KR20190088798A - GV1001 as therapeutic drugs or composites for prevention and treatment of osteoporesis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for alleviating, treating or preventing bone loss. More particularly, the present invention relates to the pharmaceutical composition and a health functional food that can alleviate, treat or prevent bone loss diseases such as osteoporosis by containing telomerase peptide as an active component.

Description

[0001] The present invention relates to pharmaceutical compositions and health functional foods for alleviating, treating or preventing bone loss,

The present invention relates to a pharmaceutical composition for alleviating, treating or preventing bone loss, and more particularly, to a pharmaceutical composition comprising a telomerase peptide as an active ingredient and capable of alleviating, treating or preventing a bone loss disease such as osteoporosis, Functional foods.

Bone is an important tissue of the body's immune system that protects important organs and stores hematopoietic cells to supply blood cells. Bone formation, growth and metabolism involve bone modeling and remodeling This plays an important role. The bone formation starts from the birth and continues until the young adult years, when the skeleton matures and the growth ends, forming the maximum bone mass from the 20s to the early 30s. After about three years, bone is removed and the bone is replenished again. After this period, bone loss due to bone resorption can not be fully followed by bone formation, resulting in bone loss of about 0.3 to 0.5% per year. Especially in women, a considerable number of bone grafts Loss.

The bone is composed of four cells: osteoblast, osteoclast, lining cell, and osteocyte. At this time, osteoblasts derived from bone marrow stromal cells are differentiated cells that synthesize bone matrix, leading to osteogenesis, and osteoclast-derived osteoblasts lead to bone resorption. Balance of bone formation and bone loss is balanced by a process that is effectively controlled by the functional balance of destruction by osteoclasts of osteoblasts and bone formation by osteoblasts. (Harada and Rodan, 2003; Teitelbaum, 2000).

Osteoporosis, one of the most common bone loss diseases, causes osteoclast activity due to changes in sex hormones caused by menopause and aging, resulting in breakage of the normal bone remodeling process, especially in postmenopausal women. Other causes include hyperthyroidism, hyperparathyroidism, chronic renal failure, and long-term adrenocortical hormone administration.

In addition, bone diseases caused by osteoclast activation include arthritis, periodontal disease, and bone cancer. Currently, osteoporosis treatment is estimated to reach $ 9.3 billion in 2009, with an average annual growth rate of 13%. The global market for osteoporosis, including not only treatments but also health foods related to osteoporosis, is expected to reach US $ 29 billion by 2012. In addition, if the market for therapeutic drugs related to arthritis, periodontal diseases and bone cancers combined, the global market size for bone diseases is increasing.

Osteoporosis is naturally occurring as the age increases, but especially in women, hormone secretion, such as menopause, is also deepened. Men are also less likely to develop osteoporosis with aging than women. Osteoporosis often occurs in younger people through disease or weakening of the immune system. Therefore, the development of therapeutic agents for osteoporosis and the development of food materials are very necessary.

Currently, bisphosphonate drugs used as osteoporosis drugs are limited in their use due to the cumbersome and low efficacy of their use. The rate of absorption is as low as 1-5%, so it is difficult to take it, and there are many side effects that cause ulcers in the esophagus, gastrointestinal tract and tubules. Thus, although there are differences in the degree of the bone disease treatment drugs being developed and sold at present, most of them have side effects.

Therefore, it is very urgent to develop an excellent therapeutic agent for preventing or treating bone loss diseases for treating or preventing bone loss diseases.

The present inventors have completed the present invention by developing a pharmaceutical composition that promotes osteoblast differentiation and inhibits osteoclast differentiation as a result of efforts to develop a therapeutic agent capable of treating bone loss diseases.

Therefore, it is an object of the present invention to provide a method for preventing bone loss by using a specific peptide, which is a fragment of a telomerase peptide, by promoting osteoblast differentiation and inhibiting osteoclast differentiation, thereby preventing, , A pharmaceutical composition for treatment or prevention, and a health functional food.

The object of the present invention is not limited to the above-mentioned objects, and although not explicitly mentioned, the object of the invention which can be recognized by a person skilled in the art from the description of the detailed description of the invention .

