Novel peptides

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 dental pulp tissues and for treating dentin-pulp disease and / or periodontal disease, polynucleotides encoding the peptides, expression vectors containing the polynucleotides, pharmaceutical compositions containing the peptides for preventing or treating dentin-pulp disease and / or periodontal disease, quasi-drug compositions for preventing or ameliorating dentin-pulp disease and / or periodontal disease, and functional health food compositions for preventing or ameliorating dentin-pulp disease and / or periodontal disease. [Background technology]

[0002] The dental pulp is the soft connective tissue that fills the pulp cavity inside the tooth, and is rich in nerves and blood vessels, reaching down to the surface of the dentin. Diseases that occur in this dental pulp are called dental pulp diseases.

[0003] The causes of dental pulp disease are extremely diverse, but most cases are caused by bacterial infection due to tooth decay, or infection into the dental pulp via perforations, fractures, cracks, or periodontal sacs. Trauma, wear, cracks in the tooth, and heat and friction from dental instruments during treatment can also cause pulpitis caused by bacterial infection can develop into apical disease and periodontal disease. Once pulp disease develops, it progresses in the following order: pulp congestion, pulpitis, and pulp necrosis. In the case of pulp necrosis, the pulp dies and loses all blood supply to the pulp, resulting in the complete loss of periodontal tissue, which can eventually lead to apical disease or even tooth abnormalities.

[0004] Pulp capping materials and root canal filling materials are used to treat pulp and root-end diseases, and calcium hydroxide, mineral trioxide aggregate (MTA), gutta-percha, etc. have commonly been used. MTA is effective in treatment due to its sealing power and biocompatibility, but it is relatively expensive as a dental treatment and can cause aesthetic problems due to discoloration. Gutta-percha is cost-effective and has favorable fluidity, but it is an unphysiological treatment method that loses pulp viability. Until now, conservative treatment methods for dentin-pulp diseases have resulted in treated teeth becoming brittle or prone to fracture, and there is a risk of reinfection.

[0005] The periodontal tissue (periodontium) is a complex organ composed of epithelial tissue, soft connective tissue, and mineralized connective tissue. Periodontal tissue structures include the gingiva, periodontal ligament (PDL), cementum, and alveolar bone. Gingival fibroblasts and periodontal ligament fibroblasts are the major cellular components of the gingival soft connective tissue and are responsible for forming and maintaining the extracellular matrix. Gingival fibroblasts are primarily involved in maintaining the gingival connective tissue, while periodontal ligament fibroblasts, through their unique functions, are known to not only form the periodontal ligament but also repair and regenerate the adjacent alveolar bone and cementum in vivo. Periodontal disease clinically manifests as gingival bleeding and swelling, periodontal pocket formation, and alveolar bone destruction, leading to tooth loss.

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

[0007] Therefore, active research is being conducted to develop therapeutic agents that can effectively treat dentin-pulp diseases. For example, Patent Document 1 discloses a composition for hard tissue formation and dentin or dental pulp tissue regeneration containing ameloblasts, apical vein cells, or a culture medium thereof as an active ingredient, while Patent Document 2 discloses new dental stem cells derived from the dental follicle and a method for culturing the same. Furthermore, Patent Document 3 discloses a composition for treating periodontal disease containing an ameloblast culture medium.

[0008] Under these circumstances, the present inventors have made intensive research efforts to develop a formulation that can more effectively treat dentin-pulp disease and / or periodontal disease that causes damage to alveolar bone and chalk, and as a result, have developed a peptide that exhibits cell therapy for promoting the regeneration of hard tissues including dentin, bone, and chalk and / or pulp tissue, and is effective in treating dentin-pulp disease and / or periodontal disease, thereby completing the present invention. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Korean Patent Publication No. 2012-0089547 [Patent Document 2] Korean Patent Publication No. 2009-0033643 [Patent Document 3] Korean Patent Publication No. 2016-0105627 Summary of the Invention [Problem to be solved by the invention]

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

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

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

[0013] It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating dentin-pulp disease and / or periodontal disease, comprising the peptide.

[0014] It is still another object of the present invention to provide a quasi-drug composition for preventing or ameliorating dentin-pulp disease and / or periodontal disease, which comprises the peptide.

[0015] It is still another object of the present invention to provide a health functional food composition for preventing or ameliorating dentin-pulp disease and / or periodontal disease, which comprises the peptide.

[0016] Another object of the present invention is to provide a method for preventing or treating dentin-pulp disease and / or periodontal disease, which comprises 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 regeneration of hard tissues, including dentin, bone, and chalk, and / or dental pulp tissue, comprising 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 other objects not described will be clearly understood by those skilled in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0019] According to one aspect of the present invention for solving the above technical problems, there is provided a peptide for promoting the regeneration of hard tissue and / or dental pulp tissue and treating dentin-pulp disease and / or periodontal disease, which comprises an amino acid sequence represented by the following general formula 1:

[0020] KY-R1-R2-R3-R4-R5-R6-YK (general formula 1) In the 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).

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

[0022] According to one embodiment, the peptide may consist 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- or C-terminally acetylated, amidated, or methylated; D-amino acid introduction; peptide bond modifications such as CH2-NH, CH2-S, CH2-S=O, or CH2-CH2; backbone modifications; or side chain modifications.

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

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

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

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

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

[0029] In another aspect, the present invention provides a pharmaceutical composition for preventing or treating periodontal disease, comprising the peptide.

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

[0031] In another aspect, the present invention provides a quasi-drug composition for preventing or ameliorating dentin-pulp disease and / or periodontal disease, comprising the peptide.

[0032] In another aspect, the present invention provides a health functional food composition for preventing or ameliorating dentin-pulp disease and / or periodontal disease, comprising the peptide.

[0033] In yet another aspect, the present invention provides a method for preventing or treating a dentin-pulp disease, comprising administering a composition containing the peptide to an individual other than a human.

