KR102118953B1 - 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, and more specifically, a pharmaceutical composition and health that can alleviate, treat or prevent bone loss diseases such as osteoporosis by including a telomerase peptide as an active ingredient. It is about functional food.

Description

{GV1001 as therapeutic drugs or composites for prevention and treatment of osteoporesis}

The present invention relates to a pharmaceutical composition for alleviating, treating or preventing bone loss, and more specifically, a pharmaceutical composition and health that can alleviate, treat or prevent bone loss diseases such as osteoporosis by including a telomerase peptide as an active ingredient. It is about functional food.

Bone protects important organs, and is an important tissue of the body's immune system that stores hematopoietic stem cells and supplies blood cells. During bone development, growth and metabolism, bone modeling and remodeling processes This plays an important role. Bone formation begins in the early stages, and then continues until the adolescence begins when the skeleton matures and grows, forming the maximum bone mass from the 20s to the early 30s. For the next three years, the bone removal process is repeated to remove the bone and replenish it. After this period, bone loss due to bone resorption cannot fully follow bone formation, resulting in a decrease in bone mass of about 0.3 to 0.5% per year, especially in women. You suffer losses.

The bone is largely composed of four cells: osteoblasts, osteoclasts, lining cells and osteoblasts. At this time, osteoblasts derived from bone marrow stromal cells are differentiated cells that synthesize bone matrix and lead to bone formation, and osteoclasts derived from hematopoietic stem cells lead to bone resorption. The balance of bone formation and bone loss is a process that is effectively controlled by the functional balance of bone formation by osteoclasts and destruction by osteoclasts of the mineralized bone matrix. (Harada and Rodan, 2003; Teitelbaum, 2000).

Osteoporosis, one of the typical bone loss diseases, is caused by the increase in osteoclast activity due to changes in sex hormones due to menopause and aging, resulting in breakage of the normal bone remodeling process, especially in postmenopausal women. In addition, it is caused by various factors such as hyperthyroidism, hyperparathyroidism, chronic renal failure, and long-term administration of adrenal cortical hormone.

In addition, bone diseases caused by activation of osteoclasts include arthritis, periodontal disease, and bone metastasis. Currently, osteoporosis treatments are expected to reach $9.3 billion in 2009 with an average annual growth rate of 13%. The global market for osteoporosis is expected to reach $29 billion by 2012, including health food related to osteoporosis as well as therapeutics. In addition, if the combined treatment markets related to arthritis, periodontal disease, and bone metastasis are combined, the world market for bone disease can be considered to be increasing in size.

Osteoporosis appears naturally as you get older, but especially in women, it is very much worsened by hormonal abnormalities such as menopause. Males are also less likely than females, but most often develop osteoporosis with aging, and sometimes appear in younger people through disease or weakening of the immune system. Therefore, the development of a therapeutic agent for osteoporosis and the development of food materials are very necessary.

Bisposphonate-based drugs that are currently used as osteoporosis treatments are subject to many limitations due to their cumbersome and low medicinal properties. The absorption rate is low at 1-5%, making it difficult to take, and there are many side effects that cause ulcers in the esophagus, stomach, and ducts. As described above, in the case of a drug for treating bone disease, which is currently being developed and sold, there is a difference in degree, but most have side effects.

Therefore, there is an urgent need to develop an excellent therapeutic agent that prevents or treats bone loss disease to treat or prevent bone loss disease.

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

Accordingly, an object of the present invention is to alleviate bone loss using a new use that certain peptides, which are some fragments of telomerase peptides, promote osteoblast differentiation and inhibit osteoclast differentiation to prevent, alleviate and treat bone loss diseases. , To provide a pharmaceutical composition and health functional food for treatment or prevention.

The object of the present invention is not limited to the above-mentioned object, and even if not explicitly mentioned, the object of the invention that can be recognized by those skilled in the art from the description of the detailed description of the invention to be described later may naturally be included. .

In order to achieve the above-described object of the present invention, the present invention first alleviates bone loss comprising a peptide 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 as an active ingredient, It is 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 the osteoblast differentiation markers BSP, collagen type I, ALP, Runx2 and Osterix.

