Pharmaceutical compositions for the prevention or treatment of periodontal disease or traumatic tooth avulsion.

Abstract

The present invention provides a pharmaceutical composition comprising a peptide containing a specific amino acid sequence for the prevention or treatment of periodontal disease or traumatic tooth avulsion, and a composition for periodontal tissue regeneration.

Description

Technical Field

[0001] This invention relates to a pharmaceutical composition for the prevention or treatment of periodontal disease or traumatic tooth avulsion, and a composition for periodontal tissue regeneration. Background Technology

[0002] The periodontal tissue is a collective term for the tissues surrounding the teeth. It consists of the alveolar bone supporting the teeth within the jawbone (dental jawbone), the periodontal ligament connecting the alveolar bone and teeth within the jawbone, cementum containing fibers of the periodontal ligament, and the gingiva (gum), the soft tissue covering the alveolar bone. Gingival fibroblasts and periodontal ligament fibroblasts are the main cellular components of the soft connective tissue of the gingiva, playing a role in forming and maintaining the extracellular matrix. Gingival fibroblasts are known to primarily participate in maintaining the gingival connective tissue, while periodontal ligament fibroblasts, due to their unique functions, not only form the periodontal ligament but also participate in the repair and regeneration of adjacent alveolar bone and cementum within the body.

[0003] The periodontal ligament is a fibrous connective tissue that connects the cementum and alveolar bone at the root of the tooth. The two ends of the periodontal ligament are embedded in the cementum and alveolar bone at the root. The fibrous bundles of the periodontal ligament embedded in the alveolar bone or cementum in this way are called Sharpey's fibers. The periodontal ligament plays a crucial role not only in maintaining the teeth but also in maintaining the structural properties of hard tissues, resisting the impact of occlusal pressure, and protecting soft tissues such as blood vessels and nerves from damage. Another important function is its role in nutrient supply and as a sensory receptor. The nerve innervation of the periodontal ligament transmits proprioception, touch, and pain sensations through the trigeminal nerve pathway, sensing and regulating the external pressure on each tooth, and playing a vital role in the neuromuscular mechanisms that regulate the masticatory muscle system. This periodontal ligament contains many fibroblasts. Due to their unique functions, these cells can not only form the periodontal ligament, but also participate in the formation and resorption of cementum and alveolar bone, induce physiological tooth movement, enable periodontal tissues to adapt to occlusal pressure, and have the function of repairing damage.

[0004] Periodontal disease can be classified into gingivitis, which is confined to the gums, and periodontitis, which involves inflammation spreading to the alveolar bone surrounding the tooth root. It is a disease where inflammation occurs in the periodontal tissues supporting the tooth, rather than damaging the tooth itself. Clinically, periodontal disease leads to tooth loss due to gingival bleeding and swelling, periodontal pocket formation, and alveolar bone destruction. The causes of periodontal disease include both local and systemic factors. Two well-known local factors are dental plaque, calculus and other local causes, and occlusal trauma. As a pathogenesis, when dental plaque mechanically accumulates in periodontal pockets, it becomes a habitat for surrounding bacteria. This habitat gradually shifts from aerobic, aerated, Gram-positive bacteria to anaerobic Gram-negative bacteria, which proliferate and migrate deeper into the periodontal pockets. At this point, the toxins and all products of the proliferating anaerobic Gram-negative bacteria directly damage tissues or stimulate the immune system, causing inflammation to be induced by the stimulated immune system through various mechanisms, along with the destruction of periodontal tissues. As a defense mechanism against this, the function of polymorphonuclear leukocytes and the immune response play a major role as systemic factors. However, it is well known that the key to the prevention and treatment of periodontal disease lies in the antibacterial and bacteriostatic effects against the anaerobic Gram-negative bacteria, which are the root cause, and in the removal and destruction of the toxic products of these bacteria, as well as the restoration of the lost periodontal tissues to their original state.

[0005] Basic treatments for periodontal disease include dental cleaning (scaling) to remove plaque and tartar, which are the contributing factors to periodontal disease, and root planing, a similar procedure to remove plaque and tartar. Root planing removes the main factors causing inflammation in the cementum at the root surface of the teeth. As inflammation worsens due to periodontal disease, alveolar bone damage may occur. When alveolar bone resorption is severe, bone grafting through regenerative periodontal surgery may be performed. Regarding the type of bone graft, the patient's own bone (autologous bone), xenogeneic bone, or synthetic bone can be used.

[0006] Therefore, in order to treat periodontal disease, it is necessary not only to remove the pathogens causing the periodontal disease, but also to carry out the process of regenerating periodontal tissue.

[0007] Currently, treatment methods for regenerating damaged periodontal tissues include bone grafting, guided tissue regeneration, and treatments using various growth factors. There are also tooth replantation procedures that focus on the regeneration of periodontal ligament cells and the formation of new cementum.

[0008] In this regard, research is actively underway to develop effective methods for regenerating periodontal tissue. For example, Korean Patent No. 1179476 discloses the following genes that are highly expressed during the mineralization process of human periodontal ligament (hPDL) cells: promyelocytic leukemia zinc finger (PLZF) gene, Fk506-binding protein 5 (FKBP5) gene, serum amyloid A1 (SAA1) gene, fatty acid-binding protein 4 (FABP4) gene, sequence similarity family 107 member A (FAM107A) gene, serine protease (CORIN) gene, ras-associated c3 botulinum toxin substrate 3 (RAC3) gene, or prolactin-inducible protein (PIP) gene; and genes that are lowly expressed during the mineralization process of human periodontal ligament (hPDL) cells: FNDC1, PTGS2, RSAD2, NPTX1, VCAM1, MX1, IFIT1, CLDN1, or WISP2 genes. Furthermore, Korean Patent No. 101788916 discloses a pharmaceutical composition for treating periodontal disease containing ameloblast culture medium.

[0009] Furthermore, when external force is applied to teeth or periodontal tissues, various forms of damage occur depending on the direction and size of the force. Dental injuries can be broadly classified into fracture injuries and dislocation injuries. Dislocation injuries refer to damage to the periodontal ligament, which can be further subdivided into concussion subluxation, extrusion dislocation, lateral dislocation, complete dislocation, and intrusion. These can occur at all ages, but are most common in children aged 8-12. The success of replantation of a completely dislocated tooth depends on the regeneration of the periodontal ligament attached to the completely dislocated tooth or the periodontal ligament remaining in the alveolar socket where the tooth was detached. If periodontal ligament regeneration does not occur, resorption of cementum and dentin at the tooth root will occur, leading to ankylosis, where the alveolar bone replaces the dentin in that area. Therefore, the ultimate goal of periodontal treatment is to stop the progression of periodontal disease and regenerate the damaged periodontal tissues, including the gums, periodontal ligaments, alveolar bone, and cementum, to restore them to their original state. However, to date, no method has been reported that can directly regenerate or differentiate the periodontal ligaments.

[0010] Against this backdrop, in order to develop a method for regenerating periodontal tissues (periodontal ligament, cementum, alveolar bone) damaged by periodontal disease, the inventors, through dedicated research and effort, identified a new use for the peptide described below, thus completing this invention. This peptide induces the differentiation of periodontal ligament cells, which can not only treat periodontal ligament damage caused by periodontal disease or traumatic tooth avulsion, but also regenerate damaged cementum and alveolar bone. Summary of the Invention

[0011] Technical problems to be solved The purpose of this invention is to provide a pharmaceutical composition for the prevention or treatment of periodontal disease or traumatic tooth avulsion.

[0012] Another object of the present invention is to provide a composition for regenerating periodontal tissue.

[0013] Another object of the present invention is to provide a composition for promoting gene expression, wherein the gene comprises any one or more of BSP (bone sialoprotein), DMP1 (Dentin matrix protein 1), CAP (Cementum attachment protein), COL3 (collagen type III), and periosteum protein.

