WO2016098935A1 - Bmp-derived peptide and use of same - Google Patents

Abstract

The present invention relates to a peptide, comprising an amino acid sequence of sequence number 1, and a composition, for promoting osteogenesis, containing the peptide as an active ingredient. A peptide, according to the present invention, promotes osseointegration and osteogenesis, increases osteoblast differentiation and dental pulp tissue proliferation and thus can be used for inhibiting inflammation due to an implant operation and oral bacteria. Moreover, the peptide promotes growth and movement of fibroblasts and thus can be utilized for skin aging inhibition and wound healing.

Description

Peptides derived from BMP and uses thereof

The present invention relates to BMP derived peptides and their use.

Bone tissue is a dynamic tissue that constantly undergoes bone formation / Osteoblast differentiation and bone resorption / Osteoclast differentiation. Osteoclasts absorb bone, while osteoblasts play a role in synthesizing and filling bone matrix. Thus, bone mass depends on the relative function of these cells. In normal adults, the amount of bone resorption and the amount of bone formation are always balanced.

In recent 10 to 20 years, with the prolonged human life and improved quality of life, the implant market at home and abroad has grown and became popular. Implants are artificial or tertiary teeth, which have been enlarged to allow for sufficient coverage, such as bone grafts and bone elongation in the jawbone of the missing / corroded area of the tooth or where the tooth is pulled out. It is a dental treatment that restores the function of natural teeth by planting a biocompatible implant body. After osteo-integration, which is a morphological, physiological, and direct connection between the jawbone where the normal function is maintained and the surface of the implanted body, the bone reconstruction of the jawbone around the implant is performed. This bone adhesion is the most important indicator of implant success. If the bone adhesion does not occur within a short period of time, the success rate of the implant is reduced due to the failure of the initial bone adhesion. Therefore, for the success of implant placement, it is necessary to enhance the ability of bone adhesion and bone formation.

The success rate of the implant depends on the initial bone fusion between the implant placement material and bone cells. Recently, bioactive substances such as BMP (Bone matrix protein), a protein belonging to the TGF-β family that promotes proliferation and differentiation of osteoblasts, have been applied directly to the surface of implants or spread around the surgical site. . It is widely reported that BMP proteins play an important role in osteoblast differentiation and growth. Since it has been reported that BMP family proteins play an important role in bone formation, bone differentiation and alveolar bone formation, many experiments have been conducted. BMP family proteins are involved in bone formation and differentiation in humans as well as animals. It has been reported to regenerate and strengthen. However, BMP is not only expensive but also rapidly decomposed and disappeared by an enzyme present in the body, so that it is difficult to apply effectively. Since the cost of the procedure rises and does not remain effective, it is difficult to apply, the half-life is short, and standardization is not achieved.

Periodontal disease is often referred to as flavour, and is classified into gingivitis and periodontitis depending on the progress of the disease. Periodontal disease, which is a relatively mild symptom and quickly recovers, is called gingivitis, and it is called periodontitis when this inflammation progresses to the gums and around the gum bone. There is a V-shaped gap between the gum and teeth, which is the periodontal disease that oral bacteria attack the lower part of the gum and damage the periodontal ligament and nearby tissues. There will be a loss of teeth.

Tooth decay accounts for most of dental diseases, from children to adults. According to the report of the Korean Dental Association, more than 90% of Korean children have experienced dental caries. In addition, more than 80% of adults have periodontitis.

There are many causes of tooth decay and periodontitis, but in general, the lactose of carbohydrates such as sugar or starch of food debris attached to teeth is decomposed by the fermentation of bacteria that reside in the oral cavity. Occurs. Accordingly, the anaerobic pathogens are proliferated in a large amount, and the gum tissue is destroyed due to the toxin component generated by the periodontitis strains, which causes the tooth to shake and drop out. Streptococcus mutans and Porphyromonas gingivalis are typical pathogenic microorganisms that cause tooth decay and periodontitis.