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 comprising osteoporosis, fracture, arthritis, periodontal disease, Paget's disease, soluble bone metastasis.

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

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

In addition, the present invention 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 sequence homology with SEQ ID NO: 1 as an active ingredient.

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

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

In addition, the present invention comprises the steps of administering 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 sequence homology with SEQ ID NO: 1 as an active ingredient. It provides a method for treating bone loss in mammals except humans derived from any one selected from the group comprising osteoporosis, fracture, arthritis, periodontal disease, Paget's disease, and soluble bone metastasis.

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

These technical effects of the present invention are not limited to the above-mentioned ranges, and even if not explicitly mentioned, the effects of the invention that can be recognized by those skilled in the art from the description of specific contents for carrying out the invention described below are also Of course it is included.

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

The terminology used in the present invention has been selected, while considering the functions in the present invention, general terms that are currently widely used are selected, but this may vary according to the intention or precedent of a person skilled in the art or the appearance of new technologies. In addition, in certain cases, some terms are arbitrarily selected by the applicant, and in this case, their meanings will be described in detail in the description of the applicable invention. Therefore, the terms used in the present invention should be interpreted based on the general meaning of the terms and the contents described throughout the specification of the present invention, not simply the names of the terms. In particular, when the terms "about", "substantially", etc. of degree are used, it can be interpreted as being used in or close to the value when manufacturing and substance tolerances unique to the stated meaning are given. .

The terms used in this specification are intended for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term in which the number is omitted before the noun is not intended to limit the quantity, but indicates that one or more of the noun articles mentioned exist. The terms “comprising”, “having,” and “containing” are to be interpreted as open terms (ie, meaning of “comprising (including but not limited to)”).

Reference to a range of values is merely a substitute for individually referring to each separate value falling within the range, and unless otherwise stated, each value is referred to herein as the same as recited in the specification individually. Applies. The limits of all ranges are included within the range and can be combined independently.

All methods mentioned herein can be performed in any suitable order unless otherwise indicated or clearly contradicted by context. The use of any one embodiment and all embodiments or exemplary languages (eg, “such as”) is for ease of description of the present invention only, unless included in the claims, and the scope of the present invention It is not intended to limit. No language in the specification should be construed as having any unclaimed component essential to the practice of the present invention.

Hereinafter, the technical configuration 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 and may be embodied in other forms. The same reference numerals used to describe the present invention throughout the specification indicate the same components.

The technical feature of the present invention is a new use that a specific peptide (known as "GV1001") composed of some fragments of the telomerase peptide promotes osteoblast differentiation and inhibits osteoclast differentiation to prevent, alleviate and treat bone loss disease. Discovery, and to develop a pharmaceutical composition for the relief, treatment or prevention of bone loss, including it as an active ingredient.

That is, as described later, GV1001 is known as an anticancer drug and is a candidate for an anticancer drug in clinical trials.

Originally, telomerase is an enzyme that extends telomere, a nucleotide sequence that is repeatedly present at the ends of chromosomes. Telomeres are known to prevent damage to the chromosome or binding to other chromosomes, and each cell divides the length of the telomer, which decreases slightly, but if the cell divides more than a certain number of times, the telomer becomes very short and the cell Is said to stop dividing and die, but it is known that prolonging telomeres prolongs the lifespan of cells.For example, cancer cells can continue to multiply without dying because cancer cells secrete telomerase and prevent telomeres from shortening. Is known.

In particular, GV1001 is a 16-length (16-mer) peptide EARPALLTSRLRFIPK (SEQ ID NO: 1), which is a fragment of the human telomerase enzyme, and GV1001 binds to various HLA class II molecules and HLA class I. The putative antigenic determinants are harbored. Thus, GV1001 has been thought to be capable of eliciting a combined T-cell response of CD4/CD8, which in turn has been shown to be important for tumor elimination and long-term memory onset. In clinical trials of patients with advanced pancreatic and lung cancer, GV1001-specific T-cell responses have been demonstrated in more than 50% of subjects without clinically significant toxicity [Kyte JA (2009) , Expert Opin Investing Drugs 18(5):687-94].