[0034] In yet another aspect, 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 yet another aspect, the present invention provides a method for promoting regeneration of hard tissues, including dentin, bone, and chalk, and / or dental pulp tissue, comprising administering a composition containing the peptide to an individual other than a human. [Effects of the Invention]

[0036] The peptide of the present invention for promoting the regeneration of hard tissue and / or dental pulp tissue and treating dentin-pulp disease and / or periodontal disease exhibits excellent effects of promoting the regeneration of hard tissue and / or dental pulp tissue, and therefore will be widely used in the development of preparations for the prevention or treatment of various dentin-pulp diseases, or for the prevention or treatment of periodontal diseases that cause damage to bone and / or chalk.

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

[0038] [Figure 1] Figure 1 shows the effect of each group of peptides on the expression of DSPP (Dentin sialophosphoprotein), a marker gene for odontoblast differentiation, in human dental pulp cells, after grouping novel peptides with substituted amino acid sequences. (These results are the average values ​​for each group, measured by quantitative real-time PCR of DSPP mRNA levels in human dental pulp cells, and the peptides were treated at a concentration of 10 μg / ml.) [Figure 2] Figure 2 shows the results of examining the effect of the new peptides on the expression of BSP and DMP1 genes, which are osteoblast and osteoblast differentiation marker genes, by treating human mesenchymal stem cells with each group of peptides and then examining the expression of BSP and DMP1 genes by real-time PCR. [Figure 3] Figure 3 shows the results of Western blot analysis of DSP and BSP protein expression after treating human dental pulp cells with the novel peptide (SEQ ID NO: 1) in a concentration-dependent manner (1 μg, 10 μg, 50 μg) to confirm the effect of the novel peptide on the expression of odontoblast, osteoblast, and leucocyte differentiation marker proteins. [Figure 4] Figure 4 shows the results of histological evaluation three weeks after damaging the dentin of a beagle dog's teeth with 10 μg of the novel peptide (SEQ ID NO: 1) to determine whether the novel peptide induces physiological dentin regeneration in a dentin damage animal model. [Figure 5]Figure 5 shows the results of the in vitro experiments shown in Figure 2 and Tables 10 and 11. To confirm the effect of the Group 1 (SEQ ID NO: 1) peptide on hard tissue formation in vivo, 10 μg of the Group 1 SEQ ID NO: 1 peptide or control (cell-only) human dental pulp cells (hDPCs) were transplanted into the subcutaneous tissue of mice with impaired immune systems. 12 weeks after transplantation, the rate of hard tissue formation was confirmed to be increased in the groups treated with the novel peptide compared to the control group (A-D: hDPCs alone; E-H: Group 1 peptide; I-L: BMP2 treatment). Size bars: 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 effect of Group 1 (SEQ ID NO: 1) peptide on periodontal ligament formation in vivo. Group 1 SEQ ID NO: 1 peptide (10 μg) or control human dental pulp cells (hDPCs) were transplanted into the subcutaneous tissue of mice with impaired immune systems, and the results were compared with those of the control group 6 weeks after transplantation (size bar: A, C: 200 μm; B, D: 100 μm). DETAILED DESCRIPTION OF THE INVENTION

[0039] The objects and advantages of the present invention, as well as the technical configurations for achieving them, will become apparent from the following detailed description of the embodiments, along with the accompanying drawings. When describing the present invention, detailed descriptions of known functions or configurations are omitted if it is deemed unnecessary and may obscure the gist of the present invention. The terms used below are defined for the purpose of describing the embodiments of the present invention, and may vary depending on the intentions or practices of users and operators.

[0040] However, the present invention is not limited to the embodiments disclosed below and can be realized in various different forms. The embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the invention. The present invention is defined only by the claims. Therefore, the definition should be based on the entire content of this specification.

[0041] The present invention will be specifically described below.

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

[0043] The newly developed peptide was created by partially substituting the amino acid sequence of a peptide that may be effective in treating dentin-pulp disease and / or periodontal disease. It is capable of increasing the expression level of the DSPP gene, an odontoblast differentiation marker gene, thereby promoting dentin regeneration, and it is also capable of increasing the expression levels of the BSP (Bone sialoprotein) gene and DMP1, which are differentiation marker genes for osteoblasts and leukocytes, thereby promoting bone and chalk regeneration.

[0044] In addition, implants containing the peptide together with human dental pulp cells were prepared and implanted into the subcutaneous tissue of mice with impaired immune systems. After 6 or 12 weeks, the implanted tissues were analyzed and found to have formed dentin-pulp-like tissue with a morphology most similar to in vivo dentin-pulp tissue, bone-like tissue with a morphology most similar to in vivo bone tissue, increased collagen formation levels, and increased expression levels of DSP, an odontoblast-specific differentiation marker gene.

[0045] Furthermore, the morphology of the implanted tissue was analyzed by scanning electron microscopy, and it was confirmed that odontoblast-like cells were observed along the formed hard tissue, and that odontoblast processes also extended in the direction of the formed hard tissue. Furthermore, it was confirmed that the formed hard tissue exhibited typical osteoblast and / or leucoblast characteristics, with cuboidal cells attached to the surface.

[0046] Therefore, it was found that the peptide of the present invention can promote the regeneration of hard tissues and / or dental pulp tissues and has an effect on dentin-pulp disease and / or periodontal disease. The peptide of the present invention exhibiting such an effect has never been reported before and is the first to be developed by the present inventors.

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

[0048] The term "dentin" as used herein refers to the yellowish-white hard tissue that constitutes the majority of teeth. Dentin is covered by enamel at the crown and chalk at the root, preventing it from being exposed on the tooth surface. However, as the enamel wears away with age, dentin can become exposed at the tip or occlusal surface of the crown. Dentin is a type of bone-like tissue, but it differs from general bone tissue in that the cells that make up dentin are located within the dental pulp, with only their processes extending into the dentin.