Currently, as a method used to suppress tooth decay and periodontitis, various antibiotics, a method of suppressing pathogenic bacteria by fluorine, and the like are used.However, antibiotics may be resistant to microorganisms. As well as causing side effects such as diarrhea and vomiting, there is a problem that the growth of the flora in the oral cavity is inhibited. The antimicrobial substances derived from natural products known to date are mostly inferior in antimicrobial activity and poor in thermal stability, and thus, the problem of sharply deteriorating antimicrobial activity is pointed out.

Thus, the present inventors came to complete the present invention by confirming through experiments that the peptide composed of LYLDENEKVVLKN has excellent bone formation effect while studying the peptide to develop a composition for bone formation.

It is an object of the present invention to provide a peptide for promoting BMP-derived bone formation.

It is also an object of the present invention to provide a polynucleotide encoding the peptide and a vector comprising the polynucleotide.

It is also an object of the present invention to provide a composition for promoting bone formation, a composition for preventing or treating periodontal disease and an anti-inflammatory composition composed of the peptide.

In order to achieve the above object, the present invention provides a peptide for promoting BMP-derived bone formation.

It is also an object of the present invention to provide a polynucleotide encoding the peptide and a vector comprising the polynucleotide.

It is also an object of the present invention to provide a composition for promoting bone formation, a composition for preventing or treating periodontal disease, and an anti-inflammatory composition consisting of the peptide.

BMP-derived peptides according to the present invention can be used for implantation and inhibition of inflammatory action by oral bacteria by promoting osteointegration and bone formation, thereby increasing osteoblast differentiation and proliferation of pulp tissue, and inhibiting the growth and migration of fibroblasts. By promoting, it can be applied to skin aging and wound healing.

1 is a diagram showing the results of measuring the binding capacity of the BMP receptor of the peptide of the present invention.

Figure 2 is a diagram showing the results of measuring the cell growth promotion rate of the peptide of the present invention.

Figure 3 is a diagram showing the results of performing the ALP staining method of the peptide of the present invention.

4 is a diagram showing the results of measuring the ALP enzyme activity of the peptide of the present invention.

Figure 5 is a diagram showing the results of measuring the expression of osteoblast differentiation-related genes of the peptide of the present invention through RT-PCR.

Figure 6 is a diagram showing the results of measuring the degree of calcification of osteoblast extracellular matrix of the peptide of the present invention.

Figure 7 is a diagram showing the results of measuring the expression of osteoblast adhesion-related genes of the peptide of the present invention via RT-PCR.

Figure 8 is a diagram showing the results measured by the ALP staining the recovery effect of early osteoblast differentiation inhibition by the caries bacteria of the peptide of the present invention.

9 is a diagram showing the results of measuring the inhibitory effect of the inflammatory cytokine increase of the peptide of the present invention via RT-PCR.

Hereinafter, the present invention will be described in detail.

The present invention provides a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 and a composition for promoting bone formation containing the same as an active ingredient.

Natural amino acids include glycine, alanine, serine, aspartic acid, glutamic acid, valine, threonine, arginine, lysine (lysine, proline, leucine, leucine, phenylalanine, isoleucine, histidine, tyrosine, methionine, cysteine, tryptopane Asparagine, glutamine and the like. The peptide of the present invention is composed of LYLDENEKVVLKN (SEQ ID NO: 1), which can be synthesized by solid phase peptide synthesis (Solid phase peptide synthesis; Merrifield, Biochemistry 3 (1964) 1385) commonly used in the art.

L, Y, D, E, N, K and V of the peptides are leucine, tyrosine, thyrosine, aspartic acid, glutamic acid, asparagine and lysine, respectively. Since (Lysine) is represented, the amino acid sequence of the peptide of the present invention is as follows.

