KR20190082697A - Composition for preventing or treating erectile dysfunction, ischemic disease or peripheral neurological disorder comprising fusion protein comprising LRG1 protein - Google Patents

Abstract

The present invention relates to a fusion protein comprising an LRG1 glycoprotein and a composition for preventing or treating impotence, ischemic diseases or peripheral nerve diseases, containing the same as an active ingredient. The LRG1 glycoprotein or an LRG1-Fc fusion protein according to the present invention increases the level of endothelial cell-specific and nerve cell-specific proteins, and thus has an excellent effect of improving impotence by inducing the regeneration of the endothelial cells and nerve cells of the corpus cavernosum and increasing penile erection, thereby being usefully used for the prevention or treatment of impotence. Further, the LRG1glycoprotein or LRG1-Fc fusion protein according to the present invention has the effect of regenerating blood vessels and nerves, and thus can be usefully used for the prevention or treatment of ischemic diseases or peripheral nerve diseases.

Description

Composition for preventing or treating erectile dysfunction, ischemic disease or peripheral neurological disorder comprising fusion protein comprising LRG1 protein}

The present invention relates to a composition for preventing or treating erectile dysfunction, ischemic disease, or peripheral neurological disease, including a fusion protein containing the LRG1 glycoprotein and the same as an active ingredient.

Erectile dysfunction is a type of male sexual dysfunction, which is a phenomenon in which a male's genitals are not erected or the erectile state does not persist and thus cannot engage in sexual activity. The causes of erectile dysfunction are largely divided into psychogenic and organic causes. Psychogenic erectile dysfunction is caused by excessive secretion of noradrenaline, an increase in corpus cavernosum smooth muscle tension, and suppression of the secretion of neurotransmitters due to the excessive action of the sympathetic nerves caused by psychological and psychological effects.

Organic erectile dysfunction is classified into neurogenic, vascular and endocrine erectile dysfunction according to its cause. The vascular and neurogenic erectile dysfunction is caused by hyperlipidemia, diabetes, hypertension, smoking, systemic cardiovascular disease, spinal cord injury, penile nerve damage, etc., resulting in damage to the corpus cavernosum, vascular endothelial cells, and penile nerve cells. It is a disorder caused by inadequate secretion of relaxing substances such as nitric oxide: NO).

Recently, research on erectile dysfunction has placed more emphasis on organic causes, and oral PDE-5 (phosphodiesterase-5) inhibitors, including viagra (component name: sildenafil), are mainly used worldwide as their treatment. These oral drugs are known to have the therapeutic effect of erectile dysfunction by increasing the concentration of cGMP by inhibition of PDE-5, which is specifically distributed in the corpus cavernosum, thereby increasing blood flow in the corpus cavernosum. However, drugs of the PDE-5 inhibitor family such as Viagra have been reported to have various side effects such as headache, hot flashes, indigestion, and heart attack, as well as to regulate transient protein expression and related factors at the molecular level. It cannot be called a fundamental treatment. Moreover, in the case of erectile dysfunction due to diabetes or penile nerve damage, there is a disadvantage in that the treatment effect does not appear well, and even if there is an effect, the effect does not last for a long time. Accordingly, there is a need for a treatment for erectile dysfunction that fundamentally treats the abnormal cavernous structure in the penis with erectile dysfunction, and also has a long-lasting efficacy.

On the other hand, ischemia refers to a state of reduced blood flow to body organs, tissues, or parts, and ultimately leads to irreversible damage, that is, necrosis of cells and tissues. In particular, the heart, kidney, and brain are body organs most sensitive to lack of blood flow.For example, when ischemia occurs on the brain tissue due to a stroke or head injury, a series of processes called ischemic cascades are triggered, causing the brain tissue to become damaged. Permanently damaged. Ischemia can be caused by a number of reasons, for example, abnormal fast beating of the heart, hypotension, pressure of blood vessels from outside the blood vessels (e.g., pressure of blood vessels due to cancer outside the blood vessels), blood clots, embolism, and transients. Ischemic symptoms can be caused by transient ischemic attacks, transient heart attacks, accidental cerebrovascular damage, cerebral blood clots, cerebral infarction, arteriovenous malformations, and peripheral arterial obstruction.

The ischemic disease is a disease that exhibits ischemic symptoms as described above, and includes cerebral ischemia, heart ischemia, retinal ischemia, critical limb ischemia, ischemic colitis, acute ischemic renal failure, and myocardial hypertrophy, depending on the body organs involved. I can. Specifically, cardiac ischemia that occurs when the blood supply to the heart muscle is insufficient can cause chest pain such as angina, cardiac failure, and cerebral ischemia caused by insufficient blood flow to the brain is acute ischemic stroke or It can cause vascular dementia due to chronic brain ischemia. Ischemia of the large intestine or small intestine may cause ischemic colitis or mesenteric ischemia, and if the blood supply to the skin decreases, spotty skin discoloration may occur. In addition, due to poor blood vessels, blood flow to the ends of the body is insufficient, which can cause severe limb ischemia, which requires limb amputation, and amyotrophic lateral sclerosis, diabetic neuropathy. (diabetic neuropathy), may include diabetic vascular heart disease. For the treatment of ischemic diseases as described above, a method of using angiogenesis has been studied to solve the state of reduced blood flow to body organs, tissues or regions.

On the other hand, neuropathy is a disease caused by structural or functional abnormalities in the nervous system. The nervous system is divided into the central nervous system, which is distributed in the brain and spinal cord and is involved in the regulation of its functions, and the peripheral nervous system, which is distributed in almost all organs in the body except for the brain and spinal cord. The peripheral nervous system is again classified into the motor nervous system, the sensory nervous system, and the autonomic nervous system. Neurites protrude from the brain and spinal cord and go to the trunk, arms, and legs, which are called the peripheral nerves. The peripheral nerves transmit sensations felt in the hands, feet, and auditory organs to the central nerves (brain and spinal cord) and transmit commands from the central nerves to the muscles.

The peripheral nerve is damaged by various causes, and this case is collectively referred to as peripheral neuropathy. A case in which only one peripheral nerve is damaged is called mono neuropathy, and a case in which several peripheral nerves are damaged to a similar degree is called multiple neuropathy. There are many causes of neuropathy, but broadly divided into metabolic diseases (diabetes, kidney failure, hypothyroidism), drug (anticancer drugs, tuberculosis drugs) or toxic substance addiction (lead, organic solvent), nutritional deficiencies (vitamin deficiency, alcoholism). , Connective tissue disease (rheumatoid arthritis, systemic lupus erythematosus, etc.), inflammatory disease (Guilang-Barre syndrome), and hereditary neuropathy. In addition, it is also associated with cancer.

On the other hand, LRG1 is a serum glycoprotein having eight leucine-rich repeats (LRR), and in the case of a protein having an LRR motif, it is known that it is involved in various intracellular functions such as signaling and cell adhesion. Unlike other LRR family proteins that have various functions in cells, the function of LRG1 glycoprotein is hardly known, and its expression is only reported to increase during the differentiation process of various inflammatory diseases and granulocytes. Recently, it has been reported that LRG1 glycoprotein acts on the TGF-mediated signaling pathway in mouse retinal blood vessels to induce angiogenesis through proliferation and migration of vascular endothelial cells (X Wang et al., Nature, 499 (7458)). , pp306-311, 2013). However, the relationship between LRG1 glycoprotein and penile erection and its effect on preventing or treating erectile dysfunction is not known, and there is no research on this.

Accordingly, the present inventors conducted research to develop a new protein drug, and as a result, the LRG1-Fc fusion protein fused with the LRG1 glycoprotein and the Fc domain induces the regeneration of the corpus cavernosum endothelial cells and nerve cells, thereby increasing penile erection, and excellent It was confirmed that the effect of regenerating blood vessels and nerves was confirmed, and the present invention was completed.

An object of the present invention is to provide an LRG1-Fc fusion protein in which an LRG1 (Leucine-Rich alpha-2-Glycoprotein 1) glycoprotein and an Fc domain are fused.

Another object of the present invention is to provide a composition for preventing or treating erectile dysfunction, ischemic disease, or peripheral neurological disease, including LRG1 glycoprotein or LRG1-Fc fusion protein.

In order to achieve the above object, the present invention provides an LRG1-Fc fusion protein in which an LRG1 (Leucine-Rich alpha-2-Glycoprotein 1) glycoprotein and an Fc domain are fused.

In addition, the present invention provides a polynucleotide encoding the LRG1-Fc fusion protein.

In addition, the present invention provides a vector comprising the polynucleotide.

In addition, the present invention provides a cell transformed with the vector.

In addition, the present invention provides a pharmaceutical composition for preventing or treating erectile dysfunction comprising the LRG1 glycoprotein or the LRG1-Fc fusion protein.

In addition, the present invention provides a food composition for preventing or improving erectile dysfunction comprising the LRG1 glycoprotein or the LRG1-Fc fusion protein.

In addition, the present invention provides a quasi-drug composition for preventing or improving erectile dysfunction comprising the LRG1 glycoprotein or the LRG1-Fc fusion protein.

In addition, the present invention provides a pharmaceutical composition for preventing or treating ischemic diseases comprising the LRG1 glycoprotein or the LRG1-Fc fusion protein.

In addition, the present invention provides a food composition for preventing or improving ischemic diseases comprising the LRG1 glycoprotein or the LRG1-Fc fusion protein.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of peripheral neurological diseases comprising the LRG1 glycoprotein or the LRG1-Fc fusion protein.

In addition, the present invention provides a food composition for preventing or improving peripheral neurological diseases comprising the LRG1 glycoprotein or the LRG1-Fc fusion protein.

The LRG1 glycoprotein or LRG1-Fc fusion protein according to the present invention has an excellent erectile dysfunction improvement effect, such as inducing regeneration of cavernous endothelial cells and nerve cells of the penile by increasing the level of endothelial cells and nerve cell-specific proteins, and increasing penile erection. Having a bar, it can be usefully used in the prevention or treatment of erectile dysfunction. In addition, the LRG1 glycoprotein or LRG1-Fc fusion protein according to the present invention has an effect of regenerating blood vessels and nerves, and thus can be usefully used in the prevention or treatment of ischemic diseases or peripheral neurological diseases.