[0049] The term "cementum" as used herein refers to a thin, slightly deformed membrane of bone that covers the roots and other parts of mammalian teeth. The chalk is composed of 50% inorganic matter and 50% organic matter, is yellowish, and exhibits lower hardness than dentin and enamel. The chalk contains the periodontal ligament fibrous tissue that secures the teeth to the alveolar bone. However, when bacteria infect the gums, the chalk surrounding the teeth degenerates, preventing the periodontal ligament fibrous tissue that connects the teeth to the alveolar bone from adhering to the degenerated chalk, resulting in loose teeth. To treat this type of chalk degeneration, methods are used to remove the degenerated chalk and promote the formation of new chalk.

[0050] The peptide provided by the present 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 leucocyte differentiation marker genes, and when transplanted into a living body together with human dental pulp cells, the human dental pulp cells can exhibit the characteristic of forming dentin / pulp tissue-like tissue and bone-like tissue.

[0051] As long as the peptides provided by the present invention can promote the regeneration of hard tissues including dentin, bone, and chalk and / or dental pulp tissue, and can be effective in treating dentin-pulp disease and / or periodontal disease, variant peptides having sequences that differ by one or more amino acid residues from the amino acid sequence that constitutes the peptides are also included in the category of peptides provided by the present invention.

[0052] In general, amino acid exchanges in proteins and polypeptides that do not change the overall activity of the molecule are known in the art. 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. Furthermore, mutations or modifications in the amino acid sequence may increase the structural stability of the peptide against heat, pH, etc., or may increase the ability to promote the regeneration 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 the amino acid side chain substitutions, such as hydrophobicity, hydrophilicity, charge, size, etc. Among the amino acids constituting the peptides of the present invention, asparagine (N) and serine (S) are hydrophilic amino acids, and therefore have high relative similarity of the amino acid side chain substitutions. Therefore, even when the amino acids constituting the peptides according to embodiments of the present invention are substituted with hydrophilic amino acids, they can exhibit similar effects due to their structural similarity.

[0054] For example, even if the acidic amino acid asparagine at the 8th position of the peptide of SEQ ID NO: 1 provided by the present invention is substituted with serine, the peptide can still exhibit the effects of the peptide provided by the present invention, and even if the basic amino acid lysine at the 5th position of the peptide of SEQ ID NO: 1 is substituted with arginine, the peptide can still exhibit the effects of the peptide provided by the present invention.

[0055] The effects of the peptides provided by the present invention can be exhibited even when the acidic or basic amino acids constituting the peptides of the present invention are substituted with different acidic or basic amino acids, respectively. Therefore, it is obvious that mutant peptides having a sequence that differs in part by one or more amino acid residues from the amino acid sequence constituting the peptides of the present invention are also included in the category of peptides provided by the present invention.

[0056] Furthermore, the peptides of the present invention can exhibit the same effects as the peptides provided by the present invention even if they have any amino acids added to their N- or C-terminus. For example, the peptides may have 1 to 300 amino acids added to their N- or C-terminus, or 1 to 100 amino acids added to their N- or C-terminus, or 1 to 24 amino acids added to their N- or C-terminus.

[0057] The peptides of the present invention may be chemically modified at their N-terminus and / or C-terminus, protected with an organic terminal, or modified by the addition of amino acids to the peptide termini, etc., to protect them from in vivo protease cleavage and increase their stability. In particular, in the case of chemically synthesized peptides, the N- and C-termini are charged. To remove such charges, N-terminus acetylation, N-terminus methylation, and / or C-terminus amidation may be performed. Modifications may also include, but are not limited to, the introduction of D-amino acids, peptide bond modifications such as CH2-NH, CH2-S, CH2-S=O, and CH2-CH2, backbone modifications, and side chain modifications. Methods for producing peptidomimetic compounds are known in the art, and reference may be made to, for example, Quantitative Drug Design, CA Ramsden Gd., Choplin Pergamon Press (1992).

[0058] The term "backbone modification" as used herein refers to the main linear or cyclic structure of the amino acids that make up a peptide, which is called the backbone. The backbone modification refers to the direct modification of the amino acids that make up the peptide backbone with amino acid analogs. An amino acid analog refers to an amino acid that has been modified by substituting a hydrogen atom at the nitrogen or α-carbon of the amino acid backbone.

[0059] The term "side chain modification" as used herein refers to the atomic group branched from the main linear or cyclic backbone of an amino acid constituting a peptide, and refers to the modification of these side chains using chemicals. Examples of peptide side chain modifications include reductive alkylation, amidination with methylacetimidate, alkylation with acetic anhydride, carbamoylation of amino groups with cyanate, trinitrobenzylation of amino acids with 2,4,6-trinitrobenzenesulfonic acid (TNBS), alkylation of amino groups with succinic anhydride, or pyridoxylation by pyridoxal-5-phosphate treatment followed by reduction with NaBH4.

[0060] The peptides of the present invention may be used alone or in combination with a pharmaceutically acceptable carrier such as an organic solvent. To enhance stability and efficacy, the peptides 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 by the present invention were synthesized, and the effects of the synthesized peptides on the expression level of the DSPP gene, an odontoblast differentiation marker gene, were examined. 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, 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 in human dental pulp cells not treated with the peptides of the present invention (control group) (FIG. 1 and Table 10).

[0062] Previous reports have shown that an increase in 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] We also investigated the effect of the synthesized peptides on the expression level of the BSP gene, an osteoblast / leucoblast differentiation marker gene. As a result, we confirmed that the mRNA level of the BSP gene, an osteoblast / leucoblast differentiation marker, in human dental pulp cells treated with the 64 peptides was 2.9-fold or more, or at least about 1.6-fold higher, and the mRNA level of the DMP1 gene was 10.6-fold or more, or at least about 5.7-fold higher, compared to the mRNA level of the BSP gene, an osteoblast / leucoblast differentiation marker, measured in human dental pulp cells (control group) not treated with the peptides of the present invention (Figure 2 and Tables 11 and 12).

[0064] It is known that an increase in the mRNA expression level of BSP promotes osteoblast / leucocyte differentiation and bone and chalk regeneration. Therefore, the 64 peptides that show the effect of increasing the mRNA level of the BSP gene were found to have the effect of promoting osteoblast / leucocyte differentiation and bone and chalk regeneration.