Leu-Tyr-Leu-Asp-Glu-Asn-Glu-Lys-Val-Val-Leu-Lys-Asn

Commonly used amino acids are used in the protected form of the N-terminus and in the protected form of the reactive side chain. As the protecting group for protecting the N-terminus, 9-fluorenylmethyloxycarbonyl group (Fmoc) may be used, and as protecting groups for protecting the reactive side chain, triphenylmethyl, t-butyl ester, butyloxycarbonyl, pentamethyl Chroman-6-sulfonyl and the like can be used. The solid resin used for the reaction may be a resin such as 2-chlorotrityl chloride (CTC), 4-benzyloxybenzyl alcohol (Wang) or trialkoxybenzhydrylamine (Rink Amide). .

The peptide of the present invention is synthesized from the C-terminus to the N-terminus direction. When coupling, the carboxyl group of the amino acid is HATU (N-[(dimethylamino-)-1H-1,2,3-triazolo [4,5-b] pyridin-1-ylmethylene] -N-methylmethanaminium hexafluorophosphate N-oxide). 1-hydroxy-7-azabenzotriazole (HOAt) or HBTU (N-[(1H-benzotriazol-1-yl) (dimethylamino) methylene] -N-methylmethanaminium hexafluorophosphate N-oxide) And DIEA (N, N-diisopropylethylamine) together with 1-hydroxybenzotriazole (HOBt), or N, N'-diisopropylcarbodiimide (DIC) and 1-hydroxy-7 It is used after activation by adding azabenzotriazole (HOAt) or 1-hydroxybenzotriazole (HOBt). After coupling, the N-terminal protecting group is removed with Piperidine, and the next amino acid coupling reaction is performed. Thus synthesized Peptidyl Resin (Peptidyl Resin) by the cleavage solution containing trifluroacetic acid (TFA), the peptide is separated from the solid resin and the protecting group bound to the side chain is removed.

The present invention also provides a polynucleotide encoding a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.

The "polynucleotide" is a polymer of deoxyribonucleotides or ribonucleotides present in single- or double-stranded form. It encompasses RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom and includes analogs of natural polynucleotides unless specifically stated otherwise.

The polynucleotide includes not only the nucleotide sequence encoding the peptide, but also a sequence complementary to the sequence. Such complementary sequences include sequences that are substantially complementary, as well as sequences that are substantially complementary. This means a sequence capable of hybridizing with the nucleotide sequence encoding the peptide of SEQ ID NO: 1 under stringent conditions known in the art.

The polynucleotide may also be modified. Such modifications include addition, deletion or non-conservative substitutions or conservative substitutions of nucleotides. The polynucleotide encoding the amino acid sequence is to be interpreted to also include a nucleotide sequence showing substantial identity to the nucleotide sequence. The substantial identity is at least 80% homology when the nucleotide sequence is aligned with any other sequence as closely as possible and the aligned sequence is analyzed using algorithms commonly used in the art. A sequence exhibiting at least 90% homology or at least 95% homology.

The present invention also provides a recombinant vector comprising the polynucleotide.

The term "vector" means a means for expressing a gene of interest in a host cell. For example, viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors are included. Vectors that can be used as the recombinant vector are plasmids often used in the art (eg, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14). , pGEX series, pET series and pUC19, etc.), phages (eg, λgt4λB, λ-Charon, λΔz1 and M13, etc.) or viruses (eg, CMV, SV40, etc.) can be produced.

The polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 in the recombinant vector may be operably linked to a promoter. The term "operatively linked" means a functional bond between a nucleotide expression control sequence (eg, a promoter sequence) and another nucleotide sequence. Thus, the regulatory sequence can thereby regulate transcription and / or translation of the other nucleotide sequence.

The recombinant vector can typically be constructed as a vector for cloning or a vector for expression. The expression vector may be a conventional one used in the art to express foreign proteins in plants, animals or microorganisms. The recombinant vector may be constructed through various methods known in the art.