1 is a diagram showing the results of confirming the isolated LRG1-Fc fusion protein through SDS-PAGE after expressing the recombinant vector in insect cells.
2 is a diagram showing the results of confirming the isolated LRG1-Fc fusion protein through SDS-PAGE after expressing the recombinant vector in mammalian cells.
3 is a schematic diagram of an LRG1-Fc fusion protein in which a human-derived LRG1 glycoprotein is linked to an Fc domain of human IgG1 by a linker (signal peptide (1-28)-human LRG1 (29-340)-linker (AAA)) (341 -343)-Fc domain (344-570, human IgG1)).
Figure 4 is a schematic diagram of an LRG1-Fc fusion protein in which a mouse-derived LRG1 glycoprotein is linked to an Fc domain of human IgG1 by a linker (signal peptide (1-28)-mouse LRG1 (29-338)-linker (AAA)) (339 -341)-Fc domain (342-568, human IgG1)).
Figure 5 is a diagram showing the results of measuring the intracavernous body pressure (erection power) according to the electrical stimulation of the penile nerve by administration of the human LRG1-Fc fusion protein in the diabetic erectile dysfunction mouse model.
6 is a diagram showing the results of measuring the intracavernous body pressure (erection power) according to the electrical stimulation of the penile nerve by administration of the human LRG1-Fc fusion protein in a mouse model of penile nerve injury and erectile dysfunction.
7 is a vascular endothelial cell-specific protein, platelet-endothelial cell adhesion molecule-1 (PECAM-1: Platelet/Endothelial Cell Adhesion Molucule-) by administration of a human LRG1-Fc fusion protein in the corpus cavernosum tissue of a diabetic erectile dysfunction mouse model. 1) and the expression level of BrdU (5-bromo-2-deoxyuridine), a proliferative cell marker, was analyzed with a confocal microscope.
8 shows the results of analyzing the expression level of neuronal nitric oxide synthase (nNOS) by administration of a human LRG1-Fc fusion protein in the dorsal nerve of a diabetic erectile dysfunction mouse model penis tissue with a confocal microscope. It is a degree.
9 is a result of analyzing the expression level of neuronal nitric oxide synthase (nNOS) by administration of a human LRG1-Fc fusion protein in the corpus cavernosum of a diabetic erectile dysfunction mouse model penile tissue with a confocal microscope. It is a diagram shown.
10 is a diagram showing the results of analyzing the expression levels of various neuronal regeneration specific proteins and their receptors by administration of human LRG1-Fc fusion protein in the corpus cavernosum of a diabetic erectile dysfunction mouse model penile tissue by Western blot.
11 is a diagram showing the results of observing the degree of tube formation according to the treatment of human LRG1-Fc fusion protein in HUVECs induced ischemia with hyperglycemia and mouse cavernous endothelial cells (MCECs).
12 is a diagram showing the results of microscopic angiogenesis according to the treatment of human LRG1-Fc fusion protein in an aortic ring induced ischemia due to hyperglycemia.
13 shows a peripheral neurological disease model by culturing a major pelvic ganglion (MPG) of a normal mouse under hyperglycemic conditions, and then neurofilament, which is a neuronal cell-specific protein treated with human LRG1-Fc fusion protein. ) And beta-III tubulin expression levels were analyzed with a confocal microscope.
14 shows a peripheral neurological disease model by culturing the dorsal root ganglion (DRG) of a normal mouse under hyperglycemic conditions, and then the beta-III tubulin, a neuronal cell-specific protein treated with human LRG1-Fc fusion protein. It is a diagram showing the result of analyzing the expression level with a confocal microscope.

Hereinafter, the present invention will be described in detail.

The present invention provides an LRG1-Fc fusion protein in which an LRG1 (Leucine-Rich alpha-2-Glycoprotein 1) glycoprotein and an Fc domain are fused.

In the present invention, “LRG1 (Leucine-Rich alpha-2-Glycoprotein 1)” refers to a serum glycoprotein having 8 leucine-rich repeats (LRR), and is derived from various mammals such as humans and mice. It includes all of the LRG1 glycoprotein, but is not limited thereto. The LRG1 glycoprotein according to the present invention is preferably derived from human or mouse and may consist of an amino acid sequence represented by, for example, SEQ ID NO: 1 or 2, and includes a functional equivalent of the protein.

The "functional equivalent" refers to at least 70% or more, preferably 80% or more, more preferably 90% or more, and more with the amino acid sequence represented by SEQ ID NO: 1 or 2 as a result of addition, substitution or deletion of amino acids. More preferably, it refers to a protein having a sequence homology of 95% or more and exhibiting substantially the same physiological activity as a protein consisting of an amino acid sequence represented by SEQ ID NO: 1 or 2.

The LRG1 glycoprotein of the present invention includes not only a protein having its native amino acid sequence, but also an amino acid sequence variant thereof. The variant of the LRG1 glycoprotein refers to a protein having a sequence different from the natural amino acid sequence of the LRG1 glycoprotein and one or more amino acid residues by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchanges in proteins and peptides that do not totally alter the activity of the molecule are known in the art. The LRG1 glycoprotein or a variant thereof may be extracted from nature or synthesized (Merrifleld, J. Amer. chem. Soc. 85:2149-2156, 1963) or prepared by a gene recombination method based on a DNA sequence (Sambrook). et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd ed., 1989).

The LRG1 glycoprotein of the present invention may be encoded with a nucleotide sequence represented by SEQ ID NO: 4 or 5, and variants capable of functioning functionally equivalent to the nucleotide are included within the scope of the present invention.

The term "functionally equivalent" means at least 70% or more, preferably 80% or more, more preferably 90% or more, even more than the nucleotide sequence represented by SEQ ID NO: 4 or 5 as a result of the addition, substitution or deletion of a base. Preferably, it means a nucleotide sequence capable of encoding a protein having a physiological activity substantially identical to that of a protein encoded by the nucleotide sequence represented by SEQ ID NO: 4 or 5, which has 95% or more sequence homology. For example, although some nucleotide sequences have been modified by deletion, substitution, or insertion, variants capable of functioning functionally the same as the nucleic acid molecule encoding the LRG1 glycoprotein are identified. It is a concept that includes.

In the present invention, "Fc domain" refers to the Fc domain of Ig (immunoglobulin, immunoglobulin). The Fc domain of Ig refers to the heavy and light chain variable regions of Ig, heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) excluding heavy chain constant region 1 (CH1) and light chain constant region (CL1), and , A hinge portion may be included in the heavy chain constant region. As long as the Fc domain of the Ig has substantially the same or improved effect with the native type, some or all of the heavy chain constant region 1 (CH1) and/or light chain constant region 1 (CL1) except only the heavy and light chain variable regions of Ig It may be an expanded Fc region including. In addition, it may be a region from which some considerably long amino acid sequences corresponding to CH2 and/or CH3 have been removed. In addition, in the Fc domain of the Ig, a site capable of forming a disulfide bond may be removed, some amino acids at the N-terminus in a native Fc may be removed, or a methionine residue may be added to the N-terminus of a native Fc. Various types of derivatives are included.

In the present invention, Ig includes IgA, IgD, IgE, IgG or IgM, and is preferably IgG. The IgG may be derived from humans or mammals such as cows, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, and the like, and is preferably derived from humans. The IgG includes IgG1, IgG2, IgG3 or IgG4, and is preferably IgG1.

In one embodiment according to the present invention, the Fc domain of human IgG1 consisting of the amino acid sequence represented by SEQ ID NO: 3 was used. The Fc domain of the human IgG1 may be encoded by the nucleotide sequence represented by SEQ ID NO: 6, and variants capable of functioning functionally equivalent to the nucleotide are included within the scope of the present invention.

The fusion protein of the present invention may have a form in which the glycoprotein of LRG1 (Leucine-Rich alpha-2-Glycoprotein 1) and the Fc domain are linked by a linker.

In the present invention, "linker" is basically two different fusion partners (for example, biological polymers, etc.) hydrogen bonding, electrostatic interaction, van der Waals force, disulfide bond, salt bridge, hydrophobic It refers to a linker that can be linked using an interaction, a covalent bond, or the like, and is preferably a peptide linker.

The peptide linker is a peptide having an arbitrary amino acid sequence, does not have a functional group, and a small amino acid is suitable, and preferably a peptide linker consisting of one or more amino acids selected from the group consisting of alanine, glycine, and combinations thereof. have. The peptide linker does not interfere with the ability to bind to the LRG1 glycoprotein and the Fc domain, and may consist of a number of amino acids that can give flexibility so as to maintain proper culture, for example, 1 to dozens of amino acids. And, preferably, it may consist of 1 to 10 amino acids, but is not limited thereto.

The LRG1-Fc fusion protein according to the present invention may consist of, for example, an amino acid sequence represented by SEQ ID NO: 7 or 8, and includes a functional equivalent of the protein.

The Fc domain according to the present invention binds to FcRN (neonatal Fc-receptor) on the surface of vascular endothelial cells, which binds to FcRN at low pH and separates at neutral pH. Due to this characteristic, the fusion protein containing the Fc domain has the characteristic that endothelial cells are endothelial cells, exocytosis again, and discharged into the blood, thereby remarkably increasing the half-life of the fusion protein in the blood. In addition, the fusion protein containing the Fc domain has the effect of improving water solubility and safety by fusion with the Fc domain, and it is easy to purify and separate from the viewpoint of drug production to save production cost. It has an advantage compared to using the bay.

In addition, the present invention provides a polynucleotide encoding the LRG1-Fc fusion protein.

The polynucleotide is included within the scope of the present invention without limitation as long as it encodes the LRG1-Fc fusion protein of the present invention through transcription and translation processes.

The polynucleotide of the present invention may additionally include a sequence encoding a signal peptide for secretion of an expressed protein. It is preferable that the signal peptide sequence is cleaved and removed after the protein expression and purification process.