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

[0066] The polynucleotide may be mutated by one or more base substitutions, deletions, insertions, or a combination thereof. When the nucleotide sequence is produced by chemical synthesis, a synthesis method known in the art, such as the method described in the literature (Engels and Uhlmann, Angew Chem Int Ed Engl., 37:73-127, 1988), can be used, such as the phosphotriester, phosphite, phosphoramidite and H-phosphate method, PCR and other autoprimer methods, oligonucleotide synthesis on a solid support, etc.

[0067] In another aspect, 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] The term "expression vector" as used herein refers to a recombinant vector capable of expressing a target peptide in a target host cell, comprising essential regulatory elements operably linked to a gene insert for expression. The expression vector includes expression regulatory elements such as an initiation codon, a termination codon, a promoter, and an operator. The initiation codon and termination codon are generally considered to be part of the nucleotide sequence encoding the polypeptide, and must be functional in an individual when the gene product is administered, and must be in frame with the coding sequence. The promoter of the vector can be constitutive or inducible.

[0069] As used herein, the term "operably linked" refers to a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest are functionally linked so as to perform their common function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA can be operably linked to affect the expression of the coding sequence. Operable linkage with an expression vector can be produced using recombinant DNA techniques known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes commonly known in the art.

[0070] Additionally, the expression vector may contain a signal sequence for peptide excretion to facilitate separation of the peptide from the cell culture medium. Specific initiation signals may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG initiation codon and adjacent sequences. In some cases, exogenous translational control signals, which may include the ATG initiation codon, must be provided. These exogenous translational control signals and initiation codons may be of various natural and synthetic origins. Expression efficiency may be increased by the introduction of appropriate transcriptional or translational enhancing factors.

[0071] In addition, the expression vector may further comprise a protein tag that can optionally be removed using an endopeptidase to facilitate detection of the peptide.

[0072] The term "tag" as used herein refers to a molecule that exhibits a quantifiable activity or property, and may be a fluorescent molecule, including a chemical fluorescein, a polypeptide fluorescein, such as green fluorescent protein (GFP) or related proteins, or an epitope tag, such as a Myc tag, Flag tag, histidine tag, leucine tag, IgG tag, or streptavidin tag. In particular, when an epitope tag is used, a peptide tag consisting of preferably 6 or more amino acid residues, more preferably 8 to 50 amino acid residues, may be used.

[0073] In the present invention, the expression vector may contain a nucleotide sequence encoding the peptide for promoting the regeneration of hard tissues including dentin, bone, and chalk and / or dental pulp tissues and for treating dentin-pulp disease and / or periodontal disease of the present invention. The vector used in this case is not particularly limited as long as it can produce the peptide, but is preferably a plasmid DNA, a phage DNA, or the like, and more preferably a commercially developed plasmid (pUC18, pBAD, pIDTSAMRT-AMP, etc.), a plasmid derived from Escherichia coli (pYG601BR322, pBR325, pUC118, pUC119, etc.), a plasmid derived from Bacillus subtilis (Bacillus The vector may be a plasmid derived from B. subtilis (e.g., pUB110 or pTP5), a yeast-derived plasmid (e.g., YEp13, YEp24, or YCp50), a phage DNA (e.g., Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, or λZAP), an animal virus vector (e.g., retrovirus, adenovirus, or vaccinia virus), or an insect virus vector (e.g., baculovirus). Since the expression vector may result in different levels of protein expression or modifications depending on the host cell, it is desirable to select and use a host cell that is most suitable for the purpose.

[0074] The transformant provided by the present invention can be prepared by transforming a host with the expression vector provided by the present invention, and can be used to produce the peptide by expressing the polynucleotide contained in the expression vector. The transformation can be carried out by various methods, including, but not limited to, CaCl precipitation, the Hanahan method in which the efficiency of CaCl precipitation is improved by using a reducing agent such as DMSO (dimethyl sulfoxide), electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, PEG-mediated transformation, dextran sulfate, lipofectamine, and desiccation / repression-mediated transformation. Furthermore, the host used to prepare the transformant is not particularly limited as long as it is capable of producing the peptide, and may be bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium, etc.; yeast cells such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, etc.; fungal cells such as Pichia pastoris, insect cells such as Drosophila and Spodoptera frugiperda Sf9 cells, etc.; animal cells such as CHO, COS, NSO, 293, and Bowes melanoma cells, or plant cells.

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

[0076] The term "culturing" as used herein refers to a method of growing microorganisms under appropriately artificially controlled environmental conditions. In the present invention, the method of culturing the transformant may be performed by a method known in the art. Specifically, the culture may be continuous, using a batch process, fed-batch culture, or repeated fed-batch culture process, as long as it is capable of expressing and producing the peptide of the present invention for promoting the regeneration of hard tissues including dentin, bone, and chalk and / or dental pulp tissue and for treating dentin-pulp disease and / or periodontal disease, without being particularly limited thereto.

[0077] The medium used for cultivation must meet the requirements of the specific strain by adjusting the temperature, pH, etc. under aerobic conditions in a standard medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc. Carbon sources that can be used include a mixed sugar 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 alone or in combination. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate, as well as amino acids such as glutamic acid, methionine, and glutamine, and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its hydrolyzed products, and defatted soybean cake or its hydrolyzed products. These nitrogen sources can be used alone or in combination. The medium can contain potassium monophosphate, potassium diphosphate, and the corresponding sodium-containing salts as phosphorus sources. Potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or the corresponding sodium-containing salts, can be used as phosphorus sources. Inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate. Finally, essential growth substances such as amino acids and vitamins can be used in addition to the above substances.

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

[0079] Additionally, foam formation can be suppressed by using an antifoaming agent such as a fatty acid polyglycol ester. To maintain an aerobic state, oxygen or an oxygen-containing gas (e.g., air) is injected into the culture. The temperature of the culture is usually 27°C to 37°C, preferably 30°C to 35°C. The culture is continued until the maximum amount of the peptide is produced. To achieve this goal, the culture is usually continued for 10 to 100 hours.