In order to achieve the object of the present invention described above, the present invention provides a method for alleviating bone loss comprising as an active ingredient a peptide having an amino acid sequence of SEQ ID NO: 1 or a peptide having a sequence homology of 80% And to provide a pharmaceutical composition for treatment or prevention.

In a preferred embodiment, the peptide promotes osteoblast differentiation and inhibits osteoclast differentiation.

In a preferred embodiment, the peptide is a concentration-dependent increase in mRNA of BSP, collagen type I, ALP, Runx2 and Osterix, which are osteoblast differentiation markers.

In a preferred embodiment, the osteoblast differentiation marker is an osteoblast differentiation marker induced by Pin1 (peptidylprolyl cis / trans isomerase, NIMA-interacting 1).

In a preferred embodiment, the content of the peptide is 0.1 μg / mg to 1 mg / mg.

In a preferred embodiment, the bone loss is derived from any one selected from the group including osteoporosis, fracture, arthritis, periodontal disease, Paget's disease, soluble bone metastasis.

In a preferred embodiment, it further comprises an osteoblast differentiation promoting substance.

In a preferred embodiment, the osteoblast differentiation promoting substance is BMP2 or BMP4.

The present invention also provides a health functional food for alleviating or preventing bone loss comprising a peptide having an amino acid sequence of SEQ ID NO: 1 or a peptide having 80% or more of sequence homology with SEQ ID NO: 1 as an active ingredient.

In a preferred embodiment, it further comprises an osteoblast differentiation promoting substance.

In a preferred embodiment, the osteoblast differentiation promoting substance is BMP2 or BMP4.

The present invention also provides a pharmaceutical composition for alleviating, treating or preventing bone loss comprising a peptide having an amino acid sequence of SEQ ID NO: 1 or a peptide having 80% or more of sequence homology with SEQ ID NO: 1 as an active ingredient The present invention provides a method of treating osteoporosis in a mammal other than a human, which is derived from any one selected from the group consisting of osteoporosis, fracture, arthritis, periodontal disease, Paget's disease and soluble bone metastasis.

The pharmaceutical composition according to the present invention is effective for the prevention and treatment of bone loss diseases and is excellent in safety without cytotoxicity and can be very useful for alleviating bone loss, treating or preventing bone loss.

These technical advantages of the present invention are not limited to the above-mentioned technical scope, and even if not explicitly mentioned, the effect of the invention which can be recognized by a person skilled in the art from the description of the concrete contents for carrying out the invention Of course.

Figure 1 shows the results of an experiment on the effect of GV1001 on alkaline phosphatase activity.
FIGS. 2A and 2B show the results of experiments on the effect of GV1001 on osteoblast differentiation markers.
FIG. 3 shows the results of an experiment on the effect of GV1001 on the transcription factor.
FIG. 4 shows the results of experiments on the effect of GV1001 on the protein stability of Runx.
5A to 5C show the results of experiments on the effect of GV1001 on the transcriptional activity of Runx2.
FIGS. 6A and 6B show experimental results on the effect of the GV 1001 on the Wnt signal.
Fig. 7 shows the results of experiments on the effect of Pin1 on BMP2-induced osteoblast differentiation.
8A and 8B show the results of experiments on the effect of GV1001 on Pin1-induced osteoblast differentiation.
FIGS. 9A and 9B show the results of experiments on the effect of Sh-Pin1 on GV1001-induced osteoblast differentiation.
FIG. 10 is a graph showing that Pin1 restores the activity of GV1001 in Pin1 - / - MEF cells as an experimental result for confirming the activity of Sh-pin1.
FIGS. 11A and 11B show the results of experiments on the effect of Sh-RNA on GV1001-induced ALP activity and mineralization in C2C12 cells.
12A and 12B show the results of an experiment on the effect of Sh-RNA on the mRNA of the GV1001-induced osteoblast differentiation marker.
Fig. 13 shows the results of an experiment on the effect of Sh-Pin1 on the protein level of the GV1001-induced osteoblast differentiation marker.
14A is a photograph of TRAP assay at RAW 264.7 cells, and FIG. 14B is a photograph of TRAP assay at macrophages cells derived from bone marrow.
FIGS. 15A to 16D are photographs showing the results of experiments on the effect on GV1001 osteoclast differentiation.
FIG. 17 shows experimental results in which GV1001 inhibits osteoclast differentiation derived from mouse bone marrow.