In the present invention, the above compounds were applied for the purpose of alleviating, treating or preventing bone loss, not for anti-cancer use. In particular, it was confirmed that it promotes osteoblast differentiation and inhibits osteoclast differentiation and completed this invention.

Accordingly, the pharmaceutical composition for relieving, treating or preventing bone loss of the present invention is 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% or more with the SEQ ID NO: 1 It is included as an active ingredient.

In this case, the peptide may include a peptide having a sequence homology of 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more. In addition, in the present invention, the peptide is a peptide or a fragment thereof comprising SEQ ID NO: 1 and one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, six or more amino acids, or 7 or more amino acids may include a modified peptide. In general, amino acid substitutions that do not alter specific activity are known in the art, and the most common exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser /Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly, and vice versa to be.

GV1001 or a peptide having a sequence homology of 80% or more with GV1001 promotes osteoblast differentiation and inhibits osteoclast differentiation, as can be seen in the experimental examples described below. In particular, the osteoblast differentiation markers BSP, collagen type I, ALP, Runx2 and Osterix increase the concentration-dependent mRNA, and these osteoblast differentiation markers are induced by Pin1 (peptidylprolyl cis/trans isomerase, NIMA-interacting 1). will be. Pin1 (peptidylprolyl cis/trans isomerase) is a member of the peptidyl-prolyl cis-trans isomer superfamily, isomerization of cis-trans conformations of fixed peptide bonds in the proline backbone Catalyzes and changes its conformation accordingly.

Due to the activity of GV1001 or a peptide having 80% or more sequence homology with GV1001, the pharmaceutical composition of the present invention may effectively act in 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 effectively act on lowered bone loss. In particular, osteoporosis is glucocorticoid-induced osteoporosis, hyperthyroidism osteoporosis, fixation-induced osteoporosis, heparin-induced osteoporosis, immune suppression-induced osteoporosis, osteoporosis following renal failure, inflammatory osteoporosis, osteoporosis according to Cushing's syndrome, and rheumatoid osteoporosis It can be any one selected from osteoporosis, the pharmaceutical composition of the present invention may be useful for osteoporosis caused by degenerative osteoporosis, diabetes complications, and the like.

In addition, the pharmaceutical composition of the present invention may further include an osteoblast differentiation promoter to assist the activity of a peptide having 80% or more sequence homology with the active ingredient GV1001 or GV1001. Osteoporosis differentiation promoting material may be any known material, BMP2 or BMP4 may be used as an embodiment.

Pharmaceutical compositions comprising peptides having 80% or more sequence homology with GV1001 or GV1001 of the present invention are powders, granules, tablets, capsules, suspensions, emulsions, syrups, according to a conventional method for each purpose of use, It can be formulated and used in various forms, such as oral formulations such as aerosols and injections of sterile injectable solutions, and can be administered orally or through various routes including intravenous, intraperitoneal, lymph node injection, subcutaneous, rectal, and topical administration. have.

In the present invention, "administration" means providing a predetermined substance to a patient in any suitable way, and the route of administration of the pharmaceutical composition of the present invention is oral or parenteral through all general routes as long as it can reach the target tissue. It can be administered orally. In addition, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells. Here, "patient", "object" or "subject" is not particularly limited, for example, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, mouse, Rabbit or guinea pigs, preferably mammals, more preferably humans.

The pharmaceutical composition may further include a carrier, excipient, or diluent, and examples of suitable carrier, excipient, or diluent that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil 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, a preservative, and the like.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the pharmaceutical composition, for example, starch, calcium carbonate, sucrose, lactose, gelatin The mixture is formulated by mixing. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients. As an exemplary embodiment, a liquid preparation for oral use may include suspending agents, intravenous solutions, emulsions, syrups, etc., and various excipients, such as water and liquid paraffin, which are commonly used simple diluents, such as wetting agents, sweeteners, and fragrances , Preservatives, and the like.

Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, suppositories, and the like. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. In particular, injections may include 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, "a 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 is the type of patient's disease, severity, activity of the drug, Sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-drugs and other factors well known in the medical arts. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect 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 GV1001 or a peptide having 80% or more sequence homology with GV1001 at 0.1 µg/mg to 1 mg/mg, and 1 µg/mg to 0.5 mg/mg It may be, and may be included in an amount of 10 ㎍ / ㎎ to 0.1 ㎎ / ㎎.

The effective amount in the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, and body weight, and the daily dosage thereof is, for example, specifically 1 μg/kg/day to 10 mg/kg/day, more specifically As 10 μg/kg/day to 1 mg/kg/day, more specifically 50 μg/kg/day to 100 μg/kg/day, but is not limited thereto, and the age of the subject to be administered , Health condition, complications, etc.

The health functional food of the present invention alleviates or prevents bone loss by including a peptide having an amino acid sequence of SEQ ID NO: 1 (GV1001) or a peptide having 80% or more sequence homology with SEQ ID NO: 1. The description of GV1001 is the same as that described above, so the description is omitted.

There are no particular restrictions on the types of dietary supplements for the relief or prevention of bone loss. Examples include dairy products, confectionery, seasonings, beverages and drinks, snacks, candies, ice cream and frozen desserts, breakfast cereals, nutrition bars, snack bar chocolate products, processed foods, grain products and pasta, soups, sauces and dressings, confectionery products , Oil and fat products, dairy drinks and milk beverages, soy milk and soy dairy-like products, frozen foods, cooked and alternative foods, meat products, cheese, yogurt, bread and buns, yeast products , May be any one selected from the group consisting of cakes and cookies and crackers, but is not limited thereto, and includes all healthy foods in a general sense.

Health functional foods can be formulated into capsules, tablets, powders, liquid suspensions, pills and granules, etc., and usually mixed with health functional foods or excipients, binders, disintegrants, etc. in health functional foods. It is made into granules by the method, and the particles are evened as much as possible, and a flavoring agent, mating agent, etc. can be added as necessary. When the health functional food is a health functional food of a beverage formulation, it may contain various flavoring agents or natural carbohydrates, etc., as additional components, like a normal beverage. Examples of natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetener, natural sweeteners such as taumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used. Functional ingredients such as deer antler extract, fructooligosaccharide, acacia honey, natural preservative complex golden extract, and sediment-improving thickener gellan gum, which help defecation and calcium absorption, can be added, but are not limited to health functional foods. Suitable ingredients can be used as appropriate.

On the other hand, the bone loss treatment method of the present invention is a peptide 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 as an active ingredient for the relief, treatment or prevention of bone loss Including the step of administering the composition, it is particularly effective in the treatment of bone loss in mammals except humans derived from any one selected from the group comprising osteoporosis, fractures, Paget's disease, soluble bone metastasis.

Example

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

Experimental Examples 1 to 13 to be described later experimented with the effect of GV1001 on osteoblast differentiation and showed the results.

Experimental Example 1

In order to evaluate the effect of the pharmaceutical composition containing GV1001 on alkaline phosphatase activity, BMP4 was treated in the C2C12 cell line to induce osteoblast differentiation, while the concentration of GV1001 was treated differently, and then the osteoblast differentiation marker alkaline phosphatae (ALP) staining and enzyme activity The results are measured and shown in FIG. 1.

It can be seen from FIG. 1 that the ALP enzyme activity increases as the concentration of GV1001 contained in the pharmaceutical composition increases.

Experimental Example 2

After treating BMP4 in the C2C12 cell line to induce osteoblast differentiation, the effect of GV1001 on different osteoblast differentiation markers was investigated by RT-PCR and luciferase assay, and the results are shown in FIGS. 2A to 2C.

From 2a to 2c, GV1001 increased the mRNA of BSP, collagen type I and ALP, which are osteoblast differentiation markers, in a concentration-dependent manner, and the activity of BSP and ALP promoters when examined using a Luciferase vector containing BSP and ALP promoters. It was confirmed that increases.