[0080] Furthermore, the step of recovering the peptide from the culture can be carried out by a method known in the art. Specifically, the recovery method is not particularly limited as long as it can be used to recover the produced peptide, but preferably includes centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobic, and size exclusion), etc.

[0081] In another aspect, the present invention provides a pharmaceutical composition for preventing or treating dentin-pulp diseases, comprising the peptide.

[0082] As described above, the peptide of the present invention for promoting the regeneration of hard tissues including dentin, bone, and chalk and / or dental pulp tissue and for treating dentin-pulp disease and / or periodontal disease can promote the formation of dentin / pulp tissue-like tissue by the human dental pulp cells when transplanted into a living body together with the human dental pulp cells, and therefore can be used as an active ingredient in a pharmaceutical composition for treating dentin-pulp disease caused by damage to dental pulp tissue.

[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 repeatedly linked two or more times, or in the form of a conjugate in which a drug exhibiting a therapeutic effect on dentin-pulp disease is bound to the N-terminus or C-terminus of the peptide.

[0084] The term "dentin-pulp disease" as used herein means a disease that develops when the dental pulp tissue and the dentin connected thereto are damaged due to damage to the dental pulp tissue.

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

[0086] In another aspect, the present invention provides a pharmaceutical composition for preventing or treating periodontal disease, comprising the peptide.

[0087] As described above, the peptide of the present invention for promoting the regeneration of hard tissues including dentin, bone, and chalk and / or dental pulp tissue and for treating dentin-pulp disease and / or periodontal disease can promote the formation of bone-like tissue by human dental pulp cells when transplanted into a living body together with the human dental pulp cells, and therefore can be used as an active ingredient in a pharmaceutical composition for treating periodontal disease that causes 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 conjugate 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" as used herein refers to a disease caused by bacteria infecting the gap between the gums and teeth, damaging the periodontal ligament 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 tissues and forming periodontal pockets. The more severe the periodontitis, the deeper the periodontal pockets become. As the periodontal pockets deepen, inflammation of the periodontal ligament occurs, ultimately leading to bone loss.

[0090] In the present invention, the periodontal disease is not particularly limited as long as the peptide of the present invention shows a therapeutic effect, but examples thereof include gingivitis, periodontitis, periodontal pockets, and periodontal abscesses.

[0091] The term "prevention" as used herein means any action of inhibiting or delaying the onset of dentin-pulp disease by administering a pharmaceutical composition for preventing or treating dentin-pulp disease containing the peptide of the present invention, or any action of inhibiting or delaying the onset of periodontal disease by administering a pharmaceutical composition for preventing or treating periodontal disease containing the peptide of the present invention.

[0092] The term "treatment" in the present invention means any action 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, thereby treating the dental pulp disease by promoting the regeneration of dentin or dental pulp tissue, or any action of administering a pharmaceutical composition containing the peptide of the present invention as an active ingredient to an individual seeking treatment for periodontal disease, thereby treating the periodontal disease by promoting the regeneration of bone and / or chalk.

[0093] The pharmaceutical composition of the present invention may be prepared in the form of a pharmaceutical composition for treating dentin-pulp disease and / or periodontal disease, further comprising a suitable carrier (natural or non-natural carrier), excipient, or diluent commonly used in preparing pharmaceutical compositions of the peptide. Specifically, the pharmaceutical composition may be formulated into a sterile injectable solution that can be administered to the site of induced dentin-pulp disease and / or periodontal disease by a conventional method. Examples of carriers, excipients, and diluents that may be included in the pharmaceutical composition 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, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, collagen, etc. When formulated, they may be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. In particular, these may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, ointments (e.g., dental pulp liner, etc.), etc. Examples of non-aqueous solvents and suspensions that may be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Examples of suppository bases that may be used include witepsol (registered trademark), macrogol (registered trademark), Tween (registered trademark) 61, cocoa butter, lauric butter, and glycerogelatin.

[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 wt %, more preferably 0.01 to 20 wt %, based on the total weight of the final composition.

[0095] The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein refers to an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dose level may be determined based on factors including the severity of the disease, the activity of the drug, the patient's age, weight, health status, sex, and sensitivity to the drug, the administration time, route of administration, and excretion rate of the pharmaceutical composition of the present invention used, the duration of treatment, drugs used in combination with or concomitantly with the pharmaceutical composition of the present invention, and other factors known in the medical field. The pharmaceutical composition of the present invention may be administered alone or in combination with a known pharmaceutical composition for treating dentin-pulp disease and / or periodontal disease. Taking all of the above factors into consideration, it is important to administer an amount that provides maximum efficacy at a minimum dose without causing side effects.

[0096] The dosage of the pharmaceutical composition of the present invention can be determined by one skilled in the art, taking into consideration the purpose of use, severity of the disease, the patient's age, weight, sex, medical history, and the type of substance used as the active ingredient. For example, the pharmaceutical composition of the present invention can be administered at about 0.1 ng / kg to about 100 mg / kg, preferably 1 ng / kg to about 10 mg / kg, per adult. The administration frequency of the pharmaceutical composition of the present invention is not particularly limited, but it can be administered once a day or in divided doses administered several times a day. The above dosages do 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 administering a pharmaceutically effective amount of the pharmaceutical composition to a non-human individual suffering from dentin-pulp disease.In yet another aspect, the present invention provides a method for treating periodontal disease, comprising administering a pharmaceutically effective amount of the pharmaceutical composition to a non-human individual suffering from periodontal disease.

[0098] The term "individual" as used herein may include, without limitation, humans seeking treatment for dentin-pulp disease and / or periodontal disease, or mammals other than humans, including rats, livestock, and the like.

[0099] The pharmaceutical composition of the present invention for treating dentin-pulp disease and / or periodontal disease 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 via routes such as oral administration or oral injection, depending on the purpose, but is not limited thereto.

[0100] In another aspect, the present invention provides a quasi-drug composition for preventing or ameliorating dentin-pulp disease, which contains the peptide, or a quasi-drug composition for preventing or ameliorating periodontal disease, which contains the peptide.

