Composition for preventing or treating dental caries

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating dental caries and its use. The pharmaceutical composition according to the present invention comprises a peptide having an amino acid sequence represented by the following general formula 1 as an active ingredient. KY-R1-R2-R3-R4-R5-R6-R7-R8 (general formula 1) In the general formula 1, R1 is arginine (R), lysine (K) or glutamine (Q), R2 is arginine (R) or glutamine (Q), R3, R4 and R5 are arginine (R) or lysine (K), R6 is asparagine (N) or serine (S), and R7 and R8 are lysine (K) or tyrosine (Y).

Description

[Technical field]

[0001] The present invention relates to a pharmaceutical composition, and more particularly to a pharmaceutical composition for preventing or treating dental caries without the removal of infected tooth tissue. [Background technology]

[0002] Dental caries is one of the most common chronic diseases, affecting 3.5 billion people worldwide (Dye 2017; Kassebaum et al. 2015; World Health Organization 2017). According to a 2013 study by the Ministry of Health and Welfare of the Republic of Korea, the prevalence of caries in permanent teeth was about 87% for adult men and about 90% for women, indicating a high prevalence rate. As the average life expectancy of the population has increased due to technological developments in medicine, the importance of oral health management to improve quality of life is increasing, and the importance of preserving natural teeth is being emphasized above all else. Early caries does not cause symptoms, but as it progresses it causes various pains, resulting in tooth loss, and eventually edentulousness can induce eating disorders, greatly impairing the overall quality of life (Bortoluzzi et al. 2012).

[0003] Generally, when dentin damage occurs due to non-infectious causes, the only treatment required is to repair the lost tooth structure, but when caries is present, the effects of the diseased tooth structure and the bacteria that cause the infection must be minimized, which distinguishes it from the treatment methods for dentin diseases caused by other non-infectious causes. Unlike other dentin and dental pulp diseases, caries begins with the infection of the tooth structure by caries-causing bacteria. Since caries is an infectious disease caused by bacteria, it is important to minimize the effects of the bacteria by removing the diseased tooth structure. Therefore, conventional caries treatment generally involves removing the infected tooth structure, physically removing the source of bacterial infection, and then applying a treatment method to repair the area of ​​the removed tooth structure.

[0004] Current common caries treatment methods have problems such as delays in treatment due to fear of dental treatment, and problems caused by the use of artificial dental materials.

[0005] A tooth consists of hard tissues, enamel, dentin, and cementum, and an internal soft tissue called the dental pulp. The dental pulp has developed nerves and blood vessels, and odontoblasts that form dentin are located at the boundary between the dental pulp and dentin. Considering the presence or absence of erosion of the tooth's constituents, dental caries can be divided into five stages according to the degree of progression. Specifically, the first stage of dental caries involves slight damage limited to the enamel, with almost no subjective symptoms; the second stage involves damage near the boundary between the enamel and dentin, and is accompanied by hypersensitive symptoms; the third stage involves damage to the dentin, with pain felt from external stimuli; the fourth stage involves damage to the dental pulp, with inflammation occurring even in the absence of external stimuli; and the fifth stage involves necrosis of the dental pulp and extreme pain. Generally, people first become aware of caries in the early stages, stages 2 and 3, which may be the appropriate time to treat caries by minimizing the effects of cariogenic bacteria.

[0006] Although early dental treatment can be received due to symptoms, there are many cases where the appropriate treatment stage is missed, which is due to the traditional method of treating caries. Current caries treatment involves mechanically removing weakened tooth structure and filling the area with new material, so most dental patients tend to delay treatment due to anxiety, worry, fear, and tension regarding dental treatment. As a result, the appropriate treatment time is missed, and there are cases where a considerable part of the tooth structure has to be removed during the treatment process. Anxiety or worry regarding caries treatment appears in various age groups, from young children to the elderly. It is known that young children (0-14 years old), who are not yet psychologically and physically mature, are particularly afraid of dental treatment. In 1984, Berggren and Meynert reported on a survey of adults aged 20 to 40 years old on the causes of dental treatment avoidance, and found that approximately 85% of the subjects had developed a fear of dental treatment due to a traumatic dental treatment experience in childhood, and that the main causes of dental treatment fear were pain experienced during dental cavity formation, anesthesia, or tooth extraction. Fear of dental treatment formed in childhood can continue into adulthood and can be a factor in missing appropriate treatment timing.

