Novel peptide

Abstract

The present invention relates to novel peptides, polynucleotides encoding the peptides, expression vectors containing the polynucleotides, and pharmaceutical compositions, quasi-drug compositions, and functional health food compositions each containing the peptide.

Description

Technical Field

[0001] The present invention relates to novel peptides, and more specifically, to 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 dentin-pulp diseases and / or periodontal diseases.

Background Art

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

[0003] The causes of pulp diseases are very diverse. In most cases, they are caused by bacterial infections due to dental caries and infections into the pulp through dental perforations, fractures, cracks, and periodontal sacs. Also, trauma, abrasion, tooth cracks, heat and friction from dental instruments during treatment, etc. can also cause them. Pulpitis caused by bacterial infection can spread to periapical diseases and periodontal diseases. When a pulp disease occurs, it progresses in the order of pulp congestion, pulpitis, and pulp necrosis. In the case of pulp necrosis, since the pulp dies and there is no blood supply to the pulp, the entire periodontal tissue disappears, and ultimately, periapical diseases or abnormalities of the entire tooth may result.

[0004] For the treatment of pulp and apical diseases, pulp capping material and root canal filling material 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 presents a problem of relatively high cost as a dental treatment material and discoloration can cause aesthetic problems. In the case of gutta-percha, the cost is economical and fluidity is desirable, but it is an unphysiological treatment method that destroys the vitality of the pulp. Until now, conservative treatment methods for dentin-pulp diseases have resulted in treated teeth becoming brittle or easily fractured and carrying a risk of reinfection.

[0005] The periodontal tissue is a complex organ composed of epithelial tissue, soft connective tissue, and calcified connective tissue. The structure of the 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. Gingival fibroblasts are primarily involved in maintaining gingival connective tissue, while periodontal ligament fibroblasts, due to their unique functions, are known to not only form the periodontal ligament 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 regenerating the periodontal ligaments that support the alveolar bone, but also regenerating the 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 diseases. For example, Patent Document 1 discloses a composition for hard tissue formation and dentin or pulp tissue regeneration containing ameloblasts, apical bard cells, or their culture media as active ingredients, and Patent Document 2 discloses a novel dental sac-derived dental stem cell and a method for culturing the same. Furthermore, Patent Document 3 discloses a composition for treating periodontal disease containing ameloblast culture media.

[0008] Against this backdrop, the inventors diligently researched and developed a formulation that could more effectively treat dentin-pulp disease and / or periodontal disease causing 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 for 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] The object of the present invention is to provide peptides for promoting the regeneration of hard tissue and / or dental pulp tissue and for treating 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] The objects of the present invention are not limited to those described above, and any other objects not described will be clearly understood by a person with ordinary skill in the art from the following description. [Means for solving the problem]

[0019] According to one aspect of the present invention for solving the aforementioned technical problems, a peptide is provided for promoting the regeneration of hard tissue and / or dental pulp tissue and for treating dentin-pulp disease and / or periodontal disease, comprising the amino acid sequence of the following general formula 1.

[0020] K - Y - R1 - R2 - R3 - R4 - R5 - R6 - Y - K (General Formula 1) In the above General Formula 1, R1 and R2 are each lysine (K), alanine (A), or arginine (R), R3, R4, and R5 are each lysine (K) or arginine (R), and R6 is asparagine (N) or serine (S).

[0021] According to one embodiment, the peptide may be composed of any one of the amino acid sequences of SEQ ID NOs: 1 to 64.

[0022] According to one embodiment, the peptide may be composed of any one of the amino acid sequences of SEQ ID NOs: 1 to 8.

[0023] According to one embodiment, the peptide may be N - terminal or C - terminal acetylated, amidated, or methylated; D - amino acid introduced; peptide bond modified such as CH2 - NH, CH2 - S, CH2 - S = O, or CH2 - CH2; backbone modified; or side - chain modified.

[0024] According to one embodiment, the hard tissue may include dentin, bone, and chalky substance.

[0025] In another aspect, the present invention provides a polynucleotide encoding the peptide.

[0026] In yet another aspect, the present invention provides an expression vector containing the polynucleotide.

[0027] In yet another aspect, the present invention provides a pharmaceutical composition for preventing or treating dentin - pulp diseases containing the peptide.

[0028] According to one embodiment, the dentin - pulp disease may be dentin hypersensitivity, pulp hyperemia, pulpitis, pulp degeneration, or necrosis and gangrene of the pulp.

[0029] In another embodiment, the present invention provides a pharmaceutical composition for the prevention or treatment of periodontal disease, comprising the peptide.

[0030] According to one embodiment, the periodontal disease may be gingivitis, periodontitis, periodontal pocket, or periodontal abscess.

[0031] In another embodiment, the present invention provides a quasi-drug composition for the prevention or improvement of dentin-pulp disease and / or periodontal disease, comprising the peptide.

[0032] 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.

[0033] 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 other than a human.

[0034] 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 other than a human.

[0035] In another embodiment, 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 other than a human. [Effects of the Invention]

[0036] The peptide for promoting the regeneration of hard tissue and / or dental pulp tissue and for treating dentin-pulp disease and / or periodontal disease of the present invention exhibits excellent regeneration-promoting effects on hard tissue and / or dental pulp tissue, and is therefore 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 causes damage to bone and / or chalk.