The recombinant vector may be constructed using prokaryotic or eukaryotic cells as hosts. For example, when the vector used is an expression vector and the prokaryotic cell is a host, a strong promoter (for example, a pLλ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.) capable of promoting transcription It is common to include ribosome binding sites and transcription / detox termination sequences for initiation of translation. In the case of using a eukaryotic cell as a host, replication origins that operate in eukaryotic cells included in the vector include f1 origin, SV40 origin, pMB1 origin, adeno origin, AAV origin, CMV origin, and BBV origin. Including but not limited to. In addition, promoters derived from the genome of mammalian cells (eg, metallothionine promoters) or promoters derived from mammalian viruses (eg, adenovirus late promoters, vaccinia virus 7.5K promoters, SV40 promoters, Cytomegalovirus (CMV) promoter and tk promoter of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

The peptide synthesized as described above is a peptide that specifically binds to the BMP receptor, and exhibits strong binding ability to the BMP receptor, promotes bone adhesion and bone formation through the growth and differentiation of osteoblasts and fibroblasts, and protects against oral bacteria. Action.

Accordingly, the present invention provides a composition for promoting bone formation, a composition for preventing or treating periodontal disease and an anti-inflammatory composition comprising the peptide, polynucleotide or recombinant vector as an active ingredient.

The composition for preventing or treating periodontal disease includes a pharmaceutical composition or quasi-drug composition, and the quasi-drug composition may be, for example, in the form of toothpaste or mouthwash, but is not limited thereto.

The periodontal disease includes, but is not limited to, gingivitis, periodontitis, periodontal pocket or periodontal abscess.

The periodontal disease is caused by oral bacteria, preferably may be caused by s.mutans or P.gingivalis , but is not limited thereto.

The composition of the present invention may be prepared by including at least one pharmaceutically acceptable carrier in addition to the peptide, polynucleotide or recombinant vector. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components, as necessary. And other conventional additives such as bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate injectable formulations, injection solutions, pills, capsules, granules, powders or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, the method described in Remington's Pharmaceutical Science (Recent Edition) or Mack Publishing Company, Easton PA can be formulated according to each disease or component according to the appropriate method in the art.

The composition of the present invention may be administered orally or parenterally (eg, intravenously, subcutaneously or topically) according to the desired method, and the dosage may be weight, age, sex, health condition, diet, administration of the patient. The range varies depending on the time, the method of administration, the rate of excretion and the severity of the disease. As used herein, the term "administration" means providing a subject with any of the compositions of the present invention in any suitable manner.

The pharmaceutical composition of the present invention may be used to determine the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as thought by a researcher, veterinarian, doctor or other clinic, i.e., the symptoms of a disease or disorder being treated. It may be administered in a therapeutically effective amount that is an amount that induces remission. It will be apparent to those skilled in the art that the therapeutically effective dosages and frequency of administrations for the pharmaceutical compositions of the invention will vary depending upon the desired effect. Therefore, the optimal dosage to be administered can be readily determined by one skilled in the art and includes the type of disease, the severity of the disease, the amount of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, general health of the patient. It can be adjusted according to various factors including the condition, sex and diet, time of administration, route of administration and rate of composition, duration of treatment, and drugs used simultaneously. For the desired effect, the pharmaceutical composition of the present invention may be administered in an amount of 0.05 to 0.1 mg / kg / day, preferably 0.01 to 0.1 mg / kg / day, or may be administered once a day, or It may be administered in divided doses.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.

In addition, the present invention provides a bone graft material and a support for tissue engineering is coated on the surface of the composition for promoting bone formation comprising the peptide, polynucleotide or recombinant vector.

 The bone graft material and the support for use in the present invention may be any kind and type of bone graft material and polymer support used in the art, preferably bio-derived bone mineral powder and porous block derived from autologous bone, bovine bone and pork bone , Synthetic Apatite Powder and Porous Block, Tricalcium Phosphate Powder and Porous Block, Monocalcium Phosphate Powder and Porous Block, Bone Graft Material of Silicon Dioxide Particles and porous support, titanium and three-dimensional porous support and the like made of a biocompatible polymer comprising a, but is not particularly limited thereto. At this time, the surface of the bone graft material and the support is preferably modified surface to facilitate the attachment of the active peptide.