[0007] Conventionally, dental materials used in the treatment of caries include various dental materials such as amalgam, glass ionomer cement, composite resin, gold, and porcelain, which are used for restoring and prosthetic restorations to restore the structure and function of teeth from damage. The restorative materials used in conventional dental treatments are highly likely to cause microleakage over the long term, and chemical irritation and microleakage of dental materials for restoring and prosthetic restorations can cause or worsen pulp lesions. This leads to necrosis of the pulp as the inflammatory reaction progresses, causing loss of tooth structure due to root canal treatment to remove the pulp and additional tooth removal. Root caries, which often occurs in old age, is difficult to prevent moisture, ensure visibility, and access during restoration, and the margins of the cavity are located in the cementum and dentin, so root caries is less resistant to acid corrosion than enamel caries, and it has been reported that caries recurs frequently due to incomplete margins. In addition, if the restoration is located in an area where it is difficult to maintain its shape and the restoration is prone to falling off, the tooth root does not have a thick dentin layer that reaches the pulp, resulting in a high incidence of post-treatment pain, and the pulp is easily exposed during caries removal, which means that root canal treatment is often required, making it difficult to preserve the natural tooth.

[0008] In the early stages of caries, you may feel a little pain, or you may not feel any pain at all. Since the caries has not progressed to the pulp, where the nerves and blood vessels are located, only the infected part is removed and filled with dental materials. However, if the caries has progressed to the pulp, root canal treatment, also known as nerve treatment, is usually required. Root canal treatment is a treatment in which all the nerve and blood vessel tissue in the pulp is removed and filled with dental materials. After root canal treatment, the removed part of the tooth is filled with dental materials. Teeth that have undergone root canal treatment are brittle and easily broken with filling treatment alone, so after filling treatment, a metal tube made by casting metal is placed over the tooth. If caries continues for a long time after progressing to the pulp, a pus bag will form in the bone at the root end of the tooth, and the pus will flow through the bone into the gums, causing swelling. In this case, root canal treatment is also available, but if the pus bag in the bone becomes too large, surgery to remove the pus bag or tooth extraction may be necessary. When root canal treatment is performed, which removes dental pulp cells rather than protecting them, the chewing sensation of natural teeth is lost and patients experience the inconvenience of a foreign body sensation. In addition, in relation to the immune response in the tooth, the autophagy of odontoblasts is known to be related to the differentiation of new odontoblasts, cell survival rate, and protection from cell death. From the perspective of the protection and adaptation of odontoblasts, it is expected that autophagy of odontoblasts can provide a beneficial effect on the survival of pulp cells and new odontoblasts, but since odontoblasts are also removed in conventional root canal treatment methods, it is difficult to expect beneficial effects from odontoblasts.

[0009] Under these circumstances, the inventors have made intensive research efforts to develop a formulation that can treat dental caries more effectively, and as a result have developed a new use of peptides for promoting the regeneration of dentin or dental pulp tissue for the prevention, amelioration or treatment of dental caries, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide an application for improving or treating dental caries, including root caries, caries in baby teeth that are about to fall out, and caries in erupting teeth, and pain caused by dental caries.

[0011] Another object of the present invention is to provide an improving or treating effect on dental caries, which increases the healing ability of dental pulp damaged by dental caries and reduces the expression of inflammatory factors, thereby preventing the progression of the disease to irreversible pain.