While the present invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not limited to the disclosed embodiments. Also, in certain cases, there may be a term selected arbitrarily by the applicant, in which case the meaning thereof will be described in detail in the description of the corresponding invention. Accordingly, the terms used in the present invention should not be construed to be mere terms, but should be interpreted based on the ordinary meanings of the terms and contents described throughout the specification of the present invention. In particular, where the use of the terms " about ", "substantially" or " about " is used, it can be interpreted that the manufacturing and material tolerances inherent in the stated meanings are used in the numerical value or in close proximity to the numerical value .

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The abbreviated term in front of a noun is not intended to limit the quantity but to indicate that there is more than one noun item mentioned. The terms " comprising ", "having ", and" containing "are to be construed as open terms (i.e., the word " comprising "

Reference to a range of values is intended to be taken to refer to each separate value that falls within its scope individually, and unless otherwise stated, each value is individually referred to in the specification . All ranges of limits are contained within that range and are independently combinable.

All methods mentioned herein may be performed in any suitable order unless otherwise indicated or clearly contradicted by context. It is to be understood that the use of any embodiment and all of the embodiments or example language (e.g., "such as") is for the purpose of facilitating description of the invention, Is not intended to limit. No language in the specification should be construed as requiring any non-claimed component to be essential to the practice of the invention.

Hereinafter, the technical structure of the present invention will be described in detail with reference to the accompanying drawings and preferred embodiments.

However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Like reference numerals used to describe the present invention throughout the specification denote like elements.

It is a technical feature of the present invention that a specific peptide (also known as "GV1001") composed of some fragments of a telomerase peptide promotes osteoblast differentiation and inhibits osteoclast differentiation to prevent, And to provide a pharmaceutical composition for alleviating, treating or preventing bone loss containing the same as an active ingredient.

That is, as described later, GV1001 is known as an anticancer agent and is an anticancer drug candidate undergoing clinical trials.

Originally, telomerase is an enzyme that elongates telomere, a nucleotide sequence that repeatedly exists at the end of a chromosome. Telomere is known to prevent damage to the chromosomes or to other chromosomes, and the length of the telomere is slightly shortened each time the cell divides. Telomere becomes very short if there is cell division more than a certain number of times, It is known that lengthening telomeres prolongs the life of the cells. For example, in cancer cells, telomerase is secreted to prevent shortening of telomeres, so that cancer cells can continue to multiply without dying It is known.

In particular, GV1001 is a 16-mer peptide EARPALLTSRLRFIPK (SEQ ID NO: 1) which is a fragment of human telomerase enzyme, GV1001 binds to various HLA class II molecules and has HLA class I And harbors the putative eptiope. Thus, GV1001 has been shown to induce CD4 / CD8-bound T-cell responses, which ultimately are important for tumor eradication and long-term memory initiation. In clinical trials of patients with advanced pancreatic cancer and pulmonary cancer, GV1001-specific T-cell responses have been demonstrated to occur in more than 50% of patients without clinically significant toxicity [Kyte JA (2009) , Expert Opin Investing Drugs 18 (5): 687-94].

In the present invention, the above compounds were applied to alleviate bone loss, to treat or prevent bone loss, and to promote osteoblast differentiation and inhibit osteoclast differentiation.

Accordingly, the pharmaceutical composition for alleviating, treating or preventing bone loss of the present invention comprises a peptide having an amino acid sequence of SEQ ID NO: 1 (hereinafter abbreviated as "GV1001") or a peptide having a sequence homology of 80% As an active ingredient.

At this time, the peptide may include peptides having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more of sequence homology. In addition, the peptide of the present invention may comprise a peptide comprising the peptide of SEQ ID NO: 1 or a fragment thereof and at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, And may include peptides in which 7 or more amino acids have been changed. Generally, amino acid substitutions that do not alter specific activity are known in the art, the most common occurring interactions being Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu and Asp / to be.