In addition, it is preferable to chemically bond the composition for promoting bone formation to the surface of the bone graft material and the support so that 0.01 ~ 1 μM per cm 2 coated on the surface, more preferably 0.05 ~ per cm 2 surface of the bone graft material and the support Coating 0.5 μM, most preferably coating 0.1 μM per surface cm 2 of bone graft material and support, but is not particularly limited thereto.

As described above, when the composition of the present invention is fixed to the surface of a bone graft material and a support or a shielding membrane or an implant, and used in a procedure, a composition having a desired concentration can be present locally, and the therapeutic effect can be enhanced.

The present invention also provides an anti-aging cosmetic composition comprising the peptide, polynucleotide or recombinant vector as an active ingredient. The cosmetic composition may be used to prevent skin aging and to improve skin wrinkles. Preferably, the cosmetic composition may be in the form of skin, lotion, cream, essence, ointment, gel, stick, powder, spray, and the like, but is not limited thereto.

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

Example 1. Preparation of Sample

In order to prepare a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1 of the present invention, solid phase peptide synthesis (Serrifield, Biochemistry 3 (1964) 1385) was performed.

First, 700 mg of chloro trityl chloride resin (CTL resin, Nova biochem Cat No. 01-64-0021) was added to a reaction vessel, and 10 ml of methylene chloride (MC) was added thereto, and stirred for 3 minutes. 10 ml of dimethylformamide (DMF) was added thereto, stirred for 3 minutes, and then the solvent was removed again. Then, 10 ml of dichloromethane (DCM) solution was added to the reaction vessel, 200 mmole of Fmoc-Asn (Trt) -OH (Bachem, Swiss) and 400 mmole of diisopropyl ethylamine (DIEA) were added thereto, stirred, and dissolved. The reaction was stirred for 1 hour. After the reaction, methanol and DIEA (2: 1) were dissolved in DCM, reacted for 10 minutes, washed with excess DCM / DMF (1: 1), the solution was removed, 10 ml of DMF was added, stirred for 3 minutes, and then the solvent was again used. Was removed. Thereafter, 10 ml of a deprotection solution (20% of piperidine / DMF) was added to the reaction vessel, stirred at room temperature for 10 minutes, and then the solution was removed. The same amount of deprotection solution was added and the reaction was again maintained for 10 minutes, and then the solution was removed and washed 3 times with DMF, once with MC and once with DMF, respectively, to prepare Asn-CTL resin. 10 ml of DMF solution was added to a new reactor, 200 mmole of Fmoc-Lys (Boc) -OH (Bachem, Swiss), 200 mmole of HoBt and 200 mmole of Bop were stirred and dissolved in the reactor, followed by 400 mmole DIEA (N, N). -Diisopropylethylamine) was added twice in fractions and stirred for at least 5 minutes until all solids dissolved. The dissolved amino acid mixture solution was placed in a reaction vessel with deprotected resin and reacted with stirring at room temperature for 1 hour. After removing the reaction solution, stirring with DMF solution three times for 5 minutes, and then removing the solution again, a small amount of the reaction resin was taken to check the degree of reaction using a Nihydrin test. Thereafter, the deprotection reaction was performed twice with the deprotection solution in the same manner as above to prepare Lys-Asn-CTL resin. The prepared resin was sufficiently washed with DMF and MC, and once again subjected to a Kaiser test, the following amino acid attachment was performed in the same manner as above. Fmoc-Val, Fmoc-Val, Fmoc-Lys (Boc), Fmoc-Glu (OtBt), Fmoc-Asp (OtBu), Fmoc-Leu, Fmoc-Tyr (tBu) and Fmoc-Leu based on selected amino acid sequences The chain reaction was carried out in the order of (tBu), and the Fmoc-protecting group was reacted twice with a deprotection solution twice for 10 minutes and then washed. After acetylation with acetic anhydride, DIEA and HoBt (Hydroxybenzotriazole) for 1 hour, the prepared peptidyl resin was washed three times with DMF, MC and methanol, and dried slowly with nitrogen air. After drying under reduced pressure under vacuum under phosphorus (Phosphorus pentoxide, P ₂ O ₅ ), 30 ml of fugitive solution [TFA (Trifluroacetic acid) 95%, distilled water 2.5%, Thianisole 2.5%] was added occasionally. The reaction was shaken for 2 hours, filtered to filter the resin, the resin washed with a small amount of solution and combined with the mother liquor. This was distilled off using a reduced pressure to half the total volume, 50 ml of cold ether was added to precipitate the oil by segregating the precipitate, washed twice with cold ether. The mother liquor was removed and dried sufficiently under nitrogen to synthesize 0.09 g of NH ₂ -Leu-Tyr-Leu-Asp-Glu-Asn-Glu-Lys-Val-Val-Leu-Lys-Asn-OH peptide 1 before purification (yield). : 91.9%). The peptide was measured using a molecular weight meter to confirm that the molecular weight was 1574 (theoretical value: 1576.8 Da).

