 있는 공초점 현미경으로 발현 정도를 분석하였고, 그 결과를 도 7에 나타내었다. The present inventors examined the expression level of PECAM-1, a protein specific for vascular endothelial cells, to investigate the changes in vascular endothelial cells after HGF protein administration to endothelial cells obtained from penile tissues. Fluorescence immunostaining with anti-PECAM-1 antibody by the method specified above, and after the final washing, a mounting solution containing DAPI containing a staining nucleus is added and covered with a cover slide. After that, the expression level was analyzed by a confocal microscope that can identify a fluorescence microscope or a fluorescent substance, and the results are shown in FIG. 7.
도 7에 나타난 바와 같이, NG군과 NG+HGF군은 PECAM-1 발현의 차이가 없었다. HG군에서는NG군에 비해 PECAM-1 발현이 현저하게 감소되었으며, HG+HGF군에서는 대조군(NG군) 수준으로 회복됨을 알 수 있었다. 이는 고혈당 조건에 의해서 감소된 내피세포의 양이 HGF 단백질에 의하여 회복된다는 것을 증명하는 결과이다.As shown in FIG. 7, there was no difference in PECAM-1 expression between the NG group and the NG + HGF group. In the HG group, PECAM-1 expression was significantly reduced compared to the NG group, and the HG + HGF group was found to recover to the control (NG group) level. This proves that the amount of endothelial cells reduced by hyperglycemic conditions is recovered by the HGF protein.
(3) 신생혈관형성 과정 중 튜브 형성 정도를 평가(3) evaluating the degree of tube formation during angiogenesis
혈관신생은 혈관내피세포의 단순한 증식만이 아닌, 여러 단계의 복잡하고 순차적인 과정으로 구성되며, 각각 단계는 혈관 네트워크의 구축에 반드시 필요하다. 혈관신생 과정을 크게 세가지로 나누어 살펴보면, 증식(proliferation), 이동(migration), 튜브 형성(tube formation) 과정으로 나뉠 수 있다. 튜브 형성 단계는 혈관신생 과정 중 이동, 증식한 내피세포들이 분열을 계속하여 속이 빈 튜브 모양으로 성장하여 최종 혈관으로 분화하고 여기에 혈액이 들어가 혈관 생성이 완성되는 단계이다. 본 발명자들은 튜브 형성 분석를 통해 HGF 단백질이 혈관신생과정 중 튜브 형성에 관여하는지 여부를 관찰하기 위해 다음의 실험을 진행하였다.Angiogenesis is not only a simple proliferation of vascular endothelial cells, but also consists of several complex and sequential processes, each of which is essential for the construction of a vascular network. The angiogenesis process can be divided into three types: proliferation, migration, and tube formation. The tube formation step is a step in which endothelial cells migrated and proliferated during angiogenesis continue to divide and grow into hollow tube shapes, differentiate into final blood vessels, and blood enters to complete blood vessel formation. The inventors conducted the following experiment to observe whether the HGF protein is involved in tube formation during angiogenesis through tube formation analysis.
음경 조직을 일차배양 해서 얻은 내피세포에 혈청 기아(serum starvation)를 24시간 한 후 상기에 명시된 4개의 군으로 나누어 각각 글루코스와 HGF 단백질을 처리하여 48-well 세포 배양 접시의 마트리겔 위에 시딩하고 37℃, 5% CO2 인큐베이터에서 배양한다. 4, 8, 24시간 후 튜브 형성 정도를 현미경을 통해 관찰한 결과를 도 8에 나타내었다. Endothelial cells obtained by primary culture of penile tissues were subjected to serum starvation for 24 hours, then divided into four groups as described above, treated with glucose and HGF proteins, respectively, and seeded on matrigel in a 48-well cell culture dish. Incubate in 5% CO 2 incubator. After 4, 8, 24 hours, the degree of tube formation was observed through a microscope.
도 8에 나타난 바와 같이, 튜브는 8시간 이후부터 뚜렷하게 생성되었고, HG군에서 튜브 형성이 현저하게 감소되었다가 HG+HGF군에서 대조군 수준으로 회복됨을 관찰할 수 있었다. 이는 고혈당 조건에 의해서 감소된 튜브의 형성이 HGF 단백질에 의하여 회복된다는 것을 증명하는 결과이다.As shown in FIG. 8, the tube was clearly generated after 8 hours, and the tube formation was significantly reduced in the HG group, and then recovered to the control level in the HG + HGF group. This is the result demonstrating that the formation of tubes reduced by hyperglycemic conditions is restored by HGF protein.
