Novel peptide

Abstract

The present invention provides a peptide for promoting the regeneration of hard tissue and / or dental pulp tissue and for treating dentin-pulp disease and / or periodontal disease, as well as a pharmaceutical composition, a quasi-drug composition, and a functional health food composition containing the peptide. [Solution] In one embodiment, the present invention provides a peptide comprising an amino acid sequence of the general formula KY-R1-R2-R3-R4-R5 (R1 and R2 are arginine, lysine, glutamine, or asparagine, respectively, and R3, R4, and R5 are arginine or lysine, respectively), which has been modified by acetylation, amidation, or methylation at the N-terminus or C-terminus, introduction of a D-amino acid, modification of a peptide bond of CH2-NH, CH2-S, CH2-S=O, or CH2-CH2, backbone modification, and side chain modification; and a pharmaceutical composition, a quasi-drug composition, and a functional health food composition each containing the peptide.

Description

Technical Field

[0001] The present invention relates to novel peptides, and more specifically, peptides for promoting the regeneration of hard tissues and / or pulp tissues and for treating dentin-pulp diseases and / or periodontal diseases, polynucleotides encoding the peptides, expression vectors containing the polynucleotides, and pharmaceutical compositions for preventing or treating dentin-pulp diseases and / or periodontal diseases containing the peptides, quasi-drug compositions for preventing or improving dentin-pulp diseases and / or periodontal diseases, and health functional food compositions for preventing or improving the same.

Background Art

[0002] The pulp is a soft connective tissue that occupies the pulp cavity inside the tooth, is rich in nerves and blood vessels, and refers to the part that reaches the surface of the dentin. Such lesions occurring in the pulp are called pulp diseases.

[0003] The causes of pulp diseases are very diverse, but in most cases, they occur due to bacterial infection by dental caries or infection into the pulp through dental perforation, fracture, crack, or periodontal pocket. Also, trauma, abrasion, tooth crack, heat or friction from dental instruments during treatment can also induce pulp diseases. Pulpitis caused by bacterial infection can spread to periapical diseases and periodontal diseases. When a pulp disease occurs, it progresses in order to pulp congestion, pulpitis, and pulp necrosis. In the case of pulp necrosis, the pulp dies and blood supply to the pulp becomes impossible, and the entire pulp tissue is lost, which may ultimately lead to periapical diseases or abnormalities of the entire tooth.

[0004] For the treatment of pulp and apical diseases, pulp capping materials and root canal filling materials are used, and generally, calcium hydroxide, MTA (Mineral Trioxide Aggregate), and gutta-percha have been used. In the case of MTA, it is effective in treatment due to its sealing ability and biocompatibility, but it has the problem of being relatively high cost as a dental treatment material and aesthetic problems due to discoloration. Gutta-percha is relatively low cost and has good fluidity, but it is a physiologically unsuitable method because it causes loss of pulp viability. Until now, conservative treatment methods for dentin-pulp diseases have resulted in treated teeth becoming brittle or easily fractured, and there is a risk of reinfection.

[0005] Periodontal tissue is a complex organ composed of epithelial tissue, soft connective tissue, and calcified connective tissue. The structure of periodontal tissue includes the gingiva, periodontal ligament (PDL), cementum, and alveolar bone. Gingival fibroblasts and periodontal ligament fibroblasts are major cellular components of gingival soft connective tissue, forming and maintaining the extracellular matrix. While gingival fibroblasts are primarily involved in maintaining gingival connective tissue, periodontal ligament fibroblasts, due to their unique functions, are known to not only form periodontal ligaments but also to be involved in the repair and regeneration of adjacent alveolar bone and cementum within the body. When periodontal disease develops, it clinically leads to tooth loss due to gingival bleeding and swelling, periodontal pocket formation, and alveolar bone destruction.

[0006] The ultimate goal of treating periodontal disease is to restore damaged connective tissue, chalk, and alveolar bone. This requires not only the regeneration of periodontal ligaments that support the alveolar bone, but also the regeneration of alveolar bone and chalk to which the periodontal ligaments can attach.

[0007] As a result, research is actively being conducted to develop therapeutic agents that can effectively treat the aforementioned dentin-pulp disease or periodontal disease. For example, Patent Document 1 discloses a composition for hard tissue formation and dentin or pulp tissue regeneration containing ameloblasts, apical bud cells, or culture media thereof as active ingredients, and Patent Document 2 discloses novel dental stem cells derived from dental follicles and a method for culturing them. Furthermore, Patent Document 3 discloses a therapeutic composition for periodontal disease containing ameloblast culture media.

[0008] Against this backdrop, the inventors diligently conducted research to develop a formulation that can more effectively treat dentin-pulp disease and / or periodontal disease that induces damage to alveolar bone and chalk. As a result, they developed a cell therapy for promoting the regeneration of hard tissues including dentin, bone, and chalk, and / or pulp tissue, as well as a peptide that exhibits therapeutic effects on dentin-pulp disease and / or periodontal disease, thus completing the present invention. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Korean Published Patent Publication No. 2012-0089547 [Patent Document 2] Korean Published Patent Publication No. 2009-0033643 [Patent Document 3] Korean Published Patent Publication No. 2016-0105627 [Overview of the project] [Problems that the invention aims to solve]

[0010] One objective of the present invention is to provide peptides for promoting the regeneration of hard tissue and / or dental pulp tissue, and for the treatment of dentin-pulp disease and / or periodontal disease.

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

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

[0013] Another object of the present invention is to provide a pharmaceutical composition comprising the peptide for the prevention or treatment of dentin-pulp disease and / or periodontal disease.

[0014] Another object of the present invention is to provide a quasi-drug composition comprising the peptide for the prevention or improvement of dentin-pulp disease and / or periodontal disease.

[0015] Another object of the present invention is to provide a health functional food composition comprising the peptide for the prevention or improvement of dentin-pulp disease and / or periodontal disease.

[0016] Another object of the present invention is to provide a method for preventing or treating dentin-pulp disease and / or periodontal disease, comprising 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, including dentin, bone, and chalk, and / or dental pulp tissue, comprising the step of administering a composition containing the peptide to an individual other than a human.

[0018] Another object of the present invention is to provide the peptide for use in promoting the regeneration of hard tissue and / or dental pulp tissue.

[0019] Another object of the present invention is to provide the peptide for use in the prevention or treatment of dentin-pulp disease or periodontal disease.

[0020] The objects of the present invention are not limited to those mentioned above, and any other objects not mentioned can be clearly understood by a person of ordinary skill in the art from the following description. [Means for solving the problem]

[0021] As one embodiment for achieving the above object, the present invention provides a peptide containing an amino acid sequence of the following general formula (1).

[0022] K - Y - R1 - R2 - R3 - R4 - R5 ··· General formula (1) In the general formula (1), R1 and R2 are each arginine (R), lysine (K), glutamine (Q), or asparagine (N), and R3, R4, and R5 are each arginine (R) or lysine (K).

[0023] According to one example, the peptide may contain any one of the amino acid sequences of SEQ ID NOs: 1 to 128, or may be composed of any one of the amino acid sequences of SEQ ID NOs: 1 to 128.

[0024] According to one example, the peptide may contain any one of the amino acid sequences of SEQ ID NOs: 1 to 24, or may be composed of any one of the amino acid sequences of SEQ ID NOs: 1 to 24.

[0025] According to one example, in the general formula (1), R1 may be glutamine (Q) and R2 may be arginine (R).

[0026] According to one example, in the general formula (1), R1 may be arginine (R) or lysine (K), and R2 may be glutamine (Q).

[0027] According to one example, the peptide may contain any one of the amino acid sequences of SEQ ID NOs: 25 to 48, or may be composed of any one of the amino acid sequences of SEQ ID NOs: 25 to 48.

[0028] According to one example, in the general formula (1), R1 and R2 may each be glutamine (Q).

[0029] According to one embodiment, in the general formula (1) above, R1 is arginine (R) or lysine (K), and R2 may be arginine (R).

[0030] According to one embodiment, the peptide may contain any one amino acid sequence from SEQ ID NOs. 49 to 128, or may consist of any one amino acid sequence from SEQ ID NOs. 49 to 128.

[0031] According to one embodiment, in the general formula (1) above, R1 is glutamine (Q), arginine (R), or lysine (K), and R2 may be lysine (K).

[0032] According to one embodiment, in the general formula (1) above, R1 is arginine (R), lysine (K), glutamine (Q), or asparagine (N), and R2 is arginine (R), lysine (K), glutamine (Q), or asparagine (N), and at least one of R1 and R2 may be asparagine (N).

[0033] According to one example, the peptide may be acetylated, amidated, or methylated at the N-terminus or C-terminus, have a D-amino acid introduced, have peptide bond modifications such as CH2-NH, CH2-S, CH2-S=O, or CH2-CH2, have a backbone modification, or have a side chain modification.

[0034] According to one embodiment, the peptide may be used to promote the regeneration of hard tissue and / or dental pulp tissue, and for the treatment of dentin-pulp disease and / or periodontal disease.

[0035] According to one embodiment, the hard tissue may include dentin, bone, and chalk.

[0036] The aforementioned dentin-pulp diseases may include dentin hypersensitivity, pulp congestion, pulpitis, pulp degeneration, and / or necrotic or gangrenous pulp.

[0037] The aforementioned periodontal diseases may include gingivitis, periodontitis, periodontal pockets, and / or periodontal abscesses.

[0038] In another embodiment, the present invention provides a polynucleotide encoding the peptide.

[0039] In another embodiment, the present invention provides an expression vector containing the polynucleotide.

[0040] In another embodiment, the present invention provides a composition containing the peptide.

[0041] In one embodiment, the composition may be a pharmaceutical composition for the prevention or treatment of dentin-pulp disease.

[0042] In one embodiment, the composition may be a pharmaceutical composition for the prevention or treatment of periodontal disease.

[0043] In another embodiment, the present invention provides a quasi-drug composition containing the peptide for the prevention or improvement of dentin-pulp disease and / or periodontal disease.

[0044] In another embodiment, the present invention provides a health functional food composition containing the peptide for the prevention or improvement of dentin-pulp disease and / or periodontal disease.