Experimental Example 1. Confirmation of BMP receptor specific binding capacity

In order to measure the binding capacity of the peptide prepared in Example 1 with the BMP receptor, a BMP receptor binding assay was performed, and the results are shown in FIG. 1.

As shown in Figure 1, it was confirmed that the peptide of the present invention specifically binds to the BMP receptor as compared to the negative control. This indicates that the peptide of the present invention can bind to the BMP receptor and activate the downstream signaling system of the BMP receptor.

Experimental Example 2. Confirmation test for growth of pulp tissue and osteoblast growth

The growth promoting effect of pulp tissue fibroblasts (HPLF, Human periodontal ligament fibroblast) and osteoblasts (C2C12, murine osteoblast) of the peptide prepared in Example 1 was tested.

First, each cell was placed in a well of a 96-well plate at 2 × 10 ³ cells / well (final media volume 100 ml), incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator, and then the medium of each well was removed, and Change to serum free medium. Each well was treated with the peptides by concentration, followed by incubation for 72 hours in an incubator. After incubation, 10 ml of MTT solution (5mg / ml in DW) was added to each well and incubated for 4 hours more, the cultures were removed, and 100 ml of DMSO solution was added to dissolve the generated formazin. . Absorbance was measured at 540 nm of the solution using a spectrophotometer. At this time, the cell survival rate (%) of each cell line based on the negative control group treated with distilled water instead of the sample was converted by Equation 1 below, and the results are shown in FIG. 2.

[Equation 1]

% Cell viability = [(OD _sample ) / OD _control ] x 100

It was confirmed that the peptide of the present invention increased the growth of pulp ligament fibroblasts and osteoblasts.

Experimental Example 3. Confirmation of osteoblast differentiation promoting ability

3-1. Confirmation of early osteoblast differentiation ability

In order to confirm the osteoblast initial differentiation promoting ability of the peptide prepared in Example 1 was measured ALP (Alkaline phosphatase) staining and activity of the early differentiation marker protein of osteoblasts. First, dissociate osteoblasts (C2C12) into ⁴ wells in a 24-well plate and incubate for 1 day, replace the medium (including 10% FBS) and simultaneously treat the peptides for 3 days at 37 ° C and 5% CO ² . Incubated. For ALP staining, the cells were fixed with a fixed solution (25 mM citrate buffer, 3% formaldehyde, 65% acetone), and 200 ml of staining reagent (sigma 85C-1kit) was added thereto and reacted at 37 ° C. for 30 minutes. Staining was observed under an optical microscope, and the results are shown in FIG.

In order to measure ALP enzyme activity, the peptide was added and incubated for 3 days, dissolved in cytolysis buffer (10 mM Tris, pH8.0, 1 mM MgCl 2, 0.5% Triton X-100), followed by phosphatase, a substrate of ALP. 10 mM of phosphatase substrate was reacted, and then absorbance was measured at 405 nm. The results are shown in FIG. 4.