[0014] Another object of the present invention is to provide a method for treating periodontal disease or traumatically avulsed teeth in an individual, the method comprising the step of administering an effective amount of a peptide or a pharmaceutically acceptable salt thereof to an individual who requires treatment for periodontal disease or traumatically avulsed teeth.

[0015] Another object of the present invention is to provide the use of the above-mentioned peptide or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of periodontal disease or traumatic tooth avulsion.

[0016] The purpose of this invention is not limited to the contents mentioned above. Those skilled in the art to which this invention pertains can clearly understand other purposes not mentioned from the following description.

[0017] Technical solution One embodiment of the present invention, which aims to solve the above-mentioned technical problems, provides a pharmaceutical composition for the prevention or treatment of periodontal disease or traumatic avulsion of teeth, comprising a peptide comprising an amino acid sequence of the following general formula 1 or a pharmaceutically acceptable salt thereof.

[0018] KYKQ-X5-X6-X7-X8-YK (General Formula 1) In the above general formula 1, X5 to X7 are arginine (R) or lysine (K) respectively; X8 is either asparagine (N) or serine (S).

[0019] Regarding the aforementioned peptides, mutant peptides whose constituent amino acid sequences and one or more amino acid residues have different sequences are also included in the scope of peptides provided in this invention, as long as they can exhibit the effect of preventing or treating periodontal disease or traumatic tooth avulsion.

[0020] Throughout this specification, conventional single-letter and three-letter symbols are used for the naturally occurring amino acids that constitute peptides. Furthermore, amino acids described in abbreviations in this specification are referred to according to the IUPAC-IUB nomenclature.

[0021] Alanine (Ala, A) and Arginine (Arg, R) Asparagine Asn, N-aspartic acid Asp, D Cysteine ​​(Cys, C) and Glutamate (Glu, E) Glutamine (Gln, Q) and glycine (Gly, G) Histidine (His, H) and Isoleucine (Ile, I) Leucine (Leu, L), Lysine (Lys, K) Methionine (Met, M) and Phenylalanine (Phe, F) Proline (Pro, P) and Serine (S) Threonine (Thr), T; Tryptophan (Trp), W Tyrosine (Tyr, Y) and Valine (V) The inventors have confirmed that the above-mentioned peptides can treat periodontal diseases or traumatic tooth avulsion by promoting the regeneration of periodontal ligaments or their differentiation into osteoblasts and cementoblasts, as well as the regeneration of alveolar bone and cementum, thereby completing the present invention.

[0022] It was confirmed that the aforementioned peptides increased the expression of BSP, DMP1, and CAP, which are marker genes for osteoblast and cementoblast differentiation, in human periodontal ligament cells, and also increased the expression of periosteal protein and COL3, which are marker genes for periodontal ligament differentiation. Furthermore, it was confirmed that the aforementioned peptides increased the expression of BSP and DMP1, which are marker genes for osteoblast and cementoblast differentiation, in human mesenchymal stem cells.

[0023] Furthermore, experimental results from animal models of periodontal tissue injury confirmed that the aforementioned peptides regenerate alveolar bone, cementum, and periodontal ligament. Specifically, the peptides of the present invention have the effect of regenerating alveolar bone, forming new cementum-like tissue and new periodontal ligament-like tissue, and the new periodontal ligament formed by the peptides of the present invention is embedded in the newly formed alveolar bone and cementum.

[0024] In one instance, the peptide described above may be a case where one or more amino acids undergo a conservative substitution.

[0025] "Conservative substitution" refers to the replacement of one amino acid with another amino acid having similar structure and / or chemical properties. The peptide retains the same or similar biological activities, and may exhibit more than one conserved substitution. Such amino acid substitutions typically occur based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues. For example, positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include glutamic acid and aspartic acid; aromatic amino acids include phenylalanine, tryptophan, and tyrosine; and hydrophobic amino acids include alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. Furthermore, amino acids can be classified into those with electrically charged side chains and those with uncharged side chains. Amino acids with charged side chains include aspartic acid, glutamic acid, lysine, arginine, and histidine. Amino acids with uncharged side chains can be further classified into nonpolar and polar amino acids. Specifically, nonpolar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, and proline, while polar amino acids include serine, threonine, cysteine, asparagine, and glutamine. Conservative substitutions to amino acids with similar properties as described above are expected to exhibit the same or similar activities.

[0026] Thus, even if the acidic, basic, or aromatic amino acids constituting the peptides of the present invention are replaced by different acidic, basic, neutral, or aromatic amino acids, they can still exhibit the effects of the peptides provided in the present invention. Therefore, mutant peptides with sequences having one or more amino acid residues different from the amino acid sequence constituting the peptides of the present invention are also included in the scope of the peptides provided in the present invention, which is obvious.

[0027] Furthermore, the peptides of the present invention, even if they have any amino acid appended to their N-terminus or C-terminus, can directly exhibit the effects of the peptides provided in the present invention, and are therefore included in the scope of the peptides provided in the present invention. As one example, the peptide may have 1 to 300 amino acids appended to its N-terminus or C-terminus; as another example, it may have 1 to 100 amino acids appended to its N-terminus or C-terminus; and as yet another example, it may have 1 to 24 amino acids appended to its N-terminus or C-terminus.

[0028] In one example, the peptide described above may contain any of the amino acid sequences of sequence numbers 1 to 16.

[0029] In another example, the peptide described above is essentially composed of any of the amino acid sequences in sequence numbers 1 to 16, or the peptide described above may be composed of any of the amino acid sequences in sequence numbers 1 to 16.

[0030] Even if described in this invention as a "peptide composed of a specific sequence number," when it has the same or corresponding activity as a peptide composed of amino acid sequences of the corresponding sequence number, it does not exclude meaningless sequence additions or naturally occurring mutations or silent mutations before or after the amino acid sequence of the corresponding sequence number. Such sequence additions or mutations are also within the scope of this invention, which is obvious. That is, even if there are partial sequence differences, if a certain level of homology is exhibited and the same or similar activity is observed, it falls within the scope of this invention. Specifically, the peptides of this invention may comprise amino acid sequences of sequence numbers 1 to 16 and amino acid sequences having 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of homology or identity, but are not limited thereto.

[0031] "Homology" or "identity" refers to the degree of relevance to two given amino acid sequences or base sequences, expressed as a percentage. The terms "homology" and "identity" are sometimes used interchangeably.

[0032] Whether any two peptide sequences are homologous, similar, or identical can be determined, for example, by using default parameters such as those in Pearson et al. (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444, and by using well-known computer algorithms such as the "FASTA" program. Alternatively, as performed in the Needleman program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) is used to determine (including the GCG package (Devereux, J. et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ et al., J MOLEC BIOL 215]: 403 (1990); Guideto Huge Computers, Martin J. Bishop [ed.] Academic Press, Santiago, 1994, and [CARILLO] ETA / .](1988) SIAM J Applied Math 48:1073). For example, BLAST or ClustalW from the National Bioengineering Information Data Center can be used to determine homology, similarity, or identity.

[0033] The homology, similarity, or identity of peptides can be determined by comparing sequence information, for example, using a computer program such as GAP (Growth Aptitude Test) as described, for example, as published in Smith and Waterman, Adv. Appl. Math (1981) 2:482, J Mol Biol. 48:443. In short, the GAP program is defined as the number of similarly arranged symbols (i.e., amino acids) divided by the total number of symbols in the shorter of the two sequences. The default parameters for the GAP procedure may include (1) a unary comparison matrix (containing a value of 1 for identity and a value of 0 for non-identity) and a weighted comparison matrix as disclosed in Schwartz and Dayhoff, Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), Gribskov et al. (1986) Nucl. Acids Res. 14: 6745 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) replacement matrix); (2) a penalty of 3.0 for each vacancy and an additional penalty of 0.10 for each symbol of each vacancy (or a penalty of 10 for vacancy opening and 0.5 for vacancy extension); and (3) no penalty for terminal vacancy. Therefore, the term "homology" or "identity" as used in this invention refers to the relevance between sequences.