Peptides having 80% or more sequence homology with GV1001 or GV1001 promote osteoblast differentiation and inhibit osteoclast differentiation, as will be seen in the experimental examples described below. In particular, the osteoblast differentiation markers BSP, collagen type I, ALP, Runx2 and Osterix mRNA are increased in a concentration-dependent manner. These osteoblast differentiation markers are induced by Pin1 (peptidylprolyl cis / trans isomerase, NIMA-interacting 1) will be. Pin 1 (peptidylprolyl cis / trans isomerase) is a member of the peptidyl-prolyl cis-trans isomer (PPIase) superfamily and isomerization of the cis-trans donor conformations of the peptide bonds immobilized on the proline backbone And changes its seating configuration accordingly.

Due to the activity of the peptide having 80% or more of sequence homology with GV1001 or GV1001, the pharmaceutical composition of the present invention can be effectively used for the prevention or treatment of all diseases related to bone loss, particularly osteoporosis, fracture, arthritis, periodontal disease, Paget's disease, soluble bone metastasis, etc. can be effective in reducing the bone loss. Particularly, the osteoporosis is selected from the group consisting of glucocorticoid-induced osteoporosis, thyroid glandular osteoporosis, fixed-induced osteoporosis, heparin-induced osteoporosis, immunosuppressive osteoporosis, osteoporosis due to renal failure, inflammatory osteoporosis, osteoporosis due to Cushing's syndrome and rheumatoid osteoporosis , And the pharmaceutical composition of the present invention may be useful for degenerative osteoporosis, osteoporosis induced by diabetic complications, and the like.

In addition, the pharmaceutical composition of the present invention may further comprise an osteoblast differentiation promoting substance to assist the activity of a peptide having 80% or more of sequence homology with the active ingredient GV1001 or GV1001. As the osteogenic vertical differentiation promoting substance, any known substance can be used, and in one embodiment, BMP2 or BMP4 can be used.

The pharmaceutical composition comprising a peptide having a sequence homology of 80% or more with GV1001 or GV1001 of the present invention may be formulated into a powder, granule, tablet, capsule, suspension, emulsion, syrup, Aerosol and the like, sterile injectable solution, and the like, and they can be administered by various routes including oral administration or intravenous, intraperitoneal, intraperitoneal injection, subcutaneous, rectal, topical administration and the like have.

In the present invention, "administration" means providing a predetermined substance to a patient by any suitable method, and the administration route of the pharmaceutical composition of the present invention is either oral or non-oral May be administered orally. The composition of the present invention may also be administered using any device capable of delivering an effective ingredient to a target cell. The term "patient", "subject" or "subject" includes, but is not limited to, a human, a monkey, a cow, a horse, a sheep, a pig, a chicken, a turkey, a quail, a cat, a dog, Rabbit or guinea pig, preferably a mammal, more preferably a human.

Such pharmaceutical compositions may further comprise carriers, excipients or diluents, and examples of suitable carriers, excipients or diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, But are not limited to, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, amorphous cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, And the like. In addition, the pharmaceutical composition of the present invention may further include a filler, an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin And the like. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Examples of the oral liquid preparation include suspensions, solutions, emulsions, syrups, and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, , Preservatives, and the like.

Examples of the preparation for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried agents, suppositories and the like. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. In particular, injections may contain conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives and the like.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, The sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

The pharmaceutical composition of the present invention may contain 0.1 μg / mg to 1 mg / mg of peptide having 80% or more sequence homology with GV1001 or GV1001, and may be one containing 1 μg / mg to 0.5 mg / And may be contained in an amount of 10 mu g / mg to 0.1 mg / mg.

The effective dose in the pharmaceutical composition of the present invention may vary depending on the age, sex, and body weight of the patient, and its daily dose may be, for example, 1 μg / kg / day to 10 mg / kg / May be 10 μg / kg / day to 1 mg / kg / day, more particularly 50 μg / kg / day to 100 μg / kg / day, but is not limited to the age , Health status, complications, and other factors.

The health functional food of the present invention comprises a peptide (GV1001) having an amino acid sequence of SEQ ID NO: 1 or a peptide having a sequence homology of 80% or more with SEQ ID NO: 1 to alleviate or prevent bone loss. The description of the GV1001 is the same as that described above, so the description thereof will be omitted.