[0045] In another embodiment, the present invention provides a method for preventing or treating dentin-pulp disease, comprising the step of administering a composition containing the peptide to an individual having dentin-pulp disease.

[0046] In another embodiment, the present invention provides a method for preventing or treating periodontal disease, comprising the step of administering a composition containing the peptide to an individual having periodontal disease.

[0047] In another aspect, the present invention provides a method for promoting the regeneration of hard tissues, including dentin, bone, and chalk, and / or dental pulp tissue, comprising the step of administering a composition containing the peptide to an individual.

[0048] The aforementioned individual may be a human or an individual other than a human.

[0049] In another aspect, the present invention provides the peptide for the prevention or treatment of dentin-pulp disease.

[0050] In another aspect, the present invention provides the peptide for use in the prevention or treatment of periodontal disease.

[0051] In another aspect, the present invention provides applications for promoting the regeneration of hard tissues, including dentin, bone, and chalk, and / or dental pulp tissue. [Effects of the Invention]

[0052] The peptides of the present invention, which promote the regeneration of hard tissue and / or dental pulp tissue, and for the treatment of dentin-pulp disease and / or periodontal disease, exhibit excellent regeneration-promoting effects on hard tissue and / or dental pulp tissue, and can therefore be widely used in the development of formulations for the prevention or treatment of various dentin-pulp diseases, or in the development of formulations for the prevention or treatment of periodontal disease that induces bone and / or chalk damage. [Brief explanation of the drawing]

[0053] [Figure 1a] Figure 1a is a graph showing the results of comparing the expression levels of the Dspp (Dentin sialophosphoprotein) gene, an odontoblast differentiation marker, in human dental pulp cells (hDPCs) treated with the novel peptide of the present invention. [Figure 1b] Figure 1b is another graph showing the results of comparing the expression levels of the Dspp gene, an odontoblast differentiation marker, in human dental pulp cells (hDPCs) treated with the novel peptide of the present invention. [Figure 1c] Figure 1c is a graph showing the results of comparing the expression levels of the Nestin gene, an odontoblast differentiation marker, in human dental pulp cells (hDPCs) treated with the peptide of the present invention. [Figure 2a] Figure 2a is a graph showing a comparison of the expression levels of the BSP gene, a differentiation marker for bone and chalk, in human mesenchymal stem cells (hBMSCs) treated with the novel peptide of the present invention. [Figure 2b] Figure 2b is another graph showing the results of comparing the expression levels of the BSP gene, a differentiation marker for bone and chalk, in human mesenchymal stem cells (hBMSCs) treated with the novel peptide of the present invention. [Figure 3] Figure 3 shows the results of measuring the amount of newly formed hard tissue using human dental pulp cells (hDPCs) in vivo over a 6-week period. [Figure 4] Figure 4 shows micrographs of histologically analyzed hard tissue formed in vivo for 6 weeks using human dental pulp cells (hDPCs). A-D show control implants prepared by mixing hDPCs and 100 mg of HA / TCP in 0.5% fibrin gel, while E-H show the results of implanting implants prepared by mixing hDPCs and 100 mg of HA / TCP with 10 μg of group 3 peptide in 0.5% fibrin gel into immune-weakened mice for 6 weeks (size bars: A, E 500 μm, B, F 200 μm, C, G 100 μm, D, H 50 μm). [Figure 5] Figure 5 shows micrographs illustrating the level of collagen formation in hard tissue formed in vivo using human dental pulp cells (hDPCs) for 6 weeks. A-D show the results of implants prepared by mixing hDPCs and 100 mg of HA / TCP in 0.5% fibrin gel for the control group, while E-H show the results of implants prepared by mixing hDPCs and 100 mg of HA / TCP with 10 μg of group 3 peptide in 0.5% fibrin gel for 6 weeks in mice with weakened immune systems (size bars: A, E 500 μm, B, F 200 μm, C, G 100 μm, D, H 50 μm). [Figure 6]Figure 6 shows immunohistochemical staining images of hard tissue formed in vivo using human dental pulp cells (hDPCs) for 6 weeks, analyzing the expression level of DSP, an odontoblast differentiation marker, by immunohistochemical staining. A shows the results of implanting a control group implant prepared by mixing hDPCs and 100 mg of HA / TCP in 0.5% fibrin gel, and B shows the results of implanting implants prepared by mixing hDPCs and 100 mg of HA / TCP with 10 μg of group 3 peptide in 0.5% fibrin gel in mice with weakened immune systems for 6 weeks. A and B show the formed hard tissue immunostained with anti-DSP antibody. C is the negative control group for immunohistochemical analysis treated only with secondary antibody. The arrows in A and B indicate DSP expression in the newly formed calcified tissue. The size bars are 50 μm. [Figure 7] Figure 7 shows the results of measuring the amount of newly formed hard tissue using human dental pulp cells (hDPCs) in vivo over a 12-week period. [Figure 8] Figure 8 shows micrographs of histologically analyzed hard tissue formed in vivo for 12 weeks using human dental pulp cells (hDPCs). A-D show the results of implants prepared by mixing hDPCs and 100 mg of HA / TCP in 0.5% fibrin gel for the control group, while E-H show the results of implants prepared by mixing hDPCs and 100 mg of HA / TCP with 10 μg of group 3 peptide in 0.5% fibrin gel for 12 weeks in mice with weakened immune systems (size bars: A, E 500 μm, B, F 200 μm, C, G 100 μm, D, H 50 μm). [Figure 9]Figure 9 is a micrograph showing the level of collagen formation in hard tissue formed in vivo using human dental pulp cells (hDPCs) for 12 weeks. A-D show the results of implants prepared by mixing hDPCs and 100 mg of HA / TCP in 0.5% fibrin gel for the control group, while E-H show the results of implants prepared by mixing hDPCs and 100 mg of HA / TCP with 10 μg of group 3 peptide in 0.5% fibrin gel for 12 weeks in mice with weakened immune systems (size bars: A, E 500 μm, B, F 200 μm, C, G 100 μm, D, H 50 μm). [Figure 10] Figure 10 shows immunohistochemical staining images of hard tissue formed in vivo for 12 weeks using human dental pulp cells (hDPCs), analyzing the expression level of DSP, an odontoblast differentiation marker, by immunohistochemical staining. A shows the results of implanting a control group implant prepared by mixing hDPCs and 100 mg of HA / TCP in 0.5% fibrin gel, and B shows the results of implanting implants prepared by mixing hDPCs and 100 mg of HA / TCP with 10 μg of group 3 peptide in 0.5% fibrin gel in mice with weakened immune systems for 12 weeks. A and B show the formed hard tissue immunostained with anti-DSP antibody. C is the negative control group for immunohistochemical analysis treated only with secondary antibody. The arrows in A and B indicate DSP expression in the newly formed calcified tissue. The size bars are 50 μm. [Figure 11] Figure 11 is an SEM image showing the results of scanning electron microscopy (SEM) analysis of hard tissue formed in vivo using human dental pulp cells (hDPCs) for 12 weeks. A shows the results of implanting a control group implant prepared by mixing hDPCs and 100 mg of HA / TCP in 0.5% fibrin gel, while B and C show the results of implanting implants prepared by mixing hDPCs and 100 mg of HA / TCP with 10 μg of group 3 peptide in 0.5% fibrin gel, respectively, into mice with weakened immune systems for 12 weeks. The size bar is 10 μm. [Figure 12] Figure 12 shows the results of scanning electron microscopy observation of the regeneration and closure of damaged dentinal tubules with physiological dentin. Specifically, Figures 12B, C, and D are magnified images of Figure 12A, respectively. Also, Figures 12F, G, and H are magnified images of Figure 12E, respectively (size bars A: 1 mm, B: 50 μm, C: 20 μm, D: 10 μm, E: 1 mm, F: 50 μm, G: 20 μm, H: 10 μm). [Figure 13] Figure 13 shows the results of scanning electron microscopy observation of dentinal tubules exposed on the surface of damaged dentin being closed by physiological remineralization. Specifically, Figures 13B, C, and D are magnified images of Figure 13A, respectively. Also, Figures 13F, G, and H are magnified images of Figure 13E, respectively (size bars A: 1 mm, B: 50 μm, C: 20 μm, D: 10 μm, E: 1 mm, F: 50 μm, G: 20 μm, H: 10 μm). [Modes for carrying out the invention]

[0054] The inventors conducted various studies to develop a formulation that can more effectively treat dentin-pulp disease and / or periodontal disease, and as a result, developed a novel peptide containing or composed of seven amino acids.

[0055] The novel peptide developed as described above was created by substituting a portion of the amino acid sequence of a peptide that can exhibit therapeutic effects on dentin-pulp disease and / or periodontal disease. It can increase the expression levels of the Dspp and Nestin genes, which are odontoblast differentiation markers, thereby promoting dentin regeneration. It can also increase the expression level of the BSP (Bone sialoprotein) gene, which is a differentiation marker for osteoblasts and chalkblasts, thereby promoting bone and chalk regeneration.

[0056] Furthermore, implants containing the peptide along with human dental pulp cells were fabricated, and these implants were transplanted into the subcutaneous tissue of mice with damaged immune systems. After 6 or 12 weeks, the transplanted tissue was analyzed, and it was confirmed that dentin-pulp-like tissue with a morphology most similar to in vivo dentin-pulp tissue was formed, as was bone-like tissue with a morphology most similar to in vivo bone tissue, collagen formation levels increased, and the expression level of the DSP gene, a specific differentiation marker for odontoblasts, increased.

[0057] Furthermore, analysis of the shape of the transplanted tissue using a scanning electron microscope revealed the presence of odontoblast-like cells along the formed hard tissue, and it was confirmed that the odontoblast processes also extended in the direction of the formed hard tissue. It was also confirmed that the tissue exhibited typical osteoblast and / or cementoblast characteristics, with cuboidal cells attached to the surface of the formed hard tissue.

[0058] Therefore, the peptides of the present invention have been found to promote the regeneration of hard tissue and / or dental pulp tissue, and to be effective against dentin-pulp disease and / or periodontal disease. Peptides of the present invention exhibiting such effects have never been reported before and were first developed by the inventors.

[0059] As one embodiment, the present invention provides a peptide comprising the amino acid sequence of the following general formula (1) for promoting the regeneration of hard tissue and / or dental pulp tissue, and for the treatment of dentin-pulp disease and / or periodontal disease.