[0037] The effects of the present invention are not limited to those mentioned above, and any other effects not mentioned will be clearly understood by a person of ordinary skill in the art to which the present invention pertains from the following description. [Brief explanation of the drawing]

[0038] [Figure 1] Figure 1 shows the results of grouping novel peptides with substituted amino acid sequences and demonstrating the effect of each peptide group on the expression of DSPP (Dentin sialophosphoprotein), an odontoblast differentiation marker gene, in human dental pulp cells (these are the average values ​​of DSPP mRNA levels measured by quantitative real-time PCR in human dental pulp cells for each group, and the peptides were treated at a concentration of 10 μg / ml). [Figure 2] Figure 2 shows the results of real-time PCR testing of BSP and DMP1 gene expression after treating human-derived mesenchymal stem cells with the novel peptides from each group, in order to confirm the effects of the novel peptides on the expression of BSP and DMP1 genes, which are differentiation marker genes for osteoblasts and leucoblasts. [Figure 3] Figure 3 shows the results of Western blot analysis of DSP and BSP protein expression after treating human dental pulp cells with a novel peptide (SEQ ID NO: 1) in a concentration-dependent manner (1 μg, 10 μg, 50 μg) to confirm the effects of the novel peptide on the expression of odontoblast, osteoblast, and cyanamide differentiation marker proteins. [Figure 4] Figure 4 shows the results of histological evaluation three weeks after applying 10 μg of the novel peptide (SEQ ID NO: 1) to the dentin of a beagle dog's tooth following dentin damage, in order to confirm whether the novel peptide induces physiological dentin regeneration in an animal model of dentin injury. [Figure 5]Figure 5 shows the results of an in vitro experiment based on the results shown in Figure 2 and Tables 10-11. To confirm the effect of the group 1 (sequence number 1) peptide on hard tissue formation in vivo, either the group 1 peptide (10 μg) or human dental pulp cells (hDPCs) from the control group (cell only) were transplanted into the subcutaneous tissue of mice with impaired immune systems. After 12 weeks, the hard tissue formation rate was confirmed to be increased in the group treated with the novel peptide compared to the control group (A-D: hDPCs alone; E-H: group 1 peptide; I-L: BMP2 treatment / size bar; A, E, I: 500 μm; B, F, J: 200 μm; C, G, K: 100 μm; D, H, L: 50 μm). [Figure 6] Figure 6 shows the results of a study in vivo to confirm the effect of the group 1 (sequence number 1) peptide on periodontal ligament formation. The study involved transplanting either the group 1 (sequence number 1) peptide (10 μg) or control group human dental pulp cells (hDPCs) into the subcutaneous tissue of mice with impaired immune systems, and comparing the results to the control group 6 weeks after transplantation (size bars; A, C: 200 μm; B, D: 100 μm). [Modes for carrying out the invention]

[0039] The objectives and effects of the present invention, and the technical configurations for achieving them, will become clear when referring to the embodiments described in detail below, along with the accompanying drawings. In describing the present invention, if it is determined that a specific description of a known function or configuration would obscure the gist of the invention unnecessarily, such detailed description will be omitted. Furthermore, the terms used below are defined for the purpose of describing the embodiments of the present invention and may change depending on the intent or conventions of the user or operator.

[0040] However, the present invention is not limited to the embodiments disclosed below and can be realized in a variety of different forms. These embodiments are provided solely to ensure the complete disclosure of the present invention and to fully inform those ordinary skill in the art of which the invention pertains, and the invention is defined solely by its claims. Therefore, its definition should be based on the content throughout this specification.

[0041] The present invention will be described in detail below.

[0042] As a result of various studies conducted to develop a formulation that can more effectively treat dentin-pulp disease and / or periodontal disease, the inventors have developed a novel peptide consisting of 10 amino acids.

[0043] The novel peptide developed as described above was created by partially substituting 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 level of the DSPP gene, which is an odontoblast differentiation marker gene, thereby promoting dentin regeneration. It can also increase the expression levels of the BSP (Bone sialoprotein) gene and DMP1, which are differentiation marker genes for osteoblasts and cyanoblasts, thereby promoting bone and cyanoblast regeneration.

[0044] Furthermore, when a graft containing the peptide was prepared together with human dental pulp cells, and the prepared graft was transplanted into the subcutaneous tissue of mice with damaged immune systems, and the transplanted tissue was analyzed after 6 or 12 weeks, it was confirmed that dentin-pulp-like tissue with a morphology most similar to in vivo dentin-pulp tissue was formed, bone-like tissue with a morphology most similar to in vivo bone tissue was formed, the level of collagen formation increased, and the expression level of DSP, an odontoblast-specific differentiation marker gene, increased.

[0045] Furthermore, scanning electron microscopy analysis of the transplanted tissue revealed the presence of odontoblast-like cells along the formed hard tissue, and 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 cyanobacteria characteristics, with cuboidal cells attached to the surface of the formed hard tissue.

[0046] Therefore, it has been found that the peptide of the present invention can promote the regeneration of hard tissue and / or dental pulp tissue and have an effect on dentin-pulp disease and / or periodontal disease. No peptide of the present invention exhibiting such effects has been reported before, and it is the first to be developed by the inventors.

[0047] In this invention, the term "hard tissue" refers to relatively hard skeletal tissue, including bone, hyaline cartilage, and fibrocartilage. In one embodiment of the present invention, the hard tissue may include dentin, bone, and chalk.

[0048] In this invention, 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 not exposed on the tooth surface, as 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.

[0049] In this invention, the term "cementum" refers to a thin, slightly deformed membrane of bone that covers the tooth root and other parts of a mammal's tooth. This cementum is composed of 50% inorganic and 50% water-organic matter, is yellowish in color, and exhibits a 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 of the cementum surrounding the tooth occurs, and the periodontal ligament fibers that connect the tooth to the alveolar bone cannot adhere to the degenerated cementum, causing the tooth to become loose. To treat such degeneration of cementum, methods are used to remove the degenerated cementum and promote the formation of new cementum.

[0050] The peptide provided in this invention can increase the expression levels of the DSPP gene, which is an odontoblast differentiation marker gene, and the BSP and DMP1 genes, which are osteoblast and cyanoblast differentiation marker genes. When transplanted into vivo together with human dental pulp cells, the human dental pulp cells may exhibit the characteristic of forming dentin / pulp tissue-like tissue and bone-like tissue.