Experimental Example 3

Among the transcription factors related to osteoblast differentiation, the effects on the mRNA and protein of the master transcription factors Runx2 and Osterix were investigated using RT-PCR, Western blot and Luciferase assay, and the results are shown in FIG. 3.

It can be seen from FIG. 3 that GV1001 increased the amount of Runx2 and Osterix proteins in a concentration-dependent manner.

Experimental Example 4

As GV1001 increased the protein amount of Runx2 as in Experimental Example 3, the effect on the promotion of ALP activity by Runx2 and the effect on protein stability were tested using ALP staining assay and a half-life measurement method using cycloheximide and the results It is shown in FIG. 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 to increase the amount, the transcriptional activity was also investigated by performing RT-PCR of luciferase assay and differentiation markers using ALP-Luc and BSP-Luc and examining the results. It is shown in 5c.

It can be observed from FIG. 5A to FIG. 5C that GV1001 increased the transcriptional activity of Runx2 and also increased the mRNA amount of differentiation markers.

Experimental Example 6

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

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

Experimental Example 7

C2C12 cells induce osteoblast differentiation with BMP2 and increase the concentration of Pin1 to confirm the effect of ALP activity and the effect of juglone, a Pin1 inhibitor on ALP activity, inducing osteoblast differentiation of C2C12 cells with BMP2 and Pin1 After increasing the concentration or increasing the amount of juglone, RNA was obtained after 2 days of culturing, and the osteoblast differentiation markers were observed with RT-PCR for quantitative changes, and a promoter assay for bone cell differentiation was performed, and the results are shown in FIG.

From FIG. 7, when C2C12 cells induce osteoblast differentiation with BMP2 and increased the concentration of Pin1, the activity of ALP increased, and juglone, a Pin1 inhibitor, can be observed to decrease the activity of ALP, so Pin1 is osteoblast differentiation Although the expression of markers was increased, it was confirmed that juglone decreased the expression of osteoblast differentiation markers in a concentration-dependent manner. In addition, it was observed that Pin1 increases the activity of the promoter in a concentration-dependent manner.

The above results show that Pin1 promotes osteoblast differentiation and Pin1 inhibitor inhibits osteoblast differentiation.

Experimental Example 8

Pin1 increases mRNA expression of osteoblast differentiation markers. At this time, the effect of GV1001 is confirmed by real-time PCR to observe whether GV1001 further increases osteoblast differentiation induced by Pin1 and the results are shown in FIGS. 8A and 8A. It is shown in 8b.

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

Experimental Example 9

Whether the activity decreases when knock-down of Pin1 is investigated by examining the effect of GV1001 on osteoblast differentiation marker promoter activity with and without Sh-Pin1, and the results are shown in FIGS. 9A and 9B.

9A and 9B show that GV1001 further increases osteoblast differentiation induced by Pin1, and it was confirmed that GV1001 decreases activity promoting osteoblast differentiation by Sh-Pin1, and this result shows that GV1001 is partially It was confirmed that it promotes osteoblast differentiation through Pin1.

Experimental Example 10

To confirm the activity of Sh-pin1, we tested whether the effect of GV1001 on the activity of ALP-luc using Pin1 -/- MEF cells and the effect of GV1001 on osteoblasts when Pin1 was transfected and recovered. It is shown in Figure 10.

Fig. 10 shows that in the absence of Pin1, GV1001 was inactive and Pin1 was introduced again, and its activity was restored.

Experimental Example 11

The effect of GV1001 on ALP activity and mineralization in the Pin1 knock-down state was investigated by ALP staining assay and Alizarin red staining, and the results are shown in FIG. 11.

From FIG. 11, it was observed that the activity and mineralization of ALP increased by GV1001 decreased when Pin1 was knocked down.

Experimental Example 12

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

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

Experimental Example 13

As described above, the protein amount of RUNX2, an osteoblast-related transcription factor, and Col1A1, which is an Osterix differentiation marker, is increased by GV1001, and the quantitative change of these proteins is observed by western blot after Pin1 knock-down using Sh-Pin1. The results are shown in FIG. 13.