[0060] KY-R1-R2-R3-R4-R5 General formula (1) In the general formula (1) above, R1 and R2 are arginine (R), lysine (K), glutamine (Q), or asparagine (N), respectively, and R3, R4, and R5 are arginine (R) or lysine (K), respectively. Also, in the general formula (1) above, K represents lysine and Y represents tyrosine.

[0061] In this invention, the term "hard tissue" refers to relatively hard bone tissue, including bone, hyaline cartilage, and fibrocartilage. In one embodiment of the present invention, the hard tissue may include dentin, bone, and chalk.

[0062] In this specification, the term "dentin," also known as "tooth structure," refers to the hard, yellowish-white tissue that makes up the majority of the tooth. Although dentin is covered by enamel in the crown and chalk in the root, and therefore does not appear on the tooth surface, as the enamel wears away with age, dentin may be exposed at the tip of the crown and on the occlusal surface. While dentin is a type of bone-like tissue, it is distinguished from general bone tissue in that the main body of the cells that make up dentin is located in the pulp, and only their projections extend into the dentin.

[0063] In this invention, the term "cementum" refers to a thin membrane that is a slightly deformed form of bone covering the tooth root and other parts of a mammal. This cementum is composed of 50% inorganic and 50% water-organic matter, has a yellowish tint, and exhibits lower hardness than dentin or enamel. The cementum contains periodontal ligament fibers that fix the tooth to the alveolar bone. However, when bacteria infect the gums, degeneration occurs in the cementum surrounding the tooth. In the degenerated cementum, the periodontal ligament fibers that connect the tooth to the alveolar bone can no longer adhere, causing the tooth to become loose. To treat such degeneration of cementum, methods are used that remove the degenerated cementum and promote the formation of new cementum.

[0064] The peptide provided in this invention can increase the expression levels of the Dspp gene and Nestin gene, which are odontoblast differentiation markers, and the BSP gene, which is an osteoblast and chalkblast differentiation marker. When transplanted into vivo together with human dental pulp cells, the human dental pulp cells may exhibit the characteristic of forming dentin / pulp-like tissue and bone-like tissue.

[0065] The peptides provided in this invention are also included in the category of peptides provided in this invention, as long as they can promote the regeneration of hard tissues including dentin, bone, and chalk, and / or pulp tissue, and have therapeutic effects on dentin-pulp diseases and / or periodontal diseases, and include mutant peptides having a sequence in which one or more amino acid residues differ from the amino acid sequence that constitutes them.

[0066] Generally, amino acid exchanges in proteins and polypeptides that do not alter the overall molecular activity are known in the field. The most common exchanges are 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, and Asp / Gly. These may also include peptides whose structural stability to heat, pH, etc., has been increased by mutations or modifications in the amino acid sequence, or peptides whose ability to promote the regeneration of hard tissues, including dentin, bone, and chalk, and / or dental pulp tissue has been increased.

[0067] Amino acid mutations are made based on the relative similarity of amino acid side-chain substitutions, such as hydrophobicity, hydrophilicity, charge, and size. Since all seven amino acids constituting the peptide of the present invention are hydrophilic amino acids, the relative similarity of the amino acid side-chain substitutions is high. Therefore, even if the amino acids constituting the peptide of Sequence ID No. 1 are substituted with various amino acids that have hydrophilic properties, the peptide can still exhibit the effects provided by the present invention due to its structural similarity.

[0068] For example, even if the glutamine located at the third position of the peptide of Sequence ID No. 1 provided in the present invention is replaced with asparagine or with the basic amino acid arginine or lysine, the peptide will still exhibit the same effects; even if the arginine, the basic amino acid located at the fourth position of the peptide of Sequence ID No. 1, is replaced with the basic amino acid lysine or with glutamine or asparagine, the peptide will still exhibit the same effects; even if the arginine, the basic amino acid located at the fifth position of the peptide of Sequence ID No. 1, is replaced with the basic amino acid lysine, the peptide will still exhibit the same effects; and even if the lysine, the basic amino acid located at the sixth or seventh position of the peptide of Sequence ID No. 1, is replaced with the basic amino acid arginine, the peptide will still exhibit the same effects.

[0069] Thus, even if the acidic amino acids or basic amino acids constituting the peptide of the present invention are substituted with different acidic amino acids or basic amino acids, the effects of the peptide provided by the present invention can still be observed. Therefore, it is obvious that peptide variants having a sequence that differs from the amino acid sequence constituting the peptide of the present invention by one or more amino acid residues are also included in the category of peptides provided by the present invention.

[0070] Furthermore, even if the peptide of the present invention has any amino acids attached to its N-terminus or C-terminus, it can still exhibit the same effects as the peptide provided by the present invention, and is therefore included in the category of peptides provided by the present invention. For example, the peptide may have 1 to 300 amino acids attached to its N-terminus or C-terminus. Another example is that the peptide may have 1 to 100 amino acids attached to its N-terminus or C-terminus. Yet another example is that the peptide may have 1 to 24 amino acids attached to its N-terminus or C-terminus.

[0071] The peptides of the present invention may be in a form in which their N-terminus and / or C-terminus are chemically modified, protected with organic compounds, or modified by adding amino acids to the peptide terminus, in order to protect them from protein-cutting enzymes in vivo and increase their stability. In particular, in the case of chemically synthesized peptides, the N- and C-terminus are charged, so to remove such charges, the N-terminus may be acetylated, the N-terminus may be methylated and / or the C-terminus may be amidated, or this may include, but is not limited to, the introduction of D-amino acids, peptide bond modifications such as CH2-NH, CH2S, CH2S=O, CH2-CH2, backbone modifications, and side chain modifications. Methods for preparing peptide mimetic compounds are known in the art, for example, as described in Quantitative Drug Design, CA Ramsden Gd., Choplin Pergamon Press (1992).

[0072] In this invention, the term "backbone modification" refers to the direct modification of amino acids that make up a peptide by replacing them with amino acid analogs. The main chain-like or cyclic skeleton of the amino acids that constitute the peptide is called the backbone (main chain). An amino acid analog is an amino acid in which the nitrogen or α-carbon hydrogen atom of the amino acid backbone has been modified by substitution.

[0073] In this invention, the term "side-chain modification" refers to the modification of amino acid side chains, which are atomic groups branching off from the main chain-like or cyclic skeleton of the amino acids that constitute a peptide. Examples of peptide side-chain modifications include reductive alkylation reactions, amidation with methyl acetylamide, alkylation with acetic anhydride, carbamoylation of the amino group with cyanate, trinitrobenzylation of the amino acid with 2,4,6-trinitrobenzenesulfonic acid (TNBS), alkylation of the amino group with succinic anhydride, or pyridoxylation by reaction with pyridoxal-5-phosphate followed by reduction with NaBH4, and other modifications of the amino group.

[0074] Furthermore, while the peptides of the present invention may be used alone, they may also be used in combination with a carrier permitted as a drug, such as an organic solvent, and may also be used in combination with carbohydrates such as glucose, sucrose, or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers to increase stability and efficacy.

[0075] According to one embodiment of the present invention, 128 peptides corresponding to the general formula (1) provided in the present invention were synthesized, and the effect of the synthesized peptides on the expression level of the Dspp gene, an odontoblast differentiation marker, was investigated. As a result, it was confirmed that the mRNA level of the Dspp gene, an odontoblast differentiation marker, measured from human dental pulp cells treated with the 128 peptides was 8 times or more, 6 times or more, 3 times or more, or at least about 1.5 times higher than the mRNA level of the Dspp gene measured from human dental pulp cells that were not treated with the peptides of the present invention (control group) (Figures 1a, 1b, and Tables 18 to 33).

[0076] Previous studies have shown that increasing the mRNA expression level of Dspp promotes odontoblast differentiation and dentin regeneration. Therefore, the 128 peptides that increase the mRNA level of the Dspp gene were found to promote 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).

[0077] Furthermore, the effect of the synthesized peptides on the expression level of the BSP gene, an osteoblast / chrythroblast differentiation marker, was investigated. As a result, it was confirmed that the mRNA level of the BSP gene, an osteoblast / chrythroblast differentiation marker, in human dental pulp cells treated with the 128 peptides was 13 times or more, 12 times or more, 9 times or more, or at least approximately 3 times higher than the mRNA level of the BSP gene measured in human dental pulp cells that were not treated with the peptides of the present invention (control group) (Figures 2a and 2b).

[0078] As a result, it is known that an increase in BSP mRNA expression levels promotes osteoblast / chalstroblast differentiation and bone and chalk regeneration. Therefore, it was found that the 128 peptides that increase the mRNA level of the BSP gene also promote osteoblast / chalstroblast differentiation and bone and chalk regeneration.

[0079] As another example, the present invention provides a polynucleotide encoding the peptide.

[0080] The polynucleotide may be modified by substitution, deletion, insertion, or combination thereof of one or more bases. When the nucleotide sequence is prepared by chemical synthesis, synthetic methods known in the art, such as those described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), can be used, and synthesis can be carried out using methods such as phosphite triester, phosphoramidite and H-phosphonate methods, PCR, and other autoprimer methods, as well as oligonucleotide synthesis methods on solid supports. For example, the polynucleotide encoding the peptide of the present invention may contain the base sequence of SEQ ID NO: 4.

[0081] In 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.

[0082] As used herein, the term "expression vector" means a recombinant vector capable of expressing a target peptide from a host cell, and a gene construct containing essential regulatory elements operably linked to the gene insert for expression. The expression vector contains expression regulatory elements such as start codons, stop codons, promoters, and operators, wherein the start and stop codons are typically considered part of the nucleotide sequence encoding the polypeptide, must always exert an effect in the organism when the gene construct is administered, and must always be in frame with the coding sequence. The promoter of the vector may be constitutive or inductive.

[0083] In this specification, the term "operably linked" means a state in which a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked so that they can perform their general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA can be operably linked to influence the expression of the coding sequence. The operably linked expression vector can be created using genetic engineering techniques known in the art, and site-specific DNA cleavage and linking can be performed using enzymes and other methods commonly known in the art.