(4) 세포 예정사 정도를 알 수 있는 TUNEL 분석(4) TUNEL analysis to know the degree of cell death
TUNEL 분석은 TdT(terminal deoxynucleotidyl transferase)라는 효소를 이용해 디곡시제닌(digoxigenin)이 표지된 UTP(uridine triphosphate)를 DNA 조각 말단에 붙여 DNA 단편화가 일어났는지 여부를 파악하는 실험방법이다. 세포핵 안의 DNA가 분해되는 과정에서 DNA 단편화가 일어나면 2중 나선 구조에 분열이 생기고 N 말단의 3'-OH기가 노출되게 되어 N 말단에 TUNEL이 결합하는 방법으로 분석한다. TUNEL 분석 결과를 도 9에 나타내었다. 이때 세포의 핵을 염색하기 위하여 DAPI(4',6-diamidino-2-phenylindole)를 사용하였다. TUNEL analysis is an experimental method to determine whether DNA fragmentation has occurred by attaching digoxigenin-labeled uridine triphosphate (UTP) to the end of a DNA fragment using an enzyme called terminal deoxynucleotidyl transferase (TdT). When DNA fragmentation occurs in the process of decomposing DNA in the cell nucleus, splitting occurs in the double helix structure and the 3'-OH group is exposed at the N-terminus. TUNEL analysis results are shown in FIG. 9. At this time, DAPI (4 ', 6-diamidino-2-phenylindole) was used to stain the nuclei of cells.
도 9에 나타난 바와 같이, HG군에서 NG군, NG+HGF군에 비해 세포사멸사가 증가 하였고, HG+HGF군에서 세포사멸사가 현저히 감소하였다. 이는 고혈당 조건에 의해서 증가된 세포사멸사가 HGF 단백질에 의하여 감소된다는 것을 증명하는 결과이다.As shown in FIG. 9, apoptosis was increased in the HG group compared to NG and NG + HGF groups, and apoptosis was significantly reduced in the HG + HGF group. This is a result demonstrating that apoptosis increased by hyperglycemic conditions is reduced by HGF protein.
이상의 실험 결과를 통하여, 본 발명에 따른 HGF 단백질은 당뇨에 의해 감소된 혈관내피세포, 평활근세포, 혈관주위세포의 재생을 유도하고, 혈관내피세포, 평활근세포, 혈관주위세포 특이적 단백질인 PECAM-1, SMA, PDGF-beta의 발현을 증가시킴을 확인하였다. 또한 HGF 단백질은 혈관내피세포에서 튜브생성 촉진 및 세포사멸사 억제효과를 보였다. 상기 기전을 통해서 HGF 단백질은 음경해면체 내압을 상승시킴으로써 발기부전에 대하여 우수한 발기력 개선 효과가 있어, 발기부전의 예방 또는 치료에 유용하게 이용될 수 있음을 확인하였다. Based on the above experimental results, HGF protein according to the present invention induces the regeneration of vascular endothelial cells, smooth muscle cells, perivascular cells reduced by diabetes mellitus, PECAM- specific proteins, vascular endothelial cells, smooth muscle cells, perivascular cells 1, SMA, PDGF-beta was confirmed to increase the expression. In addition, HGF protein has been shown to promote tube production and inhibit cell death in vascular endothelial cells. Through the above mechanism, HGF protein has been found to have an excellent effect on improving erectile dysfunction by increasing intracavernosal pressure, and thus it may be usefully used for the prevention or treatment of erectile dysfunction.
이하 본 발명의 약학적 조성물 및 식품 조성물의 제제예를 설명하나, 이는 본 발명을 한정하고자 함이 아니다.Hereinafter, the preparation examples of the pharmaceutical composition and the food composition of the present invention, but this is not intended to limit the present invention.