[0034] In one instance, the aforementioned peptide can be used either as a peptide-independent form or as a polypeptide formed by repeating the aforementioned peptide two or more times.

[0035] Therefore, the above pharmaceutical composition may contain polypeptides with repeated peptide linkages.

[0036] In one example, a combination of various methods for preparing diverse peptides can be used to prepare peptides suitable for the present invention, comprising an amino acid sequence of general formula 1.

[0037] The peptides of the present invention, depending on their length, can be synthesized using methods well-known in the relevant field, for example, by using an automated peptide synthesizer, or produced by gene modification techniques. Specifically, the peptides of the present invention can be prepared using standard synthetic methods, recombinant expression systems, or any other methods in the relevant field. Therefore, the peptides according to the present invention can be synthesized, for example, by a variety of methods including those described below, but are not limited thereto.

[0038] (a) A method for synthesizing peptides in stages or by fragment assembly using solid-phase or liquid-phase methods, and ultimately separating and purifying the peptide products; or (b) A method for expressing a nucleic acid construct encoding a peptide in a host cell and recovering the expression product from the host cell culture; or (c) A method for performing in vitro cell-free expression of a nucleic acid construct encoding a peptide and recovering the expression product; or A method for obtaining peptide fragments by any combination of (a), (b) and (c), then ligating the fragments to obtain peptides, and recovering the corresponding peptides.

[0039] Furthermore, the preparation of the aforementioned peptides includes modification using L- or D-type amino acids and / or non-natural amino acids; and / or modification through natural sequence alteration, such as modification of side chain functional groups, intramolecular covalent bonding, such as cyclization between side chains, methylation, acylation, ubiquitination, phosphorylation, aminohexaneization, biotinylation, etc. Moreover, the aforementioned modifications include substitution to form non-natural compounds.

[0040] The substituted or added amino acids used for the above modifications can be not only the 20 amino acids commonly found in human proteins, but also atypical or non-naturally occurring amino acids. Commercial sources of atypical amino acids may include, but are not limited to, Sigma-Aldrich, ChemPep, and Genzyme Pharmaceuticals. Peptides containing these amino acids and typical peptide sequences can be synthesized and purchased from commercial peptide synthesis companies, such as American Peptide Company or Bachem in the United States, or Anygen in South Korea, but are not limited to these.

[0041] Amino acid derivatives can also be obtained in a similar manner; for example, 4-imidazoleacetic acid can be used.

[0042] Furthermore, regarding the peptides according to the invention, the N-terminus and / or C-terminus may be unmodified. For protection against protein-cleaving enzymes in vivo and to increase stability, the scope of peptides according to the invention may include forms where the N-terminus and / or C-terminus are chemically modified or protected with organic ends, or modified by adding amino acids to the peptide terminus. When the C-terminus is unmodified, the peptides according to the invention have a free carboxyl group at the end, but this is not particularly limiting.

[0043] In particular, in the case of chemically synthesized peptides, since the N- and C-termini are charged, the N-terminus can be acetylated and / or the C-terminus can be amidated to remove this charge, but this is not a particular limitation.

[0044] The aforementioned peptides may include peptides with increased structural stability related to heat, pH, etc., through mutations or modifications in their amino acid sequences; peptides with increased efficacy in preventing or treating periodontal disease or traumatic tooth avulsion; or peptides with increased regenerative capacity of periodontal ligament tissue.

[0045] The aforementioned peptides include the peptide itself, its salts (e.g., pharmaceutically acceptable salts of the aforementioned peptides), or its solvates.

[0046] The types of salts mentioned above are not particularly limited. However, a form that is safe and effective for individuals such as mammals is preferred, but not particularly limited thereto.

[0047] Moreover, the aforementioned peptides can be in any pharmaceutically acceptable form.

[0048] "Pharmaceutical acceptable" means a sufficient amount that can demonstrate a therapeutic effect without causing side effects. This can be easily determined by a person skilled in the art based on medically well-known factors such as the type of disease, the patient's age, weight, health, gender, the patient's sensitivity to the drug, the route of administration, the method of administration, the frequency of administration, the duration of treatment, and any drugs that are mixed or used concurrently.

[0049] In one instance, the aforementioned peptide can be in the form of its pharmaceutically acceptable salt. This salt includes conventional acid addition salts used in the pharmaceutical field, such as in the field of periodontal disease; salts derived from inorganic acids such as hydrochloric acid, bromic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, or nitric acid; and salts derived from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, citric acid, maleic acid, malonic acid, methanesulfonic acid, tartaric acid, malic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, oxalic acid, or trifluoroacetic acid. Furthermore, the aforementioned salt can be a base addition salt such as ammonium, dimethylamine, monomethylamine, monoethylamine, or diethylamine. Moreover, the aforementioned salt includes conventional metal salt forms, salts derived from metals such as lithium, sodium, potassium, magnesium, or calcium. The aforementioned acid addition salts, base addition salts, or metal salts can be prepared by conventional methods. Pharmaceutically acceptable salts and general methodologies for their preparation are well known in the relevant technical fields. For example, see references to [P. Stahl et al. Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd revised edition (Wiley-VCH, 2011)]; [SM Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Sciences, Vol. 66, No. 1, January 1977].

[0050] To protect the condensation of amino acids or peptides, various activating agents that facilitate peptide synthesis are used, with triphosphonium salts, tetramethylurea, and carbodiimide being particularly preferred. Examples of triphosphonium salts include benzotriazol-1-yloxytris(pyrrolidinyl)phosphonium hexafluorophosphate (PyBOP), bromotris(pyrrolidinyl)phosphonium hexafluorophosphate (PyBroP), and 7-azabenzotriazol-1-yloxytris(pyrrolidinyl)phosphonium hexafluorophosphate (PyAOP). Examples of tetramethylurea include 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU), 2-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HATU), and 2-(1H-benzotriazolyl)-1,1,3,3-tetramethylurea hexafluorophosphate (HATU). Examples of carbodiimides include triazol-1-yl)-1,1,3,3-tetramethylureon tetrafluoroborate (TBTU), 2-(5-norbornene-2,3-dicarboxylimide)-1,1,3,3-tetramethylureon tetrafluoroborate (TNTU), and O-(N-succinimide)-1,1,3,3-tetramethylureon tetrafluoroborate (TSTU). Other examples of carbodiimides include N,N'-dicyclohexylcarbodiimide (DCC), N,N-diisopropylcarbodiimide (DIPCDI), and N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl). For use in condensation, it is preferable to add a racemic inhibition inhibitor [e.g., N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (HONB), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt), ethyl 2-oxime nitrile (Oxyma), etc.]. The solvent used for condensation can be suitably selected from those known solvents used in peptide condensation reactions. For example, amides such as anhydrous or aqueous N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone can be used; halogenated hydrocarbons such as dichloromethane and chloroform; alcohols such as trifluoroethanol and phenol; sulfoxides such as dimethyl sulfoxide; tertiary amines such as pyridine; ethers such as dioxane and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; esters such as methyl acetate and ethyl acetate; and suitable mixtures thereof can be used. The reaction temperature is appropriately selected from the known temperature range used in peptide binding reactions, typically from approximately -20°C to 90°C. An excess of 1.5 to 6 times the amount of the activated amino acid derivative is usually used. In solid-phase synthesis, when experiments using ninhydrin show insufficient condensation, complete condensation can be achieved by repeating the condensation reaction without removing the protecting group. If condensation is still insufficient after repeated reactions, unreacted amino acids can be acetylated using acid anhydrides, acetylated imidazoles, etc., thus avoiding any influence on subsequent reactions.