[0084] Furthermore, the expression vector may include a signal sequence for the release of the peptide to facilitate its separation from the cell culture. A specific start signal may also be necessary for the efficient translation of the inserted nucleic acid sequence. These signals include an ATG start codon and adjacent sequences. In some cases, an exogenous translational regulatory signal, which may include an ATG start codon, should be provided. These exogenous translational regulatory signals and start codons may come from a variety of natural and synthetic sources. Expression efficiency may be increased by the introduction of appropriate transcription or translational enhancement factors.

[0085] In addition, the expression vector may further include a protein tag that can be optionally removed using an endopeptidase to facilitate the detection of the peptide.

[0086] In this specification, the term "tag" means a molecule that exhibits quantifiable activity or properties, and may be a fluorescent molecule including a chemifluorescent substance (fluoracer) such as fluorescein, a polypeptide fluorescent substance such as green fluorescent protein (GFP) or an associated protein, or an epitope tag such as a Myc tag, Flag tag, His tag, leucine tag, IgG tag, or streptavidin tag. In particular, when using an epitope tag, a peptide tag consisting of 6 or more amino acid residues is preferably used, and more preferably 8 to 50 amino acid residues.

[0087] In the present invention, the expression vector may contain a nucleotide sequence encoding a peptide for promoting the regeneration of hard tissues including dentin, bone, and white aromatic material and / or pulp tissue, as well as for the treatment of dentin-pulp disease and / or periodontal disease, as described above. Furthermore, the vector used in this process is not particularly limited as long as it can produce the peptide, but is preferably plasmid DNA, phage DNA, etc., and more preferably commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), plasmids derived from Escherichia coli (pYG601BR322, pBR325, pUC118, pUC119, etc.), plasmids derived from Bacillus subtilis (pUB110, pTP5, etc.), plasmids derived from yeast (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.). Since the expression vector exhibits different protein expression levels and modifications depending on the type of host cell, it is desirable to select and use the host cell that is most suitable for the intended purpose.

[0088] The transformants provided in the present invention can be produced by introducing the expression vector provided in the present invention into a host and performing transformation, and can be used to express the polynucleotides contained in the expression vector and produce the peptide. The transformation can be carried out by various methods, including the CaCl2 precipitation method, the Hanahan method which is improved in efficiency by using DMSO (dimethyl sulfoxide) as a reducing agent in the CaCl2 precipitation method, electroporation, calcium phosphate precipitation, plasmofusion, stirring method using silicon carbide fibers, transformation mediated by agrobacteria, transformation using PEG, transformation mediated by dextran sulfate, lipofectamine, and drying / inhibition, but is not particularly limited to these as long as the peptide can be produced. Furthermore, the host used in the production of the transformants is also a bacterial cell such as Escherichia coli, Streptomyces, Salmonella typhimurium, or Saccharomyces cerevisiae. c Yeast cells such as *Erythrina erevisiae* and *Schizosaccharomyces pombe*; fungal cells such as *Pichia pastoris*; insect cells such as *Drosophila* and *Spodoptera frugiperda* Sf9 cells; animal cells such as CHO, COS, NSO, 293, and Bowes melanoma cells; or plant cells, but not particularly limited to these, as long as the peptide can be produced.

[0089] The transformants may also be used in a method for producing peptides of the present invention for promoting the regeneration of hard tissues including dentin, bone, and white algae and / or pulp tissue, and for treating dentin-pulp disease and / or periodontal disease. Specifically, a method for producing peptides of the present invention for promoting the regeneration of hard tissues including dentin, bone, and white algae and / or pulp, and for treating dentin-pulp disease and / or periodontal disease may include (a) culturing the transformants to obtain a culture, and (b) recovering the peptides of the present invention from the culture.

[0090] In this specification, the term "culture" means a method of growing microorganisms under appropriately artificially controlled environmental conditions. In the present invention, the method of culturing the transformants can be carried out by methods known in the art. Specifically, the culture can be carried out by batch processing, fed-batch culture, or repeated fed-batch process, but is not particularly limited to these, as long as it can express and produce peptides for promoting the regeneration of hard tissues including dentin, bone, and white aromatic material and / or pulp tissue, and for the treatment of dentin-pulp disease and / or periodontal disease.

[0091] The culture medium used for cultivation must meet the requirements of the specific strain by adjusting the temperature, pH, etc., under aerobic conditions within a standard culture medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc., using appropriate methods. Possible carbon sources include a mixture of glucose and xylose as the main carbon source, as well as sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances can be used individually or in mixtures. Furthermore, possible nitrogen sources include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate, and organic nitrogen sources such as amino acids and peptones, NZ-amines, meat extracts, yeast extracts, malt extracts, corn maceration, casein hydrolysates, fish or their decomposition products, defatted soy cake or its decomposition products. These nitrogen sources may be used individually or in mixtures. The culture medium may contain monopotassium phosphate, dispotassium phosphate, and corresponding sodium-containing salts as phosphorus sources. Possible phosphorus sources include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium-containing salts. Inorganic compounds such as sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate may be used. In addition to the above substances, growth-essential substances such as amino acids and vitamins may be used.

[0092] Furthermore, appropriate precursors may be used in the culture medium. The raw materials may be added to the culture in a batch, fed-batch, or continuous manner during the culture process, but are not limited to these methods. The pH of the culture can be adjusted using basic compounds such as sodium hydroxide, potassium hydroxide, and ammonia, or acidic compounds such as phosphoric acid or sulfuric acid.

[0093] Furthermore, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble formation. To maintain aerobic conditions, oxygen or an oxygen-containing gas (e.g., air) is injected into the culture. The culture temperature is typically 27°C to 37°C, preferably 30°C to 35°C. Culturing is continued until the amount of peptide produced reaches the desired level. For this purpose, this is usually achieved in 10 to 100 hours.

[0094] Furthermore, the step of recovering the peptide from the culture is carried out by methods known in the art. Specifically, the recovery method may preferably include methods such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), and chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion), but is not particularly limited to these as long as it can be used to recover the produced peptide.

[0095] In another embodiment, the present invention provides a pharmaceutical composition comprising the peptide for the prevention or treatment of dentin-pulp disease.

[0096] As described above, the peptides of the present invention for promoting the regeneration of hard tissues including dentin, bone, and white aromatic material and / or pulp tissue, and for treating dentin-pulp disease and / or periodontal disease, when transplanted into vivo together with human pulp cells, can promote the formation of dentin / pulp-like tissue by human pulp cells. Therefore, they can be used as active ingredients in pharmaceutical compositions for treating dentin-pulp disease resulting from damage to pulp tissue.

[0097] The peptide contained in the pharmaceutical composition may be used in the form of a peptide alone, in the form of a polypeptide in which the peptide is repeated two or more times, or in the form of a complex in which a drug exhibiting therapeutic effects for dentin-pulp disease is bound to the N-terminus or C-terminus of the peptide.

[0098] In this specification, the term "dentin-pulp disease" refers to a disease that develops as a result of damage to the pulp tissue and the dentin bound to it, caused by damage to the pulp tissue.

[0099] In the present invention, the dentin-pulp diseases include, for example, dentin hypersensitivity, pulp congestion, pulpitis, pulp degeneration, pulp necrosis, and gangrenous pulp, but are not particularly limited to these as long as the peptide of the present invention exhibits a therapeutic effect.

[0100] In another embodiment, the present invention provides a pharmaceutical composition for the prevention or treatment of periodontal disease, comprising the peptide.

[0101] As described above, the peptides of the present invention for promoting the regeneration of hard tissues and / or pulp tissue, including dentin, bone, and chalk, and for treating dentin-pulp diseases and / or periodontal diseases, when transplanted into vivo together with human pulp cells, can promote the formation of bone-like tissue by said human pulp cells. Therefore, they can be used as active ingredients in pharmaceutical compositions for treating periodontal diseases that induce bone and / or chalk damage.

[0102] The peptide contained in the pharmaceutical composition may be used in the form of a peptide alone, in the form of a polypeptide in which the peptide is repeatedly bonded two or more times, or in the form of a complex in which a drug exhibiting therapeutic effects for periodontal disease is bonded to the N-terminus or C-terminus of the peptide.

[0103] In this invention, the term "periodontal disease" refers to a disease in which bacteria infect the space between the gums and teeth, damaging the periodontal ligaments and adjacent tissues. It is classified into gingivitis and periodontitis depending on the severity of the disease. When a periodontal disease develops, as inflammation progresses, more tissues are damaged and a periodontal pocket is formed. The more severe the periodontitis, the deeper the periodontal pocket becomes. It is known that as the periodontal pocket deepens, inflammation occurs in the periodontal ligaments, ultimately inducing bone loss.

[0104] In the present invention, the periodontal disease includes, for example, gingivitis, periodontitis, periodontal pocket, or periodontal abscess, but is not particularly limited to these as long as the peptide of the present invention exhibits a therapeutic effect.

[0105] In this invention, the term "prevention" means all actions that inhibit or delay the onset of dentin-pulp disease by administering a pharmaceutical composition for the prevention or treatment of dentin-pulp disease containing the peptide of the present invention, or all actions that inhibit or delay the onset of periodontal disease by administering a pharmaceutical composition for the prevention or treatment of periodontal disease containing the peptide of the present invention.

[0106] In this invention, the term "treatment" means all actions that enable the treatment of dentin-pulp disease by 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 disease, thereby promoting the regeneration of dentin or pulp tissue, or all actions that enable the treatment of periodontal disease by administering a pharmaceutical composition containing the peptide of the present invention as an active ingredient to an individual in need of treatment for periodontal disease, thereby promoting the regeneration of bone and / or chalk.

[0107] The pharmaceutical compositions of the present invention may be prepared in the form of pharmaceutical compositions for the treatment of dentin-pulp disease and / or periodontal disease, further comprising, in addition to the peptide, a suitable carrier (natural or non-natural carrier), excipient, or diluent commonly used in the preparation of pharmaceutical compositions. Specifically, each of the pharmaceutical compositions may be used in the form of a sterile injection solution that can be administered to the site where dentin-pulp disease and / or periodontal disease is induced, by conventional methods. Examples of carriers, excipients, and diluents that may be included in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, collagen, and the like. When formulated, these can be prepared using commonly used fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients. In particular, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, suppositories, and ointments (e.g., pulp liners) may be used. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, tween 61, cocoa butter, lauric acid butter, and glycerol gelatin.