[0012] Another object of the present invention is to provide a use for improving or treating dental caries by increasing autophagy of odontoblasts, protecting the dental pulp, and reactivating odontoblasts in an environment where the dental pulp has been damaged by dental caries.

[0013] The objects of the present invention are not limited to those mentioned above, and other objects not mentioned will be clearly understood by those having ordinary skill in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0014] As a result of various studies, the present inventors have developed a preparation for preventing, improving and treating dental caries, which contains a peptide consisting of 10 amino acids, and uses thereof.

[0015] It was confirmed that the peptide can increase the expression levels of odontoblast differentiation marker genes Dspp, Dmp1 and Nestin, thereby promoting dentin regeneration while not exerting any cytotoxicity on dental pulp tissue cells.

[0016] In addition, it was confirmed that the peptide forms dentin-pulp-like tissue that is most similar in morphology to dentin-pulp tissue in vivo, increases collagen formation levels, and increases the expression level of DSP, an odontoblast-specific differentiation marker gene.

[0017] In one embodiment for achieving the above object, the present invention provides a pharmaceutical composition for preventing or treating dental caries, comprising a peptide having an amino acid sequence represented by the following general formula 1:

[0018] KY-R1-R2-R3-R4-R5-R6-R7-R8 (general formula 1) In the general formula 1, R1 is arginine (R), lysine (K) or glutamine (Q); R2 is arginine (R) or glutamine (Q); R3, R4 and R5 are arginine (R) or lysine (K), R6 is asparagine (N) or serine (S), and R7 and R8 are lysine (K) or tyrosine (Y).

[0019] The term "dentine" as used herein means a yellowish-white hard tissue that constitutes the majority of teeth. The dentine is covered by enamel at the crown and cementum at the root, and is not exposed on the surface of the tooth. However, as the enamel wears away with age, the dentine may be exposed at the tip or occlusal surface of the crown. The dentine is a type of bone tissue, but is distinct from general bone tissue in that the cells that make up the dentin are located in the dental pulp, and only their processes extend into the dentin.

[0020] The term "dental pulp" as used herein means the soft connective tissue that fills the pulp cavity inside a tooth. Anatomically, it is rich in nerves and blood vessels and can refer to the area extending down to the surface of the dentin.

[0021] The peptides contained in the formulation provided by the present invention do not exhibit cytotoxicity, but can increase the expression levels of odontoblast differentiation marker genes Dspp, Dmp1 and Nestin genes, and when transplanted into a living body together with dental pulp tissue cells, the dental pulp tissue cells can exhibit the characteristic of forming dentin / dental pulp tissue-like tissue.

[0022] The peptides contained in the formulation provided by the present invention include mutant peptides having a sequence that differs by one or more amino acid residues from the amino acid sequence that constitutes the formulation, as long as they are effective in treating dental caries.

[0023] 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. Also included may be peptides that have increased structural stability against heat, pH, etc., or increased ability to promote regeneration of dentin or dental pulp tissue due to mutations or modifications in amino acid sequence.

[0024] For example, the peptide of the present invention can show the same effect even if the acidic amino acid glutamine at the third position of the peptide of SEQ ID NO: 1 is replaced by the basic amino acid lysine or arginine; the peptide of the present invention can show the same effect even if the basic amino acid arginine at the fourth or fifth position of the peptide of SEQ ID NO: 1 is replaced by the acidic amino acid glutamine or the basic amino acid lysine; the peptide of the present invention can show the same effect even if the basic amino acid lysine at the sixth, seventh or ninth position of the peptide of SEQ ID NO: 1 is replaced by the basic amino acid arginine or the aromatic amino acid tyrosine; the peptide of the present invention can show the same effect even if the acidic amino acid asparagine at the eighth position of the peptide of SEQ ID NO: 1 is replaced by the neutral amino acid serine; and the peptide of the present invention can show the same effect even if the aromatic amino acid tyrosine at the tenth position of the peptide of SEQ ID NO: 1 is replaced by the basic amino acid lysine.