[0051] 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, even if the mutant peptides have a sequence that differs from the amino acid sequence of the peptides provided in this invention by one or more amino acid residues.

[0052] In general, amino acid exchanges in proteins and polypeptides that do not alter the overall molecular activity are known in the field. The most commonly occurring 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. Furthermore, mutations or modifications in the amino acid sequence may increase the structural stability of the peptide against heat, pH, etc., or increase the regenerative capacity of hard tissues, including dentin, bone, and chalk, and / or dental pulp tissue.

[0053] Amino acid mutations are performed based on the relative similarity of amino acid side-chain substitutions, such as hydrophobicity, hydrophilicity, charge, and size. Among the amino acids constituting the peptide of the present invention, asparagine (N) and serine (S) are hydrophilic amino acids, and therefore the relative similarity of the amino acid side-chain substitutions is high. As a result, even if the amino acids constituting the peptide according to the examples of the present invention are substituted with hydrophilic amino acids, they can exhibit similar effects due to their structural similarity.

[0054] For example, asparagine, the acidic amino acid located at position 8 of the peptide of Sequence ID No. 1 provided in the present invention, can still exhibit the effects of the peptide provided in the present invention even if it is substituted with serine, and lysine, the basic amino acid located at position 5 of the peptide of Sequence ID No. 1, can still exhibit the effects of the peptide provided in the present invention even if it is substituted with arginine, which is a basic amino acid.

[0055] Since the effects of the peptide provided by the present invention can be exhibited 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, it is obvious that mutant peptides having a sequence in which one or more amino acid residues differ from the amino acid sequence constituting the peptide of the present invention are also included in the category of peptides provided by the present invention.

[0056] 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 the peptide having 1 to 100 amino acids attached to its N-terminus or C-terminus; and yet another example is the peptide having 1 to 24 amino acids attached to its N-terminus or C-terminus.

[0057] The peptides of the present invention may be in a form in which their N-terminus and / or C-terminus are chemically modified, protected by organic terminals, or modified by the addition of 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 the N-terminus can be acetylated, the N-terminus can be methylated and / or the C-terminus can be amidated to remove such charges, or this may include, but is not limited to, D-amino acid introduction, peptide bond modifications such as CH2-NH, CH2-S, CH2-S=O, CH2-CH2, backbone modifications, and side chain modifications. Methods for producing peptide mimetic compounds are known in the art, and can be found, for example, in Quantitative Drug Design, CA Ramsden Gd., Choplin Pergamon Press (1992).

[0058] In this invention, the term "backbone modification" refers to the direct modification of amino acids constituting a peptide into amino acid analogs. The main chain-like or cyclic skeleton of the amino acids that make up a peptide is called the backbone (main chain). An amino acid analog is an amino acid in which a hydrogen atom has been altered by substitution at the nitrogen or α-carbon of the amino acid backbone.

[0059] 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. The modification of these side chains using chemical substances is called side-chain modification. Examples of peptide side-chain modifications include reductive alkylation reactions, amidinization with methyl acetimidate, alkylation with acetic anhydride, carbamoylation of amino groups with cyanates, trinitrobenzylation of amino acids with 2,4,6-trinitrobenzenesulfonic acid (TNBS), alkylation of amino groups with succinic anhydride, or modification of amino groups such as pyridoxylation, which is achieved by pyridoxal-5-phosphate treatment followed by reduction with NaBH4.

[0060] Furthermore, while the peptides of the present invention may be used alone, they may also be used in combination with a carrier approved as a pharmaceutical agent, such as an organic solvent. For increased stability and efficacy, they 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.

[0061] According to one embodiment of the present invention, 64 peptides corresponding to 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 gene, was investigated. As a result, it was confirmed that the mRNA level of the DSPP gene, an odontoblast differentiation marker, in human dental pulp cells treated with the 64 peptides was 9.7 times or more, or 6 times or more, or 3 times or more, or at least about 1.5 times higher, 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) (Figure 1 and Table 10).

[0062] Previous studies have shown that increasing the mRNA expression level of DSPP promotes odontoblast differentiation and dentin regeneration. Therefore, the 64 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): 171178, 2003).

[0063] Furthermore, the effect of the synthesized peptides on the expression level of the BSP gene, an osteoblast / chlorite differentiation marker gene, was investigated. As a result, it was confirmed that, compared to the mRNA level of the BSP gene, an osteoblast / chlorite differentiation marker, measured in human dental pulp cells that were not treated with the peptides of the present invention (control group), the mRNA level of the BSP gene in human dental pulp cells treated with the 64 peptides was 2.9 times or more, or at least approximately 1.6 times, and the mRNA level of the DMP1 gene was 10.6 times or more, or at least approximately 5.7 times (Figure 2 and Tables 11 and 12).

[0064] As a result, it is known that an increase in BSP mRNA expression levels promotes osteoblast / chrythroblast differentiation and bone and chalk regeneration. Therefore, it was found that the 64 peptides that show the effect of increasing the mRNA level of the BSP gene also show the effect of promoting osteoblast / chrythroblast differentiation and bone and chalk regeneration.

[0065] In another embodiment, the present invention provides a polynucleotide encoding the peptide.

[0066] The aforementioned polynucleotides may be mutated by substitution, deletion, insertion, or combination thereof of one or more bases. When nucleotide sequences are produced by chemical synthesis, synthetic methods known to 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 phosphotriester, phosphite, phosphoramidite and H-phosphate methods, PCR and other autoprimer methods, and oligonucleotide synthesis methods on solid supports.