The LRG1-Fc fusion protein according to the present invention may consist of, for example, a nucleotide sequence represented by SEQ ID NO: 9 or 10, and variants capable of functioning functionally equivalent to the polynucleotide are included within the scope of the present invention. .

In addition, the present invention provides a vector comprising a polynucleotide.

The vector of the present invention is preferably an expression vector, and the expression vector is a recombinant vector capable of expressing a protein of interest in a host cell of interest, and a gene construct comprising an essential regulatory element operably linked so that the gene insert is expressed. it means. The expression vector includes expression control elements such as a start codon, a stop codon, a promoter, and an operator, and the start and stop codons are generally considered to be part of the nucleotide sequence encoding the polypeptide, and when the gene construct is administered, the individual It must exhibit an action in and must be in frame with the coding sequence. The promoter of the vector can be constitutive or inducible.

In order to facilitate detection of the fusion protein, the expression vector may additionally include a protein tag that can be removed by optionally using an endopeptiase.

In the present invention, "tag" refers to a molecule that exhibits quantifiable activity or properties, and refers to a chemical fluorescent substance such as fluorescein, a fluorescent protein (GFP), or a polypeptide fluorescent substance such as a related protein. It may be a fluorescent molecule including; It may be an epitope tag such as a Myc tag, a Flag tag, a histidine tag, a leucine tag, an IgG tag, or a strapavidin tag. In particular, when using an epitope tag, preferably a peptide tag consisting of 6 or more amino acid residues, more preferably 8 to 50 amino acid residues may be used.

Vectors that can be used in the present invention include plasmids often used in the art (pUC18, pBAD, pIDTSAMRT-AMP, etc.), plasmids derived from E. coli (pYG601BR322, pBR325, pUC118, pUC119, etc.), plasmids derived from Bacillus subtilis (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vectors (retrovirus, adenovirus, etc.) ), vacinia virus, etc.), insect virus vectors (baculovirus, etc.).

The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), This document is incorporated herein by reference.

In addition, the present invention provides a cell transformed with the vector.

The transformation may be performed by various methods, and as long as it can produce the fusion protein of the present invention, it is not particularly limited thereto, but a reducing material called dimethyl sulfoxide (DMSO) is used in the _{CaCl 2} precipitation method and the CaCl _{2 precipitation method.} Hanahan method, electroporation method, calcium phosphate precipitation method, protoplasm fusion method, stirring method using silicon carbide fiber, Agrobacteria mediated transformation method, transformation method using PEG, dextran sulfate, Lipofectamine and drying/inhibition mediated transformation methods and the like can be used.

The cells are not particularly limited as long as they can produce the fusion protein of the present invention, but include bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium; Yeast cells such as Saccharomyces cerevisiae and Schizocaromyces pombe; Fungal cells such as Pichia pastoris; Insect cells such as Drozophila and Spodoptera Sf9 cells; Animal cells such as CHO, COS, NSO, 293, and Bow melanoma cells; Or it could be a plant cell.

The LRG1 glycoprotein or LRG1-Fc fusion protein according to the present invention has an excellent erectile dysfunction improvement effect, such as inducing regeneration of cavernous endothelial cells and nerve cells of the penile by increasing the level of endothelial cells and nerve cell-specific proteins, and increasing penile erection. Have. In addition, the LRG1 glycoprotein or LRG1-Fc fusion protein according to the present invention has vascular and nerve regeneration effects. Accordingly, the LRG1 glycoprotein or LRG1-Fc fusion protein of the present invention can be usefully used in the prevention or treatment of erectile dysfunction, ischemic disease or peripheral neurological disease.

Accordingly, the present invention provides a composition for preventing or treating erectile dysfunction comprising LRG1 glycoprotein as an active ingredient.

In addition, the present invention provides a composition for preventing or treating ischemic diseases comprising the LRG1 glycoprotein or the LRG1-Fc fusion protein.

In addition, the present invention provides a composition for preventing or treating peripheral neurological diseases comprising the LRG1 glycoprotein or the LRG1-Fc fusion protein.

The composition includes a pharmaceutical composition, a quasi-drug composition, or a food composition.

In the present invention, the term "prevention" is used to block the symptoms of erectile dysfunction, ischemic disease or peripheral neurological disease using a composition comprising the LRG1 glycoprotein or LRG1-Fc fusion protein of the present invention, or erectile dysfunction, ischemic disease or peripheral It refers to any action that suppresses or delays the symptoms of neurological disease.

In the present invention, the term "treatment" refers to any action that improves or benefits symptoms of erectile dysfunction, ischemic disease, or peripheral neurological disease by using a composition containing the LRG1 glycoprotein or LRG1-Fc fusion protein of the present invention.

In the present invention, the term "erectile dysfunction" means a state in which an erection is not sufficiently maintained or maintained for sexual life, and is generally defined as erectile dysfunction when such a state persists for 3 months or more. The erectile dysfunction can be broadly divided into psychogenic erectile dysfunction due to psychological factors and organic erectile dysfunction due to physical disorders, and the LRG1 glycoprotein or LRG1-Fc fusion protein of the present invention ultimately improves the function of the penis. To treat both psychogenic and organic erectile dysfunction. The organic erectile dysfunction may be due to diseases such as diabetes, penile tissue damage, coronary artery disease, nephropathy, neuropathy, high blood pressure, arteriosclerosis or hyperlipidemia, but is not limited to the above examples and may affect erection. Any disease that is present is included without limitation.

In the present invention, the term "cavernous body" is an erectile tissue that forms the main body of the penis or clitoris of a mammal, and the periphery is stacked with a membrane of thick and sturdy connective tissue containing elastic fibers. It is fulfilling. Men's penis has two cavernous bodies on the left and right and one urethral cavernous body below it, and females have a clitoral cavernous body and a urethral cavernous body having a structure similar to that of the penile cavernous body. When the cavernous body is filled with venous blood, the penis becomes erect. The corpus cavernosum and the blood vessels of the penis are normally in a contracted state by the adrenaline sympathetic nerve, and after an erectile response by relaxation, they return to the contracted state again by stimulation of the adrenergic sympathetic nervous system. In this way, the erectile tissues are usually kept in a contracted state, but during sexual arousal, the erectile tissue is relaxed, the erection begins, and the erection is initiated by signals sent from the brain center, stimulation of the nervous system, and various neurotransmitters and hormones in the body and erectile tissue. Is maintained. After the erectile reaction is over, the erectile tissue is maintained in a contracted state again.

In the present invention, the term "ischemic disease" refers to a disease indicating a state of reduced blood flow to a body organ, tissue or part, and depending on the body organ or cause involved, myocardial infarction, cerebral infarction, ischemic acute renal failure, ischemic acute liver failure , Diabetic foot ulcer, diabetic nephropathy, ischemic colitis, myocardial hypertrophy, ischemic disease or organ tissue damage caused by side effects of surgery, but is not limited thereto. Ischemic diseases caused by side effects of the surgical operation include, but are not limited to, ischemic heart failure, ischemic renal failure, ischemic liver failure, or ischemic stroke. The organ tissue damage includes organ surgery or transplantation accompanied by reperfusion after ischemia, or due to traumatic amputated limb rejoint. The organs include, but are not limited to, kidney, liver, pancreas, lungs, or heart.

In the present invention, the term "peripheral neuropathy" refers to a disease caused by damage or death of a peripheral nerve, and includes peripheral nerve damage, diabetic neuropathy (diabetic peripheral neuropathy), and the like, but is not limited thereto. Does not. The diabetic neuropathy is one of the complications of diabetes and is caused by abnormalities in the metabolism of nerve cells or disorders of nutrient blood vessels associated with hyperglycemia. Symmetrical peripheral polyneuropathy, diabetic muscle atrophy, mononeuropathy neuropathy, autonomic neuropathy. , Painless myocardial ischemia, movement disorders, and other symptoms.

When the composition of the present invention is formulated into a pharmaceutical composition for the purpose of preventing or treating erectile dysfunction, ischemic disease or peripheral neurological disease, it may further contain an appropriate carrier, excipient, and diluent commonly used in the manufacture of pharmaceutical compositions. I can. In addition, it can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories, and sterile injectable solutions according to a conventional method. Suitable formulations known in the art are preferably those disclosed in Remington's Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose , Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. When the composition is formulated, it is prepared using a diluent or excipient, such as a filler, extender, binder, wetting agent, disintegrant, or surfactant, which is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient in the composition such as starch, calcium carbonate, sucrose, lactose, It is prepared by mixing gelatin or the like. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.

In the present invention, the term "administering" means providing a given composition of the present invention to an individual in any suitable manner.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the patient, the severity of the disease, the form of the drug, the route and duration of administration, but may be appropriately selected by those skilled in the art. Administration of the composition may be administered once a day or may be divided several times.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be expected and may be administered, for example, by oral, rectal, intravenous, intramuscular, subcutaneous, or intracerebrovascular injection. However, when administered orally, since the protein is digested, the oral composition is preferably coated with an active agent or formulated to be protected from degradation in the stomach. The composition of the present invention may be preferably administered in the form of an injection.

The composition of the present invention may further contain at least one known active ingredient having an effect of preventing or treating erectile dysfunction, ischemic disease, or peripheral neurological disease together with LRG1 glycoprotein.

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

The composition of the present invention may be added to a quasi-drug composition for the purpose of preventing or improving erectile dysfunction, ischemic disease or peripheral neurological disease.

When the composition of the present invention is used as a quasi-drug composition, the active ingredient may be added as it is or used with other quasi-drug ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment).

In addition, the composition of the present invention may be added to health functional foods for the purpose of preventing or improving erectile dysfunction, ischemic diseases or peripheral neurological diseases.

In the present invention, the term'health functional food' refers to a food that has a biological control function such as prevention and improvement of diseases, biological defense, immunity, recovery after illness, and aging suppression, and should be harmless to the human body when taken for a long time.

The health functional food may contain food additives acceptable for food, and may further include suitable carriers, excipients, and diluents commonly used in the manufacture of health functional foods.