[0025] Thus, even if the acidic amino acids, basic amino acids, or aromatic amino acids constituting the peptides of the present invention are replaced with different acidic amino acids, basic amino acids, neutral amino acids, or aromatic amino acids, the effects of the peptides provided by the present invention can be exhibited as is, and therefore mutant peptides having a sequence that differs by one or more amino acid residues from the amino acid sequence constituting the peptides of the present invention can also be included in the category of the peptides provided by the present invention.

[0026] Furthermore, the peptides contained in the formulations 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-terminus or C-terminus, and are therefore included in the category of peptides provided by the present invention. As an example, the peptides may have 1 to 300 amino acids added to their N-terminus or C-terminus, as another example, the peptides may have 1 to 100 amino acids added to their N-terminus or C-terminus, as another example, the peptides may have 1 to 24 amino acids added to their N-terminus or C-terminus.

[0027] The 96 types of peptides corresponding to general formula 1 provided in the present invention significantly increased the mRNA level of Dspp gene, an odontoblast differentiation marker, in MDPC-23 cells treated with the 96 types of peptides, compared to the mRNA level of Dspp gene, which is an odontoblast differentiation marker, measured in MDPC-23 cells (control group) not treated with the peptides.

[0028] According to previous reports, it is known that an increase in the mRNA expression level of DSPP promotes odontoblast differentiation and dentin regeneration, and therefore, the 96 peptides that have the effect of increasing the mRNA level of the Dspp gene are known to have the effect of promoting odontoblast differentiation and dentin regeneration (Taduru Sreenath et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 278, No. 27, Issue of July 4, pp. 24874-24880, 2003; William T. Butler et al., Connective Tissue Research, 44(Suppl. 1):171-178, 2003).

[0029] In another aspect, the present invention provides a polynucleotide encoding the peptide.

[0030] The polynucleotide may be mutated by one or more bases being substituted, deleted, inserted, or a combination thereof. When preparing the nucleotide sequence by chemical synthesis, the synthesis method known in the art, for example, the method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), can be used, and can be synthesized using triester, phosphite, phosphoramidite, and H-phosphonate methods, PCR and other autoprimer methods, oligonucleotide synthesis methods on solid support, etc. For example, the polynucleotide encoding the peptide of the present invention may include the base sequence of SEQ ID NO:4.

[0031] In still another aspect, the present invention provides an expression vector comprising the polynucleotide, a transformant comprising the expression vector, and a method for producing the peptide using the transformant.

[0032] The term "expression vector" as used herein refers to a recombinant vector capable of expressing a target peptide in a desired host cell, and a gene product containing the necessary regulatory elements operably linked to the gene insert for expression. The expression vector contains expression regulatory elements such as start codons, stop codons, promoters, and operators, and the start codons and stop codons are generally considered to be part of the nucleotide sequence encoding a 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.

[0033] The term "operably linked" as used herein means that a nucleic acid expression control sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked to each other so as to perform a 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. The operably linked expression vector can be prepared using recombinant gene technology known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes or the like commonly known in the art.

[0034] In addition, the expression vector may contain a signal sequence for the excretion of the peptide to facilitate the 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, need to 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.

[0035] In addition, the expression vector may further comprise a protein tag, which may optionally be removed using an endopeptidase, to facilitate detection of the peptide.

[0036] The term "tag" as used herein means 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, a Flag tag, a histidine (His) tag, a leucine tag, an IgG tag, or a 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.

[0037] In the present invention, the expression vector may contain a nucleotide sequence encoding the peptide for promoting regeneration of dentin or dental pulp tissue and for treating dentin hypersensitivity 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, etc., 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 subtilis-derived plasmids (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vectors (retrovirus, adenovirus, vaccinia virus, etc.), and insect virus vectors (baculovirus, etc.). The expression vectors vary in protein expression level and modification depending on the host cell, so it is desirable to select and use the most suitable host cell for the purpose.

