Novel peptides

Abstract

The present invention relates to a novel peptide, a polynucleotide encoding the peptide, an expression vector comprising the polynucleotide, and a pharmaceutical composition, a foreign medicine composition and a health functional food composition comprising the peptide.

Description

Technical Field

[0001] The present invention relates to a novel peptide, and more particularly, to a peptide for promoting the regeneration of hard tissues and / or dental pulp tissues and for treating dentin-pulp diseases and / or periodontal diseases, a polynucleotide encoding the peptide, an expression vector comprising the polynucleotide, a pharmaceutical composition comprising the peptide for preventing or treating dentin-pulp diseases and / or periodontal diseases, a pharmaceutical external composition for preventing or improving dentin-pulp diseases and / or periodontal diseases, and a health functional food composition for preventing or improving dentin-pulp diseases and / or periodontal diseases. Background Art

[0002] The dental pulp is a soft connective tissue filling the pulp cavity inside the tooth, rich in nerves and blood vessels, and extending to the inner surface layer of dentin. Lesions occurring in this dental pulp are called pulp diseases.

[0003] The causes of pulp diseases are very diverse. In most cases, they occur based on bacterial infections of dental caries and infections caused by perforations, fractures, cracks in the teeth, or invasion into the dental pulp through periodontal pockets. Also, trauma, abrasion, tooth cracks, heat and friction generated in dental equipment during treatment, etc. can also induce pulp diseases. Pulpitis based on bacterial infection can progress to apical diseases and periodontal diseases. When a pulp disease occurs, it proceeds in the order of pulp hyperemia, pulpitis, and pulp necrosis. In the case of pulp necrosis, since the pulp dies and blood supply to the pulp is completely cut off, it causes the disappearance of the entire periodontal tissue, and ultimately can lead to apical diseases or abnormalities of the entire tooth.

[0004] In the treatment of pulp and apical diseases, pulp capping agents and root canal filling materials are used. Commonly used are calcium hydroxide, mineral trioxide aggregate (MTA), gutta-percha, etc. In the case of mineral trioxide aggregate, it has sealing power and biocompatibility, thus showing effects in treatment. However, relatively speaking, it has the problem of high cost as a dental treatment agent, and there are aesthetic problems due to discoloration. In the case of gutta-percha, it is cost-effective and has good fluidity, but it is a non-physiological treatment method that causes the loss of pulp viability. Until now, in terms of conservative treatment methods for dentin-pulp diseases, the treated teeth become weak or brittle and have a risk of reinfection.

[0005] The periodontium is a composite organ composed of epithelial tissue, soft tissue connective tissue, and calcified connective tissue. The structures of the periodontium include the gingiva, periodontal ligament (PDL), cementum, and alveolar bone. Gingival fibroblasts and periodontal ligament fibroblasts are the main cellular components of the soft tissue connective tissue of the gingiva, performing the functions of forming and maintaining the extracellular matrix. Among them, it is known that gingival fibroblasts are mainly involved in maintaining the gingival connective tissue. On the contrary, due to their unique functions, periodontal ligament fibroblasts can not only form the periodontal ligament but also participate in the repair and regeneration of adjacent alveolar bone and cementum in vivo. If periodontal diseases occur, clinically, tooth loss may result from gingival bleeding and swelling, the formation of periodontal pockets, and alveolar bone destruction, etc.

[0006] The ultimate result of treating periodontal diseases is to restore the damaged connective tissue, cementum, and alveolar bone. For this purpose, not only the regeneration of the periodontal ligament that supports the alveolar bone is required, but also the regeneration of the alveolar bone and cementum to which the periodontal ligament can attach.

[0007] Therefore, in order to develop a therapeutic agent capable of effectively treating the dentin-pulp diseases, relevant research is being actively carried out. For example, in Korean Patent Publication No. 2012-0089547, a composition for hard tissue formation and dentin or pulp tissue regeneration containing ameloblasts, apical bud cells, or their culture solutions as active ingredients is disclosed. In Korean Patent Publication No. 2009-0033643, a novel dental follicle-derived dental stem cell and its culture method are disclosed. Moreover, in Korean Patent Publication No. 2016-0105627, a composition for treating periodontal diseases containing ameloblast culture solution is disclosed.

[0008] Against this background, the inventors of the present invention, in order to develop a preparation capable of more effectively treating periodontal diseases that induce dentin-pulp diseases and / or damage to alveolar bone and cementum, conducted intensive research and efforts, and as a result, developed a cell therapy for promoting the regeneration of hard tissues including dentin, bone, and cementum and / or pulp tissue, and a peptide showing the therapeutic effect on dentin-pulp diseases and / or periodontal diseases, and completed the present invention. Summary of the Invention

[0009] Technical Problem

[0010] One object of the present invention is to provide a peptide for promoting the regeneration of hard tissues and / or dental pulp tissues and for treating dentin-pulp diseases and / or periodontal diseases.

[0011] Another object of the present invention is to provide a polynucleotide encoding the peptide.

[0012] Still another object of the present invention is to provide an expression vector containing the polynucleotide.

[0013] Yet another object of the present invention is to provide a pharmaceutical composition containing the peptide for preventing or treating dentin-pulp diseases and / or periodontal diseases.

[0014] Another object of the present invention is to provide a quasi-drug composition containing the peptide for preventing or improving dentin-pulp diseases and / or periodontal diseases.

[0015] Another object of the present invention is to provide a health functional food composition containing the peptide for preventing or improving dentin-pulp diseases and / or periodontal diseases.

[0016] Another object of the present invention is to provide a method for preventing or treating dentin-pulp diseases and / or periodontal diseases, which includes the step of administering a composition containing the peptide to an individual other than a human.

[0017] Another object of the present invention is to provide a method for promoting the regeneration of hard tissues and / or dental pulp tissues, which includes dentin, bone, and cementum, and which includes the step of administering a composition containing the peptide to an individual other than a human.

[0018] The objects of the present invention are not limited to the above-mentioned content, and those with ordinary knowledge in the technical field to which the present invention pertains can clearly understand other objects not mentioned from the following description.

[0019] Technical Solution

[0020] To solve the above technical problem, according to an embodiment of the present invention, there is provided a peptide for promoting the regeneration of hard tissues and / or dental pulp tissues and for treating dentin-pulp diseases and / or periodontal diseases, which contains an amino acid sequence represented by the following general formula 1.

[0021] K-Y-R1-R2-R3-R4-R5-R6-Y-K (General formula 1)

[0022] In the general formula 1, R1 and R2 are each lysine K, alanine A, or arginine R, R3, R4, and R5 are each lysine K or arginine R, and R6 is asparagine N or serine S.

[0023] According to one embodiment, the peptide may be composed of any one of the amino acid sequences of SEQ ID NO: 1 to 64.

[0024] According to one embodiment, the peptide may be composed of any one of the amino acid sequences of SEQ ID NO: 1 to 8.

[0025] According to one embodiment, the peptide may be formed by acetylation, amidation or methylation at the N-terminus or C-terminus; introduction of D-amino acids; modification of peptide bonds such as CH2-NH, CH2-S, CH2-S=O or CH2-CH2; modification of the main chain; or modification of the side chain.

[0026] According to one embodiment, the hard tissue may include dentin, bone and cementum.

[0027] As another embodiment, the present invention provides a polynucleotide encoding the peptide.

[0028] As yet another embodiment, the present invention provides an expression vector comprising the polynucleotide.

[0029] As yet another embodiment, the present invention provides a pharmaceutical composition comprising the peptide for preventing or treating dentin-pulp diseases.

[0030] According to one embodiment, the dentin-pulp disease may be dentin hypersensitivity, pulp hyperemia, pulpitis, pulp degeneration or necrosis and gangrene of the pulp.

[0031] As yet another embodiment, the present invention provides a pharmaceutical composition comprising the peptide for preventing or treating periodontal diseases.

[0032] According to one embodiment, the periodontal disease may be gingivitis, periodontitis, periodontal pocket or periodontal abscess.

[0033] As yet another embodiment, the present invention provides a pharmaceutical external composition comprising the peptide for preventing or improving dentin-pulp diseases and / or periodontal diseases.

[0034] As yet another embodiment, the present invention provides a health functional food composition comprising the peptide for preventing or improving dentin-pulp diseases or periodontal diseases.

[0035] As yet another embodiment, the present invention provides a method for preventing or treating dentin-pulp diseases, comprising the step of administering a composition comprising the peptide to an individual other than a human.