When the composition of the present invention is used as a food additive, the active ingredient may be added as it is or may be used with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for health control purposes, it may be less than the above range, and there is no problem in terms of safety, so the active ingredient may be used in an amount greater than the above range.

The composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonated beverages It may contain a carbonation agent, etc. used for. Although the proportion of these additives is not very important, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, preferred examples and experimental examples are presented to aid in understanding of the present invention. However, the following Examples and Experimental Examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the Examples and Experimental Examples.

Example One. LRG1 Preparation of glycoprotein

The LRG1 glycoprotein was synthesized and purified based on the amino acid sequence information of a known human-derived LRG1 glycoprotein or a mouse-derived LRG1 glycoprotein. The amino acid sequence of the human-derived LRG1 glycoprotein used in the experiment is represented by SEQ ID NO: 1, and the amino acid sequence of the mouse-derived LRG1 glycoprotein is represented by SEQ ID NO: 2. At this time, the sequence of the original signal peptide of the LRG1 glycoprotein is MSSWSRQRPKSPGGIQPHVSRTLFLLLLLAASAWG (SEQ ID NO: 13).In order to facilitate expression, the signal peptide of the expression vector, MVSYWDTGVLLCALLSCLLLTGSSSGGS (SEQ ID NO: 11) is attached to the amino acid sequence of SEQ ID NO: Was used.

Example 2. LRG1 Glycoprotein and Fc Preparation of a fusion protein containing a domain

The Fc domain binds to FcRN (neonatal Fc-receptor) on the surface of vascular endothelial cells, which binds to FcRN at low pH and separates at neutral pH. Due to this characteristic, the fusion protein containing the Fc domain has the characteristic that endothelial cells are endothelial cells, exocytosis again, and discharged into the blood, thereby remarkably increasing the half-life of the fusion protein in the blood. In addition, the fusion protein containing the Fc domain has the effect of improving water solubility and safety by fusion with the Fc domain, and it is easy to purify and separate from the viewpoint of drug production to save production cost. It has an advantage compared to using the bay.

Under the above background, in order to increase the possibility of utilizing the LRG1 glycoprotein of Example 1 as a pharmaceutical use, a fusion protein with an Fc domain was prepared, and for this purpose, the following experiment was performed.

2-1. Expression and isolation in insect cells

First, a gene containing a signal peptide, an LRG1 glycoprotein, and an Fc domain of human IgG1 was inserted into the pVL1391 vector. At this time, the LRG1 glycoprotein and the Fc domain of human IgG1 were linked with a linker (AAA). As the signal peptide, the signal peptide of the expression vector, MVSYWDTGVLLCALLSCLLLTGSSSGGS (SEQ ID NO: 11) was used. A baculovirus for producing a fusion protein of the LRG1-Fc domain was constructed using a recombinant vector and SF9 cells into which the recombinant gene encoding the fusion protein was inserted. The recombinant baculovirus was infected with Hi5 cells for protein expression. After 4 days, the fusion protein of the LRG1-Fc domain expressed in Hi5 cells was secreted into the medium, which was isolated and purified. More specifically, the culture medium of Hi5 cells containing the fusion protein of the LRG1-Fc domain was loaded onto the Protein A resin. The fusion protein of the LRG1-Fc domain bound to the resin was eluted with 0.1 M glycine buffer (pH 2.7). The eluted fraction containing the fusion protein of the LRG1-Fc domain was neutralized with 1M Tris buffer (pH 8.0) (the concentration of the final Tris pH 8.0 buffer was 100 mM). The fusion protein of the purified and separated LRG1-Fc domain was concentrated, and buffer exchange was performed with PBS using a desalting column. Finally, a fusion protein of the LRG1-Fc domain at a concentration of 2 mg/ml was used in the subsequent experiment. After eluting the fusion protein of the LRG1-Fc domain bound to the resin, it was confirmed through SDS-PAGE. This is shown in FIG. 1.

2-2. Expression and isolation in mammalian cells

First, a gene containing a signal peptide, an LRG1 glycoprotein, and an Fc domain of human IgG1 was inserted into a pcDNA3.1 vector. At this time, the LRG1 glycoprotein and the Fc domain of human IgG1 were linked with a linker (AAA). As the signal peptide, the signal peptide of the expression vector, MVSYWDTGVLLCALLSCLLLTGSSSGGS (SEQ ID NO: 11) was used. The recombinant vector into which the recombinant gene encoding the fusion protein was inserted was transfected into Expi293F cells. After 4 days, the fusion protein of the LRG1-Fc domain expressed in Expi293F cells was secreted into the medium, which was isolated and purified. More specifically, the culture medium of Expi293F cells containing the fusion protein of the LRG1-Fc domain was loaded onto the Protein A resin. The fusion protein of the LRG1-Fc domain bound to the resin was eluted with 0.1 M glycine buffer (pH 2.7). The eluted fraction containing the fusion protein of the LRG1-Fc domain was neutralized with 1M Tris buffer (pH9.0) (the concentration of the final Tris pH9.0 buffer was 100 mM). The purified and isolated LRG1-Fc domain fusion protein was exchanged with PBS through dialysis, and finally concentrated to a concentration of 2.8 mg/ml and used in subsequent experiments. The eluted LRG1-Fc domain fusion protein was confirmed through SDS-PAGE. This is shown in Figure 2.

2-3. Sequence analysis

In the same manner as in Example 2-1 or 2-2, a fusion protein containing human-derived LRG1 glycoprotein (SEQ ID NO: 1) and human IgG1 Fc domain (SEQ ID NO: 3), and mouse-derived LRG1 glycoprotein (SEQ ID NO: 2) and a fusion protein containing the Fc domain of human IgG1 (SEQ ID NO: 3) were prepared, respectively, and amino acid sequence analysis thereof was performed.

The fusion protein in which the human-derived LRG1 glycoprotein is linked to the Fc domain of human IgG1 with a linker is represented by the amino acid sequence of SEQ ID NO: 7, and the fusion protein in which the mouse-derived LRG1 glycoprotein is linked to the Fc domain of human IgG1 with a linker is SEQ ID NO: It is represented by the amino acid sequence of 8. The sequence does not contain a signal peptide consisting of 28 amino acids (MVSYWDTGVLLCALLSCLLLTGSSSGGS, SEQ ID NO: 11), and the signal peptide is located in front of the N-terminus of the LRG1 glycoprotein. A schematic diagram of the fusion protein is shown in FIGS. 3 and 4.

Experimental example One. LRG1 Erectile dysfunction treatment effect verification of glycoprotein

1-1. Diabetic Erectile Dysfunction Animal Model Design

Two-month-old male mice (C57BL/6) were administered streptozotocin to induce erectile dysfunction due to diabetes, and the experiment was divided into four groups as follows (N = 10/group). :

Group 1-normal mice;

Group 2-Induction of diabetes using streptozotocin [mouse 8 weeks old after intraperitoneal administration at a concentration of 50 mg/kg for 5 consecutive days];

Group 3-Induction of diabetes using streptozotocin [mouse 8 weeks old after intraperitoneal administration at a concentration of 50 mg/kg for 5 consecutive days] + mice administered phosphate-buffered solution (PBS) [20 μL] to the corpus cavernosum;

Group 4-Induction of diabetes using streptozotocin [mouse 8 weeks old after intraperitoneal administration for 5 consecutive days at a concentration of 50 mg/kg] + fusion protein of the human LRG1-Fc domain obtained in Example 2 was twice added to the corpus cavernosum Injection [day -3, 0; 5 μg/20 μL] one mouse.

1-2. Design of an Animal Model of Penile Nerve Damage Erectile Dysfunction

A 3-month-old male mouse (C57BL/6) was subjected to compressive damage to both penile nerves for about 2 minutes with forceps to induce erectile dysfunction due to penile nerve damage, and the experiment was divided into three groups as follows ( N = 6/group):

Group 1-sham operation mice;

Group 2-Penile nerve injury mice + mice administered phosphate-buffered solution (PBS) [20 μL] to the corpus cavernosum;

Group 3-Penile nerve injury mice + penile cavernous bodies were injected with a fusion protein of the human LRG1-Fc domain obtained in Example 2 twice [day -3, 0; 5 μg/20 μL] one mouse.

1-3. Erectile power measurement according to electrical stimulation

As in Examples 1-1 and 1-2, a fusion protein of the human LRG1-Fc domain was injected into the corpus cavernosum of an erectile dysfunction animal model, and two weeks later, nerves of the corpus cavernosum were stimulated and erectile power was measured. More specifically, in order to measure erectile power, the left epidermal part was opened in the lower abdomen of each mouse, and the corpus cavernosum nerve (penis nerve) located at the posterior and external sides of the prostate was prepared to be clearly visible. For electrical stimulation of the penile nerve, a platinum electrode was placed on the penile nerve, and electrical stimulation was applied for about 1 minute at an intensity of 5 volts and 12 hertz. When electric stimulation is applied, the penis has an erection. At this time, the pressure inside the penis during erection (intracavernosal pressure) was measured using a pressure transmitter (Biospac system, USA) connected to a computer through a catheter inserted into the corpus cavernosum. The results are shown in FIGS. 5 and 6.

In Figs. 5A and 6A, the vertical axis ICP (Intracavernous Pressure: intracavernous pressure) refers to the pressure inside the penis (intracavernous body pressure) during an erection, and is generally an index indicating erection. 5A and 6A, the horizontal axis represents the time after electric stimulation, and the 1 minute electric stimulation period is indicated by a black bar on the horizontal axis.

In addition, Figures 5B and 6B show the value obtained by dividing the maximum intracavernous body pressure (Maximal ICP) by the average systolic blood pressure (MSBP), and Figures 5C and 6C are the area of the corpus cavernosum pressure curve (Total ICP [area under the curve]) It represents the value divided by the mean systolic blood pressure (MSBP), which is a value that is checked to measure erectile power because blood pressure itself can affect the corpus cavernosum pressure.