[0038] 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 express the polynucleotide contained in the expression vector to produce the peptide. The transformation can be performed by various methods, and is not particularly limited as long as the peptide can be produced. Examples of the transformation include CaCl2 precipitation, Hanahan method in which the efficiency of CaCl2 precipitation is improved by using a reducing substance called DMSO (dimethyl sulfoxide), electroporation, calcium phosphate precipitation, protoplast fusion, stirring method using silicon carbide fiber, Agrobacterium-mediated transformation, PEG-based transformation, dextran sulfate, lipofectamine, and desiccation / repression-mediated transformation. In addition, the host used to prepare the transformant is also 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, Schizosaccharomyces pombe, etc.; fungal cells such as Pichia pastoris, etc.; insect cells such as Drosophila and Spodoptera frugiperda Sf9 cells, etc.; animal cells such as CHO, COS, NSO, 293, Bowes melanoma cells, etc.; or plant cells.

[0039] The transformant may also be used in a method for producing the peptide for promoting regeneration of dentin or dental pulp tissue and treating dentin hypersensitivity of the present invention. Specifically, the method for producing the peptide for promoting regeneration of dentin or dental pulp tissue and treating dentin hypersensitivity of the present invention may include (a) culturing the transformant to obtain a culture, and (b) recovering the peptide of the present invention from the culture.

[0040] The term "culture" as used herein means a method for growing a microorganism under appropriately artificially controlled environmental conditions. In the present invention, the method for culturing the transformant can be performed by a method known in the art. Specifically, the culture is not particularly limited as long as the peptide for promoting regeneration of dentin or dental pulp tissue and for treating dentin hypersensitivity of the present invention can be expressed and produced, and the culture can be continuously cultured by a batch process, a fed batch process or a repeated fed batch process.

[0041] The medium used for the culture should be a normal medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc., and should be adjusted under aerobic conditions, such as temperature and pH, to meet the requirements of the specific strain in an appropriate manner. Carbon sources that can be used include a mixture of glucose and xylose as the main carbon source, as well as sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances can be used alone or in mixtures. Also, the 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, and organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, and peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steeping liquid, casein hydrolysate, fish or its decomposition products, defatted soybean cake or its decomposition products. These nitrogen sources can be used alone or in combination. The medium can include potassium monophosphate, potassium diphosphate, and the corresponding sodium-containing salts as phosphorus sources. The phosphorus sources that can be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or the corresponding sodium-containing salts. Also, the inorganic compounds that can be used include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate. Finally, in addition to the above substances, substances essential for growth such as amino acids and vitamins can be used.

[0042] Also, suitable precursors may be used in the culture medium. The above-mentioned raw materials may be added to the culture in a batch, fed-batch or continuous manner by an appropriate method during the culture process, but are not limited thereto. The pH of the culture may be adjusted by an appropriate method using basic compounds such as sodium hydroxide, potassium hydroxide, ammonia, or acidic compounds such as phosphoric acid or sulfuric acid.

[0043] In addition, foam formation can be suppressed by using an antifoaming agent such as 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. For this purpose, the culture is usually achieved in 10 to 100 hours.

[0044] Furthermore, the step of recovering the peptide from the culture may 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, fractional dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobic, and size exclusion), etc.

[0045] The pharmaceutical composition of the present invention can promote dental pulp tissue cells to form dentin / pulp tissue-like tissue, and therefore can be used to treat dental caries by methods of repairing dentin or dental pulp tissue.

[0046] The peptide contained in the pharmaceutical composition may be used in the form of a peptide alone, or in the form of a polypeptide in which the peptide is repeated two or more times and bound, or in the form of a complex in which a drug exhibiting an antibiotic effect against cariogenic bacteria is bound to the N-terminus or C-terminus of the peptide.

[0047] The term "dental caries" according to the present invention may refer to an infectious disease caused by bacteria that live on the tooth surfaces.