[0108] The content of the peptide contained in the pharmaceutical composition of the present invention is not particularly limited, but may be 0.0001% to 50% by weight, more preferably 0.01% to 20% by weight, based on the total weight of the final composition.

[0109] The pharmaceutical compositions of the present invention may be administered in pharmaceutically effective amounts, where "pharmaceutically effective amount" as used herein means an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dose level is determined based on the severity of the disease, the activity of the drug, the patient's age, weight, health status, sex, the patient's sensitivity to the drug, the time of administration, route of administration, and elimination ratio of the composition of the present invention used, the duration of treatment, the drugs used in combination with or concurrently with the composition of the present invention, and other factors known in the medical field. The pharmaceutical compositions of the present invention may be administered alone or in combination with known pharmaceutical compositions for the treatment of dentin-pulp disease and / or periodontal disease. Taking all of the above factors into consideration, it is important to administer an amount that provides the greatest effect with the minimum amount without side effects.

[0110] The dosage of the pharmaceutical composition of the present invention can be determined by those skilled in the art, taking into consideration the intended use, the severity of the disease, the patient's age, weight, sex, medical history, or the type of substance used as the active ingredient. For example, the pharmaceutical composition of the present invention can be administered to an adult at a dose of about 0.1 ng / kg to about 100 mg / kg, preferably 1 ng / kg to about 10 mg / kg. The frequency of administration of the composition of the present invention can be once a day or divided into doses and administered several times a day, but is not particularly limited thereto. The aforementioned dosage does not limit the scope of the present invention in any way.

[0111] In other embodiments, the present invention provides a method for treating dentin-pulp disease, comprising the step of administering the aforementioned pharmaceutical composition in a pharmaceutically effective amount to a human or non-human individual who has developed dentin-pulp disease. In yet another embodiment, the present invention provides a method for treating periodontal disease, comprising the step of administering the aforementioned pharmaceutical composition in a pharmaceutically effective amount to a human or non-human individual who has developed periodontal disease.

[0112] As used herein, the term "individual" may include, without limitation, humans or mammals other than humans, including mice, livestock, etc., that require treatment for dentin-pulp disease and / or periodontal disease.

[0113] The pharmaceutical compositions for the treatment of dentin-pulp disease and / or periodontal disease of the present invention may be administered via any common route, as long as it can reach the target tissue. The pharmaceutical compositions of the present invention may be administered via routes such as oral administration or oral injection, depending on the purpose, but are not particularly limited.

[0114] In another embodiment, the present invention provides a quasi-drug composition for the prevention or improvement of dentin-pulp disease, comprising the peptide, or a quasi-drug composition for the prevention or improvement of periodontal disease, comprising the peptide.

[0115] As used herein, the term "improvement" means any action that at least reduces a parameter related to the condition being treated, such as the severity of symptoms.

[0116] In the present invention, the improvements described above can be interpreted as meaning all actions that involve 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 disease to promote the regeneration of dentin or pulp tissue, thereby improving or benefiting the symptoms of dentin-pulp disease, or all actions that involve administering a pharmaceutical composition containing the peptide of the present invention as an active ingredient to an individual in need of treatment for periodontal disease to promote the regeneration of bone and / or chalk, thereby improving or benefiting the symptoms of periodontal disease.

[0117] In this specification, the term "quasi-drug" means an article used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing diseases in humans or animals, which has a milder effect than pharmaceuticals. For example, according to the Pharmaceutical Affairs Law, quasi-drugs, excluding articles used for pharmaceutical purposes, include textile and rubber products used for the treatment or prevention of diseases in humans or animals, items that have a mild or no direct effect on the human body and are not instruments or machines, and similar items, as well as disinfectants and insecticides for preventing infectious diseases.

[0118] In the present invention, the type and dosage form of the quasi-drug composition containing the peptide are not particularly limited, but examples include oral disinfectant mouthwash, oral hygiene products, toothpaste, dental floss, and oral ointment.

[0119] In another embodiment, the present invention provides a health functional food composition containing the peptide for the prevention or improvement of dentin-pulp disease and / or periodontal disease.

[0120] As used herein, the term "food" includes all foods in the ordinary sense, such as meats, sausages, bread, chocolates, candies, snacks, confectionery, pizzas, ramen and other noodle products, gums, dairy products including ice cream, various soups, beverages, teas, energy drinks, alcoholic beverages, vitamin complexes, functional foods, and health foods.

[0121] The term "functional food" is the same as "food for special health use (FoSHU)," and refers to a food with high medical and therapeutic effects that has been processed to efficiently exhibit biological regulatory functions in addition to nutritional supplementation. "Functionality" means adjusting nutrients to the structure and function of the human body, or obtaining effects useful for health purposes such as physiological effects. The food of the present invention can be manufactured by methods commonly used in the industry, and may include raw materials and components commonly added in the industry. Furthermore, the dosage form of the food can also be manufactured without restriction, as long as it is a dosage form recognized as a food. The food composition of the present invention can be manufactured in various dosage forms, and unlike general pharmaceuticals, it uses food as a raw material, thus having the advantage of not having side effects that may occur with long-term use of pharmaceuticals, and is also highly portable. The food of the present invention can be taken as a supplement to enhance the preventive or ameliorative effects of dentin-pulp disease and / or periodontal disease.

[0122] The term "health food" refers to foods that have a more active effect on maintaining or promoting health compared to general foods, while "health supplement food" refers to foods intended for health supplementation. In some cases, the terms "health functional food," "health food," and "health supplement food" may be used interchangeably.

[0123] Specifically, the aforementioned health functional foods are foods produced by adding the peptide of the present invention to food ingredients such as beverages, teas, spices, gums, and confectionery, or by encapsulating, powdering, or suspending them. Ingesting these foods brings about specific health effects, but unlike general medicines, they have the advantage of not having the side effects that can occur with long-term use of medicines, because they are made from food ingredients.

[0124] Since the food composition of the present invention can be consumed on a daily basis, it can be expected to have a high effect in preventing or improving dentin-pulp disease and / or periodontal disease, and therefore can be used very effectively.

[0125] The food composition may further contain a physiologically acceptable carrier, but the type of carrier is not particularly limited, and any carrier that is commonly used in the art may be used.

[0126] Furthermore, the food composition may further contain ingredients commonly used in food compositions that can improve aroma, taste, appearance, etc. For example, it may contain vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. It may also contain minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), and copper (Cu). In addition, it may contain amino acids such as lysine, tryptophan, cysteine, and valine.

[0127] Furthermore, the food composition may contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder and high-grade bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (tar dyes, etc.), color fixatives (sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (monosodium glutamate (MSG), etc.), artificial sweeteners (dulcin, sodium cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum (potassium aluminum sulfate), potassium bitartrate, etc.), fortifiers, emulsifiers, thickeners (gelling agents), coating agents, gum bases, defoamers, solvents, and improvers. The additives may be selected according to the type of food and used in appropriate amounts.

[0128] The peptide of the present invention may be added as is or used in combination with other foods or food ingredients, and may be used as appropriate in the usual manner. The amount of active ingredient mixed may be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). In general, when manufacturing food or beverages, the food composition of the present invention may be added in an amount of 50 parts by weight or less, specifically 20 parts by weight or less, relative to the total weight of the food or beverage. However, when ingested for a long period of time for health and hygiene purposes, the active ingredient may be included in amounts below the above range, but there are no safety concerns, and therefore, amounts above the above range may also be used.

[0129] An example of the food composition of the present invention may be used as a health beverage composition, in which case various flavorings or natural carbohydrates may be included as additional ingredients, similar to ordinary beverages. The natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; or sugar alcohols such as xylitol, sorbitol, and erythritol. Sweeteners that can be used include natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame. The ratio of the natural carbohydrates is typically about 0.01g to 0.04g per 100ml of the health beverage composition of the present invention, specifically about 0.02g to 0.03g.

[0130] In addition to the above, the health beverage composition may contain various nutrients, vitamins, minerals (electrolytes), flavorings, colorings, pectin acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonation agents. It may also contain fruit pulp for the production of natural fruit juices, fruit juice beverages, or vegetable beverages. Such components may be used individually or in combination. While the proportion of such additives is not particularly important, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

[0131] The food composition of the present invention may contain various weight percentages if it can demonstrate a preventive or ameliorative effect on dentin-pulp disease and / or periodontal disease. Specifically, the peptide of the present invention may be contained in amounts from 0.00001% to 100% by weight, or from 0.01% to 80% by weight, relative to the total weight of the food composition, but is not limited thereto.

[0132] Another aspect of the present invention provides a method for preventing or treating dentin-pulp disease and / or periodontal disease, comprising the step of administering a composition containing the peptide to an individual.

[0133] In other embodiments, the present invention provides a method for promoting the regeneration of dentin or pulp tissue, and / or a method for promoting the regeneration of bone or chalk, comprising the step of administering a composition containing the peptide to an individual.

[0134] Another embodiment of the present invention provides a peptide containing the amino acid sequence of the following general formula (1) or a composition containing the peptide for use in promoting the regeneration of hard tissues including dentin, bone, and chalk, and / or pulp tissue, as well as for use in preventing or treating dentin-pulp disease or periodontal disease.

[0135] KY-R1-R2-R3-R4-R5 General formula (1) In formula (1) above, R1 and R2 are arginine (R), lysine (K), glutamine (Q), or asparagine (N), respectively, and R3, R4, and R5 are arginine (R) or lysine (K), respectively.

[0136] In another aspect, the present invention provides a peptide containing any one amino acid sequence of Sequence ID No. 1 to 128, or a composition containing said peptide, for use in promoting the regeneration of hard tissues including dentin, bone, and chalk, and / or pulp tissue, and for use in preventing or treating dentin-pulp disease and / or periodontal disease.

[0137] The present invention will be described in more detail below with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

[0138] (Example 1: Experimental Method and Materials) [Example 1-1. Synthesis of peptides for promoting the regeneration of hard tissues including dentin, bone, and chalk, and / or pulp tissue, and for treating dentin-pulp disease and / or periodontal disease] The inventors synthesized a peptide (SEQ ID NO: 1) that promotes the regeneration of hard tissues including dentin, bone, and chalk, and / or dental pulp tissue, using 9-fluorenylmethyloxycarbonyl (Fmoc), and synthesized representative groups of peptides by substituting amino acids in the synthesized peptide (Tables 1 to 16).