As shown in Figure 5, the group administered with the fusion protein of the human LRG1-Fc domain (LRG1) showed a higher effect of improving erection compared to the untreated diabetic group (NT) and the PBS-administered group (PBS), and the normal control group (Control It was confirmed that the erection was restored to about 90% of ). In particular, it was confirmed that two variables related to penile erection, namely, the peak intracavernous body pressure and the area of the penile pressure curve, showed a remarkable synergistic effect after administration of LRG1 glycoprotein.

In addition, as shown in Figure 6, the group (LRG1) to which the fusion protein of the human LRG1-Fc domain was administered, as in the result of the diabetic erectile dysfunction animal model in the penile nerve damage erectile dysfunction animal model, the highest intracavernous body pressure and penile pressure. It was confirmed that the area of the curve was increased, showing remarkably improved erection compared to the Sham surgery group.

1-4. Vascular endothelial cell-specific proteins in the corpus cavernosum and the dorsal nerve of the penis Neuronal nitric oxide synthase Analysis of the effect on expression

The penis consists of cavernous tissues responsible for the function of penile erections and small blood vessels that supply nutrients to them. However, under pathological conditions such as diabetes, hyperlipidemia, nerve damage, etc., lesions occur in cavernous tissues including blood vessels and nerves, and the structure of the tissues becomes abnormal, resulting in erectile dysfunction.

Platelet-endothelial cell adhesion molecule-1 (PECAM-1), a vascular endothelial cell-specific protein in the corpus cavernosum and penile dorsal nerve of the diabetic erectile dysfunction animal model of Example 1-1, and BrdU (5), a cell proliferation marker. -Bromo-2-deoxyuridine) and neuronal nitric oxide synthase (nNOS) expression levels after immunohistochemical staining using confocal microscopy, quantitative changes in vascular endothelial cells, labeling in the nucleus of vascular endothelial cells Quantitative changes in BrdU and neuronal nitric oxide synthase were investigated.

More specifically, after separating the penile dorsal nerve and the penile cavernous body from the penile tissue of each mouse, they were fixed in 4% paraformaldehyde at 4° C. for 24 hours. The tissue was fixed using an embedding agent for freezing, and then cut to a thickness of 7 μm in a frozen section to prepare a tissue section. Next, the prepared tissue sections were mounted on a slide and fixed in 4% paraformaldehyde for about 5 minutes for analysis of the expression of PECAM-1, BrdU and nNOS. The fixed penile tissue sections were washed three times with a washing buffer (2% FBS + 0.1% Sodium Azide in PBS), and then blocked with a non-specific protein blocking buffer (5% BSA in PBS) for 1 hour. Then, after reacting with the primary antibody (anti-PECAM-1 hamster antibody, anti-BrdU rat antibody, 1:100, anti-nNOS rat antibody) at 4° C. for 16 hours, in order to remove the remaining antibody After washing three times with a washing buffer, the reaction was performed with a secondary antibody (FITC-labeled anti-hamster antibody or TRITC-labeled anti-rat antibody, 1:1000) at room temperature for 2 hours. After the reaction is over, wash twice with washing buffer to remove the remaining antibody, add DAPI (4',6-diamidino-2-phenylindole; 2 μg/ml) and react at room temperature for 5 minutes to remove the cell nucleus. Dyed. After washing twice with washing buffer again, analysis was performed using a confocal microscope. The results are shown in FIGS. 7 to 9.

As shown in Figure 7, the number of vascular endothelial cells is significantly reduced in the corpus cavernosum tissue of the non-treated diabetic group (DM) or the PBS-administered group (DM+PBS) compared to the normal mouse (Control), but the human LRG1-Fc domain It was confirmed that the expression of PECAM-1, a vascular endothelial cell-specific protein, was increased compared to the control group (DM or DM+PBS) in the corpus cavernosum tissue of the group administered with the fusion protein of (DM+LRG1).

In addition, as shown in Figs. 8 and 9, in the penile dorsal nerve and corpus cavernosum tissues of the untreated diabetic group (DM) or the PBS-administered group (DM+PBS), neuronal nitric oxide synthase (nNOS) compared to normal mice (Control). ), but the human LRG1-Fc domain fusion protein-administered group (DM+LRG1) produced neuronal nitric oxide compared to the control group (DM or DM+PBS) in the dorsal nerve and corpus cavernosum tissues of the penile. It was confirmed that the expression of the enzyme was increased.

1-5. Analysis of the effect on the expression of proteins related to nerve regeneration in the corpus cavernosum tissue

In order to investigate the effect of the fusion protein of the human LRG1-Fc domain on nerve regeneration, nerve growth factor (NGF) and neurotroph in the corpus cavernosum tissue of the diabetic erectile dysfunction animal model of Experimental Example 1-1. The expression levels of pin-3 (neurotrophin-3, NT-3), brain-derived neutrophic factor (BDNF), and TrkA protein, a receptor for nerve growth factor, were confirmed.

More specifically, the corpus cavernosum tissue isolated from each mouse was completely washed with PBS, and then lysed in a cell lysis buffer containing a protease inhibitor cocktail (RIPA buffer), and then 13,000 at 4°C. Only the supernatant was recovered by centrifugation at rpm for 15 minutes. The protein concentration in the lysate was measured using bovine serum albumin (BSA). After electrophoresis of the extracted proteins using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), the proteins in the gel were transferred to a nitrocellulose membrane, and 5% skim milk Blocked with. And nerve growth factor (NGF), neurotrophin-3 (NT-3), brain-derived neutrophic factor (BDNF), and TrkA, a receptor for nerve growth factor. It was reacted at 4° C. for 16 hours with the primary antibody against. After that, in order to remove the remaining antibody, it is washed again 3 times with washing buffer, and then reacted with a secondary antibody (HRP-labeled anti-mouse or HRP-labeled anti-rabbit antibody, 1:200) at room temperature for 3 hours. Made it. After the reaction was over, color was developed after washing again three times with a washing buffer to remove the remaining antibody. As a housekeeping protein for quantifying protein expression, β-actin was used. The results are shown in FIG. 10.

As shown in FIG. 10, in the corpus cavernosum tissue of the PBS-administered group (DM+PBS), compared to normal mice (Control), the receptors of nerve growth factor, neurotropin-3, brain-derived neurotrophic factor and nerve growth factor, TrkA Although the expression was reduced, it was confirmed that the expression of the neuronal regeneration specific protein was remarkably increased in the corpus cavernosum tissue of the group (DM+LRG1) administered with the fusion protein of the human LRG1-Fc domain.

Through the above experimental results, it was confirmed that the LRG1 glycoprotein induces the regeneration of reduced neurons due to diabetes or penile damage, thereby exhibiting an effect of restoring erectile power.

Experimental example 2. LRG1 Verification of the effect of glycoproteins on the treatment of ischemic diseases

2-1. Analysis of the effect of inducing blood vessel formation using vascular endothelial cells

The tube formation stage is a stage in which endothelial cells that migrated and proliferated during the angiogenesis process continue to divide and grow into hollow tubes to differentiate into final blood vessels, where blood enters and blood vessel formation is completed.

In order to confirm whether the LRG1 glycoprotein can be used for the treatment of not only erectile dysfunction but also various other ischemic diseases, the effect on tube formation was analyzed. For this, experiments were performed using HUVECs and Mouse Cavernous Endothelial Cells (MCECs). Each cell was exposed for 48 hours under the conditions of 5mM glucose similar to the normal blood glucose level and 30 mM High glucose corresponding to high blood glucose levels, and the fusion protein of the human LRG1-Fc domain (0.2 ㎍). /Ml or 2 μg/ml) were treated with glucose for 48 hours simultaneously. Control group was treated with glucose and PBS at the same time. Here, high-concentration glucose is one of the causes of ischemic disease and is one of the known methods for preparing an ischemic disease model in vitro.

^{More specifically, 3 × 10 5} HUVEC and mouse corpus cavernosum endothelial cells exposed to normal concentration glucose, high concentration glucose, and fusion protein of high glucose and human LRG1-Fc domain in a cell culture dish coated with Matrigel. _{As much as cells/cm2 were put, and incubated in a 37° C., 5% CO 2} incubator for 8 hours or 16 hours, respectively, for tube formation, and observed through a microscope. The results are shown in FIG. 11.

As shown in FIG. 11, HUVECs and mouse penile corpus cavernosum endothelial cells (MCECs) exposed to high glucose were significantly reduced in tube formation compared to the normal glucose-treated group. On the other hand, tube formation was remarkably increased in HUVEC and mouse corpus cavernosum endothelial cells (MCECs) exposed to the fusion protein of high glucose and human LRG1-Fc domain. This means that the LRG1 glycoprotein influences the differentiation of vascular endothelial cells or cavernous endothelial cells whose activity is inhibited due to diabetes, thereby inducing the formation of blood vessels and cavernous bodies.

2-2. Analysis of the effect of inducing angiogenesis using aortic section

The angiogenesis effect evaluation using aortic section (arotic ring angiogenesis assay) is a widely used method to evaluate the angiogenic ability of candidates for treatment.Based on these results, it is possible to objectively evaluate whether the candidates for treatment can be used in various ischemic diseases. It is an important experimental method that can be used (Baker M et al, Nature Protocol, 7: 89, 2012).

To confirm the effect of LRG1 glycoprotein on the treatment of ischemic diseases, the effect of the aortic section on angiogenesis was analyzed. To this end, an experiment was performed using aortic section of a 2-month-old male mouse (C57BL/6). Each aortic section was exposed to conditions of 5 mM glucose similar to normal blood glucose levels and 30 mM high glucose equivalent to high blood glucose levels for 3 and 6 days. The fusion protein (0.2 μg/ml) was treated with glucose for 3 or 6 days at the same time. The control group was treated with glucose and PBS at the same time.

More specifically, in a tissue culture dish coated with Matrigel, an aortic section exposed to normal concentration glucose, high concentration glucose, and fusion protein of high concentration glucose and human LRG1-Fc domain was put, and for 3 or 6 days, respectively. After incubation in a 37° C., 5% CO ₂ incubator, micro-angiogenesis was observed from the aortic section through a microscope. The results are shown in FIG. 12.