[0048] The term "prevention" as used herein means any action of inhibiting or delaying the onset of dental caries by administering a pharmaceutical composition for preventing or treating dental caries, which contains the peptide of the present invention.

[0049] The term "treatment" as used herein means any action of administering a pharmaceutical composition containing the peptide of the present invention as an active ingredient to an individual in need of treatment for dental caries, thereby promoting the regeneration of dentin or dental pulp tissue, thereby treating dental caries.

[0050] The pharmaceutical composition of the present invention may be prepared in the form of a pharmaceutical composition for treating dental caries, which further contains a suitable carrier (natural or non-natural carrier), excipient, or diluent that is generally used in the preparation of a pharmaceutical composition, in addition to the peptide. Specifically, the pharmaceutical composition may be formulated into a sterile injection solution that can be administered to the site where dental caries is induced by a conventional method. In the present invention, examples of carriers, excipients, and diluents that may be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, collagen, etc. When the peptide of the present invention is formulated, it may be prepared using a diluent or excipient that is generally used, such as a filler, extender, binder, wetting agent, disintegrant, or surfactant. In particular, it may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried agents, suppositories, ointments (e.g., pulp liner, etc.), etc. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used. As suppository bases, witepsol (registered trademark), macrogol (registered trademark), tween (registered trademark) 61, cacao butter, laurin butter, glycerogelatin, etc. may be used.

[0051] The content of the peptide contained in the pharmaceutical composition of the present invention is not particularly limited, but may be 0.0001% by weight to 50% by weight, more preferably 0.01% by weight to 20% by weight, based on the total weight of the final composition.

[0052] The pharmaceutical composition of the present invention may be administered in a pharmaceutical effective amount, and the term "pharmaceutical effective amount" as used herein means an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the effective dose level is determined based on factors including the severity of the disease, the activity of the drug, the age, weight, health condition, sex, and sensitivity of the patient to the drug, the administration time, administration route, and excretion rate of the composition of the present invention used, the duration of treatment, and drugs used in combination with or simultaneously with the composition of the present invention used, as well as 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 dental caries. Taking all of the above factors into consideration, it is important to administer an amount that provides maximum effect at a minimum amount without side effects.

[0053] The dosage of the pharmaceutical composition of the present invention can be determined by a person skilled in the art, taking into consideration the purpose of use, the severity of the disease, the age, weight, sex, medical history, or the type of substance used as an active ingredient of the patient. 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 frequency of administration of the composition of the present invention is not particularly limited, but it can be administered once a day or in divided doses several times a day. The dosage does not limit the scope of the present invention in any aspect.

[0054] In another aspect, the present invention provides a method for treating dental caries, which comprises the step of administering a pharma- ceutically effective amount of the pharmaceutical composition to an individual, other than a human, suffering from dental caries.

[0055] The term "individual" as used herein may include, without limitation, mammals, including mice, livestock, etc., in which treatment for dental caries is desired, but may exclude humans from individuals affected by the disease.

[0056] The pharmaceutical composition for treating dental caries of the present invention may be administered via any common route as long as it can reach the target tissue. The pharmaceutical composition of the present invention may be administered via a route such as oral administration or oral injection depending on the purpose, without being particularly limited.

[0057] In another aspect, the present invention provides a quasi-drug composition for preventing or ameliorating dental caries, comprising the peptide.

[0058] The term "amelioration" according to the present invention refers to any action that at least reduces a parameter related to the treated condition, for example the severity of a symptom.

[0059] 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 dental caries, thereby promoting regeneration of dentin or dental pulp tissue, thereby improving or benefiting the symptoms of dental caries.

[0060] The term "quasi-drug" in this invention 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 drug. For example, according to the Pharmaceutical Affairs Law, quasi-drugs exclude articles used for medicinal purposes, and include fiber and rubber products used for the treatment and prevention of diseases in humans and animals, articles that have a mild or no direct effect on the human body and are not instruments or machines, and similar articles, fungicides and insecticides for preventing infectious diseases.