As shown in Figures 3 and 4, it was confirmed that the peptide significantly increases ALP staining and enzyme activity compared to the negative control.

3-2. Confirmation of late osteoblast differentiation capacity

In order to confirm the late osteoblast differentiation promoting ability of the peptide prepared in Example 1 RT-PCR was performed to compare the mRNA production of late marker differentiation of extracellular matrix proteins of osteoblasts.

The cells were cultured under the same conditions as in Experimental Example 3-1, and cells were recovered on the 5th day of culture, and RNA was extracted using Trizol® reagent (Intron). For cDNA synthesis from RNA, 5 mg RNA, 1 ml of random hexamer, and DEPC-treated water were added and reacted at 65 ° C. for 5 minutes, followed by 5 × first strand buffer, 0.1M DTT, 10 mM dNTP, and reverse transcriptase. The reaction was carried out at 42 ° C. for 1 hour in ml volume. For termination of the reaction, the mixture was finally heated to 95 ° C. for 5 minutes to obtain cDNA. Thereafter, 2 ml of cDNA was used as a template, and primers specific for each gene were added at 10 pmole, and PCR was performed by mixing 10x taq buffer, 10 mM dNTP, and i-taq DNA polymerase. Amplified 30 times under conditions of 94 ° C. 30 sec, 60 ° C. 30 sec, and 72 ° C. 30 sec., PCR products were loaded on 1% agarose gel, electrophoresed for 20 minutes, and used with an UV illuminator. It confirmed by the, and the result is shown in FIG.

As shown in FIG. 5, the peptide significantly increased the expression of alkaline phosphatase, osteopontin and osteocalcin, which are late osteoblast differentiation extracellular matrix markers, compared to the negative control group. Confirmed. The increase was found to be a significant increase in comparison with the positive control BMP2.

3-3. Confirmation of osteoblast calcification promoting ability

In order to confirm that osteoblast differentiation of osteoclasts of the peptide prepared in Example 1 proceeded well, Alizarin red S staining was performed to confirm the degree of calcification of osteoblast extracellular matrix.

The cells were cultured under the same conditions as in Experimental Example 3-1, and cells were fixed for 1 hour using 70% ethanol on day 7 of the culture, and stained with 2% Alizarin Red S solution (pH 4.1-4.3). After washing sufficiently with PBS, the resultant was observed using a microscope. In order to quantify the degree, 10% cetypyridinium chloride (in 10 mM sodium phosphate, pH7.0) solution was dissolved and absorbance was measured at 562 nm. The results are shown in FIG.

As shown in Figure 6, it was confirmed that the bone differentiation by the peptide is in an improved embodiment.

Experimental Example 4. Confirmation of osteoblast adhesion promoting ability

In order to confirm whether the peptide prepared in Example 1 increases the expression of genes related to the attachment of osteoblasts, RT-PCR was performed in the same manner as in Experiment 3-2, and the results are shown in FIG. 7. Indicated.

As shown in FIG. 7, the peptide expresses procollagen type 1 (collagen type 1), collagen 1, osteoadherin, and integrin α3, which are related genes of osteoblast adhesion. It was confirmed that significantly increased compared to the negative control. This indicates that the peptide of the present invention promotes the adhesion of osteoblasts by promoting the expression of osteoblastic adhesion-related genes.

Experimental Example 5. Periodontal Disease Treatment and Anti-inflammatory Effect

In order to determine whether the peptide prepared in Example 1 has a periodontal disease treatment effect and anti-inflammatory effect, an inflammatory environment is formed in the pulp cells using s.mutans , a representative oral bacterium causing caries and periodontal disease, Peptides were treated.

5-1. s.mutans Restoration effect of early osteoblast differentiation by bacteria

In order to confirm the recovery of early differentiation inhibition of osteoblasts was measured by ALP (Alkaline phosphatase) staining in the same manner as in Experiment 3-1, the results are shown in FIG.