[0036] As yet another embodiment, the present invention provides a method for preventing or treating periodontal diseases, comprising the step of administering a composition comprising the peptide to an individual other than a human.

[0037] As yet another embodiment, the present invention provides a method for promoting the regeneration of hard tissues including dentin, bone, and cementum and / or pulp tissue, including the step of administering a composition containing the peptide to an individual other than a human.

[0038] Advantages of the Invention

[0039] A peptide of the present invention for promoting the regeneration of hard tissues and / or pulp tissue and for treating dentin-pulp diseases and / or periodontal diseases exhibits excellent effects in promoting the regeneration of hard tissues and / or pulp tissue, and can be widely applied to the development of various preparations for preventing or treating dentin-pulp diseases, or can be widely applied to the development of preparations for preventing or treating periodontal diseases that induce damage to bone and / or cementum.

[0040] The effects of the present invention are not limited to those mentioned above, and those with ordinary knowledge in the technical field to which the present invention pertains can clearly understand other effects not mentioned from the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 To group novel peptides with amino acid base sequence substitutions and show the results of the effects of each group of peptides on the expression of dentin sialophosphoprotein (DSPP), a marker gene for odontoblast differentiation, in human dental pulp cells (as the average value of each group measured for the level of DSPP mRNA using quantitative real-time polymerase chain reaction (PCR) in human dental pulp cells, the peptides were treated at a concentration of 10 μg / ml).

[0042] Figure 2 To confirm the effects of novel peptides on the expression of BSP and DMP1 genes, which are marker genes for osteoblast and cementoblast differentiation, after treating each group of peptides in human-derived bone marrow mesenchymal stem cells, the results of confirming the expression of BSP and DMP1 genes by real-time PCR.

[0043] Figure 3 To confirm the effects of novel peptides on the expression of marker proteins for odontoblasts, osteoblasts, and cementoblasts, after treating novel peptide (SEQ ID NO: 1) in human dental pulp cells in a concentration-dependent manner (1, 10, 50 μg), the results of confirming the expression of DSP and BSP proteins by Western blot.

[0044] Figure 4 To confirm whether novel peptides induce physiological dentin regeneration in a dentin injury animal model, after causing damage to the dentin of the teeth of beagle dogs, 10 μg of novel peptide (SEQ ID NO: 1) was applied, and then the results of histological evaluation were performed three weeks later.

[0045] Figure 5 is to confirm the effect of the peptide of Group 1 (SEQ ID NO: 1) on hard tissue formation in vivo according to Figure 2 the in vitro experimental results shown in Tables 10 to 11, 10 μg of the peptide of SEQ ID NO: 1 of Group 1, or human dental pulp cells (hDPCs) of the control group (cell only) were transplanted into the subcutaneous tissue of immune-compromised mice. After 12 weeks of transplantation, compared with the control group, it was confirmed that the ratio of hard tissue formation increased in the group treated with the novel peptide (A-D: hDPCs-alone; E-H: peptide of Group 1; I-L: BMP2 treatment / Scale bar: A, E, I: 500 μm; B, F, J: 200 μm; C, G, K: 100 μm; D, H, L: 50 μm).

[0046] Figure 6 is to confirm the effect of the peptide of Group 1 (SEQ ID NO: 1) on periodontal ligament formation in vivo. 10 μg of the peptide of SEQ ID NO: 1 of Group 1 or human dental pulp cells (hDPCs) of the control group (cell only) were transplanted into the subcutaneous tissue of immune-compromised mice. After 6 weeks of transplantation, the results of comparison with the control group are shown (Scale bar: A, C: 200 μm; B, D: 100 μm). Detailed Description of the Invention

[0047] Refer to the embodiments described in detail later together with the attached Figure 1 drawings, the object and effect of the present invention, and the technical configuration for achieving these will become clear. In describing the present invention, when it is judged that a detailed description of a well-known function or configuration may unnecessarily obscure the gist of the present invention, such detailed description will be omitted. Moreover, the following terms, which are defined in consideration of their functions in the present invention, may vary depending on the intention or convention of the user, operator, etc.

[0048] However, the present invention is not limited to the embodiments disclosed below and can be embodied in various different forms. However, these embodiments are provided to make the disclosure of the present invention more complete and to fully explain the scope of the invention to those of ordinary skill in the art to which the present invention pertains, and the present invention is only defined by the scope of the claims. Therefore, this definition should be based on the entire content of this specification.

[0049] Next, the present invention will be specifically described.

[0050] In order to more effectively develop a preparation for treating dentin-pulp diseases and / or periodontal diseases, the present inventor conducted various studies and as a result, developed a novel peptide composed of 10 amino acids.

[0051] The developed novel peptide is prepared by partially substituting the amino acid sequence of a peptide that can exhibit the therapeutic effect of dentin-pulp diseases and / or periodontal diseases. It can increase the expression level of the dentin sialophosphoprotein (DSPP) gene, which is a marker gene for odontoblast differentiation. Therefore, while showing the effect of promoting dentin regeneration, it can also increase the expression levels of the bone sialoprotein (BSP) gene and DMP1, which are marker genes for osteoblast and cementoblast differentiation, and can exhibit the effect of promoting bone and cementum regeneration.

[0052] Moreover, a graft containing the peptide together with human dental pulp cells is prepared, and the prepared graft is transplanted into the subcutaneous tissue of mice with impaired immune systems. After 6 weeks or 12 weeks, the transplanted tissue is analyzed. As a result, it is confirmed that a dentin-pulp-like tissue with a morphology most similar to that of dentin-pulp tissue in vivo is formed, a bone-like tissue with a morphology most similar to that of bone tissue in vivo is formed, the formation level of collagen is increased, and the expression level of DSP, which is a specific differentiation marker gene for odontoblasts, is increased.

[0053] Furthermore, the shape of the transplanted tissue is analyzed by scanning electron microscopy. As a result, it is confirmed that odontoblast-like cells are observed along the formed hard tissue, and the odontoblast processes also extend towards the formed hard tissue. Moreover, it is confirmed that the surface of the formed hard tissue exhibits the characteristics of typical osteoblasts and / or cementoblasts with cubic cells attached.

[0054] Therefore, it can be known that the peptide of the present invention can show the promotion of hard tissue and / or dental pulp tissue regeneration and the therapeutic effect of dentin-pulp diseases and / or periodontal diseases. So far, no report has proposed the peptide of the present invention that can show such an effect, and it was initially developed by the present inventor.

[0055] The term "hard tissue" in the present invention means relatively hard skeletal tissues including bone (skeleton), hyaline cartilage, and fibrocartilage. In one embodiment according to the present invention, the hard tissue may include dentin, bone, and cementum.

[0056] The term "dentine" of the present invention, also known as dentin, refers to the hard tissue that forms most of the tooth and is yellowish-white. The dentine is covered by enamel in the crown part of the tooth and by cementum in the root part of the tooth, so it is not exposed on the tooth surface. However, as age increases, if the enamel wears away, the dentine may be exposed at the front end or occlusal surface of the crown. Although the dentine is a kind of osseous tissue, the cell bodies of the cells that form the dentine are located in the dental pulp, and only their processes extend into the dentine. Based on this, it is different from conventional bone tissue.

[0057] The term "cementum" of the present invention refers to a thin membrane that covers the root (tooth root) of the tooth of a mammal and a slightly deformed form of bone in other parts. The cementum is composed of 50% inorganic matter and 50% water-organic matter, is yellow in color, and has a lower hardness than dentine or enamel. The cementum contains the periodontal ligament fibrous tissue that fixes the tooth to the alveolar bone. If the gums are infected with bacteria, it will cause the degeneration of the cementum surrounding the tooth, and the periodontal ligament fibrous tissue that connects the tooth and the alveolar bone cannot adhere tightly to the degenerated cementum, resulting in tooth loosening. To treat this degeneration of the cementum, a method of removing the degenerated cementum and promoting the formation of new cementum is used.

[0058] The peptide provided in the present invention can increase the expression levels of the DSPP gene, which is a marker gene for odontoblast differentiation, and the BSP and DMP1 genes, which are marker genes for osteoblast and cementoblast differentiation. When co-transplanted with human dental pulp cells into a living body, it can show the characteristics that the human dental pulp cells form dentin / pulp tissue-like tissue and osseous tissue-like tissue.

[0059] The peptide provided in the present invention, as long as it can exhibit the effects of promoting the regeneration of hard tissues including dentine, bone and cementum and / or dental pulp tissue and treating dentine-pulp diseases and / or periodontal diseases, mutant peptides with different sequences from the amino acid sequence of the peptide by more than one amino acid residue are also included in the scope of the peptide provided in the present invention.