제제예 1. 약학적 조성물의 제조Formulation Example 1 Preparation of Pharmaceutical Composition
1-1. 산제의 제조1-1. Manufacture of powder
HGF 단백질 20 mgHGF Protein 20 mg
유당 100 mg Lactose 100 mg
탈크 10 mg Talc 10 mg
상기의 성분들을 혼합하고 기밀포에 충진하여 산제를 제조한다.The above ingredients are mixed and filled in an airtight cloth to prepare a powder.
1-2. 정제의 제조1-2. Manufacture of tablets
HGF 단백질 10 mg HGF protein 10 mg
옥수수전분 100 mg Corn starch 100 mg
유당 100 mg Lactose 100 mg
스테아린산 마그네슘 2 mg2 mg magnesium stearate
상기의 성분들을 혼합한 후 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조한다.After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.
1-3. 캡슐제의 제조1-3. Preparation of Capsule
HGF 단백질 10 mg HGF protein 10 mg
결정성 셀룰로오스 3 mg3 mg of crystalline cellulose
락토오스 14.8 mgLactose 14.8 mg
마그네슘 스테아레이트 0.2 mgMagnesium Stearate 0.2 mg
통상의 캡슐제 제조방법에 따라 상기의 성분을 혼합하고 젤라틴 캡슐에 충전하여 캡슐제를 제조한다.According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.
1-4. 주사제의 제조1-4. Preparation of Injectables
HGF 단백질 10 mg HGF protein 10 mg
만니톨 180 mgMannitol 180 mg
주사용 멸균 증류수 2974 mgSterile distilled water for injection 2974 mg
Na2HPO4·2H2O 26 mgNa 2 HPO 4 · 2H 2 O 26 mg
통상의 주사제의 제조방법에 따라 1 앰플당 (2 ml) 상기의 성분 함량으로 제조한다.According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).
1-5. 액제의 제조1-5. Preparation of liquid
HGF 단백질 20 mgHGF Protein 20 mg
이성화당 10 g10 g of isomerized sugar
만니톨 5 g5 g of mannitol
정제수 적량Purified water
통상의 액제의 제조방법에 따라 정제수에 각각의 성분을 가하여 용해시키고 레몬향을 적량 가한 다음 상기의 성분을 혼합한 다음 정제수를 가하여 전체를 정제수를 가하여 전체 100 ml로 조절한 후 갈색병에 충진하여 멸균시켜 액제를 제조한다.After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding a proper amount of lemon aroma, and then mixing the above components, adding purified water and adjusting the whole to 100 ml by adding purified water and filling into a brown bottle. The solution is prepared by sterilization.
제제예 2. 식품 조성물의 제조Formulation Example 2 Preparation of Food Composition
2-1. 건강식품의 제조2-1. Manufacture of health food
HGF 단백질 100 mg HGF Protein 100 mg
비타민 혼합물 적량Vitamin mixture proper amount
비타민 A 아세테이트 70 μg 70 μg of Vitamin A Acetate
비타민 E 1.0 mgVitamin E 1.0 mg
비타민 B1 0.13 mgVitamin B1 0.13 mg
비타민 B2 0.15 mgVitamin B2 0.15 mg
비타민 B6 0.5 mgVitamin B6 0.5 mg
비타민 B12 0.2 μg 0.2 μg of vitamin B12
비타민 C 10 mg Vitamin C 10 mg
비오틴 10 μg Biotin 10 μg
니코틴산아미드 1.7 mgNicotinamide 1.7 mg
엽산 50 μg Folic acid 50 μg
판토텐산 칼슘 0.5 mgCalcium Pantothenate 0.5 mg
무기질 혼합물 적량Mineral mixture
황산제1철 1.75 mgFerrous Sulfate 1.75 mg
산화아연 0.82 mgZinc Oxide 0.82 mg
탄산마그네슘 25.3 mgMagnesium carbonate 25.3 mg
제1인산칼륨 15 mg15 mg potassium monophosphate
제2인산칼슘 55 mgDicalcium Phosphate 55 mg
구연산칼륨 90 mgPotassium Citrate 90 mg
탄산칼슘 100 mg Calcium Carbonate 100 mg
염화마그네슘 24.8 mgMagnesium chloride 24.8 mg
상기의 비타민 및 미네랄 혼합물의 조성비는 비교적 건강식품에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 그 배합비를 임의로 변형 실시하여도 무방하며, 통상의 건강식품 제조방법에 따라 상기의 성분을 혼합한 다음, 과립을 제조하고, 통상의 방법에 따라 건강식품 조성물 제조에 사용할 수 있다.Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Claims (12)

  1. HGF(hepatocyte growth factor) 단백질 또는 그를 코딩하는 폴리뉴클레오티드를 유효성분으로 포함하는 내피세포 재생용 조성물.An endothelial cell regeneration composition comprising an HGF (hepatocyte growth factor) protein or a polynucleotide encoding the same as an active ingredient.