[0067] In another embodiment, the present invention provides an expression vector containing the polynucleotide, a transformant containing the expression vector, and a method for producing the peptide using the transformant.

[0068] In this invention, the term "expression vector" refers to a recombinant vector capable of expressing a target peptide in target host cells, and is a gene crop containing essential regulatory elements operably linked to the expression of a gene insert. The expression vector includes expression regulatory elements such as a start codon, a termination codon, a promoter, and an operator, wherein the start and termination codons are generally considered to be part of the nucleotide sequence encoding the polypeptide, and must exert their effect in the organism when the gene crop is administered, and must be in frame with the coding sequence. The promoter of the vector may be constitutive or inductive.

[0069] In this invention, the term "operably linked" refers to a state in which a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked to perform a 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. Operational linking with an expression vector can be produced using genetic engineering techniques known in the art, and site-specific DNA cleavage and linking can be performed using enzymes or other methods commonly known in the art.

[0070] Furthermore, the expression vector may include a signal sequence for peptide efflux to facilitate the separation of the peptide from the cell culture medium. Specific start signals may also be necessary for 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, may be required. These exogenous translational regulatory signals and start codons can come from a variety of natural and synthetic sources. Expression efficiency may be increased by introducing appropriate transcription or translational enhancers.

[0071] 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.

[0072] In this invention, the term "tag" refers to a molecule that exhibits quantifiable activity or properties, and may be a fluorescent molecule containing a chemical fluorescent 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, histidine 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 can be used.

[0073] In the present invention, the expression vector may contain a nucleotide sequence encoding the peptide described above 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. However, the vector used 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.), or Bacillus subtilis. Plasmids derived from subtilis (e.g., pUB110, pTP5), plasmids derived from yeast (e.g., YEp13, YEp24, YCp50), phage DNA (e.g., Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP), animal virus vectors (e.g., retrovirus, adenovirus, vaccinia virus), and insect virus vectors (e.g., baculovirus). Since the expression levels and modifications of the aforementioned expression vectors differ depending on the host cell, it is desirable to select and use a host cell that is best suited to the purpose.

[0074] The transformants provided in the present invention are 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, but is not particularly limited as long as the peptide can be produced. Possible methods include CaCl2 precipitation, the Hanahan method which improves efficiency by using a reducing agent called DMSO (dimethyl sulfoxide) in addition to CaCl2 precipitation, electroporation, calcium phosphate precipitation, plasmofusion, stirring method using silicon carbide fibers, agrobacteria-mediated transformation, PEG-mediated transformation, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation. Furthermore, the host used to produce the transformants is not particularly limited as long as it can produce the peptide, but may be bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium; yeast cells such as Saccharomyces cerevisiae and Schizosaccharomyces pombe; fungal cells such as Pichia pastoris; insect cells such as Drosophila and Spodoptera frμgiperda Sf9 cells; animal cells such as CHO, COS, NSO, 293, and Bowes melanoma cells; or plant cells.

[0075] The transformants may also be used in a method for producing peptides for promoting the regeneration of hard tissues and / or pulp tissues, including dentin, bone, and chalk, and for treating dentin-pulp disease and / or periodontal disease according to the present invention. Specifically, the method for producing peptides for promoting the regeneration of hard tissues and / or pulp tissues, including dentin, bone, and chalk, and for treating dentin-pulp disease and / or periodontal disease according to the present invention may include (a) culturing the transformants to obtain a culture; and (b) recovering the peptides of the present invention from the culture.

[0076] In this invention, the term "culture" refers to a method of growing microorganisms under appropriately artificially controlled environmental conditions. In this invention, the method of culturing the transformants can be carried out by methods known in the art. Specifically, the culture is not particularly limited as long as it can produce 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, but it can be carried out continuously by batch processing, fed-batch culture, or repeated fed-batch process.

[0077] 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 degradation products, defatted soy cake or its degradation products. These nitrogen sources may be used individually or in combination. 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. Finally, in addition to the above substances, growth-essential substances such as amino acids and vitamins may be used.

[0078] Furthermore, appropriate precursors may be used in the culture medium. The aforementioned raw materials may be added to the culture in batch, fed-batch, or continuous mode by an appropriate method during the culture process, but are not limited to these methods. The pH of the culture may be adjusted by using basic compounds such as sodium hydroxide, potassium hydroxide, or ammonia, or acidic compounds such as phosphoric acid or sulfuric acid by an appropriate method.

[0079] Furthermore, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble formation. To maintain aerobic conditions, oxygen or oxygen-containing gas (e.g., air) is injected into the culture medium. The culture temperature is typically 27°C to 37°C, preferably 30°C to 35°C. Culturing is continued until the maximum amount of peptide is produced. For this purpose, this is usually achieved in 10 to 100 hours.

[0080] Furthermore, the step of recovering the peptide from the culture can be carried out by methods known to the art. Specifically, the recovery method is not limited to these, as long as it can be used to recover the produced peptide, but preferably 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) can be used.

[0081] In another embodiment, the present invention provides a pharmaceutical composition for the prevention or treatment of dentin-pulp disease, comprising the peptide.

[0082] 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 disease and / or periodontal disease, can be used as active ingredients in pharmaceutical compositions for treating dentin-pulp disease that arises from damage to pulp tissue, because when transplanted into vivo together with human pulp cells, they can promote the formation of dentin / pulp tissue-like tissue by the human pulp cells.

[0083] 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 a dentin-pulp disease therapeutic effect is bound to the N-terminus or C-terminus of the peptide.

[0084] In this invention, the term "dentin-pulp disease" refers to a disease that develops when the pulp tissue and the dentin attached to it are damaged as a result of damage to the pulp tissue.

[0085] In the present invention, the dentin-pulp disease is not particularly limited as long as it exhibits therapeutic effects with the peptide of the present invention, but examples include dentin hypersensitivity, pulp congestion, pulpitis, pulp degeneration, pulp necrosis, and gangrene.