[0060] Generally, the exchange of amino acids in proteins and polypeptides that does not change the activity of the molecule as a whole is well-known in the technical field to which the present invention pertains. The most commonly occurring exchanges are those between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly. Moreover, it may include peptides whose structural stability related to heat, pH, etc. is increased or whose ability to promote the regeneration of hard tissues including dentin, bone, and cementum and / or pulp tissue is increased through mutations or modifications in the amino acid sequence.

[0061] Amino acid mutations are achieved based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Among the amino acids constituting the peptides of the present invention, asparagine N and serine S belong to hydrophilic amino acids, so the relative similarity of amino acid side chain substituents is high. Therefore, even if the amino acids constituting the peptides according to the embodiments of the present invention are substituted with amino acids having hydrophilic properties, due to their structural similarity, they can exhibit similar effects.

[0062] For example, in the present invention, asparagine, an acidic amino acid at the 8th position of the peptide of SEQ ID NO: 1, can directly exhibit the effects of the peptides provided in the present invention even when substituted with serine, and lysine, a basic amino acid at the 5th position of the peptide of SEQ ID NO: 1, can exhibit the effects of the peptides provided in the present invention even when substituted with arginine, a basic amino acid.

[0063] Even if the acidic or basic amino acids constituting the peptides of the present application invention are respectively substituted with acidic or basic amino acids different from them, they can exhibit the effects of the peptides provided in the present invention. Therefore, it is obvious that mutant peptides having a sequence different by more than one amino acid residue from the amino acid sequence of the peptides constituting the present invention also fall within the scope of the peptides provided in the present invention.

[0064] Moreover, even if the peptides of the present invention have a form in which arbitrary amino acids are attached to their N-terminus or C-terminus, they can directly exhibit the effects of the peptides provided in the present invention and thus are included within the scope of the peptides provided in the present invention. As an example, it can be a form in which 1 to 300 amino acids are attached to the N-terminus or C-terminus of the peptide. As another example, it can be a form in which 1 to 100 amino acids are attached to the N-terminus or C-terminus of the peptide. As yet another example, it can be a form in which 1 to 24 amino acids are attached to the N-terminus or C-terminus of the peptide.

[0065] In order to be protected from proteolytic enzymes in an organism and to increase stability, the peptides of the present invention may be in a form that is chemically modified at its N-terminus and / or C-terminus, etc., or protected with organic groups, or modified by adding amino acids at the peptide terminus, etc. In particular, in the case of chemically synthesized peptides, since the N- and C-termini are charged, in order to remove such charges, acetylation may be performed on the N-terminus, methylation may be performed on the N-terminus, and / or amidation may be performed on the C-terminus, or it may include the introduction of D-amino acids, peptide bond modifications such as CH2-NH, CH2-S, CH2S=O, CH2-CH2, backbone modifications, and side-chain modifications, but is not limited thereto. The method for preparing peptidomimetic compounds is a method known in the technical field of the present invention. For example, reference may be made to the content described in Quantitative Drug Design, C.A. Ramsden Gd., Choplin Pergamon Press (1992).

[0066] The term "backbone modification" of the present invention means that the linear or cyclic skeleton of the amino acids constituting the peptide is called the backbone (backbone), and the amino acids constituting the peptide backbone are directly modified into amino acid analogs, which is called backbone modification. Amino acid analogs refer to amino acids in which a hydrogen atom is modified by substitution at the nitrogen or α-carbon of the amino acid backbone.

[0067] The term "side-chain modification" of the present invention means that the atomic groups branching out like branches from the linear or cyclic skeleton of the amino acids constituting the peptide are called the side-chains of the amino acids (side-chains), and these side-chains are modified with chemical substances, which is called side-chain modification. Examples of peptide side-chain modification include the following modifications of amino groups: such as reductive alkylation reaction; amidation based on methyl iminoacetate; alkylation based on acetic anhydride; carbamylation of amino groups based on cyanate ester; trinitrobenzylation of amino acids based on 2,4,6-trinitrobenzenesulfonic acid (TNBS); alkylation of amino groups based on succinic anhydride; or pyridoxylation such as reduction with NaBH4 after treatment with pyridoxal-5-phosphate.

[0068] In addition, the peptides of the present invention can be used alone or in combination with carriers (such as organic solvents) approved as pharmaceuticals, and may also contain carbohydrates such as glucose, sucrose or dextran for enhancing stability and efficacy; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins or other stabilizers, etc.

[0069] According to an embodiment of the present invention, 64 peptides corresponding to the general formula 1 provided in the present invention were synthesized, and the effects of the synthesized peptides on the expression level of the DSPP gene, which is a marker gene for odontoblast differentiation, were examined. As a result, it was confirmed that the mRNA level of the DSPP gene in human dental pulp cells treated with the 64 peptides showed a value of 9.7 times or more, or 6 times or more, or 3 times or more, or at least about 1.5 times or more compared to the mRNA level of the DSPP gene, which is a marker for odontoblast differentiation, measured in human dental pulp cells (control group) not treated with the peptides of the present invention. Figure 1 and Table 10).

[0070] According to the reports so far, it is known that if the expression level of DSPP mRNA increases, odontoblast differentiation and dentin regeneration are promoted. Therefore, it can be known as follows: the 64 peptides showing the effect of increasing the mRNA level of the DSPP gene exhibit the effects of promoting odontoblast differentiation and dentin regeneration (Taduru Sreenath et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol.278, No.27, Issue of July 4, pp.24874 - 24880, 2003; William T. Butler et al, Connective Tissue Research, 44(Suppl.1):171 - 178, 2003).

[0071] Moreover, the effects of the synthesized peptides on the expression level of the BSP gene, which is a marker gene for osteoblast / cementoblast differentiation, were examined. As a result, it was confirmed that the mRNA level of the BSP gene in human dental pulp cells treated with the 64 peptides showed a value of 2.9 times or more, or at least about 1.6 times or more, and the mRNA level of the DMP1 gene showed a value of 10.6 times or more, or at least about 5.7 times or more compared to the mRNA level of the BSP gene, which is a marker for osteoblast / cementoblast differentiation, measured in human dental pulp cells (control group) not treated with the peptides of the present invention.Figure 2 and Tables 11 and 12).

[0072] Accordingly, since it is known that if the mRNA expression level of BSP increases, it promotes the differentiation of osteoblasts / cementoblasts and the regeneration of bone and cementum, the 64 peptides showing the effect of increasing the mRNA level of the BSP gene may exhibit the effect of promoting the differentiation of osteoblasts / cementoblasts and the regeneration of bone and cementum.

[0073] As another embodiment, the present invention provides a polynucleotide encoding the peptide.

[0074] Regarding the polynucleotide, one or more bases may be mutated by substitution, knockout, insertion, or a combination thereof. In the case of preparing a nucleotide sequence by chemical synthesis, synthesis methods known in the technical field to which the present invention pertains can be used, such as the methods described in the literature (Engels and Uhlmann, Angew Chem Int Ed Engl., 37:73-127, 1988), and methods such as the triester, phosphite, phosphoramidite, and H-phosphate methods, polymerase chain reaction (PCR), and other automated primer methods, oligonucleotide synthesis methods on solid supports, etc. can be utilized for synthesis.

[0075] As yet another embodiment, the present invention provides an expression vector containing the polynucleotide, a transformant containing the expression vector, and a method for preparing the peptide using the transformant.

[0076] The term "expression vector" of the present invention refers to a recombinant vector that can express a target peptide in a target host cell, and means a gene construct containing essential regulatory elements operably linked to express a gene insert. The expression vector contains expression regulatory elements such as a start codon, a stop codon, a promoter, an operator gene, etc. The start codon and the stop codon are generally regarded as part of the nucleotide sequence encoding a polypeptide, and must function when the gene construct is given, and need to be in frame with the coding sequence. The promoter of the vector can be constitutive or inducible.

[0077] The term "operably linked" in the present invention refers to a state in which a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked for performing normal functions. For example, operably linking a promoter and a nucleic acid sequence encoding a protein or RNA can affect the expression of the coding sequence. The operable linkage with an expression vector can be prepared using genetic recombination techniques well known in the technical field to which the present invention pertains, and site-specific DNA cleavage and ligation can be performed using enzymes and the like that are generally well known in the technical field to which the present invention pertains.