There is no particular limitation on the kind of health functional food for alleviating or preventing bone loss. Examples include dairy products, confectionery, seasonings, beverages and drinks, snacks, candies, ice cream and frozen desserts, breakfast cereals, nutrition bars, snack bars, chocolate products, processed foods, cereal products and pasta, soups, sauces and dressings, Dairy products and dairy products, dairy drinks and milk drinks, soy dairy-like products, frozen foods, cooked foods and alternative foods, meat products, cheese, yogurt, bread and buns, yeast products , A cake, a cookie, and a cracker, but is not limited thereto, and includes all health foods in a conventional sense.

The health functional foods can be formulated into capsules, tablets, powders, liquid suspensions, pills, granules and the like. Usually, the health functional foods are mixed with an excipient, binder, disintegrant, It is made by granulating the granules as much as possible, and it is possible to add the flavoring agent, mating agent and so on as needed. When the health functional food is a health functional food of a beverage formulation, it may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of natural carbohydrates are monosaccharides such as glucose and fructose, polysaccharides such as disaccharides such as maltose and sucrose, dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. But are not limited to, folic acid extracts, fructooligosaccharides that aid in the absorption of calcium and calcium, acacia honey, complex gold extract as a natural preservative, and gellan gum, Suitable components may be suitably used.

Meanwhile, the bone loss treating method of the present invention comprises a peptide having an amino acid sequence of SEQ ID NO: 1 or a pharmacological agent for alleviating, treating or preventing bone loss comprising, as an active ingredient, a peptide having a sequence homology of 80% Which is effective for treating bone loss in mammals other than humans derived from any one selected from the group including osteoporosis, fracture, Paget's disease, and soluble bone metastasis.

Example

GV1001, a peptide having the sequence of SEQ ID NO: 1, was prepared according to a conventional solid phase peptide synthesis method, and its molecular weight was confirmed by LC / MS and frozen to prepare a powder form. GV1001 prepared in powder form was dissolved in PBS to a certain concentration to prepare the pharmaceutical composition of the present invention.

Experimental Examples 1 to 13 described below test the effects of GV1001 on osteoblast differentiation and show the results.

Experimental Example 1

To evaluate the effect of GV1001 on the alkaline phosphatase activity, C2C12 cell line was treated with BMP4 to induce osteoblast differentiation, treated with different concentrations of GV1001, and then incubated with alkaline phosphatase (ALP) staining, an osteoblast differentiation marker, And the results are shown in Fig.

From FIG. 1, it can be seen that as the concentration of GV1001 contained in the pharmaceutical composition increases, the ALP enzyme activity is increased.

Experimental Example 2

C2C12 cell lines were treated with BMP4 to induce osteoblast differentiation, and the effect of different concentrations of GV1001 on osteoblast differentiation markers was examined by RT-PCR and luciferase assay, and the results are shown in Figs. 2a to 2c.

As shown in FIGS. 2A and 2C, when GV1001 increased the concentration of osteoblast differentiation markers such as BSP, collagen type I and ALP in a concentration-dependent manner, and when the Luciferase vector containing BSP and ALP promoter was used, the activity of BSP and ALP promoter Of the total population.

Experimental Example 3

RT-PCR, Western blot and Luciferase assay were used to investigate the effect of Runx2 and Osterix mRNA and protein in the osteoblast differentiation-related transcription factors. The results are shown in FIG.

From FIG. 3, it can be seen that GV1001 increased protein levels of Runx2 and Osterix in a concentration-dependent manner.

Experimental Example 4

Since GV1001 increased the amount of protein of Runx2 as in Experimental Example 3, the effect on the stimulation of ALP activity by Runx2 and the effect on protein stability were tested using the ALP staining assay and the half-life measurement method using cycloheximide, 4.

From FIG. 4, it was confirmed that GV1001 increased ALP activity increased by Runx2 in a concentration-dependent manner and increased half-life.

Experimental Example 5

Since GV1001 increased the protein stability of Runx2 and increased the amount of transcription, the luciferase assay using ALP-Luc and BSP-Luc and the RT-PCR of differentiation markers were performed to determine whether the transcription activity was increased. 5c.