[0101] The term "amelioration" according to the present invention refers to any action that at least reduces a parameter related to the condition being treated, such as the severity of the symptoms.

[0102] In the present invention, the improvement can be interpreted as meaning any action 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, thereby promoting the regeneration of dentin or dental pulp tissue, thereby improving or benefiting the symptoms of dentin-pulp disease, or any action of administering a pharmaceutical composition containing the peptide of the present invention as an active ingredient to an individual seeking treatment for periodontal disease, thereby promoting the regeneration of bone and / or chalk, thereby improving or benefiting the symptoms of periodontal disease.

[0103] The term "quasi-drug" as used herein means an article used for the purpose of diagnosing, curing, improving, mitigating, treating, or preventing a disease in humans or animals, which has a milder effect than a pharmaceutical product. For example, according to the Pharmaceutical Affairs Law, quasi-drugs exclude articles used for pharmaceutical purposes, and include fiber and rubber products used to treat or prevent diseases in humans and animals, items that have a mild effect on the human body or do not have a direct effect on the human body and are not tools or machines, or items similar to them, and disinfectants and insecticides used to prevent infectious diseases.

[0104] In the present invention, the type and dosage form of the quasi-drug composition containing the peptide are not particularly limited, and examples thereof include oral disinfectants, oral cleaning products, toothpaste, floss, and oral ointments.

[0105] In yet another aspect, the present invention provides a health functional food composition for preventing or ameliorating dentin-pulp disease and / or periodontal disease, comprising the peptide.

[0106] The term "food" as used herein includes all foods in the usual sense, such as meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, drinking water, tea, health supplements, alcoholic beverages, vitamin complexes, functional health foods, and health foods.

[0107] The term "functional food" refers to a food with high medical and therapeutic value that is processed to efficiently exert bioregulatory functions in addition to providing nutrients. The term "functional" refers to the ability to regulate nutrients or achieve beneficial health effects, such as physiological effects, on the structure and function of the human body. The food of the present invention can be prepared by methods commonly used in the art, and may be prepared by adding raw materials and ingredients commonly used in the art. The food may also be prepared in any dosage form that is recognized as a food. The food composition of the present invention can be prepared in various dosage forms. Unlike general pharmaceuticals, the food composition is made from food ingredients, which has the advantage of being free of side effects that can occur with long-term pharmaceutical use, and is highly portable. The food of the present invention can be taken as an adjuvant to enhance the effects of preventing or improving dentin-pulp disease and / or periodontal disease.

[0108] The term "health food" refers to food that has more active health maintenance and promotion effects than general foods, and "health supplement food" refers to food for the purpose of health supplementation. In some cases, the terms "health functional food," "health food," and "health supplement" may be used interchangeably.

[0109] Specifically, the health functional foods are foods in which the peptides of the present invention are added to food ingredients such as beverages, teas, spices, gums, and confectioneries, or are manufactured as capsules, powders, suspensions, etc., and when ingested, they bring about specific health benefits. However, unlike general medicines, they have the advantage of being made from food ingredients and therefore free from side effects that can occur when taking medicines for a long period of time.

[0110] The food composition of the present invention can be taken on a daily basis and is therefore expected to be highly effective in preventing or improving dentin-pulp disease and / or periodontal disease, making it highly useful.

[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] The food composition may also contain additional ingredients commonly used in food compositions to improve smell, taste, visual appearance, etc. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, etc. Minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chromium (Cr) may also be included. Amino acids such as lysine, tryptophan, cysteine, and valine may also be included.

[0113] The food compositions may further contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder, high-strength bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), etc.), colorants (tar dyes, etc.), color formers (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 D-bitartrate, etc.), fortifiers, emulsifiers, thickeners (thickening agents), coating agents, gum bases, antifoaming agents, solvents, and improvers. The additives may be selected according to the type of food and used in appropriate amounts.

[0114] The peptides of the present invention can be added directly or in combination with other foods or food ingredients, and can be used appropriately in a conventional manner. The amount of active ingredient to be added can be determined appropriately depending on the intended use (prevention, health, or therapeutic treatment). Generally, when producing a food or beverage, the food composition of the present invention can be added in an amount of 50 parts by weight or less, specifically 20 parts by weight or less, per 100 parts by weight of the food or beverage composition. However, when taking the food or beverage for long-term health and hygiene purposes, the active ingredient can be added in an amount below the above range, and there is no problem from the standpoint of safety, so the active ingredient can be used in an amount above the above range.

[0115] One example of the food composition of the present invention may be a health drink composition. In this case, similar to conventional beverages, various flavorings or natural carbohydrates may be added as additional ingredients. 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. Examples of sweeteners that may be used include natural sweeteners such as thaumatin and stevia extract; and synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrates per 100 mL of the health drink composition of the present invention may typically be about 0.01 g to 0.04 g, specifically about 0.02 g to 0.03 g.

[0116] In addition to the above, the health drink composition may contain various nutrients, vitamins, electrolytes, flavorings, coloring agents, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonation agents. Fruit pulp may also be included for the production of natural fruit juice, fruit juice drinks, or vegetable drinks. These ingredients may be used independently or in combination. The proportion of these additives is not critical, but is typically selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health drink composition of the present invention.

[0117] The food composition of the present invention may contain the peptide of the present invention in various weight percentages as long as it can exhibit the effect of preventing or improving dentin-pulp disease and / or periodontal disease. Specifically, the food composition may contain the peptide of the present invention in an amount of 0.00001 to 100 weight % or 0.01 to 80 weight % relative to the total weight of the food composition, but is not limited thereto.

[0118] In another aspect, the present invention provides a method for preventing or treating dentin-pulp disease and / or periodontal disease, which comprises administering a composition containing the peptide to an individual.

[0119] In yet another aspect, the present invention provides a method for promoting the regeneration of dentin or dental pulp tissue, and / or bone or chalk, comprising administering to an individual a composition comprising the peptide.