[0078] Furthermore, in order to facilitate the separation of the peptide from the cell culture solution, the expression vector may contain a signal sequence for discharging the peptide. Specific initiation signals are also required for effective translation of the inserted nucleic acid sequence. These signals include the ATG initiation codon and adjacent sequences. In some cases, it is necessary to provide exogenous translation regulatory signals that may contain the ATG initiation codon. These exogenous translation regulatory signals and initiation codons can be of various natural and synthetic origins. The expression efficiency can be increased by introducing appropriate transcriptional or translational enhancers.

[0079] Meanwhile, in order to facilitate the detection of the peptide, the expression vector may further contain a protein tag that can be optionally removed using an endopeptidase.

[0080] The term "tag" in the present invention refers to a molecule that exhibits a quantifiable activity or property, and can be a fluorescent molecule including a chemical fluoracer (such as fluorescein) and a polypeptide fluorophore (such as a fluorescent protein (GFP) or a related protein); it can also be an epitope tag such as a Myc tag, a Flag tag, a histidine tag, a leucine tag, an IgG tag, a streptavidin tag, etc. In particular, in the case of using an epitope tag, it is preferably a peptide tag composed of 6 or more amino acid residues, and more preferably a peptide tag composed of 8 to 50 amino acid residues.

[0081] In the present invention, the nucleotide sequence encodes the peptide of the present invention for promoting the regeneration of hard tissues including dentin, bone and cementum and / or pulp tissue and for treating dentin-pulp diseases and / or periodontal diseases. The expression vector may contain the nucleotide sequence. At this time, the vector used is preferably plasmid DNA, phage DNA, etc., and more preferably commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), plasmids derived from Escherichia coli (pYG601 BR322, pBR325, pUC118, pUC119, etc.), plasmids derived from Bacillus subtilis (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vectors (retrovirus, adenovirus, vaccinia virus, etc.), insect virus vectors (baculovirus, etc.). However, as long as the peptide can be produced, it is not particularly limited thereto. Since the expression vector exhibits different protein expression levels and modifications depending on the host cell, it is preferably selected and used in a host cell most suitable for the purpose.

[0082] The expression vector provided in the present invention can be introduced into a host for transformation, thereby preparing the transformant provided in the present invention. By expressing the polynucleotide contained in the expression vector, it can be used for the production of the peptide. The transformation can be carried out by various methods, such as the CaCl2 precipitation method, the Hanahan method that uses a reducing substance called dimethyl sulfoxide (DMSO) to improve efficiency in the CaCl2 precipitation method, electroporation, calcium phosphate precipitation method, protoplast fusion method, stirring method using silicon carbide fibers, Agrobacterium-mediated transformation method, transformation method using polyethylene glycol (PEG), dextran sulfate, liposome, and desiccation / inhibition-mediated transformation method, etc. However, as long as the peptide can be produced, it is not particularly limited thereto. Also, the host used in the preparation of the transformant is not particularly limited as long as the peptide can be produced, but it can be a bacterial cell, such as Escherichia coli, Streptomyces, Salmonella typhimurium, etc.; a yeast cell, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, etc.; a fungal cell, such as Pichia pastoris, etc.; an insect cell, such as Drosophila, Sf9 cells of Spodoptera, etc.; an animal cell, such as CHO cells, COS cells, NSO cells, 293 cells, human melanoma (Bowes melanoma) cells, etc.; or a plant cell.

[0083] The transformant can also be used in the method for producing the peptide of the present invention for promoting the regeneration of hard tissues including dentin, bone, and cementum and / or pulp tissue and for treating dentin-pulp diseases and / or periodontal diseases. Specifically, the method for producing the peptide of the present invention for promoting the regeneration of hard tissues including dentin, bone, and cementum and / or pulp tissue and for treating dentin-pulp diseases and / or periodontal diseases may include: step (a), obtaining a culture by culturing the transformant; and step (b), recovering the peptide of the present invention from the culture.

[0084] The term "cultivation" in the present invention means a method of growing and developing microorganisms under appropriately artificially controlled environmental conditions. In the present invention, the method for cultivating the transformant can be carried out by using methods well-known in the technical field to which the present invention pertains. Specifically, regarding the cultivation, in a batch process or a fedbatch or repeated fed batch process, continuous cultivation can be carried out, but it is not particularly limited thereto as long as production can be carried out by expressing the peptides of the present invention for promoting the regeneration of hard tissues including dentin, bone, and cementum and / or pulp tissue and for treating dentin-pulp diseases and / or periodontal diseases.

[0085] The medium used for cultivation needs to be adjusted in terms of temperature, pH, etc. under aerobic conditions in a normal medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc., and meet the conditions of specific strains in an appropriate manner. As the carbon source that can be used, a mixed sugar of glucose and xylose is used as the main carbon source. In addition, it includes: sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, cellulose; and oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, etc.; and fatty acids such as palmitic acid, stearic acid, linoleic acid; and alcohols such as glycerol, ethanol; and organic acids such as acetic acid. These substances can be used individually or as a mixture. As the nitrogen source that can be used, inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate can be used; amino acids such as glutamic acid, methionine, glutamine; and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its decomposition products, defatted soybean cake or its decomposition products, etc. These nitrogen sources can be used alone or in combination. In the said medium, as the phosphorus source, monopotassium phosphate, dipotassium phosphate, and the corresponding sodium-containing salts can be included. As the phosphorus source that can be used, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts are included. And as inorganic compounds, sodium chloride, calcium chloride, ferric chloride, magnesium sulfate, ferric sulfate, manganese sulfate, and calcium carbonate, etc. can be used. Finally, essential growth substances such as amino acids and vitamins can be used on the basis of the said substances.

[0086] Moreover, appropriate precursors can be used in the medium. The said raw materials can be added to the culture in a batch, fed-batch, or continuous manner in an appropriate way during the cultivation process, but it is not particularly limited thereto. The pH of the culture can be adjusted by using alkaline compounds such as sodium hydroxide, potassium hydroxide, ammonia, etc.; acidic compounds such as phosphoric acid or sulfuric acid, etc. in an appropriate way.

[0087] Moreover, the generation of bubbles can be suppressed by using an antifoaming agent such as polyethylene glycol fatty acid ester. To maintain an aerobic state, oxygen or an oxygen-containing gas (e.g., air) is injected into the culture. Generally, the temperature of the culture is from 27°C to 37°C, preferably from 30°C to 35°C. And the culture is continuously carried out until the production amount of the peptide reaches the maximum value. For this purpose, it can usually be achieved within 10 hours to 100 hours.

[0088] Moreover, the step of recovering the peptide from the culture can be carried out by a method well-known in the technical field to which the present invention pertains. Specifically, as long as it is applicable to the recovery of the peptide to be produced, the recovery method is not particularly limited. Preferably, methods such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion) can be used.

[0089] As yet another embodiment, the present invention provides a pharmaceutical composition for preventing or treating dentin-pulp diseases, which comprises the peptide.

[0090] As described above, the peptide of the present invention for promoting the regeneration of hard tissues including dentin, bone, and cementum and / or pulp tissue and for treating dentin-pulp diseases and / or periodontal diseases, when transplanted into a living body together with human dental pulp cells, can promote the formation of dentin / pulp tissue-like tissue by the human dental pulp cells. Therefore, it can be used as an active ingredient of a pharmaceutical composition for treating dentin-pulp diseases caused by damaged pulp tissue.

[0091] The peptide contained in the pharmaceutical composition can be used in the form of the peptide alone, can also be used in the form of a polypeptide in which the peptide is repeatedly linked two or more times, and can also be used in the form of a complex in which a drug showing a therapeutic effect on dentin-pulp diseases is combined with the N-terminus or C-terminus of the peptide.

[0092] The term "dentin-pulp disease" in the present invention means a disease in which the pulp tissue and the dentin combined therewith are damaged due to the damage of the pulp tissue.

[0093] In the present invention, the dentin-pulp disease can be, for example, dentin hypersensitivity, pulp hyperemia, pulpitis, pulp degeneration, necrosis and gangrene of the pulp, etc., but is not particularly limited thereto as long as it can show a therapeutic effect based on the peptide of the present invention.

[0094] As yet another embodiment, the present invention provides a pharmaceutical composition for preventing or treating periodontal diseases, which comprises the peptide.

[0095] As described above, when the peptide of the present invention for promoting the regeneration of hard tissues including dentin, bone, and cementum and / or pulp tissue and for treating dentin-pulp diseases and / or periodontal diseases is transplanted into a living body together with human dental pulp cells, it can promote the formation of osteoid tissue by the human dental pulp cells. Therefore, it can be used as an active ingredient of a pharmaceutical composition for treating periodontal diseases that induce damage to bone and / or cementum.