[0139] N-KYQRRKK-C (Sequence ID 1) First, the peptides in Group 1 were synthesized by substituting the peptide of SEQ ID NO: 1 or the 5th to 7th amino acids of the peptide of SEQ ID NO: 1 with lysine or arginine (Table 1).

[0140] [Table 1]

[0141] Next, the peptides in Group 2 were synthesized by substituting the third amino acid of the peptide of Sequence ID No. 1 with arginine, the fourth amino acid with glutamine, and the fifth to seventh amino acids with lysine or arginine (Table 2).

[0142] [Table 2]

[0143] Next, the peptides in Group 3 were synthesized by substituting the third amino acid of the peptide of Sequence ID No. 1 with lysine, the fourth amino acid with glutamine, and the fifth to seventh amino acids with lysine or arginine (Table 3).

[0144] [Table 3]

[0145] Next, the peptides in Group 4 were synthesized by substituting the fourth amino acid of the peptide of Sequence ID No. 1 with glutamine, and substituting the fifth to seventh amino acids with lysine or arginine (Table 4).

[0146] [Table 4]

[0147] Next, the peptides in Group 5 were synthesized by substituting the third amino acid of the peptide of Sequence ID No. 1 with arginine, and substituting the fifth to seventh amino acids with lysine or arginine (Table 5).

[0148] [Table 5]

[0149] Next, the peptides in Group 6 were synthesized by substituting the third amino acid of the peptide of Sequence ID No. 1 with lysine, and substituting the fifth to seventh amino acids with lysine or arginine (Table 6).

[0150] [Table 6]

[0151] Next, the peptides in Group 7 were synthesized by substituting the fourth amino acid of the peptide of Sequence ID No. 1 with lysine, and substituting the fifth to seventh amino acids with lysine or arginine (Table 7).

[0152] [Table 7]

[0153] Next, the peptides in group 8 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with asparagine, the fourth amino acid with lysine, and the fifth to seventh amino acids with lysine or arginine (Table 8).

[0154] [Table 8]

[0155] Next, the peptides in group 9 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with asparagine and the fifth to seventh amino acids with lysine or arginine (Table 9).

[0156] [Table 9]

[0157] Next, the peptides in group 10 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with arginine, the fourth amino acid with asparagine, and the fifth to seventh amino acids with lysine or arginine (Table 10).

[0158] [Table 10]

[0159] Next, the peptides in group 11 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with lysine, the fourth amino acid with asparagine, and the fifth to seventh amino acids with lysine or arginine (Table 11).

[0160] [Table 11]

[0161] Next, the peptides in group 12 were synthesized by substituting the fourth amino acid of the peptide of Sequence ID No. 1 with asparagine and the fifth to seventh amino acids with lysine or arginine (Table 12).

[0162] [Table 12]

[0163] Next, the peptides in group 13 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with asparagine, the fourth amino acid with glutamine, and the fifth to seventh amino acids with lysine or arginine (Table 13).

[0164] [Table 13]

[0165] Next, the peptides of group 14 were synthesized by substituting the third and fourth amino acids of the peptide of SEQ ID NO: 1 with asparagine, and the fifth to seventh amino acids with lysine or arginine (Table 14).

[0166] [Table 14]

[0167] Next, the peptides in group 15 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with arginine, the fourth amino acid with lysine, and the fifth to seventh amino acids with either lysine or arginine (Table 15).

[0168] [Table 15]

[0169] Finally, the peptides in group 16 were synthesized by substituting the third and fourth amino acids of the peptide of SEQ ID NO: 1 with lysine, and substituting the fifth to seventh amino acids with lysine or arginine (Table 16).

[0170] [Table 16]

[0171] [Examples 1-2. Cell Culture] Human bone marrow mesenchymal stem cells (hBMSCs) were cultured in humid air containing 5% CO2 at 37°C and used in the experiment. The hBMSCs were purchased from LONZA Group AG (Switzerland). The hBMSCs were cultured in α-MEM (Invitrogen) medium supplemented with 10% heat-inactivated bovine serum.

[0172] [Examples 1-3. Isolation and culture of human-derived dental pulp cells] Human dental pulp cells (hDPCs) (i.e., human-derived 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 from the hospital's Institutional Review Board and informed consent from the patients. Based on the method of Jung HS et al (J Mol Histol (2011)), wisdom teeth were cut to expose the pulp, and the pulp was isolated with forceps. The isolated pulp was finely chopped with a double-edged knife, placed in a 60 mm dish, covered with a coverslip, and cultured in DMEM (Dulbecco's Modified Eagle Medium). Human dental pulp cells are known to be able to differentiate into odontoblasts, osteoblasts, chlorite blasts, and periodontal ligament cells under various conditions (Tissue Eng Part A. 2014 Apr; 20(7-8):1342-51).

[0173] [Examples 1-4. Reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR analysis] Total RNA was isolated from human dental pulp cells (hDPCs) and human bone marrow mesenchymal stem cells (hBMSCs) using TRIzol reagent. cDNA was synthesized using 2 μg of total RNA, 1 μl of reverse transcriptase, and 0.5 μg of oligonucleotide (dT). The synthesized cDNA was used for real-time polymerase chain reaction (RMC). The RMC was performed using SYBR GREEN PCR Master Mix (TAKARA, Japan) and an ABI PRISM 7500 sequencing detection system (provided by Biosystems). The RMC was performed under the following conditions: 94°C, 1 minute; 95°C, 15 seconds; 60°C, 34 seconds, repeated for 40 cycles. The results were evaluated using the comparative cycle threshold (CT) method, and the primer sequences are listed in Table 17 below.

[0174] [Table 17]

[0175] [Examples 1-5. In vivo transplantation and histological analysis] Human dental pulp cells (hDPCs) were isolated and used in in vivo transplantation experiments. hDPCs (2 × 10⁻¹⁶) 6 Cells were mixed with 100 mg of hydroxyapatite / tricalcium phosphate (HA / TCP) ceramic powder (Zimmer, USA) alone, or with 10 μg of the peptide of the present invention, in a 0.5% fibrin gel, and then transplanted into mice with impaired immune systems (NIH-bg-nu-xid; Harlan Laboratories, Indianapolis, IN) for 6 and 12 weeks.

[0176] Subsequently, samples were harvested, fixed with 4% paraformaldehyde, decalcified with 10% EDTA (pH 7.4), embedded in paraffin, and stained with hematoxylin-eosin (HE) (Vector Labs) or subjected to immunohistochemical analysis. For immunohistochemical analysis, proteins were detected using anti-DSP antibody diluted 1:150 as the primary antigen and biotin-labeled goat anti-rabbit IgG (Vector Labs) as the secondary antigen.

[0177] Collagen staining (Masson's Trichrome Stain) was performed according to the experimental procedure provided by Polysciences' Masson's Trichrome Stain Kit (Cat. 25088-100).

[0178] Quantitative analysis of newly formed hard tissue was performed using the LSstarter program (OLYMPUS Soft Imaging Solution, Muster, Germany). The proportion of newly formed hard tissue was calculated as a percentage of the total area.

[0179] [Examples 1-6. Scanning Electron Microscope Analysis] The sample tissue was fixed in 2.5% glutaraldehyde / 0.1M cacodylate buffer for 30 minutes, and then reacted with a solution containing 1% osmium tetroxide in 0.1M cacodylate buffer for 1 hour. After that, the samples were rapidly dehydrated and dried using ethanol, coated with gold, and observed using a scanning electron microscope (S-4700, Hitachi, Japan).

[0180] [Examples 1-7. Statistical Analysis] Statistical analysis was performed using student t-tests. All statistical analyses were performed using SPSS software ver. 19.0.

[0181] (Example 2: Experimental Results) [Example 2-1. Effects of peptides for promoting dentin or pulp tissue regeneration and treating dentin-pulp diseases on the expression level of the Dspp gene, an odontoblast differentiation marker] The Dspp gene is used as an odontoblast differentiation marker and is known to be an important gene for dentin calcification. Therefore, we investigated whether the peptide of the present invention increases the expression of the Dspp gene, which is an odontoblast differentiation marker, and has the effect of promoting odontoblast differentiation and dentin formation.

[0182] Human dental pulp cells (hDPCs) cultured using the methods of Examples 1-3 were treated with the peptides from each group synthesized in Example 1-1 at a concentration of 10 μg / ml, cultured for 48 hours, and then the mRNA levels of the Dspp gene, an odontoblast differentiation marker expressed in the human dental pulp cells, were measured. The measured mRNA levels of each Dspp gene were then converted to a relative ratio to the mRNA levels of the Dspp gene measured in the control group (Tables 18-33).

[0183] Furthermore, the average mRNA levels of the Dspp gene measured by the peptides in each group described in Tables 1 to 3 were compared for each group (Figure 1a). Specifically, the novel peptides of the present invention, which have amino acid base sequence substitutions or partial sequence deletions, were grouped as shown in Tables 1 to 3. As a result of showing the effect of each group's novel peptide on the expression of the Dspp gene, an odontoblast differentiation marker, in human dental pulp cells, Figure 1a shows a graph of the average values ​​of Dspp mRNA levels for each group, measured by quantitative real-time PCR in human dental pulp cells. In this case, human dental pulp cells that had not been treated with the peptides of the present invention were used as the control group.

[0184] Furthermore, the average mRNA levels of the Dspp gene measured by each group of peptides in Tables 4 to 16 were compared for each group (Figure 1b). Specifically, the novel peptides of the present invention, which have amino acid base sequence substitutions or partial sequence deletions, were grouped as shown in Tables 4 to 16. As a result of showing the effect of each group's novel peptide on the expression of the Dspp gene, an odontoblast differentiation marker, in human dental pulp cells, Figure 1b shows a graph of the average values ​​of Dspp mRNA levels for each group, measured by quantitative real-time PCR in human dental pulp cells. In this case, human dental pulp cells that had not been treated with the peptides of the present invention were used as the control group.