[0120] In yet another aspect, the present invention provides a peptide comprising an amino acid sequence represented by the following general formula 1 or a composition comprising the peptide for promoting the regeneration of hard tissues including dentin, bone, and chalk and / or dental pulp tissue, and for preventing or treating dentin-pulp disease or periodontal disease. KY-R1-R2-R3-R4-R5-R6-YK (general formula 1) In the 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 yet another aspect, the present invention provides a peptide comprising any one of the amino acid sequences 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 dental pulp tissue, and for use in preventing or treating dentin-pulp disease and / or periodontal disease.

[0122] Furthermore, as one embodiment of the present invention, the present invention provides a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 8 or a composition comprising the peptide for promoting the regeneration of hard tissues including dentin, bone, and chalk and / or dental pulp tissue, and for 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 Methods and Materials Example 1-1: Synthesis of peptides for promoting regeneration of hard tissues including dentin, bone, and chalk and / or dental pulp tissues and for treating dentin-pulp disease and / or periodontal disease The inventors synthesized a peptide (SEQ ID NO: 1) that exhibits the effect of promoting the regeneration of hard tissues including dentin, bone, and chalk and / or dental pulp tissue using the 9-fluorenylmethyloxycarbonyl (Fmoc) method, and then synthesized peptides of each group by substituting the amino acids of the synthesized peptide (Tables 1 to 8). N-KYKAKKKNYK-C (SEQ ID NO: 1)

[0125] First, peptides in Group 1 were synthesized by substituting the peptide of SEQ ID NO: 1 or the fifth to seventh amino acids of the peptide of SEQ ID NO: 1 with arginine (Table 1).

[0126] [Table 1]

[0127] Next, peptides in Group 2 were synthesized by substituting the 8th amino acid of the peptide of SEQ 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 SEQ ID NO: 1 with arginine, and 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 SEQ 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 SEQ 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 SEQ 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 SEQ 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 SEQ ID NO: 1 with alanine, the fourth amino acid with arginine, the eighth amino acid with serine, and the fifth through seventh amino acids with arginine (Table 8).

[0138] [Table 8]

[0139] [Example 1-2: Cell culture] The cells were cultured in a humidified atmosphere containing 5% CO2 at 37°C and used in the experiments. Human mesenchymal stem cells (hBMSCs) were purchased from LONZA (Switzerland). hBMSCs were cultured in α-MEM (Invitrogen, registered trademark) medium supplemented with 10% heat-inactivated bovine serum.

[0140] [Example 1-3: Isolation and culture of human dental pulp cells] Human dental pulp cells were isolated from wisdom teeth of 10 adults (aged 18-22) at Seoul National University Dental Hospital. Specifically, all experiments were approved by the hospital's Institutional Review Board and conducted with patient consent. Based on the method described by Jung HS et al. (J Mol Histol. (2011)), wisdom teeth were amputated, the pulp was exposed, and the pulp was isolated with forceps. The isolated pulp was minced with a double-sided blade, placed in a 60 mm dish, covered with a coverslip, and cultured in Dulbecco's modified Eagle's medium (DMEM). Human dental pulp cells are known 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 was isolated from human dental pulp cells and mesenchymal stem cells using TRIzol reagent. cDNA was synthesized using 2 μg of total RNA, 1 μl of reverse transcriptase, and 0.5 μg of oligo (dT). The synthesized cDNA was used for real-time polymerase chain reaction (PCR). Real-time PCR was performed using SYBR GREEN PCR Master Mix (Takara, Japan) on an ABI PRISM 7500 sequence detection system (Biosystems). The PCR conditions were 94°C for 1 minute, 95°C for 15 seconds, and 60°C for 34 seconds, repeated 40 times. Results were analyzed using the comparative cycle threshold (CT) method. Primer sequences are listed in Table 9.

[0142] [Table 9]

[0143] [Examples 1-5: In vivo implantation and histological analysis] Human dental pulp cells (hDPCs) were isolated and used for in vivo transplantation experiments. hDPCs (2 × 10 6 The cells were mixed with 100 mg of hydroxyapatite / tricalcium phosphate (HA / TCP) ceramic powder (Zimmer, USA) alone or with 10 μg of peptide in 0.5% fibrin gel and then implanted into immune-compromised mice (NIH-bg-nu-xid; Harlan Laboratories, Indianapolis, IN) for 6 and 12 weeks. Samples were then harvested, fixed in 4% paraformaldehyde, decalcified in 10% EDTA (pH 7.4), embedded in paraffin, and stained with hematoxylin and eosin (HE).

[0144] [Example 1-6: Effect of a novel peptide on dentin regeneration in a dentin damage model] Three beagle dogs (12-16 kg; 6-8 weeks old) were anesthetized with inhaled Gerolan and intravenously administered Zoletil (5 mg / kg) and xylazine (0.2-0.5 mg / kg), followed by lidocaine treatment (2% lidocaine with 1:80,000 epinephrine). Dentin damage was created in the premolars and molars of the beagle dogs' mandibles using a dental bur, and 10 μg of Group 1 SEQ ID NO: 1 peptide per tooth was administered. After 6 weeks, the beagle dogs were sacrificed by administering an overdose of pentobarbital (90-120 mg / kg). The teeth of the beagle dogs were removed and fixed in 10% formalin, and then calcium was removed by adding 5% formic acid. The tissue was then molded and embedded in paraffin to obtain 5 μm-thick tissue sections. The tissue sections were stained with hematoxylin-eosin and analyzed using a light microscope (LEICA DM750, Germany) equipped with a digital camera (LEICA ICC50 camera, Germany).

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

[0146] [Effects of peptides for promoting dentin or dental pulp tissue regeneration and treating 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 mineralization. Therefore, in this study, we synthesized a novel peptide that increases the expression of the DSPP gene, an odontoblast differentiation marker gene, and promoted odontoblast differentiation and dentin formation, and confirmed its effectiveness.

[0147] The effect of each peptide group on DSPP mRNA expression was confirmed by real-time PCR (Table 10).