[0096] The peptide contained in the pharmaceutical composition can be used in the form of the peptide alone, in the form of a polypeptide in which the peptide is repeatedly linked two or more times, or in the form of a complex in which a drug showing a therapeutic effect on periodontal diseases is combined with the N-terminus or C-terminus of the peptide.

[0097] The term "periodontal disease" in the present invention, also known as pyorrhea, refers to a disease in which the periodontal ligament and adjacent tissues are damaged due to bacterial infection in the gap between the gums (gum beds) and the teeth. It is divided into gingivitis and periodontitis according to the degree of the disease. When the periodontal disease occurs, more tissues are damaged as the inflammation progresses, and a periodontal pocket is formed. It is well known that the deeper the periodontal pocket is as the periodontitis becomes more severe, and as the periodontal pocket deepens, the periodontal ligament becomes inflamed, eventually inducing bone loss.

[0098] In the present invention, the periodontal disease can be, for example, gingivitis, periodontitis, periodontal pocket, or periodontal abscess, etc., but is not particularly limited thereto as long as it can exhibit a therapeutic effect based on the peptide of the present invention.

[0099] The term "prevention" in the present invention refers to all actions of inhibiting or delaying the occurrence of dentin-pulp diseases by administering a pharmaceutical composition for preventing or treating dentin-pulp diseases containing the peptide of the present invention, or refers to all actions of inhibiting or delaying the occurrence of periodontal diseases by administering a pharmaceutical composition for preventing or treating periodontal diseases containing the peptide of the present invention.

[0100] The term "treatment" in the present invention refers to all actions of treating pulp diseases by promoting the regeneration of dentin or pulp tissue by administering a pharmaceutical composition containing the peptide of the present invention as an active ingredient to an individual in need of treating dentin-pulp diseases, or refers to all actions of treating periodontal diseases by promoting the regeneration of bone and / or cementum by administering a pharmaceutical composition containing the peptide of the present invention as an active ingredient to an individual in need of treating periodontal diseases.

[0101] The pharmaceutical composition of the present invention can be prepared in the form of a pharmaceutical composition for treating dentin-pulp diseases and / or periodontal diseases. In addition to the peptide, the pharmaceutical composition further comprises a suitable carrier (natural or unnatural carrier), excipient or diluent commonly used in the preparation of pharmaceutical compositions. Specifically, the pharmaceutical composition can be formulated into a sterilized injection solution in a conventional manner and used to be administered to the site where dentin-pulp diseases and / or periodontal diseases are induced. In the present invention, examples of the carrier, excipient and diluent that can be included in the pharmaceutical composition include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, mineral oil, collagen, etc. During formulation, commonly used fillers, brighteners, binders, wetting agents, disintegrants, surfactants and other diluents or excipients can be used for preparation. In particular, it can include sterilized aqueous solutions, non-aqueous solvents, suspending agents, emulsions, lyophilized agents, suppositories, ointments (such as dental pulp transplantation materials, etc.). As non-aqueous solvents and suspending agents, vegetable oils such as propylene glycol, polyethylene glycol, olive oil, etc. can be used; esters that can be injected such as ethyl oleate, etc. As the base of suppositories, synthetic fatty acid esters (witepsol), polyethylene glycol, tween 61, cocoa butter, lauric acid glyceride fat, glycerogelatin, etc. can be used.

[0102] Regarding the content of the peptide in the pharmaceutical composition of the present invention, there is no particular limitation. However, based on the total weight of the final composition, the peptide can be included in an amount of 0.0001 to 50% by weight, and more preferably 0.01 to 20% by weight of the peptide.

[0103] The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" in the present invention means an amount sufficient for the treatment or prevention of a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dosage level can be determined according to the severity of the disease, the activity of the drug, the age, weight, health, gender of the patient, the sensitivity of the patient to the drug, the administration time of the composition of the present invention used, the administration route and excretion ratio during the treatment period, the elements of the drugs used in combination with or included in the composition of the present invention and other elements well-known in the medical field. The pharmaceutical composition of the present invention can be administered independently or in combination with a known pharmaceutical composition for treating dentin-pulp diseases and / or periodontal diseases. Importantly, considering all the above elements, it should be administered in an amount that has no side effects and can achieve the maximum effect with the least amount.

[0104] Those of ordinary skill in the art to which the present invention pertains may consider factors such as the purpose of use, the degree of disease poisoning, the age, weight, sex, previous medical history of the patient, or the type of substance used as the active ingredient to determine the dosage of the pharmaceutical composition of the present invention. For example, for each adult, the pharmaceutical composition of the present invention can be administered at about 0.1 ng to about 100 mg / kg, preferably, it can be administered at 1 ng to about 10 mg / kg. The administration frequency of the composition of the present invention is not particularly limited thereto, but it can be administered once a day or in divided doses multiple times. The dosage does not limit the scope of the present invention in any way.

[0105] As another embodiment, the present invention provides a method for treating dentin-pulp diseases, which includes the step of administering the pharmaceutical composition to an individual other than a human being suffering from dentin-pulp diseases in a pharmaceutically effective amount. As yet another embodiment, the present invention provides a method for treating periodontal diseases, which includes the step of administering the pharmaceutical composition to an individual other than a human being suffering from periodontal diseases in a pharmaceutically effective amount.

[0106] The term "individual" of the present invention refers to, without limitation, mammals such as humans or non-human mice, livestock, etc. that require treatment for dentin-pulp diseases and / or periodontal diseases.

[0107] The administration route of the pharmaceutical composition of the present invention for treating dentin-pulp diseases and / or periodontal diseases can be administered through any usual route as long as it can reach the target tissue. The pharmaceutical composition of the present invention can be administered through routes such as intraoral administration or intraoral injection according to the purpose, but it is not particularly limited thereto.

[0108] As yet another embodiment, the present invention provides a pharmaceutical cosmetic composition for preventing or improving dentin-pulp diseases containing the peptide, or a pharmaceutical cosmetic composition for preventing or improving periodontal diseases containing the peptide.

[0109] The term "improve" of the present invention refers to all actions that at least reduce the parameters (such as the degree of symptoms) related to the treated state.

[0110] In the present invention, the improvement can be interpreted as follows: administering a pharmaceutical composition containing the peptide of the present invention as an active ingredient to an individual in need of treatment for dentin-pulp diseases to promote the regeneration of dentin or pulp tissue, thereby improving or favorably changing the symptoms of dentin-pulp diseases, or administering a pharmaceutical composition containing the peptide of the present invention as an active ingredient to an individual in need of treatment for periodontal diseases to promote the regeneration of bone and / or cementum, thereby improving or favorably changing the symptoms of periodontal diseases.

[0111] The term "quasi-drug" in the present invention means an article that has a milder effect compared to pharmaceuticals among articles used for the purpose of diagnosing, treating, ameliorating, alleviating, managing, or preventing diseases in humans or animals. For example, according to the Pharmaceutical Affairs Law, quasi-drugs refer to articles other than those used for pharmaceutical purposes, including fiber / rubber products for treating or preventing diseases in humans / animals, articles that are not instruments or machinery and have a mild or non-direct effect on the human body and similar articles, bactericides / insecticides for preventing infectious diseases, etc.

[0112] In the present invention, the type or dosage form of the quasi-drug composition containing the peptide is not particularly limited. For example, it can be an oral disinfectant cleaner, oral cleaning product, toothpaste, dental floss, oral ointment, etc.

[0113] As another embodiment, the present invention provides a health functional food composition containing the peptide for preventing or ameliorating dentin-pulp diseases and / or periodontal diseases.

[0114] The term "food" in the present invention includes meat, sausage, bread, chocolate, confectionery, fast food, cookies, pizza, instant noodles, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, rehydration solutions, alcoholic beverages, vitamin complexes, health functional foods, and health foods, etc., including all foods in the conventional sense.

[0115] The health functional food, which is the same term as "food for special health use" (FoSHU), refers to a food with high medical and therapeutic effects that is processed to effectively exhibit biological regulatory functions in addition to providing nutrition. Among them, the so-called "functional" means regulating nutrients (for the structure and function of the human body) or being able to achieve favorable effects in health uses (such as physiological effects, etc.). The food of the present invention can be prepared by methods commonly used in the technical field to which the present invention pertains, and raw materials and ingredients commonly added in the technical field can be added during the preparation. And as the dosage form of the food, as long as it can be recognized as a food dosage form, it can be prepared without limitation. The food composition of the present invention has the following advantages: it can be prepared into various dosage forms, different from ordinary drugs, food can be used as a raw material, so there are no side effects that may occur when taking drugs for a long time, and it has excellent portability. Therefore, the food of the present invention can be ingested as an adjuvant for enhancing the effect of preventing or ameliorating dentin-pulp diseases and / or periodontal diseases.