As shown in FIG. 8, it was confirmed that osteoblast differentiation was decreased by s.mutans , whereas inhibition of osteoblast initial differentiation by s.mutans was increased in the group treated with the peptide.

5-2. s.mutans Inhibitory Effect of Osteoblast Inflammation by Bacteria

S.mutans causes an increase in cytokine associated with inflammation of osteoblasts. This increase is also known to affect the formation of bone tissue and greatly affect the collapse of the entire periodontal tissue. RT-PCR test was carried out in the same manner as in Experiment 3-2 to confirm whether the osteoblasts inhibit inflammation, and the results are shown in FIG. 9.

As shown in FIG. 9, the inflammatory cytokines COX2, IL-1β, IL-8, and TNF-α were increased by the s.mutans bacteria, but the inflammatory factors were significantly reduced by the peptide. This indicates that the peptide of the present invention may be helpful in the prevention and treatment of periodontal disease by inhibiting the inflammatory response.

Hereinafter, the formulation examples of the composition of the present invention will be described, which is intended to explain in detail only and not intended to limit the present invention.

Formulation Example 1 Pharmaceutical Formulation

1-1. Manufacture of powder

20 mg of peptide of the present invention

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

1-2. Manufacture of tablets

10 mg of peptide of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

1-3. Preparation of Capsules

10 mg of peptide of the present invention

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

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

1-4. Preparation of Injectables

10 mg of peptide of the present invention

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

1-5. Preparation of liquid

10 mg of peptide of the present invention

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding a proper amount of lemon aroma, and then mixing the above components, adding purified water and adjusting the whole to 100 ml by adding purified water and filling into a brown bottle. The solution is prepared by sterilization.

Formulation Example 2 Cosmetic Formulation

2-1. Preparation of Soft Cosmetics

10 mg of peptide of the present invention

Glycerin 30 g

20 g of butylene glycol

20 g of propylene glycol

10 g of carboxyvinyl polymers

100 g of ethanol

10 g of triethanolamine

Trace amount appropriate

Trace amount of water

2-2. Preparation of Nutritional Cream

10 mg of peptide of the present invention

100 g of beeswax

15 g of polysorbate 60

50 g of sorbitan sesquioleate

100 g of liquid paraffin

50 g of squalane

50 g of caprylic / capric triglycerides

20 g of triethanolamine

Trace amount appropriate

Trace amount of water

Claims (11)

1. Peptides for promoting bone formation, consisting of the amino acid sequence represented by SEQ ID NO: 1.
2. A polynucleotide encoding the peptide of claim 1.
3. A recombinant vector comprising the polynucleotide of claim 2.
4. The composition for promoting bone formation comprising the peptide of claim 1, a polynucleotide encoding the peptide, or a recombinant vector comprising the polynucleotide as an active ingredient.
5. The bone graft material of claim 4 coated on the surface.
6. The support for tissue engineering, wherein the composition of claim 4 is coated on the surface.
7. The pharmaceutical composition for preventing or treating periodontal disease, comprising the peptide of claim 1, the polynucleotide encoding the peptide or a recombinant vector comprising the polynucleotide as an active ingredient.
8. According to claim 7, wherein the periodontal disease (gingivitis), periodontitis (periodontitis), periodontal pockets (periodontal pocket) or periodontal abscess (periodontal abscess), characterized in that the pharmaceutical composition for the prevention or treatment of periodontal disease.
9. According to claim 7, wherein the periodontal disease is characterized by oral bacteria, periodontal disease preventive or therapeutic pharmaceutical composition.
10. The quasi-drug composition for prevention or improvement of periodontal disease, comprising the peptide of claim 1, the polynucleotide encoding the peptide or a recombinant vector comprising the polynucleotide as an active ingredient.
11. Claim 1 peptide, anti-inflammatory pharmaceutical composition comprising a polynucleotide encoding the peptide or a recombinant vector comprising the polynucleotide as an active ingredient.

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