As shown in FIG. 12, it was found that the degree of microangiogenesis was significantly reduced in the aortic section (PBS) exposed to high glucose concentration compared to the normal glucose treatment group. On the other hand, it was confirmed that microangiogenesis was remarkably increased in the aortic section exposed to the fusion protein of high glucose and human LRG1-Fc domain.

Through the above experimental results, it was confirmed that the LRG1 glycoprotein has an excellent angiogenesis-inducing effect, and thus can be used not only for the treatment of erectile dysfunction but also for the treatment of various ischemic diseases.

Experimental example 3. LRG1 Verification of the effect of glycoprotein in treating peripheral neurological diseases

The penile nerve that governs the erectile tissue of the penis is derived from the major pelvic ganglion (MPG), which plays a major role in signal transmission related to the erectile signal. On the other hand, the dorsal root ganglion of the spinal cord is a central afferent nerve pathway that is widely used to evaluate the nerve regeneration or protective ability of candidates for treatment. It is an important experimental method that can objectively evaluate whether it can be used for neurological diseases (Jin GZ et al., Neuroscience Letter, 501: 10, 2011).

In order to confirm whether the LRG1 glycoprotein can be used for the treatment of not only erectile dysfunction but also various other ischemic diseases, the effect of nerve regeneration in the main pelvic ganglion and dorsal muscle ganglion was confirmed. To this end, the main pelvic ganglion and the dorsal muscle ganglion were isolated from normal mice. The extracted main pelvic ganglion and dorsal muscle ganglion were placed on a culture dish, covered with matrigel, and incubated for 15 minutes in an incubator under conditions of _{37°C and 5% CO 2 to solidify matrigel.} When the matrigel is hardened, it is treated with normal concentration glucose (5 mM glucose, normal glucose) or PBS and human LRG1-Fc domain fusion protein (0.2 μg/mL) under high glucose (30 mM glucose, high glucose), respectively. I did. Here, high glucose concentration is one of the causes of peripheral neuropathy and is one of the known methods for preparing a peripheral neuropathy model in vitro. Here, neurobasal medium containing 2% B-27 serum-free supplement and 0.5 mM GlutaMAX-I supplement was added, and then cultured for 3 days (main pelvic ganglion) or 7 days (posterior muscle ganglion). After incubation, the cells were fixed in 4% paraformaldehyde for about 15 minutes to analyze the expression of neurofibrillation and βIII-tubulin. After fixation, blocking was performed with a non-specific protein blocking buffer (5% BSA in PBS) for 1 hour. After that, the reaction was performed at 4°C for 16 hours with the primary antibody (anti-neurofilament mouse antibody, anti-βIII-tubulin chicken antibody), and then washed three times with a washing buffer to remove the remaining antibody, and then the second It was reacted with an antibody (FITC-labeled anti-mouse antibody or TRITC-labeled anti-chicken antibody, 1:200) at room temperature for 2 hours. After the reaction was over, it was washed twice with a washing buffer to remove the remaining antibody, and then analyzed using a confocal microscope. The results are shown in Figs. 13 and 14.

As shown in FIG. 13, it was confirmed that the proliferation of nerve cells was significantly reduced in the main pelvic ganglion exposed to high glucose, compared to the normal glucose-treated group. On the other hand, in the main pelvic ganglion exposed to the fusion protein of high glucose and human LRG1-Fc domain, the proliferation of nerve cells was remarkably increased.

In addition, as shown in FIG. 14, it was confirmed that the proliferation of nerve cells was significantly reduced in the dorsal muscle ganglion exposed to high glucose, compared to the normal glucose-treated group. On the other hand, the proliferation of neurons was remarkably increased in the dorsal root ganglion exposed to the fusion protein of high glucose and human LRG1-Fc domain. This means that the LRG1 glycoprotein influences the differentiation of neurons inhibited by diabetes and induces the regeneration of neurons.

Through the above experimental results, it was confirmed that the LRG1 glycoprotein inhibits nerve damage and death induced by high concentration of glucose, and thus can be used not only for the treatment of erectile dysfunction but also for the treatment of various peripheral neurological diseases.

Overall, the fusion protein in which the LRG1 glycoprotein and the Fc domain according to the present invention are fused is excellent in erectile dysfunction, such as inducing the regeneration of the corpus cavernosum endothelial cells and nerve cells and increasing the penile erection by increasing the level of endothelial cells and neuronal specific proteins It has an improvement effect. In addition, the fusion protein in which the LRG1 glycoprotein and the Fc domain according to the present invention are fused has vascular and nerve regeneration effects. Accordingly, the fusion protein in which the LRG1 glycoprotein and the Fc domain according to the present invention are fused can be used for the prevention or treatment of erectile dysfunction, ischemic diseases, and peripheral neurological diseases.

Hereinafter, examples of preparation of the pharmaceutical composition and food composition according to the present invention will be described, but this is not intended to limit the present invention, but is intended to be described in detail .

Formulation example 1. Preparation of pharmaceutical composition

1-1. Preparation of capsules

LRG1 glycoprotein 10 mg

3 mg of crystalline cellulose

14.8 mg lactose

Magnesium stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare a capsule.

1-2. Preparation of injections

LRG1 glycoprotein 10 mg

Mannitol 180 mg

2974 mg of sterile distilled water for injection

Na ₂ HPO ₄ 2H ₂ O 26 mg

It is prepared with the above ingredients per ampoule (2 ml) according to a conventional injection preparation method.

1-3. Liquid Produce

LRG1 glycoprotein 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water appropriate amount

According to the usual preparation method of the liquid formulation, add and dissolve each component in purified water, add an appropriate amount of lemon flavor, mix the above ingredients, add purified water, add purified water, adjust the total to 100 ml, and fill in a brown bottle. It is sterilized to prepare a liquid formulation.

Formulation example 2. Preparation of food composition

2-1. Manufacture of health food

*LRG1 glycoprotein 100 mg

The right amount of vitamin mixture

Vitamin A acetate 70 μg

1.0 mg of vitamin E

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

0.5 mg of vitamin B6

Vitamin B12 0.2 μg

10 mg of vitamin C

Biotin 10 μg

1.7 mg of nicotinic acid amide

50 μg folic acid

0.5 mg of calcium pantothenate

Suitable amount of inorganic mixture

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dicalcium phosphate 55 mg

90 mg of potassium citrate

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is relatively suitable for health food, but it may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. , Granules can be prepared, and used for preparing health food compositions according to conventional methods.