[0116] The so-called health food means a food that has a positive effect on maintaining or improving health compared to ordinary food, and the health supplement food means a food for the purpose of health assistance. Depending on the situation, the terms of health functional food, health food, and health supplement food can be used interchangeably.

[0117] Specifically, the health functional food means a food in which the peptide of the present invention is added to food materials such as beverages, teas, spices, chewing gums, biscuits, etc., or prepared into capsules, powders, suspensions, etc. When consuming such food, it will bring specific effects in terms of health. However, different from ordinary drugs, since food is used as the raw material, the advantage is that there are no side effects that may occur when taking drugs for a long time.

[0118] The food composition of the present invention can be taken daily, so good effects can be expected in preventing or improving dentin-pulp diseases and / or periodontal diseases, and thus it can be used very effectively.

[0119] The food composition may also contain a physiologically acceptable carrier. The type of the carrier is not particularly limited, and any carrier that is commonly used in the technical field to which the present invention belongs can be arbitrarily used.

[0120] Moreover, the food composition may contain additive ingredients that are commonly used in food compositions to improve odor, taste, visual appearance, etc. For example, it may contain vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. And it may contain minerals such as Zn, Fe, Ca, Mg, Mn, Cu, Cr, etc. And it may contain amino acids such as lysine, tryptophan, cysteine, valine, etc.

[0121] Moreover, the food composition may contain food additives, such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), fungicides (bleaching powder and high-efficiency bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (tar pigments, etc.), color developers (sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclohexylsulfamate, saccharin, sodium, etc.), flavors (vanillin, lactones, etc.), swelling agents (alum, potassium bitartrate, etc.), fortifiers, emulsifiers, thickeners (pastes), film-forming agents, gum bases, foam inhibitors, solvents, modifiers, etc. The appropriate amount of the additives can be selected and used according to the type of food.

[0122] The peptide of the present invention can be directly added or used together with other foods or food ingredients, and can be appropriately used according to conventional methods. The mixing amount of the active ingredient can be appropriately determined according to its use purpose (prevention, health or therapeutic treatment). Generally, when preparing a food or beverage, the food composition of the present invention can be added in an amount of 50 parts by weight or less, specifically, an amount of 20 parts by weight or less, relative to the food or beverage. However, in the case of long-term ingestion for health and hygiene purposes, an amount below the above range can be included, and an amount above the above range can also be used as an active ingredient since there are no problems in terms of safety.

[0123] As an example of the food composition of the present invention, it can be used as a health beverage composition. In this case, like ordinary beverages, it can contain various flavoring agents or natural carbohydrates, etc. as added ingredients. The natural carbohydrates can be monosaccharides, such as glucose, fructose; disaccharides, such as maltose, sucrose; polysaccharides, such as dextrin, cyclodextrin; sugar alcohols, such as xylitol, sorbitol, erythritol, etc. Natural sweeteners, such as thaumatin, stevia extract, can be used as sweeteners; synthetic sweeteners, such as saccharin, aspartame, etc. The ratio of the natural carbohydrates in every 100 mL of the health beverage composition of the present invention can generally be about 0.01 g to 0.04 g, specifically, about 0.02 g to 0.03 g.

[0124] In addition to the above, the health beverage composition may contain various nutritional agents, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, pectate, alginic acid, alginate, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerol, ethanol or carbonating agents, etc. In addition, it may contain the pulp for preparing natural fruit juice, fruit juice beverage or vegetable beverage. Such components can be used independently or in combination. Although the ratio of such additives is not particularly important, for every 100 parts by weight of the health beverage composition of the present invention, it is generally selected in the range of 0.01 to 0.1 parts by weight.

[0125] Regarding the food composition of the present invention, if it can exhibit the effect of preventing or improving dentin-pulp diseases and / or periodontal diseases, the peptide of the present invention can be contained in various weight percentages. Specifically, based on the total weight of the food composition, 0.00001 to 100% by weight or 0.01 to 80% by weight of the peptide of the present invention can be contained, but it is not limited thereto.

[0126] As another embodiment of the present invention, there is provided a method for preventing or treating dentin-pulp diseases, and / or a method for preventing or treating periodontal diseases, which includes the step of administering a composition containing the peptide to an individual.

[0127] As yet another embodiment, there is provided a method for promoting the regeneration of dentin or pulp tissue, and / or a method for promoting the regeneration of bone or cementum, which includes the step of administering a composition containing the peptide to an individual.

[0128] As another embodiment of the present invention, there is provided a use of a peptide including the amino acid sequence of General Formula 1 below or a composition containing the peptide for promoting the regeneration of hard tissues including dentin, bone and cementum and / or pulp tissue, and for preventing or treating dentin-pulp diseases or periodontal diseases.

[0129] K-Y-R1-R2-R3-R4-R5-R6-Y-K (General Formula 1)

[0130] In General Formula 1, R1 and R2 are each lysine K, alanine A or arginine R, R3, R4, R5 are each lysine K or arginine R, and R6 is asparagine N or serine S.

[0131] As yet another embodiment, the present invention provides a use of a peptide including any one of the amino acid sequences of SEQ ID NOs: 1 to 64 or a composition containing the peptide for promoting the regeneration of hard tissues including dentin, bone and cementum and / or pulp tissue, and for preventing or treating dentin-pulp diseases and / or periodontal diseases.

[0132] Moreover, as an embodiment of the present invention, the present invention provides a use for promoting the regeneration of hard tissues including dentin, bone, and cementum and / or pulp tissue, and a use for preventing or treating dentin-pulp diseases and / or periodontal diseases, of a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 8 or a composition comprising the peptide.

[0133] The present invention will be described in more detail below by way of examples. However, these examples are only used to illustrate the present invention exemplarily, and the scope of the present invention is not limited to these examples.

[0134] Example 1: Experimental Methods and Materials

[0135] Example 1-1. Synthesis of a peptide for promoting the regeneration of hard tissues including dentin, bone, and cementum and / or pulp tissue and for treating dentin-pulp diseases and / or periodontal diseases

[0136] The present inventors synthesized a peptide (SEQ ID NO: 1) showing an effect of promoting the regeneration of hard tissues including dentin, bone, and cementum and / or pulp tissue by the 9-fluorenylmethyloxycarbonyl (Fmoc) method, and synthesized peptides for each group (Tables 1 to 8) by substituting the amino acids of the synthesized peptide.

[0137] N-KYKAKKKNYK-C (SEQ ID NO: 1)

[0138] First, the peptide of Group 1 is the peptide of SEQ ID NO: 1, or is synthesized by substituting the 5th to 7th amino acids of the peptide of SEQ ID NO: 1 with arginine (Table 1).

[0139]

Table 1

[0140] Peptide of Group 1

[0141] Sequence Number Amino Acid Sequence (N-C) 1 KYKAKKKNYK 2 KYKAKKRNYK 3 KYKAKRKNYK 4 KYKARKKNYK 5 KYKAKRRNYK 6 KYKARRKNYK 7 KYKARRRNYK 8 KYKARKRNYK

[0142] Then, the 8th amino acid of the peptide of SEQ ID NO: 1 was substituted with serine, and the 5th to 7th amino acids were substituted with arginine, thereby synthesizing the peptide of Group 2 (Table 2).

[0143]

Table 2

[0144] Peptide of Group 2

[0145] Sequence Number Amino Acid Sequence (N-C) 9 KYKAKKKSYK 10 KYKAKKRSYK 11 KYKAKRKSYK 12 KYKARKKSYK 13 KYKAKRRSYK 14 KYKARRKSYK 15 KYKARRRSYK 16 KYKARKRSYK

[0146] The 3rd amino acid of the peptide of SEQ ID NO: 1 was substituted with arginine, and the 5th to 7th amino acids were substituted with arginine, thereby synthesizing the peptide of Group 3 (Table 3).

[0147]

Table 3

[0148] Peptides of Group 3

[0149] Sequence Number Amino Acid Sequence (N-C) 17 KYRAKKKNYK 18 KYRAKKRNYK 19 KYRAKRKNYK 20 KYRARKKNYK 21 KYRAKRRNYK 22 KYRARRKNYK 23 KYRARRRNYK 24 KYRARKRNYK

[0150] The amino acid at the 3rd position of the peptide of SEQ ID NO: 1 was replaced with arginine, the amino acid at the 8th position was replaced with serine, and the amino acids at the 5th to 7th positions were replaced with arginine, thereby synthesizing the peptides of Group 4 (Table 4).