From FIGS. 5A to 5C, it can be seen that GV1001 increased the transcriptional activity of Runx2 and increased the mRNA levels of differentiation markers.

Experimental Example 6

After transfection of GV1001 alone or transfection of b-catenin, TCF transcription activity was measured using TOP-flash with TCF binding site WT and FOP-flash as a mutant, and the results are shown in FIGS. 6A and 6B.

6A and 6B, it was confirmed that the transcription activity was increased by GV1001.

Experimental Example 7

To elucidate the effect of ALP activity and Pin1 inhibitor juglone on the activity of ALP when inducing osteoblast differentiation by inducing osteoblast differentiation with BMP2 in C2C12 cells, C2C12 cells induced osteoblast differentiation with BMP2, and Pin1 And the amount of Juglone was increased. After 2 days from the incubation, RNA was obtained and the osteoblast differentiation markers were quantitatively changed by RT-PCR and the osteoclast differentiation-related promoter assay was performed. The results are shown in FIG.

From FIG. 7, it can be seen that the activity of ALP is increased when the concentration of Pin1 is increased by inducing osteoblast differentiation by C2C12 cells with BMP2, and that the Pin1 inhibitor juglone reduces the activity of ALP, Juglone decreased the expression of osteoblast differentiation markers in a concentration dependent manner, while increasing the expression of markers. In addition, Pin1 was found to increase the activity of the promoter in a concentration-dependent manner.

These results suggest that Pin1 promotes osteoblast differentiation and Pin1 inhibitor inhibits osteoblast differentiation.

Experimental Example 8

Pin1 increased the mRNA expression of osteoblast differentiation markers. The effect of GV1001 on the osteoblast differentiation markers was confirmed by real-time PCR, and it was observed whether GV1001 further increased Pin1-induced osteoblast differentiation. The results are shown in FIGS. 8A and 8B 8b.

8A and 8B show that GV1001 promotes ALP activity and mineralization induced by BMP4 by Pjn1, and that GV1001 further increases the expression of markers that are significantly increased by Pin1.

Experimental Example 9

The effect of GV1001 on the osteoblast differentiation marker promoter activity in the absence or presence of Sh-Pin1 was investigated to determine whether the activity decreased when knock-down of Pin1. The results are shown in FIGS. 9a and 9b.

FIGS. 9A and 9B show that GV1001 further increases osteoblast differentiation induced by Pin1. It was confirmed that Sh-Pin1 reduced GV1001 activity promoting osteoblast differentiation, and this result indicates that GV1001 is a partial To promote osteoblast differentiation through Pin1.

Experimental Example 10

In order to confirm the activity of Sh-pin1, the effect of GV1001 on the activity of ALP-luc and the effect of GV1001 on osteoblast when Pin1 was transfected was examined using Pin1 - / - MEF cells, Is shown in Fig.

Fig. 10 shows that GV1001 was not active in the absence of Pin1 and that its activity was restored when Pin1 was introduced again.

Experimental Example 11

The effect of GV1001 on ALP activity and mineralization in Pin1 knock-down state was investigated by ALP staining assay and Alizarin red staining. The results are shown in FIG.

From FIG. 11, it can be seen that when Pin 1 is knocked down, the activity of ALP and the degree of mineralization decreased by GV1001.

Experimental Example 12

The decrease in the mRNA level of osteoblast differentiation markers increased by GV1001 when Pin1 knock-down was confirmed using real-time PCR, and the results are shown in FIGS. 12A and 12B.

12A and 12B, it was found that the amount of mRNA of osteoblast markers increased by GV1001 at the time of Pin1 knock-down did not increase.

Experimental Example 13

As described above, the amount of protein of RUNX2, Osteerix differentiation marker, Col1A1, which is an osteoblast-related transcription factor, is increased by GV1001. After the Pin1 knock-down using Sh-Pin1, the quantitative change of these proteins is observed by western blot The results are shown in Fig.

From Fig. 13, it can be seen that the amount of protein increased by GV1001 by pin 1 knock-down decreases.