[0086] In another embodiment, the present invention provides a pharmaceutical composition for the prevention or treatment of periodontal disease, comprising the peptide.

[0087] 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 the human pulp cells. Therefore, they can be used as active ingredients in pharmaceutical compositions for treating periodontal diseases that cause damage to bone and / or chalk.

[0088] 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 linked two or more times, or in the form of a complex in which a drug exhibiting a periodontal disease therapeutic effect is bound to the N-terminus or C-terminus of the peptide.

[0089] The term "periodontal disease" in this invention, also known as "cold teeth," 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 periodontal disease develops, inflammation progresses, damaging more tissue and forming a periodontal pocket. The more severe the periodontitis, the deeper the periodontal pocket becomes. As the periodontal pocket deepens, inflammation occurs in the periodontal ligaments, which ultimately leads to bone loss.

[0090] In the present invention, the periodontal disease is not particularly limited as long as it exhibits therapeutic effects with the peptide of the present invention, but examples include gingivitis, periodontitis, periodontal pockets, or periodontal abscesses.

[0091] In this invention, the term "prevention" means any action that inhibits or delays 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 any action that inhibits or delays 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.

[0092] In this invention, the term "treatment" means any act of administering a pharmaceutical composition containing the peptide of the present invention as an active ingredient to an individual seeking treatment for dentin-pulp disease to promote the regeneration of dentin or pulp tissue, thereby treating the pulp disease, or any act of administering a pharmaceutical composition containing the peptide of the present invention as an active ingredient to an individual seeking treatment for periodontal disease to promote the regeneration of bone and / or chalk, thereby treating the periodontal disease.

[0093] 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 a suitable carrier (natural or non-natural carrier), excipient, or diluent commonly used in the preparation of the peptide pharmaceutical composition. Specifically, each of the pharmaceutical compositions may be used in the form of a sterile injectable solution that can be administered by conventional methods to the site where dentin-pulp disease and / or periodontal disease is induced. 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, alginic acid, 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, lyophilized agents, 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 (registered trademark), macrogol (registered trademark), tween (registered trademark) 61, cocoa butter, lauric acid butter, and glycerol gelatin.

[0094] The content of the peptide 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.

[0095] The pharmaceutical compositions of the present invention may be administered in pharmaceutically effective amounts, where the term “pharmaceutically effective amount” in the present invention 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 may be 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 pharmaceutical compositions of the present invention used, the duration of treatment, drugs compounded or used concurrently with the pharmaceutical compositions of the present invention used, 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.

[0096] 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 pharmaceutical composition of the present invention is not particularly limited, but it may be administered once a day or divided into doses and administered several times a day. The aforementioned dosage does not limit the scope of the present invention in any way.

[0097] In another aspect, the present invention provides a method for treating dentin-pulp disease, comprising the step of administering the pharmaceutical composition in a pharmaceutically effective amount to an individual other than a human who has developed dentin-pulp disease. In yet another aspect, the present invention provides a method for treating periodontal disease, comprising the step of administering the pharmaceutical composition in a pharmaceutically effective amount to an individual other than a human who has developed periodontal disease.

[0098] In this invention, the term "individual" may include, without limitation, humans or mammals other than humans, including rats, livestock, etc., who require treatment for dentin-pulp disease and / or periodontal disease.

[0099] The pharmaceutical composition 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 composition of the present invention may be administered by routes such as oral administration or oral injection, depending on the purpose, but is not limited thereto.

[0100] In another embodiment, the present invention provides a quasi-drug composition for the prevention or improvement of dentin-pulp disease, or a quasi-drug composition for the prevention or improvement of periodontal disease, comprising the peptide.

[0101] In this invention, the term "improvement" means all actions that at least reduce parameters related to the condition being treated, such as the severity of symptoms.

[0102] 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 seeking treatment for dentin-pulp disease to promote the regeneration of dentin or pulp tissue, thereby improving or benefiting from 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 seeking treatment for periodontal disease to promote the regeneration of bone and / or chalk, thereby improving or benefiting from the symptoms of periodontal disease.

[0103] In this invention, the term "quasi-drug" refers to articles used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing diseases in humans or animals, which have a milder effect than pharmaceuticals. For example, according to the Pharmaceutical Affairs Law, quasi-drugs are articles excluding those used for pharmaceutical purposes, and include textile and rubber products used for the treatment or prevention of diseases in humans and animals, items that have a mild or no direct effect on the human body and are not instruments or machines, as well as similar items, and disinfectants and insecticides for preventing infectious diseases.

[0104] 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 and cleansing agents, oral cleaning products, toothpaste, floss, and oral ointments.

[0105] In another embodiment, the present invention provides a health functional food composition comprising the peptide for the prevention or improvement of dentin-pulp disease and / or periodontal disease.

[0106] In this invention, the term "food" includes all foods in the ordinary sense, such as meats, sausages, bread, chocolates, candies, snacks, confectionery, pizzas, ramen noodles, other noodle products, gums, dairy products including ice cream, various soups, drinking water, tea, energy drinks, alcoholic beverages, vitamin complexes, health functional foods, and health foods.

[0107] The term "functional food" refers to a food with high medical and therapeutic effects that has been processed to efficiently exhibit biological regulatory functions in addition to nutrient supply. "Functionality" here refers to effects useful for health purposes, such as regulating nutrients or physiological effects, on the structure and function of the human body. The food of the present invention can be manufactured by methods commonly used in the industry, and can be manufactured by adding raw materials and components commonly used 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 has the advantage of using food as a raw material, thus avoiding side effects that can occur with long-term use of pharmaceuticals, and is highly portable. The food of the present invention can be taken as an adjunct to enhance the preventive or ameliorative effects of dentin-pulp disease and / or periodontal disease.