13, it can be confirmed that the amount of protein increased by GV1001 is decreased by pin1 knock-down.

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

Experimental Example 14

To investigate the effect of GV1001 on osteoclast differentiation, macrophage was isolated from mouse bone marrow to establish osteoclast differentiation conditions, and then treated with 20-, 50-, and 100-ng/ml M-CSF (10 ng/ml) and RANKL. Through the TRAP assay, which is an osteoclast differentiation marker, RANKL was treated with increasing concentration and the results are shown in FIGS. 14A and 14B.

From FIGS. 14A and 14B, it was confirmed that the cells with multi-nulcear increased in a concentration-dependent manner in RANKL, thereby increasing the differentiation of osteoclasts.

Experimental Example 15

In order to investigate the effect of GV1001 on osteoclast differentiation, macrophage was isolated from mouse bone marrow and treated with M-CSF (10 ng/ml) and RANKL at 50 ng/ml or 100 ng/ml to determine whether it differentiates into osteoclasts by TRAP assay. It was confirmed through and the results are shown in FIGS. 15A to 15D and 16A to 16D, respectively.

15A to 16D, it was confirmed that the osteoclast differentiation induced by M-CSF and RANKL was suppressed in a concentration-dependent manner as the concentration of GV1001 increased.

Experimental Example 16

After performing the TRAP assay to more quantitatively measure that GV1001 inhibits osteoclast differentiation, cells with three or more fusions were counted on a microscope to observe osteoclast differentiation in a concentration-dependent manner, and the results are shown in FIG. 17. .

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

The present invention has been shown and described with reference to preferred embodiments as described above, but is not limited to the above-described embodiments and is within the scope of the present invention to those skilled in the art to which the present invention pertains. By doing so, 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 peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient, the peptide promotes osteoblast differentiation, inhibits osteoclast differentiation, and the peptide is an osteoblast differentiation marker BSP, collagen type I, ALP, Runx2 And increasing the bone density by increasing the concentration-dependent mRNA of Osterix, thereby alleviating, treating, or preventing bone loss caused by any one or more of osteoporosis, fracture, and periodontal disease. Preventive pharmaceutical composition.
delete delete According to claim 1,
The osteoblast differentiation marker is a osteoblast differentiation marker induced by Pin1 (peptidylprolyl cis/trans isomerase, NIMA-interacting 1). A pharmaceutical composition for alleviating, treating or preventing bone loss.
According to claim 1,
The content of the peptide is 0.1 ㎍ / ㎎ to 1 ㎎ / ㎎ pharmaceutical composition for the alleviation, treatment or prevention of bone loss.
delete The method of any one of claims 1, 4, and 5,
A pharmaceutical composition for relieving, treating or preventing bone loss, further comprising an osteoblast differentiation promoting material.
The method of claim 7,
The osteoblast differentiation promoting material is BMP2 or BMP4, characterized in that the pharmaceutical composition for the relief, treatment or prevention of bone loss.
A peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient, the peptide promotes osteoblast differentiation, inhibits osteoclast differentiation, and the peptide is an osteoblast differentiation marker BSP, collagen type I, ALP, Runx2 And Osterix mRNA to increase the bone density by increasing the concentration-dependent health functional food for alleviating or preventing bone loss.
The method of claim 9,
A health functional food for alleviating or preventing bone loss, further comprising an osteoblast differentiation promoting material.
The method of claim 9,
The osteoblast differentiation promoting material is BMP2 or BMP4, a health functional food for alleviating or preventing bone loss.
It comprises the step of administering a pharmaceutical composition for the relief, treatment or prevention of bone loss comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient, the peptide BSP, collagen type I, ALP, osteoblast differentiation markers, Bone in mammals other than humans, characterized by alleviating, treating or preventing bone loss caused by any one or more of osteoporosis, fracture and periodontal disease, by increasing the density of bone by increasing the concentration dependent mRNA of Runx2 and Osterix How to treat loss.

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