[0185] The expression level of the Dspp gene was measured by RT-PCR and real-time PCR analysis as described in Examples 1-4. The Gapdh gene was used as an internal control group, and the mean and standard deviation values ​​were used after three repeated experiments. The primer sequences are shown in Table 17.

[0186] [Table 18]

[0187] [Table 19]

[0188] [Table 20]

[0189] [Table 21]

[0190] [Table 22]

[0191] Table 23

[0192] Table 24

[0193] Table 25

[0194] Table 26

[0195] Table 27

[0196] Table 28

[0197] Table 29

[0198] Table 30

[0199] Table 31

[0200] Table 32

[0201] Table 33

[0202] Figure 1a is a graph showing the results of comparing the expression levels of the Dspp gene, an odontoblast differentiation marker, in human dental pulp cells (hDPCs) treated with the peptides of the present invention. Referring to Figure 1a and Tables 18 to 20, it can be seen that the mRNA level of the Dspp gene, an odontoblast differentiation marker, increased by approximately 6 to 8 times or more when treated with the peptides of the present invention compared to the mRNA level of the Dspp gene, an odontoblast differentiation marker, measured in human dental pulp cells that were not treated with the peptides of the present invention (control group). In particular, the highest Dspp mRNA expression level was observed when treated with the peptides of Group 3. Figure 1b is a graph showing the results of comparing the expression levels of the Dspp gene, an odontoblast differentiation marker, in human dental pulp cells treated with the peptides of the present invention. Referring to Figure 1b and Tables 21 to 33, it can be confirmed that, compared to the mRNA level of the Dspp gene, an odontoblast differentiation marker, measured in human dental pulp cells that were not treated with the peptide of the present invention (control group), the mRNA level of the Dspp gene increased by approximately 1.5 to 3 times or more after treatment with the peptide of the present invention.

[0203] [Example 2-2. Effects of peptides for promoting dentin or pulp tissue regeneration and treating dentin-pulp diseases on the expression level of the Nestin gene, an odontoblast differentiation marker] From the results of Example 2-1, it was confirmed that the peptides of the present invention can increase the mRNA level of the Dspp gene. For example, all groups of peptides can increase the mRNA level of the Dspp gene by 1.5 times or more, and even by 3 times or more. In particular, it was confirmed that the peptides of groups 1 to 3 can increase the mRNA level of the Dspp gene by 6 times or more.

[0204] This allowed us to confirm whether the peptides from groups 1-3 could also increase the mRNA levels of the Nestin gene, another odontoblast differentiation marker.

[0205] Except for using different primers, the same or similar methods as in Example 2-1 were used to measure the effect of the peptide of the present invention on the expression level of the Nestin gene, and the average levels calculated for each group were compared (Figure 1c). In this case, human dental pulp cells that had not been treated with the peptide of the present invention were used as the control group.

[0206] Figure 1c is a graph showing a comparison of the expression levels of the Nestin gene, an odontoblast differentiation marker, in human dental pulp cells (hDPCs) treated with the peptide of the present invention. As shown in Figure 1c, it can be confirmed that the expression levels of the Nestin gene, an odontoblast differentiation marker, increased by more than five times in all groups treated with the peptide of the present invention (groups 1, 2, and 3) compared to the control group.

[0207] Since the aforementioned Dspp and Nestin genes are used as odontoblast differentiation markers and are known to be involved in the calcification process of dentin, the peptide provided in this invention was analyzed to have the effect of promoting dentin regeneration.

[0208] [Examples 2-3. Effects of peptides for promoting bone and / or chalk cell regeneration and treating periodontal disease on the expression levels of the BSP gene, a differentiation marker for osteoblasts and chalk cell cells] The BSP gene is used as a differentiation marker for osteoblasts and chalkyblasts and is known to be an important gene for the calcification of bone and chalk. Therefore, in order to confirm the effect of the novel peptide of the present invention on the expression of the BSP gene, which is a differentiation marker for osteoblasts and chalkyblasts, human-derived mesenchymal stem cells (i.e., human bone marrow mesenchymal stem cells, hBMSCs) cultured by the method of Examples 1-2 were treated with the peptides from each group, and BSP gene expression was confirmed by real-time PCR.

[0209] Generally, the same or similar methods as in Example 2-1 were used, except for the use of different primers, to measure the effect of the peptides of the present invention on the expression level of the BSP gene, and the average levels calculated for each group were compared (Figures 2a and 2b). Specifically, for the novel peptides of the present invention grouped as shown in Tables 1 to 3, Figure 2a shows the results of measuring the mRNA level of BSP (Bone sialoprotein), a differentiation marker for bone and chalk, in human mesenchymal stem cells (hBMSCs), and the graphs of the results of measuring the mRNA level of BSP in human mesenchymal stem cells by quantitative real-time PCR are shown. Similarly, for the novel peptides of the present invention grouped as shown in Tables 4 to 16, Figure 2b shows the results of measuring the mRNA level of BSP in human mesenchymal stem cells by quantitative real-time PCR, and the graphs of the results of measuring the mRNA level of BSP in human mesenchymal stem cells by quantitative real-time PCR are shown.

[0210] In this process, the peptide was treated at a concentration of 10 μg / ml. As a control group, human bone marrow mesenchymal stem cells that had not been treated with the peptide of the present invention were used.

[0211] Figure 2a is a graph showing the results of comparing the expression levels of the BSP gene, a differentiation marker for bone and chalk, in human mesenchymal stem cells (hBMSCs) treated with the peptides of the present invention. As shown in Figure 2a, it can be confirmed that the expression of the BSP gene increased by approximately 9 to 13 times or more in the groups treated with the peptides of the present invention (groups 1, 2, and 3) compared to the control group. In particular, the highest BSP mRNA expression level was observed when treated with the peptide of group 3.

[0212] Figure 2b is a graph showing the results of comparing the expression levels of the BSP gene, a differentiation marker for bone and chalk, in human mesenchymal stem cells (hBMSCs) treated with the peptides of the present invention. As shown in Figure 2b, it can be confirmed that the expression of the BSP gene increased by approximately 3 to 9 times, and even more than 12 times, in the groups treated with the peptides of the present invention (groups 4 to 16) compared to the control group. In particular, the highest BSP mRNA expression level was observed when treated with the peptide of group 11.

[0213] The aforementioned BSP gene is used as a differentiation marker for osteoblasts and chalkyblasts and is known to be involved in the calcification process of bone and chalky matter. Therefore, the peptide provided in this invention was analyzed to have the effect of promoting the regeneration of bone and chalky matter.

[0214] [Examples 2-4. Hard tissue formation of human dental pulp cells (hDPCs) in vivo using a novel peptide over 6 weeks] (1)Tissue morphological analysis Based on the in vitro experimental results shown in Figures 1a, 1b, 1c, 2a, and 2b, to measure the effect of the peptide of the present invention on hard tissue formation in vivo, implants were prepared by mixing human dental pulp cells (hDPCs) and 100 mg of hydroxyapatite / tricalcium phosphate (HA / TCP) with 10 μg of group 3 peptide (e.g., SEQ ID NO: 24) in 0.5% fibrin gel, as described in Examples 1-5 above. These implants were transplanted into the subcutaneous tissue of mice with impaired immune systems. A control group was used in which implants without the peptide of the present invention were transplanted. Six weeks after transplantation, samples were taken as described in Examples 1-5 above, and the newly formed hard tissue was quantitatively analyzed using the LS starter program, the results of which are shown in Figure 3.

[0215] Figure 3 shows the results of measuring the amount of newly formed hard tissue using human dental pulp cells (hDPCs) in vivo over a 6-week period. As shown in Figure 3, the hard tissue formation rate 6 weeks after transplantation was more than twice as high in the group treated with the novel peptide (Group 3, 29.6%) compared to the control group (13.5%).

[0216] Figure 4 shows micrographs of histologically analyzed hard tissue formed in vivo for 6 weeks using human dental pulp cells (hDPCs). A-D show the results of implants prepared by mixing hDPCs and 100 mg of HA / TCP in 0.5% fibrin gel for the control group, while E-H show the results of implants prepared by mixing hDPCs and 100 mg of HA / TCP with 10 μg of group 3 peptide in 0.5% fibrin gel for 6 weeks in mice with weakened immune systems (size bars: A, E 500 μm, B, F 200 μm, C, G 100 μm, D, H 50 μm). The formed hard tissue was stained with hematoxylin-eosin (HE).

[0217] As shown in Figure 4, histomorphological analysis by hematoxylin-eosin staining revealed that in all groups, both the control group (A-D in Figure 4) and the group (E-H in Figure 4) containing the peptide of the present invention, bone-like tissue and dentin-pulp-like tissue were formed in which cells invaginated into the calcified tissue matrix surrounding the HA / TCP particles.

[0218] (2) Collagen staining analysis Collagen is the most abundant organic matrix in dentin, bone, and chalk, and it plays a role in accommodating deposited inorganic substances. Therefore, collagen staining was performed to confirm the accumulation of collagen protein in the calcified tissues formed in each experimental group of the histomorphological analysis described above.

[0219] Figure 5 shows micrographs illustrating the level of collagen formation in hard tissue formed in vivo for 6 weeks using human dental pulp cells (hDPCs). A-D show the results of implants prepared by mixing hDPCs and 100 mg of HA / TCP in 0.5% fibrin gel for the control group, while E-H show the results of implants prepared by mixing hDPCs and 100 mg of HA / TCP with 10 μg of group 3 peptide in 0.5% fibrin gel for 6 weeks in mice with weakened immune systems (size bars: A, E 500 μm, B, F 200 μm, C, G 100 μm, D, H 50 μm). The formed hard tissue was stained with collagen stain (Masson's trichrome stain).

[0220] As shown in Figure 5, it was confirmed that the collagen formation level increased in the group containing the peptide of the present invention (Figure 5 E-H) compared with the control group (Figure 5 A-D).

[0221] (3) Immunohistochemical analysis The expression of the DSP gene, a odontoblast-specific differentiation marker, was confirmed by immunohistochemical analysis.