[0108] 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.

[0109] Specifically, the aforementioned health functional foods are foods in which the peptide of the present invention is added to food ingredients such as beverages, teas, spices, gums, and confectionery, or manufactured as encapsulated, powdered, or suspended, and which, when consumed, produce specific health effects. However, unlike general pharmaceuticals, they have the advantage of being made from food ingredients and not having the side effects that can occur with long-term use of pharmaceuticals.

[0110] Since the food composition of the present invention can be consumed on a daily basis, it is expected to have a high effect in preventing or improving dentin-pulp disease and / or periodontal disease, and therefore can be used very effectively.

[0111] The food composition may further contain a physiologically acceptable carrier, but the type of carrier is not particularly limited, and any carrier commonly used in the art may be used.

[0112] Furthermore, the food composition may contain additional ingredients commonly used in food compositions that can improve smell, 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), copper (Cu), and chromium (Cr). It may also contain amino acids such as lysine, tryptophan, cysteine, and valine.

[0113] 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, cyclamic acid, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum (potassium aluminum sulfate), potassium bitartrate, etc.), fortifiers, emulsifiers, thickeners, coating agents, gum bases, defoamers, solvents, and improvers. The additives may be selected according to the type of food and used in appropriate amounts.

[0114] The peptide of the present invention can be added as is or used in combination with other foods or food components, and can be used as appropriate in the usual manner. The amount of active ingredient mixed can be suitably determined according to its intended use (prevention, health, or therapeutic treatment). In general, when manufacturing food or beverages, the food composition of the present invention can be added in an amount of 50 parts by weight or less, specifically 20 parts by weight or less, per 100 parts by weight of the food or beverage composition. However, for long-term intake for health and hygiene purposes, the active ingredient may be included in amounts below the above range, and there are no safety concerns, so it can also be used in amounts above the above range.

[0115] One example of the food composition of the present invention may be used as a health beverage composition, in which case, like ordinary beverages, it may contain various flavorings or natural carbohydrates as additional ingredients. The aforementioned 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 aforementioned natural carbohydrates is usually about 0.01g to 0.04g per 100mL of the health beverage composition of the present invention, specifically about 0.02g to 0.03g.

[0116] In addition to the above, the health beverage composition may contain various nutrients, vitamins, 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.

[0117] The food composition of the present invention may contain various weight percentages, provided that 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 0.00001 to 100% by weight or 0.01 to 80% by weight relative to the total weight of the food composition, but is not limited thereto.

[0118] 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.

[0119] Another embodiment provides a method for promoting the regeneration of dentin or pulp tissue, and / or bone or chalk, comprising the step of administering a composition containing the peptide to an individual.

[0120] Another aspect of the present invention provides a peptide comprising the amino acid sequence of the following general formula 1, or a composition comprising the peptide, for use in promoting the regeneration of hard tissues including dentin, bone, and chalk, and / or dental pulp tissue, as well as for use in preventing or treating dentin-pulp disease or periodontal disease. KY-R1-R2-R3-R4-R5-R6-YK (general formula 1) In the above general formula 1, R1 and R2 are lysine (K), alanine (A), or arginine (R), respectively; R3, R4, and R5 are lysine (K) or arginine (R), respectively; and R6 is asparagine (N) or serine (S).

[0121] In another embodiment, the present invention provides a peptide comprising any one amino acid sequence of SEQ ID NOs: 1 to 64, or a composition comprising the 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.

[0122] Furthermore, as an embodiment of the present invention, the present invention provides a peptide containing any one amino acid sequence of SEQ ID NOs: 1 to 8, 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.

[0123] 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.

[0124] (Example 1: Experimental Method and Materials) [Example 1-1: Synthesis of peptides for promoting 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 the 9-fluorenylmethyloxycarbonyl (Fmoc) method, and synthesized peptides of each group by substituting amino acids in the synthesized peptide (Tables 1 to 8). N-KYKAKKKNYK-C (Sequence ID 1)

[0125] First, the peptides in Group 1 were synthesized by substituting the peptide of Sequence ID No. 1 or the 5th to 7th amino acids of the peptide of Sequence ID No. 1 with arginine (Table 1).

[0126] [Table 1]

[0127] Next, the peptides in Group 2 were synthesized by substituting the 8th amino acid of the peptide of Sequence ID No. 1 with serine and the 5th to 7th amino acids with arginine (Table 2).

[0128] [Table 2]

[0129] The peptides in Group 3 were synthesized by substituting the third amino acid of the peptide of Sequence ID No. 1 with arginine, and then substituting the fifth to seventh amino acids with arginine (Table 3).

[0130] [Table 3] The peptides in Group 4 were synthesized by substituting the third amino acid of the peptide of Sequence ID No. 1 with arginine, the eighth amino acid with serine, and the fifth to seventh amino acids with arginine (Table 4).

[0131] [Table 4] The peptides in Group 5 were synthesized by substituting the third amino acid of the peptide of Sequence ID No. 1 with alanine, the fourth amino acid with lysine, and the fifth to seventh amino acids with arginine (Table 5).

[0132] [Table 5]

[0133] The peptides in Group 6 were synthesized by substituting the third amino acid of the peptide of Sequence ID No. 1 with alanine, the fourth amino acid with lysine, the eighth amino acid with serine, and the fifth to seventh amino acids with arginine (Table 6).

[0134] [Table 6]

[0135] The peptides in Group 7 were synthesized by substituting the third amino acid of the peptide of Sequence ID No. 1 with alanine, the fourth amino acid with arginine, and the fifth to seventh amino acids with arginine (Table 7).