[0151] [Table 4]

[0152] Peptides of Group 4

[0153] Sequence Number Amino Acid Sequence (N-C) 25 KYRAKKKSYK 26 KYRAKKRSYK 27 KYRAKRKSYK 28 KYRARKKSYK 29 KYRAKRRSYK 30 KYRARRKSYK 31 KYRARRRSYK 32 KYRARKRSYK

[0154] The amino acid at the 3rd position of the peptide of SEQ ID NO: 1 was replaced with alanine, the amino acid at the 4th position was replaced with lysine, and the amino acids at the 5th to 7th positions were replaced with arginine, thereby synthesizing the peptides of Group 5 (Table 5).

[0155] [Table 5]

[0156] Peptides of Group 5

[0157] Sequence Number Amino Acid Sequence (N-C) 33 KYAKKKKNYK 34 KYAKKKRNYK 35 KYAKKRKNYK 36 KYAKRKKNYK 37 KYAKKRRNYK 38 KYAKRRKNYK 39 KYAKRRRNYK 40 KYAKRKRNYK

[0158] The amino acid at the 3rd position of the peptide of SEQ ID NO: 1 was replaced with alanine, the amino acid at the 4th position was replaced with lysine, the amino acid at the 8th position was replaced with serine, and the amino acids at the 5th to 7th positions were replaced with arginine, thereby synthesizing the peptides of Group 6 (Table 6).

[0159] [Table 6]

[0160] Peptides of Group 6

[0161] Sequence Number Amino Acid Sequence (N-C) 41 KYAKKKKSYK 42 KYAKKKRSYK 43 KYAKKRKSYK 44 KYAKRKKSYK 45 KYAKKRRSYK 46 KYAKRRKSYK 47 KYAKRRRSYK 48 KYAKRKRSYK

[0162] The amino acid at the 3rd position of the peptide of SEQ ID NO: 1 was replaced with alanine, the amino acid at the 4th position was replaced with arginine, and the amino acids at the 5th to 7th positions were replaced with arginine, thereby synthesizing the peptides of Group 7 (Table 7).

[0163] [Table 7]

[0164] Peptides of Group 7

[0165] Sequence Number Amino Acid Sequence (N-C) 49 KYARKKKNYK 50 KYARKKRNYK 51 KYARKRKNYK 52 KYARRKKNYK 53 KYARKRRNYK 54 KYARRRKNYK 55 KYARRRRNYK 56 KYARRKRNYK

[0166] The amino acid at the 3rd position of the peptide of SEQ ID NO: 1 was replaced with alanine, the amino acid at the 4th position was replaced with arginine, the amino acid at the 8th position was replaced with serine, and the amino acids at the 5th to 7th positions were replaced with arginine, thereby synthesizing the peptides of Group 8 (Table 8).

[0167] [Table 8]

[0168] Peptides of Group 8

[0169] Sequence Number Amino Acid Sequence (N-C) 57 KYARKKKSYK 58 KYARKKRSYK 59 KYARKRKSYK 60 KYARRKKSYK 61 KYARKRRSYK 62 KYARRRKSYK 63 KYARRRRSYK 64 KYARRKRSYK

[0170] Example 1-2. Cell Culture

[0171] Cultivation was carried out at 37 °C in humidified air containing 5% CO2 for experiments. Human bone marrow mesenchymal stem cells (hBMSCs) were purchased from Lonza (Switzerland) and used. hBMSCs were cultured in a culture medium of α-minimum essential medium (α-MEM, Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum.

[0172] Example 1-3. Isolation and Culture of Human Dental Pulp Cells

[0173] For human dental pulp cells, dental pulp cells were isolated from the wisdom teeth of 10 adults (18 - 22 years old) at Seoul National University Dental Hospital. Specifically, all experiments were conducted after approval by the hospital's Institutional Review Board and with the consent of the patients. According to the method of Jung HS et al. (J Mol Histol. (2011)), the wisdom teeth were cut to expose the dental pulp, and the dental pulp was separated using forceps. The separated dental pulp was minced with a razor blade and placed in a 60 mm petri dish. After covering with a coverslip, it was cultured in Dulbecco's modified Eagle's medium. It is well known that human dental pulp cells can differentiate into odontoblasts, osteoblasts, cementoblasts, and periodontal ligament cells under various conditions (Tissue Eng Part A. 2014 Apr;20(7 - 8):1342 - 51).

[0174] Example 1-4. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Real-Time PCR Analysis

[0175] Total RNA of human dental pulp cells and mesenchymal stem cells was isolated using TRIzol reagent. cDNA was synthesized using 2 μg of total RNA, 1 μl of reverse transcriptase, and 0.5 μg of oligo (dT). The synthesized cDNA was used for real-time polymerase chain reaction. Real-time polymerase chain reaction was performed using SYBR GREEN PCR Master Mix (Takara, Japan) in an ABIPRISM 7500 sequence detection system (Applied Biosystems). Real-time polymerase chain reaction was performed under the condition of repeating 40 cycles in the manner of 1 minute at 94 °C, 15 seconds at 95 °C, and 34 seconds at 60 °C. As a result analysis, evaluation was performed using the comparative cycle threshold (CT) method, and the base sequences of the primers are shown in Table 9 below.

[0176]

Table 9

[0177] Table 9. Nucleotide Sequences of Real-Time PCR Primers

[0178]

[0179] Examples 1-5. In Vivo Transplantation and Histological Analysis

[0180] Human dental pulp cells (hDPCs) were isolated and used for in vivo transplantation experiments. hDPCs (2x10 6 cells) were mixed alone with 100 mg of hydroxy apatite / tricalcium phosphate (HA / TCP) ceramic powder (Zimmer, USA), or together with a peptide (10 μg), and with 0.5% fibrin gel, and then transplanted into immune system-compromised mice (NIH-bg-nu-xid; Harlan Laboratories, Indianapolis, IN) for 6 weeks and 12 weeks. Thereafter, samples were harvested and fixed in 4% paraformaldehyde, decalcified in 10% ethylenediaminetetraacetic acid (EDTA) (pH 7.4), embedded in paraffin, and stained with hematoxylin-eosin (H-E) (Vector Labs).

[0181] Examples 1-6. Effects of the Novel Peptide on Dentin Regeneration in a Dentin Injury Model

[0182] Three beagle dogs (12 to 16 kg; 6 to 8 weeks old) were anesthetized by inhaling Gerolan. After intravenous injection of Zoletil (5 mg / kg) and xylazine (0.2 - 0.5 mg / kg), lidocaine (2% lidocaine with 1:80,000 epinephrine) was administered. In the mandible of the beagle dogs, for the premolars and molars, after dentin was damaged using a dental burr, 10 μg of the peptide with SEQ ID NO: 1 in Group 1 was applied to each tooth. After 6 weeks, the beagle dogs were sacrificed by administering an overdose (90 - 120 mg / kg) of pentobarbital. The tooth parts of the beagle dogs were removed, fixed with 10% formalin, then demineralized with 5% formic acid, shaped and embedded in paraffin. After obtaining tissue sections with a thickness of 5 μm, the obtained tissue sections were stained with hematoxylin and eosin, and then analyzed using an optical microscope (LEICA DM750, Germany) equipped with a digital camera (LEICA ICC50 camera, Germany).

[0183] According to the embodiments of the present invention, for statistical analysis, the Student t-test was performed using SPSS software ver. 19.0.

[0184] - Effect of the peptide for promoting the regeneration of dentin or dental pulp tissue and for treating dentin hypersensitivity on the expression level of the DSPP gene, which is a marker gene for odontoblast differentiation

[0185] It is well known that the DSPP gene is used as a marker for odontoblast differentiation and is an important gene for dentin calcification. Therefore, in this study, by increasing the expression of the DSPP gene, which is a marker gene for odontoblast differentiation, a new peptide that promotes odontoblast differentiation and dentin formation was synthesized, and its effect was confirmed.

[0186] The effect of each peptide group on the mRNA expression of DSPP was confirmed by real-time PCR (Table 10).