[0051] Examples of protecting groups for the amino group of the starting amino acid include benzyloxycarbonyl (Z), tert-butoxycarbonyl (Boc), tert-pentoxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl (Cl-Z), 2-bromobenzyloxycarbonyl (Br-Z), adamantaneoxycarbonyl, trifluoroacetyl, phthaloyl, formyl, 2-nitrobenzenesulfinyl, diphenylthiophosphino, 9-fluorenylmethoxycarbonyl (Fmoc), triphenylmethyl, etc.

[0052] As an example of the carboxyl-protecting group for the starting amino acid, in addition to the C mentioned above... 1-6 Alkyl, C 3-10 cycloalkyl, C 7-14 In addition to aralkyl groups, it includes aryl, 2-adamantyl, 4-nitrobenzyl, 4-methoxyphenyl, 4-chlorobenzyl, benzoylmethyl and benzyloxycarbonylhydrazine, tert-butoxycarbonylhydrazine, triphenylmethylhydrazine, etc.

[0053] The hydroxyl groups of serine or threonine can be protected, for example, by esterification or etherification. Examples of groups suitable for esterification include lower (C) groups such as acetyl groups. 2-4 Alkyl groups, aromatic acyl groups such as benzoyl, and groups derived from organic acids are all suitable for etherification. Furthermore, examples of groups suitable for etherification include benzyl, tetrahydropyranyl, and tert-butyl. t ), triphenylmethyl (Trt), etc.

[0054] Examples of protecting groups for the phenolic hydroxyl group of tyrosine include Bzl, 2,6-dichlorobenzyl, 2-nitrobenzyl, Br-Z, tert-butyl, etc.

[0055] Examples of imidazole protecting groups for histidine include p-toluenesulfonyl (Tos), 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), dinitrophenyl (DNP), benzyloxymethyl (Bom), tert-butoxymethyl (Bum), Boc, Trt, Fmoc, etc.

[0056] Examples of protecting groups for the guanidinium group of arginine include Tos, Z, 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), p-methoxybenzenesulfonyl (MBS), 2,2,5,7,8-pentamethylsomn-6-sulfonyl (Pmc), mesitylene-2-sulfonyl (Mts), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), Boc, Z, NO2, etc.

[0057] Examples of protecting groups for the side chain amino group of lysine include Z, Cl-Z, trifluoroacetyl, Boc, Fmoc, Trt, Mtr, 4,4-dimethyl-2,6-dioxocyclohexylene (Dde), etc.

[0058] Examples of protecting groups for the indole group of tryptophan include formyl (For), Z, Boc, Mts, Mtr, etc.

[0059] Examples of protecting groups for asparagine and glutamine include Trt, xanthyl (Xan), 4,4'-dimethoxydiphenylmethyl (Mbh), and 2,4,6-trimethoxybenzyl (Tmob).

[0060] Examples of activated carboxyl groups in starting materials include corresponding acid anhydrides, azides, and active esters [esters with alcohols (e.g., pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanoethanol, p-nitrophenol, HONB, N-hydroxysuccinimide, 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt))]. Examples of activated amino groups in starting materials include corresponding phosphoramides.

[0061] Examples of methods for removing (eliminating) protecting groups include catalytic reduction in a hydrogen stream in the presence of catalysts such as Pd-black or Pd-carbon; acid treatment using solutions of anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroacetic acid (TFA), trimethylbromosilane (TMSBr), trimethylsilyl trifluoromethanesulfonate, tetrafluoroboric acid, tri(trifluoro)boric acid, boron tribromide, or mixtures thereof; base treatment using diisopropylethylamine, triethylamine, piperidine, piperazine, etc.; and reduction with sodium in liquid ammonia. Elimination reactions using the acid treatments described above are typically carried out at temperatures from -20°C to 40°C; acid treatment is effectively performed by adding anisole, phenol, anisole sulfide, m-cresol, and p-cresol; or by adding cation scavengers such as dimethyl sulfide, 1,4-butanedithiol, 1,2-ethanedithiol, and triisopropylsilane. In addition, the 2,4-dinitrophenyl group protecting the imidazole group used as histidine is removed by treatment with thiophenol; the formyl group protecting the indole group used as tryptophan is removed by acid treatment in the presence of 1,2-ethanedithiol, 1,4-butanedithiol, etc., and by deprotection treatment with alkali such as dilute sodium hydroxide and dilute ammonia.

[0062] For the protection of functional groups that should not participate in the reaction of the starting material and the protecting group, the elimination of the protecting group, and the activation of the functional groups that participate in the reaction, appropriate choices can be made from known protecting groups and known methods.

[0063] In methods for preparing amides of peptides, amides are formed either by solid-phase synthesis using a resin for amide synthesis, or by α-carbonylation of the carboxyl-terminal amino acid and elongation of the peptide chain toward the amino side to the desired chain length. Subsequently, peptides are prepared in which only the protecting group for the N-terminal α-amino group of the peptide chain is removed, and peptides in which only the protecting group for the C-terminal carboxyl group is removed. These two peptides are then condensed in the aforementioned mixed solvent. Details regarding the condensation reaction also apply as described above. After purifying the protected peptide obtained by condensation, all protecting groups can be removed using the methods described above to obtain the desired crude peptide. This crude peptide is then purified by major fractionation and various known methods, including freeze-drying, thereby preparing the amide required for the aforementioned peptide.

[0064] In one instance, the peptide described above can be in the form of its solvate. A "solvate" refers to a complex formed by the peptide or its salt with solvent molecules.

[0065] Periodontal disease refers to inflammation of the gingiva, periodontal ligament, and alveolar bone, the tissues surrounding the teeth that support them. It is a disease caused by bacterial infection of the space between the gums (gum) and teeth, damaging the periodontal ligament and adjacent tissues. Depending on the severity, it can be divided into gingivitis and periodontitis. As is well known, inflammation leads to further tissue damage, forming periodontal pockets. The more severe the periodontitis, the deeper the periodontal pockets. As the periodontal pockets deepen, inflammation of the periodontal ligament eventually leads to osteoporosis. The fundamental treatment for this periodontal disease lies in restoring the damaged periodontal ligament connective tissue, cementum, and alveolar bone. This requires not only the regeneration of the periodontal ligament supporting the alveolar bone but also the regeneration of the alveolar bone and cementum to which the periodontal ligament attaches.

[0066] In the above pharmaceutical composition, the periodontal disease can be an inflammatory disease of the periodontal tissues.

[0067] In the above pharmaceutical composition, the periodontal disease can be gingivitis or periodontitis.

[0068] In one instance, the pharmaceutical composition described above may further comprise a drug for treating periodontal disease.

[0069] The drugs used to treat periodontal disease can be separated from the peptides, or they can be in the form of a complex formed with the peptides by binding to the N-terminus or C-terminus of the peptides.

[0070] "Dislocation injury" includes traumatic forces applied to the teeth or periodontal tissues, which, depending on the direction and size of the force, damage the teeth and periodontal tissues, resulting in damage to the periodontal ligaments. It can be further subdivided into concussion subluxation, extrusion dislocation, lateral dislocation, complete dislocation, and intrusion. Therefore, fundamental treatment of dislocation injuries or regeneration of the damaged periodontal ligaments is necessary. In particular, the success of replantation of completely avulsed teeth depends on the regeneration of the periodontal ligaments attached to the completely avulsed tooth or the periodontal ligaments remaining in the alveolar socket where the tooth was avulsed. Without periodontal ligament regeneration, cementum and dentin resorption at the tooth root will occur, leading to ankylosis, where the alveolar bone replaces the dentin in that area.

[0071] Tooth replantation is a procedure that involves performing appropriate root canal treatment after tooth extraction or replanting a tooth in the alveolar fossa before extraction. Intentional replantation can be performed in cases of failed root canal treatment, anatomical limitations, operational difficulties, tooth dislocation due to accidents, or when rapid orthodontic intervention is required.