[0136] [Table 7]

[0137] The peptides in Group 8 were synthesized by substituting the third amino acid of the peptide of Sequence ID No. 1 with alanine, the fourth amino acid with arginine, the eighth amino acid with serine, and the fifth to seventh amino acids with arginine (Table 8).

[0138] [Table 8]

[0139] [Examples 1-2: Cell Culture] Human 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 (Switzerland). The hBMSCs were cultured in α-MEM (Invitrogen, registered trademark) culture medium supplemented with 10% heat-inactivated bovine serum.

[0140] [Examples 1-3: Isolation and culture of human-derived dental pulp cells] Human dental pulp cells were isolated from 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. Wisdom teeth were cut according to the method of Jung HS et al (J Mol Histol. (2011)), the pulp was exposed, and the pulp was separated using forceps. The separated pulp was finely chopped with a double-sided blade, placed in a 60 mm dish, covered with a coverslip, and cultured in Dulbecco's modified Eagle medium (DMEM). Human dental pulp cells are known to be able to differentiate into odontoblasts, osteoblasts, leucoblasts, and periodontal ligament cells under various conditions (Tissue Eng Part A. 2014 Apr;20(7-8):1342-51).

[0141] [Examples 1-4: Reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR analysis] Total RNA from human dental pulp cells and mesenchymal stem cells was isolated using TRIzol reagent. cDNA was synthesized using 2 μg of total RNA, 1 μl of reverse transcriptase, and 0.5 μg of oligonucleotides (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) with an ABI PRISM 7500 sequence detection system (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 9.

[0142] [Table 9]

[0143] [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 0.5% fibrin gel either with 100 mg of hydroxyapatite / tricalcium phosphate (HA / TCP) ceramic powder (Zimmer, USA) alone, or with a peptide (10 μg), and then transplanted into mice with impaired immune systems (NIH-bg-nu-xid; Harlan Laboratories, Indianapolis, IN) for 6 and 12 weeks. Subsequently, samples were harvested, fixed in 4% paraformaldehyde, decalcified with 10% EDTA (pH 7.4), embedded in paraffin, and stained with HE (Hematoxylin Eosin, Vector Labs).

[0144] [Examples 1-6: Effects of novel peptides on dentin regeneration in a dentin damage model] Three beagle dogs (12-16 kg; 6-8 weeks old) were anesthetized by inhaling gerolan, intravenously injected with zoletil (5 mg / kg) and xylazine (0.2-0.5 mg / kg), and then treated with lidocaine (2% lidocaine and 1:80000 epinephrine). The premolars and molars of the mandibles of the beagle dogs were dentin-damaged using a dental bur, and then treated with 10 μg per tooth of group 1, sequence number 1 peptide. Six weeks later, the beagle dogs were sacrificed by administering an overdose (90-120 mg / kg) of pentobavital. The teeth of the aforementioned beagle dogs were excised, fixed with 10% formalin, then calcium was removed by adding 5% formic acid, shaped, and embedded in paraffin to obtain 5 μm thick tissue sections. These tissue sections were stained with hematoxylin and eosin, and then analyzed using an optical microscope (LEICA DM750, Germany) equipped with a digital camera (LEICA ICC50 camera, Germany).

[0145] In the embodiments of this invention, statistical analysis was performed using Student's t-test, with SPSS software version 19.0 used.

[0146] [Effects of peptides used to promote dentin or pulp tissue regeneration and treat dentin hypersensitivity on the expression level of the DSPP gene, an odontoblast differentiation marker gene] The DSPP gene is used as an odontoblast differentiation marker and is known to be an important gene for dentin calcification. Therefore, in this study, we synthesized a novel peptide that increases the expression of the DSPP gene, an odontoblast differentiation marker gene, and promotes odontoblast differentiation and dentin formation, and confirmed its effect.

[0147] The effects of each group's peptides on DSPP mRNA expression were examined using real-time PCR (Table 10).

[0148] [Table 10] JPEG0007874921000011.jpg233148JPEG0007874921000012.jpg81148

[0149] Figure 1 shows the average DSPP mRNA expression levels for each group in Table 10, plotted graphically. In Figure 1, DSPP mRNA expression was increased by approximately 2 to 8 times or more in all peptide groups compared to the control group, with the peptide group in Group 1 showing the highest DSPP mRNA expression level. Since each of the differentiation marker genes is known to be involved in the differentiation of odontoblasts and the calcification process of dentin, the peptides provided in this invention were analyzed to have the effect of promoting dentin regeneration.

[0150] [Effects of peptides for promoting bone or chalk regeneration and treating periodontal disease on the expression levels of the BSP gene, a marker gene for osteoblast and clotblast differentiation] The BSP and DMP1 genes are used as differentiation markers for osteoblasts and cyanoblasts, and are known to be important genes for the calcification of bone and chalk. The effects of peptide groups from each group on BSP and DMP1 mRNA expression were confirmed by real-time PCR (Tables 11 and 12).

[0151] [Table 11] JPEG0007874921000014.jpg233148JPEG0007874921000015.jpg81148

[0152] [Table 12] JPEG0007874921000017.jpg233148JPEG0007874921000018.jpg81148

[0153] In Figure 2, to confirm the effect of novel peptides on the expression of BSP and DMP1 genes, which are differentiation marker genes for osteoblasts and chlorites, human-derived mesenchymal stem cells were treated with the peptides from each group, and then BSP and DMP1 gene expression was confirmed by real-time PCR. All groups of peptides increased BSP gene expression by approximately 1.5 to 3 times and DMP1 gene expression by 6 to 10 times or more compared to the control group. In particular, the peptide group from Group 1 showed the highest BSP and DMP1 mRNA expression levels. Since the BSP and DMP1 genes are used as differentiation markers for osteoblasts and chlorites and are known to be involved in the calcification process of bone and chalk, the peptides provided in this invention were analyzed to have the effect of promoting the regeneration of bone and chalk.