<110> Inha University Research and Business Foundation Korea Advanced Institute of Science and Technology <120> Composition for preventing or treating erectile dysfunction, ischemic disease or peripheral neurological disorder comprising fusion protein comprising LRG1 protein <130> 204-1 <150> KR 1020160096355 <151> 2016-07-28 <160> 13 <170> KopatentIn 2.0 <210> 1 <211> 312 <212> PRT <213> Homo sapiens <400> 1 Val Thr Leu Ser Pro Lys Asp Cys Gln Val Phe Arg Ser Asp His Gly 1 5 10 15 Ser Ser Ile Ser Cys Gln Pro Pro Ala Glu Ile Pro Gly Tyr Leu Pro 20 25 30 Ala Asp Thr Val His Leu Ala Val Glu Phe Phe Asn Leu Thr His Leu 35 40 45 Pro Ala Asn Leu Leu Gln Gly Ala Ser Lys Leu Gln Glu Leu His Leu 50 55 60 Ser Ser Asn Gly Leu Glu Ser Leu Ser Pro Glu Phe Leu Arg Pro Val 65 70 75 80 Pro Gln Leu Arg Val Leu Asp Leu Thr Arg Asn Ala Leu Thr Gly Leu 85 90 95 Pro Ser Gly Leu Phe Gln Ala Ser Ala Thr Leu Asp Thr Leu Val Leu 100 105 110 Lys Glu Asn Gln Leu Glu Val Leu Glu Val Ser Trp Leu His Gly Leu 115 120 125 Lys Ala Leu Gly His Leu Asp Leu Ser Gly Asn Arg Leu Arg Lys Leu 130 135 140 Pro Pro Gly Leu Leu Ala Asn Phe Thr Leu Leu Arg Thr Leu Asp Leu 145 150 155 160 Gly Glu Asn Gln Leu Glu Thr Leu Pro Pro Asp Leu Leu Arg Gly Pro 165 170 175 Leu Gln Leu Glu Arg Leu His Leu Glu Gly Asn Lys Leu Gln Val Leu 180 185 190 Gly Lys Asp Leu Leu Leu Pro Gln Pro Asp Leu Arg Tyr Leu Phe Leu 195 200 205 Asn Gly Asn Lys Leu Ala Arg Val Ala Ala Gly Ala Phe Gln Gly Leu 210 215 220 Arg Gln Leu Asp Met Leu Asp Leu Ser Asn Asn Ser Leu Ala Ser Val 225 230 235 240 Pro Glu Gly Leu Trp Ala Ser Leu Gly Gln Pro Asn Trp Asp Met Arg 245 250 255 Asp Gly Phe Asp Ile Ser Gly Asn Pro Trp Ile Cys Asp Gln Asn Leu 260 265 270 Ser Asp Leu Tyr Arg Trp Leu Gln Ala Gln Lys Asp Lys Met Phe Ser 275 280 285 Gln Asn Asp Thr Arg Cys Ala Gly Pro Glu Ala Val Lys Gly Gln Thr 290 295 300 Leu Leu Ala Val Ala Lys Ser Gln 305 310 <210> 2 <211> 310 <212> PRT <213> Mus musculus <400> 2 Leu Lys Glu Cys Leu Ile Leu Gln Ser Ala Glu Gly Ser Thr Val Ser 1 5 10 15 Cys His Gly Pro Thr Glu Phe Pro Ser Ser Leu Pro Ala Asp Thr Val 20 25 30 His Leu Ser Val Glu Phe Ser Asn Leu Thr Gln Leu Pro Ala Ala Ala 35 40 45 Leu Gln Gly Cys Pro Gly Leu Arg Glu Leu His Leu Ser Ser Asn Arg 50 55 60 Leu Gln Ala Leu Ser Pro Glu Leu Leu Ala Pro Val Pro Arg Leu Arg 65 70 75 80 Ala Leu Asp Leu Thr Arg Asn Ala Leu Arg Ser Leu Pro Pro Gly Leu 85 90 95 Phe Ser Thr Ser Ala Asn Leu Ser Thr Leu Val Leu Arg Glu Asn Gln 100 105 110 Leu Arg Glu Val Ser Ala Gln Trp Leu Gln Gly Leu Asp Ala Leu Gly 115 120 125 His Leu Asp Leu Ala Glu Asn Gln Leu Ser Ser Leu Pro Ser Gly Leu 130 135 140 Leu Ala Ser Leu Gly Ala Leu His Thr Leu Asp Leu Gly Tyr Asn Leu 145 150 155 160 Leu Glu Ser Leu Pro Glu Gly Leu Leu Arg Gly Pro Arg Arg Leu Gln 165 170 175 Arg Leu His Leu Glu Gly Asn Arg Leu Gln Arg Leu Glu Asp Ser Leu 180 185 190 Leu Ala Pro Gln Pro Phe Leu Arg Val Leu Phe Leu Asn Asp Asn Gln 195 200 205 Leu Val Gly Val Ala Thr Gly Ser Phe Gln Gly Leu Gln His Leu Asp 210 215 220 Met Leu Asp Leu Ser Asn Asn Ser Leu Ser Ser Thr Pro Pro Gly Leu 225 230 235 240 Trp Ala Phe Leu Gly Arg Pro Thr Arg Asp Met Gln Asp Gly Phe Asp 245 250 255 Ile Ser His Asn Pro Trp Ile Cys Asp Lys Asn Leu Ala Asp Leu Cys 260 265 270 Arg Trp Leu Val Ala Asn Arg Asn Lys Met Phe Ser Gln Asn Asp Thr 275 280 285 Arg Cys Ala Gly Pro Glu Ala Met Lys Gly Gln Arg Leu Leu Asp Val 290 295 300 Ala Glu Leu Gly Ser Leu 305 310 <210> 3 <211> 227 <212> PRT <213> Homo sapiens <400> 3 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 4 <211> 936 <212> DNA <213> Homo sapiens <400> 4 gtcaccctga gccccaaaga ctgccaggtg ttccgctcag accatggcag ctccatctcc 60 tgtcaaccac ctgccgaaat ccccggctac ctgccagccg acaccgtgca cctggccgtg 120 gaattcttca acctgaccca cctgccagcc aacctcctcc agggcgcctc taagctccaa 180 gaattgcacc tctccagcaa tgggctggaa agcctctcgc ccgaattcct gcggccagtg 240 ccgcagctga gggtgctgga tctaacccga aacgccctga ccgggctgcc ctcgggcctc 300 ttccaggcct cagccaccct ggacaccctg gtattgaaag aaaaccagct ggaggtcctg 360 gaggtctcgt ggctacacgg cctgaaagct ctggggcatc tggacctgtc tgggaaccgc 420 ctccggaaac tgccccccgg gctgctggcc aacttcaccc tcctgcgcac ccttgacctt 480 ggggagaacc agttggagac cttgccacct gacctcctga ggggtccgct gcaattagaa 540 cggctacatc tagaaggcaa caaattgcaa gtactgggaa aagatctcct cttgccgcag 600 ccggacctgc gctacctctt cctgaacggc aacaagctgg ccagggtggc agccggtgcc 660 ttccagggcc tgcggcagct ggacatgctg gacctctcca ataactcact ggccagcgtg 720 cccgaggggc tctgggcatc cctagggcag ccaaactggg acatgcggga tggcttcgac 780 atctccggca acccctggat ctgtgaccag aacctgagcg acctctatcg ttggcttcag 840 gcccaaaaag acaagatgtt ttcccagaat gacacgcgct gtgctgggcc tgaagccgtg 900 aagggccaga cgctcctggc agtggccaag tcccag 936 <210> 5 <211> 930 <212> DNA <213> Mus musculus <400> 5 ctcaaggaat gcctgatact gcagtcggct gagggtagca ccgtctcctg ccatggtccc 60 accgagtttc cgagctccct ccctgccgac actgtccatc tgtcggtgga attctccaac 120 ctgacgcagc tgcccgccgc cgccctgcaa gggtgtccgg gtctgcggga gctgcacctc 180 tcgagcaatc gcctgcaggc gctgtctccc gagttgctgg cgcccgtgcc ccggctgcgc 240 gccctggatc tgacccgcaa tgctctccgc agcctgcccc ccgggttgtt cagcacctcg 300 gccaacctga gcaccctggt cctgagggag aaccagctgc gggaggtgag cgcgcaatgg 360 cttcagggcc tggacgccct gggccacttg gacctggcgg agaaccagct gagttcgctg 420 ccctccgggc tcctcgccag cctcggcgcc ctgcacaccc tcgaccttgg gtacaacctg 480 ctggagtcgc tgcccgaggg actcctgcgg ggcccaaggc ggctgcagcg cctgcacctg 540 gaagggaacc gactgcagag gctggaggat agcctgcttg cgccccaacc gttcctgcgc 600 gtcctgttcc tgaatgacaa ccagttggtc ggggtggcga ccggctcctt ccagggccta 660 cagcacctgg atatgttgga tctgtccaat aactctctgt ccagcacgcc cccgggcctg 720 tgggcgttcc tggggaggcc gacccgcgac atgcaggacg gcttcgacat ctcccacaac 780 ccctggatct gtgacaagaa cctggcggac ctttgccgct ggctggtcgc caaccgaaac 840 aagatgttct cacagaacga cacgcgctgt gcggggcccg aggccatgaa gggtcagcgg 900 ctgctggacg tggcagagct ggggtccctg 930 <210> 6 <211> 681 <212> DNA <213> Homo sapiens <400> 6 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 420 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660 ctctccctgt ctccgggtaa a 681 <210> 7 <211> 542 <212> PRT <213> Artificial Sequence <220> <223> Human LRG1/Linker/Human IgG1 Fc domain <400> 7 Val Thr Leu Ser Pro Lys Asp Cys Gln Val Phe Arg Ser Asp His Gly 1 5 10 15 Ser Ser Ile Ser Cys Gln Pro Pro Ala Glu Ile Pro Gly Tyr Leu Pro 20 25 30 Ala Asp Thr Val His Leu Ala Val Glu Phe Phe Asn Leu Thr His Leu 35 40 45 Pro Ala Asn Leu Leu Gln Gly Ala Ser Lys Leu Gln Glu Leu His Leu 50 55 60 Ser Ser Asn Gly Leu Glu Ser Leu Ser Pro Glu Phe Leu Arg Pro Val 65 70 75 80 Pro Gln Leu Arg Val Leu Asp Leu Thr Arg Asn Ala Leu Thr Gly Leu 85 90 95 Pro Ser Gly Leu Phe Gln Ala Ser Ala Thr Leu Asp Thr Leu Val Leu 100 105 110 Lys Glu Asn Gln Leu Glu Val Leu Glu Val Ser Trp Leu His Gly Leu 115 120 125 Lys Ala Leu Gly His Leu Asp Leu Ser Gly Asn Arg Leu Arg Lys Leu 130 135 140 Pro Pro Gly Leu Leu Ala Asn Phe Thr Leu Leu Arg Thr Leu Asp Leu 145 150 155 160 Gly Glu Asn Gln Leu Glu Thr Leu Pro Pro Asp Leu Leu Arg Gly Pro 165 170 175 Leu Gln Leu Glu Arg Leu His Leu Glu Gly Asn Lys Leu Gln Val Leu 180 185 190 Gly Lys Asp Leu Leu Leu Pro Gln Pro Asp Leu Arg Tyr Leu Phe Leu 195 200 205 Asn Gly Asn Lys Leu Ala Arg Val Ala Ala Gly Ala Phe Gln Gly Leu 210 215 220 Arg Gln Leu Asp Met Leu Asp Leu Ser Asn Asn Ser Leu Ala Ser Val 225 230 235 240 Pro Glu Gly Leu Trp Ala Ser Leu Gly Gln Pro Asn Trp Asp Met Arg 245 250 255 Asp Gly Phe Asp Ile Ser Gly Asn Pro Trp Ile Cys Asp Gln Asn Leu 260 265 270 Ser Asp Leu Tyr Arg Trp Leu Gln Ala Gln Lys Asp Lys Met Phe Ser 275 280 285 Gln Asn Asp Thr Arg Cys Ala Gly Pro Glu Ala Val Lys Gly Gln Thr 290 295 300 Leu Leu Ala Val Ala Lys Ser Gln Ala Ala Ala Asp Lys Thr His Thr 305 310 315 320 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 325 330 335 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 340 345 350 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 355 360 365 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 370 375 380 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 385 390 395 400 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 405 410 415 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 420 425 430 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 435 440 445 Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 450 455 460 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 465 470 475 480 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 485 490 495 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 500 505 510 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 515 520 525 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 530 535 540 <210> 8 <211> 540 <212> PRT <213> Artificial Sequence <220> <223> Mouse LRG1/Linker/Human IgG1 Fc domain <400> 8 Leu Lys Glu Cys Leu Ile Leu Gln Ser Ala Glu Gly Ser Thr Val Ser 1 5 10 15 Cys His Gly Pro Thr Glu Phe Pro Ser Ser Leu Pro Ala Asp Thr Val 20 25 30 His Leu Ser Val Glu Phe Ser Asn Leu Thr Gln Leu Pro Ala Ala Ala 35 40 45 Leu Gln Gly Cys Pro Gly Leu Arg Glu Leu His Leu Ser Ser Asn Arg 50 55 60 Leu Gln Ala Leu Ser Pro Glu Leu Leu Ala Pro Val Pro Arg Leu Arg 65 70 75 80 Ala Leu Asp Leu Thr Arg Asn Ala Leu Arg Ser Leu Pro Pro Gly Leu 85 90 95 Phe Ser Thr Ser Ala Asn Leu Ser Thr Leu Val Leu Arg Glu Asn Gln 100 105 110 Leu Arg Glu Val Ser Ala Gln Trp Leu Gln Gly Leu Asp Ala Leu Gly 115 120 125 His Leu Asp Leu Ala Glu Asn Gln Leu Ser Ser Leu Pro Ser Gly Leu 130 135 140 Leu Ala Ser Leu Gly Ala Leu His Thr Leu Asp Leu Gly Tyr Asn Leu 145 150 155 160 Leu Glu Ser Leu Pro Glu Gly Leu Leu Arg Gly Pro Arg Arg Leu Gln 165 170 175 Arg Leu His Leu Glu Gly Asn Arg Leu Gln Arg Leu Glu Asp Ser Leu 180 185 190 Leu Ala Pro Gln Pro Phe Leu Arg Val Leu Phe Leu Asn Asp Asn Gln 195 200 205 Leu Val Gly Val Ala Thr Gly Ser Phe Gln Gly Leu Gln His Leu Asp 210 215 220 Met Leu Asp Leu Ser Asn Asn Ser Leu Ser Ser Thr Pro Pro Gly Leu 225 230 235 240 Trp Ala Phe Leu Gly Arg Pro Thr Arg Asp Met Gln Asp Gly Phe Asp 245 250 255 Ile Ser His Asn Pro Trp Ile Cys Asp Lys Asn Leu Ala Asp Leu Cys 260 265 270 Arg Trp Leu Val Ala Asn Arg Asn Lys Met Phe Ser Gln Asn Asp Thr 275 280 285 Arg Cys Ala Gly Pro Glu Ala Met Lys Gly Gln Arg Leu Leu Asp Val 290 295 300 Ala Glu Leu Gly Ser Leu Ala Ala Ala Asp Lys Thr His Thr Cys Pro 305 310 315 320 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 325 330 335 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 340 345 350 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 355 360 365 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 370 375 380 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 385 390 395 400 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 405 410 415 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 420 425 430 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 435 440 445 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 450 455 460 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 465 470 475 480 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 485 490 495 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 500 505 510 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 515 520 525 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 530 535 540 <210> 9 <211> 1629 <212> DNA <213> Artificial Sequence <220> <223> Human LRG1/Linker/Human IgG1 Fc domain <400> 9 gtcaccctga gccccaaaga ctgccaggtg ttccgctcag accatggcag ctccatctcc 60 tgtcaaccac ctgccgaaat ccccggctac ctgccagccg acaccgtgca cctggccgtg 120 gaattcttca acctgaccca cctgccagcc aacctcctcc agggcgcctc taagctccaa 180 gaattgcacc tctccagcaa tgggctggaa agcctctcgc ccgaattcct gcggccagtg 240 ccgcagctga gggtgctgga tctaacccga aacgccctga ccgggctgcc ctcgggcctc 300 ttccaggcct cagccaccct ggacaccctg gtattgaaag aaaaccagct ggaggtcctg 360 gaggtctcgt ggctacacgg cctgaaagct ctggggcatc tggacctgtc tgggaaccgc 420 ctccggaaac tgccccccgg gctgctggcc aacttcaccc tcctgcgcac ccttgacctt 480 ggggagaacc agttggagac cttgccacct gacctcctga ggggtccgct gcaattagaa 540 cggctacatc tagaaggcaa caaattgcaa gtactgggaa aagatctcct cttgccgcag 600 ccggacctgc gctacctctt cctgaacggc aacaagctgg ccagggtggc agccggtgcc 660 ttccagggcc tgcggcagct ggacatgctg gacctctcca ataactcact ggccagcgtg 720 cccgaggggc tctgggcatc cctagggcag ccaaactggg acatgcggga tggcttcgac 780 atctccggca acccctggat ctgtgaccag aacctgagcg acctctatcg ttggcttcag 840 gcccaaaaag acaagatgtt ttcccagaat gacacgcgct gtgctgggcc tgaagccgtg 900 aagggccaga cgctcctggc agtggccaag tcccaggcgg ccgccgacaa aactcacaca 960 tgcccaccgt gcccagcacc tgaactcctg gggggaccgt cagtcttcct cttcccccca 1020 aaacccaagg acaccctcat gatctcccgg acccctgagg tcacatgcgt ggtggtggac 1080 gtgagccacg aagaccctga ggtcaagttc aactggtacg tggacggcgt ggaggtgcat 1140 aatgccaaga caaagccgcg ggaggagcag tacaacagca cgtaccgtgt ggtcagcgtc 1200 ctcaccgtcc tgcaccagga ctggctgaat ggcaaggagt acaagtgcaa ggtctccaac 1260 aaagccctcc cagcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa 1320 ccacaggtgt acaccctgcc cccatcccgg gaggagatga ccaagaacca ggtcagcctg 1380 acctgcctgg tcaaaggctt ctatcccagc gacatcgccg tggagtggga gagcaatggg 1440 cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1500 ctctacagca agctcaccgt ggacaagagc aggtggcagc aggggaacgt cttctcatgc 1560 tccgtgatgc atgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 1620 ggtaaatga 1629 <210> 10 <211> 1623 <212> DNA <213> Artificial Sequence <220> <223> Mouse LRG1/Linker/Human IgG1 Fc domain <400> 10 ctcaaggaat gcctgatact gcagtcggct gagggtagca ccgtctcctg ccatggtccc 60 accgagtttc cgagctccct ccctgccgac actgtccatc tgtcggtgga attctccaac 120 ctgacgcagc tgcccgccgc cgccctgcaa gggtgtccgg gtctgcggga gctgcacctc 180 tcgagcaatc gcctgcaggc gctgtctccc gagttgctgg cgcccgtgcc ccggctgcgc 240 gccctggatc tgacccgcaa tgctctccgc agcctgcccc ccgggttgtt cagcacctcg 300 gccaacctga gcaccctggt cctgagggag aaccagctgc gggaggtgag cgcgcaatgg 360 cttcagggcc tggacgccct gggccacttg gacctggcgg agaaccagct gagttcgctg 420 ccctccgggc tcctcgccag cctcggcgcc ctgcacaccc tcgaccttgg gtacaacctg 480 ctggagtcgc tgcccgaggg actcctgcgg ggcccaaggc ggctgcagcg cctgcacctg 540 gaagggaacc gactgcagag gctggaggat agcctgcttg cgccccaacc gttcctgcgc 600 gtcctgttcc tgaatgacaa ccagttggtc ggggtggcga ccggctcctt ccagggccta 660 cagcacctgg atatgttgga tctgtccaat aactctctgt ccagcacgcc cccgggcctg 720 tgggcgttcc tggggaggcc gacccgcgac atgcaggacg gcttcgacat ctcccacaac 780 ccctggatct gtgacaagaa cctggcggac ctttgccgct ggctggtcgc caaccgaaac 840 aagatgttct cacagaacga cacgcgctgt gcggggcccg aggccatgaa gggtcagcgg 900 ctgctggacg tggcagagct ggggtccctg gcggccgccg acaaaactca cacatgccca 960 ccgtgcccag cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc 1020 aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 1080 cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 1140 aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 1200 gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc 1260 ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag 1320 gtgtacaccc tgcccccatc ccgggaggag atgaccaaga accaggtcag cctgacctgc 1380 ctggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg 1440 gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctctac 1500 agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 1560 atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa 1620 tga 1623 <210> 11 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Signal Peptide <400> 11 Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Gly Ser 20 25 <210> 12 <211> 84 <212> DNA <213> Artificial Sequence <220> <223> Signal Peptide <400> 12 atggtcagct actgggacac cggggtcctg ctgtgcgcgc tgctcagctg tctgcttctc 60 acaggatcta gttcaggtgg atcc 84 <210> 13 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> Signal peptide <400> 13 Met Ser Ser Trp Ser Arg Gln Arg Pro Lys Ser Pro Gly Gly Ile Gln 1 5 10 15 Pro His Val Ser Arg Thr Leu Phe Leu Leu Leu Leu Leu Ala Ala Ser 20 25 30 Ala Trp Gly 35