[0072] "Prevention" refers to all actions that use the above-described composition to suppress or delay the onset of periodontal disease or traumatic tooth avulsion.

[0073] "Treatment" refers to all actions that improve or benefit the symptoms of periodontal disease or traumatically avulsed teeth by administering the above-described composition.

[0074] The above-described pharmaceutical composition can be prepared in the form of a pharmaceutical composition for treating periodontal disease, comprising, on the peptide, a suitable carrier (natural or non-natural), excipient, or diluent commonly used in the preparation of pharmaceutical compositions. The above-described pharmaceutical composition can be formulated into a sterile injectable form for administration to the site of periodontal disease by conventional methods.

[0075] In this invention, examples of carriers, excipients, and diluents that can be included in the above-mentioned pharmaceutical compositions include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, rubber arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, mineral oil, and collagen. In the case of formulation, commonly used fillers, brighteners, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients can be used. In particular, sterile aqueous solutions, non-aqueous solvents, suspending agents, emulsions, lyophilizing agents, sachets, and ointments (e.g., pulp transfer materials) can be included. Vegetable oils such as propylene glycol, polyethylene glycol, and olive oil can be used as non-aqueous solvents and suspending agents; injectable esters such as ethyl oleate can also be used. As a base for the sedative, synthetic fatty acid esters (witepsol), polyethylene glycol, Tween 61, cocoa butter, laurin fat, glycerin gelatin, etc. can be used.

[0076] The content of the peptide or its pharmaceutically acceptable salt contained in the above pharmaceutical composition is not particularly limited thereto, but may be from 0.0001% to 50% by weight, or from 0.01% to 20% by weight, based on the total weight of the final composition.

[0077] The pharmaceutical compositions of the present invention described above can be administered in pharmaceutically effective amounts. The term "pharmaceutically effective amount" in this invention refers to an amount sufficient for the treatment or prevention of a disease, with a reasonable benefit / risk ratio suitable for medical treatment or prevention. The effective dosage level can be determined based on the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the timing of administration of the composition of the present invention, the route of administration and excretion rate during treatment, the element of co-administration with or containing concurrently used drugs in the composition of the present invention, and other elements known in the pharmaceutical field. The pharmaceutical compositions of the present invention can be administered alone or in combination with known pharmaceutical compositions for the treatment of periodontal disease. Importantly, considering all the above factors, the dosage is administered in an amount that achieves maximum effect with minimal amount and without side effects.

[0078] Those skilled in the art to which this invention pertains can determine the dosage of the pharmaceutical composition by considering factors such as the intended use, the degree of disease or poisoning, the patient's age, weight, sex, pre-existing conditions, or the type of substance used as the active ingredient. For example, each adult may be given about 0.1 ng to about 100 mg / kg of the pharmaceutical composition of this invention, preferably at a dose of 1 ng to about 10 mg / kg. The frequency of administration of the composition of this invention is not particularly limited thereto, but it may be administered once daily or in divided doses multiple times. The above dosages do not limit the scope of this invention in any way.

[0079] To address the aforementioned technical challenges, according to another embodiment of the present invention, a composition for periodontal tissue regeneration is provided, comprising a peptide containing an amino acid sequence of the following general formula 1.

[0080] KYKQ-X5-X6-X7-X8-YK (General Formula 1) In the above general formula 1, X5 to X7 are arginine (R) or lysine (K) respectively, each independently. X8 is either asparagine (N) or serine (S).

[0081] In one example, the peptide described above may contain any of the amino acid sequences from sequence numbers 1 to 16.

[0082] In another example, the peptide described above is essentially composed of any of the amino acid sequences in sequence numbers 1 to 16, or the peptide described above may be composed of any of the amino acid sequences in sequence numbers 1 to 16.

[0083] The peptides mentioned above are explained as described above.

[0084] The periodontium is a complex organ composed of epithelial tissue, soft connective tissue, and calcified connective tissue. Structurally, it consists of the gingiva, periodontal ligament (PDL), cementum, and alveolar bone.

[0085] The periodontal ligament (PDL), also known as the periodontal membrane, is a connective tissue fibrous membrane in mammals that connects the cementum and alveolar bone walls of the tooth roots. The PDL consists of principal fibers and Sharpey fiber bundles. The principal fibers are collagen fibers that extend parallel or obliquely relative to the long axis of the tooth. The Sharpey fiber bundles continue the principal fibers and are embedded in hard tissue at both ends. Through the PDL, the teeth are elastically fixed to the jawbone (alveolar bone). The PDL not only cushions the pressure generated during chewing but also has a rich network of blood vessels and nerves, and is said to be related to nutrient supply and sensory function. The PDL contains various cell types, such as fibroblasts, undifferentiated mesenchymal cells, and epithelial cells. Under appropriate stimulation, periodontal ligament fibroblasts can differentiate into osteoblasts or cementoblasts, but the differentiation mechanism and differentiation-promoting proteins of periodontal ligament fibroblasts into cementoblasts are still unclear. Known markers for cementoblast differentiation include BSP (bone sialoprotein), OC (osteocalcin), and CAP (cementum attachment protein). In particular, CAP is known to play an important role in the attachment of periodontal ligament fibers to cementum.

[0086] Cementum refers to the calcified tissue that covers the root of a mammal's tooth. Cementum anchors the tooth to the alveolar bone via the periodontal ligaments. Therefore, if the gums become infected with bacteria, it causes degeneration of the cementum surrounding the tooth. The periodontal ligament fibers connecting the tooth and alveolar bone can no longer adhere tightly to the degenerated cementum, leading to tooth loosening. To treat this degeneration, new cementum needs to be formed.

[0087] The alveolar bone, part of the maxilla and mandible, surrounds and supports the roots of the teeth. It grows as teeth form and erupt, and is gradually absorbed when teeth are lost. Together with the cementum of the tooth roots, this alveolar bone anchors the periodontal ligaments, playing a crucial role in distributing and absorbing occlusal pressure during chewing, speech, and swallowing.

[0088] Periodontal ligament fibroblasts and gingival fibroblasts are the main cellular components of the soft connective tissue of the periodontal tissue. It is well known that periodontal ligament fibroblasts, due to their unique functions, not only form the periodontal ligament but also participate in the repair and regeneration of adjacent alveolar bone and cementum in the body. However, the substances that promote their function are still unclear, and they differ from gingival fibroblasts, which are involved in maintaining gingival connective tissue. Although periodontal ligament fibroblasts can differentiate into osteoblasts or cementoblasts through appropriate stimulation, the differentiation mechanism of periodontal ligament fibroblasts into cementoblasts is still unclear. BSP (bone sialoprotein) and OC (osteocalcin) are known markers of osteoblast and cementoblast differentiation, and CAP (cementum attachment protein) is known to play an important role in the attachment of periodontal ligament fibers to cementum. Furthermore, the periodontin gene is known as a marker of periodontal ligament. Recent studies have revealed that periodontin acts as an important regulator of periodontal tissue formation, promoting collagen fiber production and the migration of fibroblasts and osteoblasts, thus playing a crucial role in the regeneration of periodontal ligament and alveolar bone after periodontal surgery.

[0089] "Regeneration" can refer to all processes that repair or replenish lost or damaged cells or tissues. The aforementioned regeneration may be caused by cell differentiation.

[0090] The above-mentioned peptides were confirmed to promote the differentiation of periodontal ligament fibroblasts into cementoblasts.

[0091] The above-mentioned composition for periodontal tissue regeneration can promote the regeneration of any one or more of the gingiva, periodontal ligament, cementum, and alveolar bone.

[0092] The above-mentioned composition for periodontal tissue regeneration can be a pharmaceutical composition.

[0093] The above pharmaceutical composition is as described above.