[0154] Figure 3 shows the results of Western blot analysis of DSP and BSP protein expression after treating human dental pulp cells with a novel peptide (SEQ ID NO: 1) in a concentration-dependent manner (1 μg, 10 μg, 50 μg) to confirm the effects of the novel peptide on the expression of differentiation marker proteins in odontoblasts, osteoblasts, and cyanuloblasts.

[0155] As shown in Figure 3, the expression of DSP, an odontoblast differentiation marker protein, and BSP, an osteoblast and cyanide cell differentiation marker protein, was confirmed to increase in a concentration-dependent manner with the introduction of a novel peptide.

[0156] Figure 4 shows the results of histological evaluation three weeks after applying 10 μg of the novel peptide (SEQ ID NO: 1) to the dentin of a beagle dog's tooth following dentin damage, in order to confirm whether the novel peptide induces physiological dentin regeneration in an animal model of dentin injury.

[0157] Referring to Figure 4, no changes were observed beneath the damaged dentin in the control group, but in the test group treated with the novel peptide, physiological dentin was confirmed to have formed beneath the damaged dentin. These results suggest that hypersensitivity, dentin caries, and pain caused by tooth fracture due to damaged dentin can be treated by regenerating physiological dentin.

[0158] Figure 5 shows the results of an in vivo study to confirm the effect of the group 1 (sequence number 1) peptide on hard tissue formation, based on the in vitro experimental results shown in Figure 2 and Tables 10-11. The study involved transplanting either the group 1 (sequence number 1) peptide (10 μg) or human dental pulp cells (hDPCs) from the control group (cell only) into the subcutaneous tissue of mice with impaired immune systems. After 12 weeks, the hard tissue formation rate was confirmed to be increased in the group treated with the novel peptide compared to the control group (A-D: hDPCs alone; E-H: group 1 peptide; I-L: BMP2 treatment / size bar; A, E, I: 500 μm; B, F, J: 200 μm; C, G, K: 100 μm; D, H, L: 50 μm).

[0159] Referring to Figure 5, histological analysis by hematoxylin-eosin staining revealed the formation of bone / chalkaline-like tissue containing cells in the matrix of newly formed calcified tissue around HA / TCP particles in the groups treated with hDPCs alone, group 1 peptide, and the positive control group BMP2. However, compared to the control group, the groups treated with the novel peptide showed more similar hard tissue formation than the group treated with BMP-2. In summary, these results indicate that the novel peptide used in this experiment may have the effect of promoting the regeneration of bone / chalkaline-like tissue and dentin / pulp tissue complex.

[0160] Figure 6 shows the results of a study in vivo to confirm the effect of the group 1 (sequence number 1) peptide on periodontal ligament formation. The study involved transplanting either the group 1 (sequence number 1) peptide (10 μg) or human dental pulp cells (hDPCs) from the control group (cell only) into the subcutaneous tissue of mice with impaired immune systems, and comparing the results to the control group 6 weeks after transplantation (size bars; A, C: 200 μm; B, D: 100 μm).

[0161] Referring to Figure 6, it was confirmed that periodontal ligament fiber bundles (black triangles) were formed in the group treated with the novel peptide six weeks after transplantation, compared to the control group. Specifically, in histological analysis by hematoxylin-eosin staining, an irregular fiber arrangement was observed in hDPCs alone, but in the group treated with the novel peptide (Group 1), it was observed that periodontal ligament-like tissue containing fiber bundles was formed in the newly formed calcified tissue around the HA / TCP particles. Based on these results, we believe that the novel peptide used in this experiment may have the effect of promoting the regeneration of damaged periodontal ligaments.

[0162] This research was supported by research grants from the Ministry of Health and Welfare and the Korea Health Industry Promotion Agency in fiscal year 2022 (1465037227, "Development of a treatment technology for chronic periodontitis utilizing the bio-fusion novel material CPNE7 peptide").

[0163] This specification and drawings disclose preferred embodiments of the present invention, and although specific terms are used, these are merely general terms used to illustrate the technical content of the invention and to aid in understanding the invention, and are not intended to limit the scope of the invention. It will be obvious to those ordinary skill in the art to which the present invention pertains that other modifications based on the technical idea of ​​the present invention are also possible, in addition to the embodiments disclosed herein.

Claims

1. A peptide comprising one of the amino acid sequences from Sequence ID No. 1 to 8, for promoting the regeneration of hard tissue or dental pulp tissue and for treating dentin-pulp disease or periodontal disease.

2. The peptide according to claim 1, wherein the peptide is acetylated or amidated at its N-terminus or C-terminus.

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

4. A polynucleotide encoding the peptide described in claim 1.

5. An expression vector comprising the polynucleotide described in claim 4.

6. A pharmaceutical composition for the prevention or treatment of dentin-pulp disease, comprising the peptide described in claim 1.

7. The composition according to claim 6, wherein the dentin-pulp disease is dentin hypersensitivity, pulp congestion, pulpitis, pulp degeneration, or pulp necrosis and gangrene.

8. A pharmaceutical composition for the prevention or treatment of periodontal disease, comprising the peptide described in claim 1.

9. The composition according to claim 8, wherein the periodontal disease is gingivitis, periodontitis, periodontal pocket, or periodontal abscess.

10. A quasi-drug composition for the prevention or improvement of dentin-pulp disease or periodontal disease, comprising the peptide described in claim 1.

11. A functional health food composition for preventing or improving dentin-pulp disease or periodontal disease, comprising the peptide described in claim 1.

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

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

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

15. A peptide for promoting the regeneration of hard tissue or dental pulp tissue and for treating dentin-pulp disease or periodontal disease, according to claim 1, comprising one amino acid sequence from sequence numbers 1 to 8.

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