  2. 청구항 1에 있어서, 상기 내피세포는 음경 해면체 내피세포 또는 음경 혈관 내피세포인 것인 조성물.The composition of claim 1, wherein the endothelial cells are penile cavernous endothelial cells or penile vascular endothelial cells.
  3. HGF 단백질 또는 그를 코딩하는 폴리뉴클레오티드를 유효성분으로 포함하는 발기부전 예방 또는 치료용 약학적 조성물.Pharmaceutical composition for preventing or treating erectile dysfunction comprising HGF protein or a polynucleotide encoding the same as an active ingredient.
  4. 청구항 3에 있어서, 상기 HGF 단백질은 서열번호 1의 아미노산 서열로 이루어진 것인 조성물.The composition of claim 3, wherein the HGF protein consists of the amino acid sequence of SEQ ID NO: 1.
  5. 청구항 3에 있어서, 상기 HGF를 코딩하는 폴리뉴클레오티드는 서열번호 2의 염기서열로 이루어진 것인 조성물.The composition of claim 3, wherein the polynucleotide encoding the HGF consists of a nucleotide sequence of SEQ ID NO: 2.
  6. 청구항 3에 있어서, 상기 조성물은 음경 해면체의 내압을 증가시키는 것인 조성물.The composition of claim 3, wherein the composition increases the internal pressure of the corpus cavernosum.
  7. 청구항 3에 있어서, 상기 발기부전은 음경 내피세포 또는 평활근 세포의 손상에 의하여 야기된 것인 조성물.The composition of claim 3, wherein the erectile dysfunction is caused by damage to penile endothelial cells or smooth muscle cells.
  8. 청구항 7에 있어서, 상기 음경 내피세포 또는 평활근 세포의 손상은 고지질혈증, 당뇨병, 고혈압 및 음경신경손상으로 이루어진 군으로부터 선택된 하나 이상에 의하여 야기된 것인 조성물.The composition of claim 7, wherein the damage to the penile endothelial cells or smooth muscle cells is caused by one or more selected from the group consisting of hyperlipidemia, diabetes, hypertension and penile nerve damage.
  9. 내피세포 재생에 유효한 양의 HGF 단백질 또는 그를 코딩하는 폴리뉴클레오티드를 유효성분으로 포함하는, 내피세포 재생용 조성물을 개체에 투여하는 단계를 포함하는, 개체의 내피세포를 재생시키는 방법.A method for regenerating endothelial cells of a subject, comprising administering to the subject a composition for endothelial cell regeneration comprising an HGF protein or a polynucleotide encoding the same as an effective ingredient effective for endothelial cell regeneration.
  10. 발기부전을 치료하기에 유효한 양의 HGF 단백질 또는 그를 코딩하는 폴리뉴클레오티드를 유효성분으로 포함하는, 발기부전 예방 또는 치료용 약학적 조성물을 개체에 투여하는 단계를 포함하는, 개체의 발기부전을 예방 또는 치료하는 방법.Preventing an erectile dysfunction of an individual, comprising administering to the subject a pharmaceutical composition for preventing or treating erectile dysfunction comprising an effective amount of HGF protein or a polynucleotide encoding the same as an active ingredient for treating erectile dysfunction. How to treat.
  11. 청구항 10에 있어서, 상기 투여는 경구, 직장, 정맥, 근육, 피하 또는 음경해면체 내에 투여하는 것인 방법.The method of claim 10, wherein the administration is oral, rectal, intravenous, intramuscular, subcutaneous or corpus cavernosum.
  12. HGF 단백질 또는 그를 코딩하는 폴리뉴클레오티드를 포함하는 발기부전 예방 또는 개선용 식품 조성물.Food composition for preventing or improving erectile dysfunction comprising a HGF protein or a polynucleotide encoding the same.
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