Experimental Examples 14 to 16 described below test the effect of GV1001 on osteoclast differentiation and show the results.

Experimental Example 14

To investigate the effect of GV1001 on osteoclast differentiation, macrophages were isolated from mouse bone marrow and osteoclast differentiation conditions were established. M-CSF (10 ng / ml) and RANKL were treated at 20, 50 and 100 ng / ml RANKL was treated at increasing concentrations by TRAP assay, an osteoclast differentiation marker, and the results are shown in FIGS. 14A and 14B.

14A and 14B, it was confirmed that the cells with multi-nulcear were increased in a concentration-dependent manner by RANKL, and the osteoclast differentiation was increased.

Experimental Example 15

To investigate the effect of GV1001 on osteoclast differentiation, macrophages were isolated from mouse bone marrow and treated with 50 ng / ml or 100 ng / ml of M-CSF (10 ng / ml) and RANKL to determine osteoclast differentiation And the results are shown in Figs. 15A to 15D and Figs. 16A to 16D, respectively.

15A to 16D, it was confirmed that osteoclast differentiation induced by M-CSF and RANKL was inhibited in a concentration-dependent manner with increasing concentration of GV1001.

Experimental Example 16

To further quantitatively measure the inhibition of osteoclast differentiation by GV1001, the TRAP assay was performed. Then, cells having three or more fused cells were counted under a microscope to observe osteoclast differentiation in a concentration-dependent manner. The results are shown in Fig. 17 .

FIG. 17 clearly shows the effect of GV1001 inhibiting osteoclast differentiation induced by M-CSF and RANKL in mouse bone marrow.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation, Various changes and modifications will be possible.

<110> Industry Foundation of Chonnam National University <120> GV1001 as therapeutic drugs or composites for prevention and          treatment of osteoporesis <130> DPP-2017-0206-KR <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 16 <212> PRT <213> Homo sapiens <400> 1 Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile Pro Lys   1 5 10 15

Claims (12)

A pharmaceutical composition for alleviating, treating or preventing bone loss comprising a peptide having an amino acid sequence of SEQ ID NO: 1 or a peptide having 80% or more of sequence homology with SEQ ID NO: 1 as an active ingredient.
The method according to claim 1,
The pharmaceutical composition for alleviating, treating or preventing bone loss, wherein the peptide promotes osteoblast differentiation and inhibits osteoclast differentiation.
The method according to claim 1,
The pharmaceutical composition for relieving, treating or preventing bone loss is characterized in that the peptide increases mRNA concentration of BSP, collagen type I, ALP, Runx2 and Osterix, which are osteoblast differentiation markers, in a concentration-dependent manner.
The method of claim 3,
Wherein the osteoblast differentiation marker is an osteoblast differentiation marker induced by Pin1 (peptidylprolyl cis / trans isomerase, NIMA-interacting 1).
3. The method of claim 2,
Wherein the content of the peptide is 0.1 占 퐂 / mg to 1 mg / mg.
The method according to claim 1,
Wherein the bone loss is derived from any one selected from the group including osteoporosis, fracture, arthritis, periodontal disease, Paget's disease, and soluble bone metastasis.
7. The method according to any one of claims 1 to 6,
A pharmaceutical composition for alleviating, treating or preventing bone loss, which further comprises an osteoblast differentiation promoting substance.
8. The method of claim 7,
Wherein the osteoblast differentiation-promoting substance is BMP2 or BMP4.
A peptide having an amino acid sequence of SEQ ID NO: 1 or a peptide having 80% or more of sequence homology with SEQ ID NO: 1 as an active ingredient.
10. The method of claim 9,
Wherein the osteoblast differentiation promoting substance is further contained.
10. The method of claim 9,
Wherein the osteoblast differentiation-promoting substance is BMP2 or BMP4.
A pharmaceutical composition for alleviating, treating or preventing bone loss comprising a peptide having an amino acid sequence of SEQ ID NO: 1 or a peptide having 80% or more of sequence homology with SEQ ID NO: 1 as an active ingredient, A method for treating bone loss in a mammal other than a human, which is derived from any one selected from the group consisting of fractures, arthritis, periodontal disease, Paget's disease, and soluble bone metastases.

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