[0187]

Table 10

[0188] Effect of the peptides in Groups 1 to 8 on the mRNA level of the DSPP gene

[0189]

[0190]

[0191]

[0192] Figure 1 It is the result of averaging the mRNA expression values of DSPP shown in each group of Table 10 and presenting them in the graph. In Figure 1 , regarding the mRNA expression of DSPP, compared with the control group, it increased by about 2 to more than 8 times in the peptide groups of all groups. In particular, the peptide group of Group 1 showed the highest mRNA expression value of DSPP. As is well known, each of these differentiation marker genes is a gene involved in the differentiation of odontoblasts and the calcification process of dentin. Therefore, based on the analysis, the peptides provided in the present invention can show the effect of promoting dentin regeneration.

[0193] - Peptides for promoting cementum regeneration and for treating periodontal diseases, the influence on the expression level of the BSP gene, which is a differentiation marker gene for osteoblasts and cementoblasts

[0194] As is well known, the BSP and DMP1 genes are used as differentiation markers for osteoblasts and cementoblasts and are important genes for the calcification of bone and cementum. The influence of the peptide groups of each group on the mRNA expression of BSP and DMP1 was confirmed by real-time PCR (Tables 11 and 12).

[0195]

Table 11

[0196] The effect of the peptides of Groups 1 to 8 on the mRNA level of the BSP gene

[0197]

[0198]

[0199]

[0200]

Table 12

[0201] The effect of the peptides of Groups 1 to 8 on the mRNA level of the DMP1 gene

[0202]

[0203]

[0204]

[0205] In Figure 2In order to confirm the effect of the novel peptide on the expression of BSP and DMP1 genes, which are marker genes for osteoblast and cementoblast differentiation, after treating human-derived bone marrow mesenchymal stem cells with each group of peptides, the expression of BSP and DMP1 genes was confirmed by real-time PCR. Compared with the control group, the BSP gene expression in all groups of peptides increased by about 1.5 to 3 times, and the DMP1 gene expression increased by more than 6 to 10 times. In particular, the peptide group of Group 1 showed the highest mRNA expression values of BSP and DMP1. As is well known, the BSP and DMP1 genes are used as marker genes for osteoblast and cementoblast differentiation and are genes involved in the calcification process of bone and cementum. According to the analysis, the peptides provided in the present invention can show the effect of promoting bone and cementum regeneration.

[0206] Figure 3 To confirm the effect of the novel peptide on the expression of odontoblast, osteoblast, and cementoblast differentiation marker proteins, after treating human dental pulp cells with the novel peptide (SEQ ID NO: 1) in a concentration-dependent manner (1, 10, 50 μg), the expression of DSP and BSP proteins was confirmed by Western blot.

[0207] Reference Figure 3 , it was confirmed that the expression of DSP, which is a marker protein for odontoblast differentiation, and BSP, which is a marker protein for osteoblast and cementoblast differentiation, increased in a concentration-dependent manner through the novel peptide.

[0208] Figure 4 To confirm whether the novel peptide induces physiological dentin regeneration in a dental injury animal model, after injuring the dentin of the teeth of beagle dogs, 10 μg of the novel peptide (SEQ ID NO: 1) was applied, and then histological evaluation was performed three weeks later.

[0209] Reference Figure 4 , although there were no changes under the injured dentin in the control group, in the experimental group treated with the novel peptide, the formation of physiological dentin was confirmed under the injured dentin. Such results mean that tooth hypersensitivity, dentin caries, and pain caused by tooth fracture induced by injured dentin can be treated by physiological dentin regeneration.

[0210] Figure 5 To according to Figure 2Based on the in vitro experimental results shown in Tables 10 to 11, the peptide effect of Group 1 (SEQ ID NO: 1) on hard tissue formation in vivo was confirmed. The peptide of SEQ ID NO: 1 in Group 1 (10 μg), or human dental pulp cells (hDPCs) of the control group (cell only) were transplanted into the subcutaneous tissue of immune-compromised mice. After 12 weeks of transplantation, compared with the control group, an increased ratio of hard tissue formation was confirmed in the group treated with the novel peptide (A-D: hDPCs - untreated; E-H: treated with the peptide of Group 1; I-L: treated with BMP2 / Scale bar: A, E, I: 500 μm; B, F, J: 200 μm; C, G, K: 100 μm; D, H, L: 50 μm).

[0211] Reference Figure 5 , in the histomorphological analysis by hematoxylin-eosin staining, in the groups treated with hDPCs-untreated, the peptide of Group 1, and BMP2 as the positive control group, a cell-embedded bone / cementum-like tissue was observed to form in the matrix of newly formed calcified tissue around the HA / TCP particles. However, compared with the control group, a harder tissue more similar to the group treated with BMP-2 was observed to form in the experimental group treated with the novel peptide. In summary, from the above results, it can be seen that the novel peptide used in this experiment shows an effect of promoting the regeneration of bone / cementum-like tissue and dentin / pulp tissue complex.

[0212] Figure 6 To confirm the effect of the peptide of Group 1 (SEQ ID NO: 1) on periodontal ligament formation in vivo, the peptide of SEQ ID NO: 1 in Group 1 (10 μg) or human dental pulp cells (hDPCs) of the control group (cell only) were transplanted into the subcutaneous tissue of immune-compromised mice. After 6 weeks of transplantation, the results were compared with the control group (Scale bar; A, C: 200 μm; B, D: 100 μm).

[0213] Reference Figure 6 , after 6 weeks of transplantation, compared with the control group, periodontal ligament fiber bundles (black triangles) were confirmed to form in the group treated with the novel peptide. Specifically, in the histomorphological analysis by hematoxylin-eosin staining, in the case of hDPCs-untreated, irregular fiber arrangements were observed, but in the group treated with the novel peptide of Group 1, a periodontal ligament-like tissue embedded with fiber bundles was observed to form as the newly formed calcified tissue around the HA / TCP particles. From the above results, it can be seen that the novel peptide used in this experiment can show an effect of promoting the regeneration of damaged periodontal ligament.

[0214] This research is based on the research supported by the Ministry of Health and Welfare and the Korea Health Industry Development Institute in 2022 (1465037227, "Development of treatment technology for chronic periodontitis using peptides of bio-convergent new material CPNE7").

[0215] Preferred embodiments of the present invention are disclosed in this specification and the accompanying drawings. Although specific terms are used, these are merely used in a general sense for the purpose of easily explaining the technical content of the present invention and facilitating understanding of the invention, and are not used to limit the scope of the present invention. Other variations based on the technical idea of the present invention can also be implemented in addition to the embodiments disclosed herein, which will be self-evident to those of ordinary skill in the technical field to which the present invention pertains.

Claims

1. A peptide for promoting the regeneration of hard tissue or pulp tissue and for treating dentin-pulp diseases or periodontal diseases, wherein, It is composed of any one of the amino acid sequences of SEQ ID NO: 1 to 8.

2. The peptide according to claim 1, wherein, The peptide is formed by acetylation or amidation at the N-terminus or C-terminus.

3. The peptide according to claim 1, wherein, The hard tissue includes dentin, bone and cementum.

4. A polynucleotide encoding the peptide according to claim 1.

5. An expression vector comprising the polynucleotide according to claim 4.

6. A pharmaceutical composition comprising the peptide according to claim 1 for preventing or treating dentin-pulp diseases.

7. The composition according to claim 6, wherein, The dentin-pulp diseases are dentin hypersensitivity, pulp hyperemia, pulpitis, pulp degeneration or necrosis and gangrene of the pulp.

8. A pharmaceutical composition comprising the peptide according to claim 1 for preventing or treating periodontal diseases.

9. The composition according to claim 8, wherein, The periodontal diseases are gingivitis, periodontitis, periodontal pocket or periodontal abscess.

10. A pharmaceutical external composition comprising the peptide according to claim 1 for preventing or improving dentin-pulp diseases or periodontal diseases.

11. A health functional food composition comprising the peptide according to claim 1 for preventing or improving dentin-pulp diseases or periodontal diseases.

12. A method for preventing or treating dentin-pulp diseases, comprising the step of administering a composition containing the peptide according to claim 1 to an individual other than a human.

13. A method for preventing or treating periodontal diseases, comprising the step of administering a composition containing the peptide according to claim 1 to an individual other than a human.

14. A method for promoting the regeneration of hard tissue or pulp tissue including dentin, bone and cementum, comprising the step of administering a composition containing the peptide according to claim 1 to an individual other than a human.

15. The peptide according to claim 1, wherein, It can be composed of any one of the amino acid sequences of SEQ ID NO: 1 to 8, for promoting the regeneration of hard tissue or pulp tissue and for treating dentin-pulp diseases or periodontal diseases.

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