[0222] Figure 6 shows immunohistochemical staining images of hard tissue formed in vivo using human dental pulp cells (hDPCs) for 6 weeks, analyzing the expression level of DSP, an odontoblast differentiation marker. A shows the results of implanting a control group implant prepared by mixing hDPCs and 100 mg of HA / TCP in 0.5% fibrin gel, and B shows the results of implanting implants prepared by mixing hDPCs and 100 mg of HA / TCP with 10 μg of group 3 peptide in 0.5% fibrin gel in mice with weakened immune systems for 6 weeks. A and B show the formed hard tissue immunostained with anti-DSP antibody. C is the negative control group for immunohistochemical analysis, treated only with the secondary antibody. The arrows in A and B indicate DSP expression in the newly formed calcified tissue. The size bars are 50 μm.

[0223] As shown in Figure 6, in the control group (Figure 6A), DSP was weakly expressed in the newly formed dentin-pulp-like tissue, while in the group containing the peptide of the present invention (Figure 6B), DSP was strongly expressed in the newly formed calcified tissue. Figure 6C shows the negative control group, which was treated only with the secondary antibody in immunohistochemical analysis and was not stained with DSP.

[0224] [Examples 2-5. Hard tissue formation of human dental pulp cells (hDPCs) in vivo using a novel peptide over 12 weeks] (1)Tissue morphological analysis The methods described in Examples 2-4 were followed, with the exception of housing mice with implants for 12 weeks, to analyze the hard tissue formation of human dental pulp cells.

[0225] Figure 7 shows the results of measuring the amount of newly formed hard tissue using human dental pulp cells (hDPCs) in vivo over 12 weeks. As shown in Figure 7, the hard tissue formation rate 12 weeks after transplantation increased by more than 1.6 times in the group treated with the novel peptide (Group 3, 39.5%) compared to the control group (23.7%).

[0226] Figure 8 shows micrographs of histologically analyzed hard tissue formed in vivo for 12 weeks using human dental pulp cells (hDPCs). A-D show the results of implants prepared by mixing hDPCs and 100 mg of HA / TCP in 0.5% fibrin gel for the control group, while E-H show the results of implants prepared by mixing hDPCs and 100 mg of HA / TCP with 10 μg of group 3 peptide in 0.5% fibrin gel for 12 weeks in mice with weakened immune systems (size bars: A, E 500 μm, B, F 200 μm, C, G 100 μm, D, H 50 μm). The formed hard tissue was stained with hematoxylin-eosin (HE).

[0227] As shown in Fig. 8, as a result of histological analysis by hematoxylin-eosin staining, in both the control group not containing the peptide of the present invention (A to D in Fig. 8) and the group containing the peptide of the present invention (E to H in Fig. 8), as in the case of Fig. 4 (transplanted for 6 weeks), it was observed that osteoid tissue and dentin-pulp-like tissue in which cells were trapped in the matrix of the calcified tissue around the HA / TCP particles were formed.

[0228] (2) Collagen staining analysis In order to confirm the accumulation of collagen protein in the calcified tissue formed in each experimental group of the histological analysis in Examples 2 to 5 above, collagen staining was performed.

[0229] Fig. 9 is a micrograph showing the formation level of collagen contained in the hard tissue formed using human dental pulp cells (hDPCs) in vivo for 12 weeks. A to D are implants of the control group prepared by mixing hDPCs and 100 mg of HA / TCP in 0.5% fibrin gel, and E to H are implants prepared by mixing hDPCs and 100 mg of HA / TCP with 10 μg of the peptide of Group 3 in 0.5% fibrin gel each, showing the results of transplantation into immunocompromised mice for 12 weeks (scale bar: A, E 5,000 μm, B, F 200 μm, C, G 100 μm, D, H 50 μm). The formed hard tissue was stained by collagen staining (Masson's trichrome stain).

[0230] As shown in Fig. 9, it was confirmed that the formation level of collagen increased in the group containing the peptide of the present invention (E to H in Fig. 9) compared with the control group (A to D in Fig. 9).

[0231] (3) Immunohistochemical analysis The expression of the DSP gene, which is an odontoblast-specific differentiation marker, was confirmed by immunohistochemical analysis.

[0232] Figure 10 shows immunohistochemical staining images of hard tissue formed in vivo for 12 weeks using human dental pulp cells (hDPCs), analyzing the expression level of DSP, an odontoblast differentiation marker, by immunohistochemical staining. A shows the results of implanting a control group implant prepared by mixing hDPCs and 100 mg of HA / TCP in 0.5% fibrin gel, and B shows the results of implanting implants prepared by mixing hDPCs and 100 mg of HA / TCP with 10 μg of group 3 peptide in 0.5% fibrin gel in mice with weakened immune systems for 12 weeks. A and B show the formed hard tissue immunostained with anti-DSP antibody. C is the negative control group for immunohistochemical analysis, treated only with the secondary antibody. The arrows in A and B indicate DSP expression in the newly formed calcified tissue. The size bars are 50 μm.

[0233] As shown in Figure 10, in the control group (Figure 6A), DSP was weakly expressed in the newly formed dentin-pulp-like tissue, while in the group containing the peptide of the present invention (Figure 6B), DSP was strongly expressed in the newly formed calcified tissue. Figure 10C shows the negative control group, which was treated only with the secondary antibody in immunohistochemical analysis and was not stained with DSP.

[0234] In summary, the results of Examples 2-4 and 2-5 show that the novel peptide of the present invention has the effect of promoting the regeneration of dentin / pulp tissue complex and bone / chalky tissue.

[0235] [Examples 2-6. Cellular analysis of transplanted tissue by scanning electron microscopy] Scanning electron microscopy analysis was performed using the methods described in Examples 1-6 to confirm the differentiation of human dental pulp cells (hDPCs) into odontoblasts or osteoblasts / chaloblasts in the control group and the experimental group treated with the novel peptide 12 weeks after transplantation.

[0236] Figure 11 is a scanning electron microscope (SEM) image showing analysis of hard tissue formed in vivo using human dental pulp cells (hDPCs) for 12 weeks. A shows a control implant prepared by mixing hDPCs and 100 mg of HA / TCP in 0.5% fibrin gel, while B and C show implants prepared by mixing hDPCs and 100 mg of HA / TCP with 10 μg of group 3 peptide in 0.5% fibrin gel, respectively, after 12 weeks of implantation in mice with weakened immune systems. The size bar is 10 μm. The formed hard tissue was observed for cell analysis using a scanning electron microscope.

[0237] As shown in Figure 11, in the control group treated with hDPCs alone, some odontoblast-like cells with incompletely formed odontoblast processes were observed around the formed hard tissue (Figure 11A). In the group treated with the peptide of the present invention (e.g., the peptide of Group 3), odontoblast-like cells were observed along the formed hard tissue, and the odontoblast processes also extended in the direction of the formed hard tissue (Figure 11B). Furthermore, in the group treated with the peptide of the present invention, it was confirmed that the cells exhibited typical osteoblast / chaloblast characteristics, with cuboidal cells attached to the surface of the formed hard tissue (Figure 11C).

[0238] Therefore, it was found that the peptide of the present invention can more effectively form odontoblasts and osteoblasts / chaloblasts.

[0239] [Examples 2-7. In vivo dentinal tubule closure test] The enamel in the gum area of ​​the premolars of 12-month-old adult dogs was removed using a dental bur to expose the dentin. The premolars with exposed dentin were thoroughly cleaned to completely remove any enamel-dentin fragments generated during cavity preparation, and then dried.

[0240] 1.5 μg of peptide SEQ ID NO: 24 (Group 3) according to the present invention was applied to the opening of the dentinal tubules in the exposed dentin area. After 3 weeks, the adult dogs were euthanized and the teeth were extracted. Subsequently, test specimens of the extracted teeth were prepared using a diamond saw.

[0241] Then, to confirm the effect of the novel peptide according to the present invention on the closure of exposed dentinal tubules in damaged dentin, the dentinal tubule closure ability was evaluated using a scanning electron microscope, and the results are shown in Figure 12.

[0242] Specifically, as shown in Figures 12A and 12E, the lower part of the damaged dentin was cut, and the underside of the cut surface (box portion) was observed. Scanning electron microscopy revealed that in the control group, which received no treatment, the dentinal tubules in the lower part of the damaged dentin were exposed (Figures 12A to 12D). On the other hand, in the experimental group treated with peptides, it was confirmed that the exposed dentinal tubules were closed by physiological remineralization (Figures 12E to 12H).

[0243] [Examples 2-8. Surface observation of dentin damage sites in vivo] Test specimens of extracted adult dog teeth were prepared using the same method as in Example 2-7.

[0244] Then, in order to confirm the effect of the novel peptide according to the present invention on dentinal tubule closure on the surface of damaged dentin, the dentinal tubule closure ability of the surface area was evaluated using a scanning electron microscope, and the results are shown in Figure 13.

[0245] Scanning electron microscopy observations revealed that dentinal tubules were exposed on the damaged dentin surface of the untreated control group (Figures 13A-13D). In contrast, in the peptide-treated experimental group, the exposed dentinal tubules were almost completely closed (Figures 13E-13H).

[0246] This research was supported by the Ministry of Trade, Industry and Energy and the Korea Institute of Industrial Technology Evaluation and Management (KEIT) in 2017 (10078369, "Development of a foundational technology for treating sensitive teeth using functional peptides that induce dentin regeneration").

[0247] Although embodiments of the present invention have been described above with reference to the attached drawings, those with ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without altering its technical idea or essential features. Therefore, the embodiments described above should be understood to be illustrative and not limiting in any respect.

Claims

1. A peptide consisting of one amino acid sequence from SEQ ID NOs: 1-121 and 123-128, The peptide is a peptide that exhibits a regeneration-promoting effect on hard tissue and / or dental pulp tissue, and has undergone modifications selected from the group consisting of those in which the N-terminus is acetylated or the C-terminus is amidated.

2. A pharmaceutical composition for the prevention or treatment of dentin-pulp disease and / or periodontal disease, comprising a peptide consisting of one amino acid sequence from SEQ ID NOs: 1-121 and 123-128.

3. A quasi-drug composition for the prevention or improvement of dentin-pulp disease and / or periodontal disease, comprising a peptide consisting of one amino acid sequence from SEQ ID NOs: 1-121 and 123-128.

4. A health functional food composition for the prevention or improvement of dentin-pulp disease and / or periodontal disease, comprising a peptide consisting of one amino acid sequence from SEQ ID NOs: 1-121 and 123-128.

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