[0094] The above-mentioned composition for periodontal tissue regeneration can be a topical pharmaceutical composition. The above-mentioned topical pharmaceutical composition can be a topical pharmaceutical composition used for the prevention or improvement of periodontal disease or traumatic avulsion of teeth.

[0095] "Improvement" refers to parameters related to the state being treated, such as the extent to which all behaviors at least reduce symptoms.

[0096] The above improvements can be explained as follows: administering a pharmaceutical composition containing the aforementioned peptide as an active ingredient to an individual in need of treatment for periodontal disease promotes the regeneration of periodontal tissues, thereby improving the symptoms of periodontal disease or traumatically dislocated teeth or all behaviors that are beneficial to them.

[0097] "Non-pharmaceuticals" refers to articles used for the purpose of diagnosing, treating, improving, alleviating, treating, or preventing diseases in humans or animals, but whose effects are less than those of pharmaceuticals. For example, according to the Pharmacist Act, non-pharmaceuticals are articles excluded from use as pharmaceuticals, including fiber and rubber products used to treat or prevent diseases in humans and animals, those with minor or indirect effects on the human body and which are not instruments or machines and are similar to them, and bactericides and insecticides used to prevent infectious diseases.

[0098] There are no particular restrictions on the types or dosage forms of quasi-pharmaceutical compositions containing the above-mentioned peptides. However, as an example, such compositions may include oral disinfectants, oral hygiene products, toothpaste, dental floss, oral ointments, etc.

[0099] The above-mentioned composition for periodontal tissue regeneration can be a composition for preventing or improving periodontal disease or traumatic tooth avulsion.

[0100] The above composition may also contain a physiologically acceptable carrier. There is no particular limitation on the type of carrier. Any carrier that is commonly used in the technical field to which this invention pertains may be used.

[0101] Furthermore, the above composition may contain additional ingredients typically used to enhance odor, taste, and appearance. For example, it may contain vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folic acid, pantothenic acid, etc. It may also contain minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), and copper (Cu). Additionally, it may contain amino acids such as lysine, tryptophan, cysteine, and valine.

[0102] Furthermore, the above composition may contain food additives, such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder and high-efficiency bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (such as tar pigments), color-developing agents (sodium nitrite, sodium acetate, etc.), bleaching agents (sodium sulfite), seasonings (MSG monosodium glutamate, etc.), sweeteners (glycan, cyclosulfonates, o-benzoylsulfonylimide, sodium, etc.), flavorings (vanillin, lactone, etc.), leavening agents (alum, potassium D-tartrate, etc.), fortifying agents, emulsifiers, thickeners (paste), coating agents, gum base agents, foam inhibitors, solvents, modifiers, etc. The above additives can be screened and used in appropriate amounts.

[0103] To address the aforementioned technical challenges, according to another embodiment of the present invention, a composition for promoting gene expression is provided, comprising a peptide containing an amino acid sequence of the following general formula 1, wherein the gene is any one or more of bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), cementum attachment protein (CAP), collagen type III (COL3), and periostin.

[0104] KYKQ-X5-X6-X7-X8-YK (General Formula 1) In the above general formula 1, X5 to X7 are arginine (R) or lysine (K) respectively; X8 is either asparagine (N) or serine (S).

[0105] In one example, the peptide described above may contain any of the amino acid sequences from sequence numbers 1 to 16.

[0106] In another example, the peptide described above is essentially composed of any of the amino acid sequences in sequence numbers 1 to 16, or the peptide described above may be composed of any of the amino acid sequences in sequence numbers 1 to 16.

[0107] The peptides mentioned above are explained as described above.

[0108] It was confirmed that the aforementioned peptides increased the expression of BSP, DMP1, and CAP, which are marker genes for osteoblast and cementoblast differentiation, in human periodontal ligament cells, and also increased the expression of periosteal protein and COL3, which are marker genes for periodontal ligament differentiation. Furthermore, it was confirmed that the aforementioned peptides increased the expression of BSP and DMP1, which are marker genes for osteoblast and cementoblast differentiation, in human mesenchymal stem cells.

[0109] To address the technical challenges of this invention, according to another embodiment, a method for treating periodontal disease or traumatically avulsed teeth in an individual is provided, the method comprising the step of administering an effective amount of the aforementioned peptide or a pharmaceutically acceptable salt thereof to the individual requiring treatment for periodontal disease or traumatically avulsed teeth.

[0110] "Individual" refers to the object that needs treatment for a disease, and more specifically, to humans or non-human primates, mice, rats, dogs, cats, horses, and cattle.

[0111] "Effective dose" refers to the amount or dose of the aforementioned peptide or its pharmaceutically acceptable salt that provides the desired effect to a patient under diagnostic or therapeutic conditions when administered to the patient in a single dose or multiple doses. The effective dose can be readily determined by a physician skilled in the art, using known techniques or observing results under similar conditions. In determining the effective dose for a patient, the physician will consider numerous factors, including but not limited to: the species of mammal; its size, age, and general health status; the specific disease or disorder involved; the extent or severity of the disease or condition; the individual patient's response; the specific compound administered; the route of administration; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; the use of concomitant medications; and other relevant factors.

[0112] "Administration" refers to the delivery of a predetermined substance to a patient using a suitable method. The route of administration can be any general route that allows the substance to reach the target within the patient's body. The aforementioned administration methods include, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, local administration, intranasal administration, and rectal administration, but are not limited to these.

[0113] The treatment of the aforementioned periodontal diseases or traumatic tooth avulsions can be aided by the regeneration of periodontal tissues, but is not limited to this.

[0114] In the above method, an effective amount of the aforementioned peptide or its pharmaceutically acceptable salt may be administered simultaneously, separately, or sequentially with an effective amount of one or more other active ingredients. The aforementioned one or more other active ingredients may be one or more other formulations used to treat periodontal disease or traumatically avulsed teeth, but are not limited thereto.

[0115] To address the aforementioned technical challenges, according to another embodiment of the present invention, in the area of ​​pharmaceutical agents for the prevention or treatment of periodontal disease or traumatic tooth avulsion, the use of the aforementioned peptide or a pharmaceutically acceptable salt thereof is provided.

[0116] All values ​​disclosed in this specification may include the meaning of “about”. “About” is a range that includes all values ​​of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes, but is not limited to, all values ​​that are equivalent to or similar to the values ​​following the term “about”.

[0117] The descriptions and embodiments disclosed in this specification are also applicable to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this specification fall within the scope of this invention. Moreover, the scope of this invention is not limited to the specific descriptions below.

[0118] Invention Effects According to the present invention, a pharmaceutical composition for the prevention or treatment of periodontal disease or traumatic tooth avulsion promotes the regeneration of periodontal tissues including periodontal ligament, alveolar bone, and cementum, and the attachment of periodontal ligament, and can be used to restore connective tissue damaged by periodontal disease or traumatic tooth avulsion. Therefore, the above-mentioned peptide can be used in various ways as a pharmaceutical composition, a quasi-pharmaceutical composition, etc.

[0119] The effects of the present invention are not limited to those mentioned above. Those skilled in the art to which this invention pertains can clearly understand other effects not mentioned from the following description. Attached Figure Description

[0120] Figure 1 This is a graph showing the effect of the peptides from Groups 1 and 2 prepared in Example 1 on the expression of BSP (bone sialoprotein), DMP1 (Dentin Matrix protein 1), and CAP (Cementum attachment protein), which are differentiation marker genes for osteoblasts and cementoblasts, in human periodontal ligament cells (hPDL).

[0121] Figure 2 The results show the effects of the peptides from Groups 1 and 2 prepared in Example 1 on the expression of BSP (bone sialoprotein) and DMP1 (Dentin Matrix protein 1), which are osteoblast differentiation marker genes, in human mesenchymal stem cells (hBMSCs).