[0148] [Table 10] JPEG2025540802000012.jpg233148JPEG2025540802000013.jpg81148

[0149] Figure 1 is a graph showing the average DSPP mRNA expression levels found in each group in Table 10. In Figure 1, DSPP mRNA expression was increased by about 2 to 8 times or more in all peptide groups compared to the control group, with the highest DSPP mRNA expression level being observed in the peptide group of Group 1. Since each of the differentiation marker genes is known to be involved in the differentiation of odontoblasts and the mineralization process of dentin, the peptides provided by the present invention were analyzed to exhibit the effect of promoting dentin regeneration.

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

[0151] [Table 11] JPEG2025540802000015.jpg233148JPEG2025540802000016.jpg81148

[0152] [Table 12] JPEG2025540802000018.jpg233148JPEG2025540802000019.jpg81148

[0153] In Figure 2, to confirm the effect of the novel peptides on the expression of BSP and DMP1 genes, which are differentiation marker genes for osteoblasts and leucocytes, human mesenchymal stem cells were treated with each group of peptides, and BSP and DMP1 gene expression was examined by real-time PCR. Compared to the control group, peptides from all groups increased BSP gene expression by approximately 1.5- to 3-fold, and DMP1 gene expression by more than 6- to 10-fold. In particular, peptides 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 leucocytes and are known to be involved in the mineralization process of bone and chalk, the peptides provided by the present invention were analyzed to exhibit the effect of promoting bone and chalk regeneration.

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

[0155] Referring to FIG. 3, it was confirmed that the expression of DSP, an odontoblast differentiation marker protein, and BSP, an osteoblast and leucoblast differentiation marker protein, increased in a concentration-dependent manner by the novel peptide.

[0156] Figure 4 shows the results of histological evaluation three weeks after damaging the dentin of a beagle dog's teeth with 10 μg of the novel peptide (SEQ ID NO: 1) to determine whether the novel peptide induces physiological dentin regeneration in a dentin damage animal model.

[0157] As shown in Figure 4, the control group showed no change below the damaged dentin, while the test group treated with the novel peptide showed the formation of physiological dentin below the damaged dentin. These results indicate that hypersensitivity, dentin caries, and pain due to tooth fracture caused by damaged dentin can be treated by physiological dentin regeneration.

[0158] Figure 5 shows the results of the in vitro experiments shown in Figure 2 and Tables 10 and 11. To confirm the effect of the Group 1 (SEQ ID NO: 1) peptide on hard tissue formation in vivo, 10 μg of the Group 1 SEQ ID NO: 1 peptide or control (cell-only) human dental pulp cells (hDPCs) were transplanted into the subcutaneous tissue of mice with impaired immune systems. 12 weeks after transplantation, the rate of hard tissue formation was confirmed to be increased in the groups treated with the novel peptide compared to the control group (A-D: hDPCs alone; E-H: Group 1 peptide; I-L: BMP2 treatment). Size bars: 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 using hematoxylin-eosin staining revealed that bone / chalk-like tissue, in which cells were embedded in the newly formed mineralized tissue matrix around the HA / TCP particles, was formed in the hDPCs-only, Group 1 peptide, and positive control BMP2-treated groups. However, compared to the control group, the test group treated with the novel peptide showed more similar hard tissue formation to the BMP-2-treated group. In summary, these results suggest that the novel peptide used in this experiment has the potential to promote the regeneration of bone / chalk-like tissue and dentin / pulp tissue complexes.

[0160] Figure 6 shows the effect of Group 1 (SEQ ID NO: 1) peptide on periodontal ligament formation in vivo. Group 1 SEQ ID NO: 1 peptide (10 μg) or control group (cell only) human dental pulp cells (hDPCs) were transplanted into the subcutaneous tissue of mice with impaired immune systems, and the results were compared with those of the control group 6 weeks after transplantation (size bar: A, C: 200 μm; B, D: 100 μm).

[0161] Referring to Figure 6, 6 weeks after implantation, periodontal ligament fiber bundles (black triangles) were observed to have formed in the novel peptide-treated group compared to the control group. Specifically, histological analysis using hematoxylin-eosin staining revealed that irregular fiber arrangements were observed in hDPCs alone, whereas in Group 1, the novel peptide-treated group, periodontal ligament-like tissue containing fiber bundles was observed to have formed in the newly formed mineralized tissue around the HA / TCP particles. These results suggest that the novel peptide used in this experiment may be effective in promoting the regeneration of damaged periodontal ligaments.

[0162] This research was supported by research funds from the Ministry of Health and Welfare and the Korea Health Industry Development Institute in 2022 (1465037227, "Development of chronic periodontitis treatment technology using new bio-fusion material CPNE7 peptide").

[0163] In the present specification and drawings, preferred embodiments of the present invention are disclosed, and although specific terms are used, these are used in a general sense merely to clearly explain the technical contents of the present invention and to aid in understanding the invention, and are not intended to limit the scope of the present invention. It is obvious to those skilled in the art to which the present invention pertains that, in addition to the embodiments disclosed herein, other modifications based on the technical concept of the present invention can be implemented.

Claims

1. A peptide for promoting the regeneration of hard tissue or dental pulp tissue and for treating dentin-pulp disease or periodontal disease, which consists of any one of the amino acid sequences of SEQ ID NOs: 1 to 8.

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

3. The peptide of claim 1 , wherein the hard tissue comprises dentin, bone, and chalk.

4. A polynucleotide encoding the peptide of claim 1.

5. An expression vector comprising the polynucleotide of claim 4.

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

7. 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 preventing or treating periodontal disease, comprising the peptide of claim 1.

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

10. A quasi-drug composition for preventing or ameliorating dentin-pulp disease or periodontal disease, comprising the peptide according to claim 1.

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

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

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

14. A method for promoting regeneration of hard tissues or dental pulp tissues, including dentin, bone and chalk, comprising administering to a non-human individual a composition comprising the peptide of claim 1.

15. 2. The peptide for promoting the regeneration of hard tissue or dental pulp tissue and treating dentin-pulp disease or periodontal disease according to claim 1, which consists of any one of the amino acid sequences of SEQ ID NOs: 1 to 8.

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