Claims (16)

A Leucine-Rich alpha-2-Glycoprotein 1 (LRG1) glycoprotein consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2;
Fc domain; LRG1-Fc fusion protein with this fused, vascular or nerve regeneration effect. The LRG1-Fc fusion protein according to claim 1, wherein the LRG1 glycoprotein is encoded by the nucleotide sequence shown in SEQ ID NO: 4 or 5. 2. The LRG1-Fc fusion protein of claim 1, wherein said Fc domain is derived from IgG (Immunoglobulin G). 4. The LRG1-Fc fusion protein according to claim 3, wherein the IgG is at least one selected from the group consisting of IgG1, IgG2, IgG3 and IgG4. 4. The LRG1-Fc fusion protein according to claim 3, wherein the Fc domain of the IgG is composed of the amino acid sequence shown in SEQ ID NO: 3. 4. The LRG1-Fc fusion protein according to claim 3, wherein the Fc domain of the IgG is encoded by the nucleotide sequence shown in SEQ ID NO: 6. 2. The LRG1-Fc fusion protein of claim 1, wherein the fusion protein comprises a linker. The LRG1-Fc fusion protein according to claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 7 or 8. 9. A polynucleotide encoding the LRG1-Fc fusion protein of any one of claims 1 to 8. 10. The polynucleotide according to claim 9, wherein the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO: 9 or 10. 12. A vector comprising the polynucleotide of claim 9. 11. A cell transformed with the vector of claim 11. 9. A pharmaceutical composition for preventing or treating an ischemic disease comprising the LRG1-Fc fusion protein according to any one of claims 1 to 8. 9. A food composition for preventing or ameliorating an ischemic disease comprising the LRG1-Fc fusion protein according to any one of claims 1 to 8. 9. A pharmaceutical composition for preventing or treating peripheral neuropathy comprising the LRG1-Fc fusion protein of any one of claims 1 to 8. 9. A food composition for preventing or ameliorating peripheral neuropathy comprising the LRG1-Fc fusion protein of any one of claims 1 to 8.

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