[0122] Figure 3This demonstrates the effect of the peptides from Groups 1 and 2 prepared in Example 1 on the expression of type III collagen fiber gene (COL3) and periosteal protein gene, which are periodontal ligament marker genes, in human periodontal ligament cells.

[0123] Figure 4 The images, taken 3 months after treatment of periodontal tissue injury sites with the peptide (serial number 16) of Example 1 and stained with hematoxylin / eosin, confirm the effect of the peptide on alveolar bone regeneration. A represents the positive control group (PC); B represents the negative control group (NC) without any treatment after periodontal tissue injury; C represents the negative control group (NC+Plug) with only collagen sponge implantation after periodontal tissue injury; D represents the experimental group treated with the peptide of group 2 (serial number 16); AB refers to alveolar bone; De refers to dentin.

[0124] Figure 5 To confirm the effect of the peptide on periodontal ligament regeneration after 3 months of treatment of the periodontal tissue injury site with the peptide from Example 1 (serial number 16), staining with hematoxylin / eosin was performed. AD represents the negative control group (NC) without any treatment; EH represents the negative control group (NC+Plug) with only collagen sponge implantation; IL represents the experimental group treated with the peptide from group 2 (serial number 16). AB refers to alveolar bone; De refers to dentin; CE refers to cementum; CT refers to connective tissue; NPD refers to newly formed periodontal ligament-like tissue; NCE refers to newly formed cementum-like tissue. Scale bar: A, E, I, M are 500 mm, B, F, J, N are 200 mm, C, G, K, O are 100 mm, D, H, L, P are 50 mm.

[0125] Figure 6 To confirm the effect of the peptide on periodontal ligament regeneration after 3 months of treatment of the periodontal tissue injury site with the peptide from Example 1 (serial number 16), collagen staining (Masson's trichrome stain) was used. AB represents the negative control group (NC) without any treatment; CD represents the negative control group (NC+Plug) with only collagen sponge implantation; EF represents the experimental group treated with the peptide from group 2 (serial number 16). AB refers to alveolar bone; De refers to dentin; CE refers to cementum; CT refers to connective tissue; NPD refers to newly formed periodontal ligament-like tissue; NCE refers to newly formed cementum-like tissue. Scale bar: A, C, E, G are 100 mm, B, D, F, H are 50 mm. Detailed Implementation

[0126] The present invention will now be described in more detail through embodiments. However, these embodiments are only illustrative and the scope of the invention is not limited to these embodiments.

[0127] Example 1: Peptide Synthesis Sixteen peptides with the amino acid sequences described in Table 1 below were synthesized. Among them, peptides with asparagine (N) at the 8th amino acid were classified as group 1, and peptides with serine (S) at the 8th amino acid were classified as group 2.

[0128] Table 1 Experimental Materials and Methods 1. Cell Culture hBMSCs cells were cultured in humidified air containing 5% CO2 at 37°C and used in experiments. Human-derived bone marrow mesenchymal stem cells (hBMSCs) were purchased and used from Lonza (Switzerland). hBMSCs were cultured in α-minimum essential medium (α-MEM, Invitrogen) supplemented with 10% heat-inactivated bovine serum.

[0129] 2. Isolation and culture of human periodontal ligament cells Regarding human periodontal ligament (hPDL) cells, cells were isolated from periodontal ligament tissue in the wisdom teeth of 10 adults (18-22 years old) at Seoul National University Dental Hospital. Specifically, all experiments were conducted with the approval of the hospital's Institutional Review Board and with the consent of the patients. The periodontal ligament tissue attached to the root of the wisdom tooth was isolated and finely cut using a double-edged scalpel, placed in a 60mm dish, covered with a coverslip, and cultured on Dalberg's modified Eagle's medium.

[0130] 3. Real-time PCR analysis Total ribonucleic acid (RNA) from human periodontal ligament cells was isolated using TRIzol reagent. cDNA was synthesized using 2 μg of total RNA, 1 μl of reverse transcriptase, and 0.5 μg of oligo(dT). The synthesized human periodontal ligament cell cDNA was used in real-time polymerase chain reaction (RT-PCR) using primers listed in Table 2 below. The R-PCR was performed using the SYBR GreenPCR Master Mix (Takara, Japan) on an ABI PRISM 7500 sequence detection system (Applied Biosystems). The R-PCR was repeated for 40 cycles at 94°C for 1 min, 95°C for 15 sec, and -60°C for 34 sec. The results were analyzed using the comparative cycle threshold (CT) method.

[0131] Table 2 4. Evaluate the effects of peptides on periodontal tissue regeneration in a periodontal tissue injury model. Four beagle dogs (12 to 16 kg; 6 to 8 weeks old) were anesthetized by inhalation of gerolan, followed by intravenous administration of Zoltil (5 mg / kg) and xylazine (0.2–0.5 mg / kg), and then treated with lidocaine (2% lidocaine and 1:80,000 epinephrine). After extraction of the fourth and second molars from the lower jaws of the beagle dogs, healing was achieved after 3 months.

[0132] Three months after treatment, a three-walled periodontal defect was guided on the distal and mesial surfaces of the first molar. Subsequently, a periodontal lesion of 4x4x4mm was created with buccal, lingual, and distal bone remaining around the root. After thoroughly removing the alveolar bone at the root, root planing was performed using a curette, and a 1 / 2 ball bur was used to create a recess in the incisor.

[0133] Next, in the experimental group, 50 mg of collagen sponge containing the peptides prepared in Example 1 (sequence number 16 of group 2) was transplanted into the defect site (the peptide solution was prepared by dissolving the peptides in physiological saline at a concentration of 1 mg / mg, adding 50 mg of the peptide solution to the EP tube, and then soaking the collagen sponge for 5 minutes). Only the negative control group (NC) that did not undergo any treatment after the defect and the collagen sponge (prepared by soaking the collagen sponge in 50 mg of physiological saline for 5 minutes) were placed inside the bone wall of the bone defect site. After 3 months, the transplanted negative control group (NC+Plug) was sacrificed, and histological evaluation was performed.

[0134] Three months later, the beagles were administered an overdose (90-120 mg / kg) of pentobarbital, resulting in their death. The teeth of the beagles were removed, fixed with 10% formalin, and then calcium was removed with 5% formic acid. The tissue sections were then shaped, embedded in paraffin, and 5 mm thick were obtained.

[0135] The tissue sections obtained above were stained with hematoxylin and eosin or, to confirm periodontal ligament regeneration, with collagen staining (Masson's Trichrome Staining). They were then analyzed using an optical microscope (LEICA DM750, Germany) equipped with a digital camera (LEICA ICC50 camera, Germany).

[0136] Experimental results Experimental Example 1: Effects of peptides on the expression of differentiation marker genes in osteoblasts and cementoblasts in human periodontal ligament cells. The BSP (Bone Sialoprotein) and DMP1 (Dentin Matrix Protein 1) genes are used as markers for osteoblast and cementoblast differentiation and are known to be important genes for bone and cementum calcification. Furthermore, the CAP (Cementum Attachment Protein) gene is expressed in differentiated cementoblasts and is known to be involved in cementum attachment to periodontal ligament fiber bundles.

[0137] Figure 1 A graph illustrating the effects of the peptides from Groups 1 and 2 prepared in Example 1 on the expression of differentiation marker genes BSP, DMP1, and CAP in human periodontal ligament cells (hPDL).

[0138] like Figure 1 As shown, it was confirmed that in the experimental groups that treated the peptides in groups 1 and 2, the expression of BSP, DMP1, and CAP genes increased by more than 2 times compared to the control group.

[0139] Table 3 shows the results of real-time PCR confirmation of the effects of peptide groups 1 and 2 on BSP mRNA expression, and presents the relative mRNA expression levels compared to the control group.

[0140] Table 4 shows the results of real-time PCR confirmation of the effects of the peptide groups of group 1 and group 2 on CAP mRNA expression, illustrating the relative mRNA expression levels compared to the control group. The results shown in Tables 3 and 4 are the mean and standard deviation (SD) obtained from three repeated experiments.

[0141] Table 3 Table 4 Experiment Example 2: Effects of peptides on the expression of differentiation marker genes in osteoblasts and cementoblasts in human mesenchymal stem cells Figure 2 To demonstrate the effect of the peptides of Group 1 and Group 2 prepared in Example 1 on the expression of differentiation marker genes of osteoblasts and cementoblasts in human mesenchymal stem cells.

[0142] like Figure 2 As shown, it was confirmed that when peptides from groups 1 and 2 were treated in human mesenchymal stem cells, the expression of BSP and DMP1, which are differentiation marker genes of osteoblasts and cementoblasts, increased by about 2 to 4 times compared with the control group.

[0143] Experimental Example 3: Effects of peptides on the expression of periodontal ligament differentiation marker genes in human periodontal ligament cells Periostin, a protein first discovered in the periosteum of the periodontal ligament and bone, is known to be expressed in the periodontal ligament and interstitium of developing teeth and to participate in cell attachment. Recent studies have shown that periostin acts as a key regulator of periodontal tissue formation, promoting collagen fiber production and the migration of fibroblasts and osteoblasts, and plays a central role in the regeneration of periodontal ligament and alveolar bone after periodontal surgery to treat periodontal disease. Therefore, the effects of the peptide from Example 1 on the expression of periostin and type III collagen fiber gene (COL3), both marker genes for periodontal ligament differentiation, were confirmed.

[0144] Figure 3 To demonstrate the effect of the peptides of Group 1 and Group 2 prepared in Example 1 on the expression of type III collagen fiber gene (COL3) and periosteal protein gene, which are periodontal ligament marker genes, in human periodontal ligament cells.

[0145] from Figure 3 It was found that when peptides from groups 1 and 2 were treated in human periodontal ligament cells, the expression of type III collagen fiber gene (COL3) and periosteal protein gene increased by more than 2 times.

[0146] Table 5 shows the results of real-time PCR confirmation of the effects of the peptide groups of group 1 and group 2 on the expression of periosteal protein mRNA, illustrating the relative mRNA expression levels compared to the control group. The results shown in Table 5 are the mean and standard deviation (SD) obtained from three repeated experiments.

[0147] Table 5 Experiment Example 4: Verifying the regenerative effect of peptides on periodontal tissues in an animal model of periodontal tissue injury. After extracting the fourth premolar and second molar from the mandibles of four beagle dogs, a three-month healing periodontal defect was created. Following this, a three-walled periodontal defect was induced on the distal and mesial surfaces of the first molar. A 4x4x4mm periodontal lesion was then created, preserving buccal and lingual bone around the root. After thorough removal of the alveolar bone at the root, root planing was performed using a curette to create a concave incisor. Next, in the experimental group, 50 mg of collagen sponge containing the peptide (serial number 16) from Example 1 was transplanted into the defect site. Only the negative control group (NC), which received no post-defect treatment, and the collagen sponge were placed within the bone wall of the defect. Three months later, the transplanted control group (NC+Plug) was sacrificed, and histological evaluation was performed.

[0148] Figure 4 The results, which confirm the effect of the peptide on alveolar bone regeneration, show that the periodontal tissue injury site was treated with the peptide (serial number 16) of Example 1 and stained with hematoxylin / eosin 3 months later.

[0149] like Figure 4 As shown, it was confirmed that, compared with the negative control group, the alveolar bone in most of the damaged periodontal tissues of the experimental group was regenerated.

[0150] Figure 5 The results of treating the periodontal tissue injury site with the peptide (serial number 16) of Example 1 and staining with hematoxylin / eosin after 3 months were used to confirm the effect of the peptide on the regeneration of periodontal ligament tissue.

[0151] like Figure 5 As shown, the negative control group (without any treatment) Figure 5 AD) or a negative control group that only received collagen sponge implantation ( Figure 5 EH), no new periodontal ligament-like tissue (NPD) was observed along the root surface, but connective tissue (CT) was observed. Furthermore, dentin resorption was observed at the site of tooth adjustment. However, peptide (serum number 16) of treatment group 2 ( Figure 5In the IL (intraocular ligament) experimental group, not only was new cementum-like tissue observed along the root surface, but also the formation of new periodontal ligament-like tissue. The new periodontal ligament is embedded in the newly formed alveolar bone and cementum.

[0152] Figure 6 The results of treating the periodontal tissue injury site with the peptide (serial number 16) of Example 1 and confirming the effect of the peptide on periodontal ligament tissue regeneration by collagen staining (Masson's trichrome stain) 3 months later were obtained.

[0153] like Figure 6 As shown, the negative control group (without any treatment) Figure 6 AB) or the negative control group that only received collagen sponge implantation ( Figure 6 CD), along the root surface, most of the tissue formed (CT) was observed, with some areas showing newly formed periodontal ligament-like tissue (NPD), but with irregularly arranged periodontal ligament fiber bundles. Furthermore, in the negative control group, some areas of newly formed cementum were observed along the root. However, the peptide (serial number 16) in treatment group 2 ( Figure 6 In the EF experimental group, not only was new cementum-like tissue observed along the root surface, but also the formation of new periodontal ligament-like tissue. The new periodontal ligament fiber bundles were vertically embedded in the newly formed alveolar bone and cementum.

[0154] In this way, the peptides of the present invention have the effect of regenerating alveolar bone, forming new cementum-like tissue and new periodontal ligament-like tissue, and it has been confirmed that the new periodontal ligament formed by the peptides of the present invention is embedded in the newly formed alveolar bone and cementum. Therefore, it is understood that the peptides of the present invention can be used for regenerating periodontal tissue, for preventing or treating periodontal diseases, and for preventing or treating traumatic tooth avulsion.

[0155] Preferred embodiments of the invention have been disclosed in this specification and accompanying drawings. Although specific terminology has been used, it is used in a general sense only for the purpose of readily illustrating the technical content of the invention and aiding in understanding the invention, and is not intended to limit the scope of the invention. Other modifications based on the technical concept of the invention may be implemented in addition to the embodiments disclosed herein, which will be apparent to those skilled in the art.

Claims

1. Use of a peptide or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for the treatment of traumatically avulsed teeth, wherein, The peptides described above consist of any one of the amino acid sequences from sequence number 1 to 16.

2. The use according to claim 1, wherein, The above-mentioned pharmaceutical composition comprises a polypeptide formed by repeated linkage of the above-mentioned peptides.

3. The use according to claim 1, wherein, The above pharmaceutical composition promotes the gene expression of type III collagen (COL3) or periosteal protein.

4. The use according to claim 1, wherein, The aforementioned traumatic dislocations are caused by damage to the periodontal ligament, resulting in concussion subluxation, extrusion, lateral dislocation, or complete dislocation or intrusion.

5. The use according to claim 1, wherein, The above pharmaceutical composition promotes the expression of any one or more genes of osteosialin (BSP), dentin matrix protein 1 (DMP1), cementum attachment protein (CAP), type III collagen (COL3), and periosteal protein in human periodontal ligament cells.

6. Use of the peptide in the preparation of a composition for treating periodontal disease with periodontal tissue damage, wherein the periodontal tissue includes periodontal ligament, cementum and / or alveolar bone, and wherein the peptide consists of any one of the amino acid sequences of serial numbers 1 to 16.

7. The use according to claim 6, wherein, The above composition promotes the regeneration of cementum or alveolar bone.

8. The use according to claim 6, wherein, The above composition is a pharmaceutical composition.

9. The use according to claim 6, wherein, The above composition is a quasi-pharmaceutical composition.

10. Use of peptides in the preparation of compositions for treating periodontal diseases accompanied by cementum or alveolar bone damage, wherein, The peptides described above consist of any one of the amino acid sequences from sequence number 1 to 16.

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