[0061] In the present invention, the type and dosage form of the quasi-drug composition containing the peptide are not particularly limited, but examples thereof may include oral disinfectant mouthwash, oral hygiene products, toothpaste, dental floss, and oral ointments.

[0062] In another aspect, the present invention provides a health functional food composition for preventing or improving dental caries, comprising the peptide.

[0063] The term "food" as used herein includes all foods in the ordinary sense of the term, such as meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, energy drinks, alcoholic beverages, vitamin complexes, health functional foods, and health foods.

[0064] The term "functional food" is the same as food for special health use (FoSHU), and refers to a food with high medical and medical effects that is processed to efficiently exhibit a biological regulation function in addition to nutritional supplementation. In addition, "functional" means to adjust nutrients to the structure and function of the human body or to obtain a useful effect for health purposes such as physiological action. The food of the present invention can be produced by a method commonly used in the industry, and during the production, raw materials and ingredients commonly added in the industry can be added. In addition, the dosage form of the food can be produced without any restrictions as long as it is a dosage form recognized as a food. The food composition of the present invention can be produced in various dosage forms, and unlike general medicines, it has the advantage of being made of food as a raw material and having no side effects that may occur when taking medicines for a long period of time, and is also highly portable, and the food of the present invention can be taken as a supplement to enhance the effect of preventing or improving dental caries.

[0065] The term "health food" refers to food that has a positive effect on maintaining or improving health compared to general foods, and "health supplement food" refers to food for the purpose of supplementing health. In some cases, the terms "health functional food," "health food," and "health supplement" may be used interchangeably.

[0066] Specifically, the health functional food is a food product produced by adding the peptide of the present invention to food ingredients such as beverages, teas, spices, gums, and sweets, or by encapsulating, powdering, suspending, etc., and ingesting the food product brings about a specific health effect. However, unlike general medicines, since the food product is used as a raw material, the health functional food product has the advantage of being free of side effects that can occur when taking medicines for a long period of time.

[0067] The food composition of the present invention can be ingested on a daily basis, and is therefore expected to be highly effective in preventing or improving dental caries, making it highly useful.

[0068] 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.

[0069] The food composition may also contain additional ingredients that are commonly used in food compositions and may improve the aroma, taste, appearance, etc. For example, the food composition may contain vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, etc. The food composition may also contain minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), etc. Furthermore, the food composition may contain amino acids such as lysine, tryptophan, cysteine, and valine.

[0070] In addition, the food composition may contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), antioxidants (butyl hydroxyanisole (BHA), butyl hydroxytoluene (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.), flavors (vanillin, lactones, etc.), leavening agents (alum (potassium aluminum sulfate), potassium D-hydrogen tartrate, etc.), strengthening agents, 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 an appropriate amount.

[0071] The peptide of the present invention can be added as it is or with other foods or food ingredients, and can be used appropriately by a conventional method. The amount of the active ingredient mixed can be suitably determined according to the purpose of use (prevention, health or therapeutic treatment). In general, 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. However, when taking it for a long period of time for health and hygiene purposes, it may be contained in an amount below the above range, and there is no problem in terms of safety, so the active ingredient can be used in an amount above the above range.

[0072] One example of the food composition of the present invention may be used as a health drink composition, and in this case, various flavorings or natural carbohydrates may be contained as additional ingredients, as in ordinary drinks. The natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. The sweetener may be a natural sweetener such as thaumatin or stevia extract; or a synthetic sweetener such as saccharin or aspartame. The ratio of the natural carbohydrates may be generally about 0.01 g to 0.04 g, specifically about 0.02 g to 0.03 g, per 100 mL of the health drink composition of the present invention.

[0073] In addition to the above, the health drink composition may contain various nutrients, vitamins, minerals (electrolytes), flavorings, colorings, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonation agents. In addition, it may contain fruit pulp for the production of natural fruit juice, fruit juice beverages, or vegetable beverages. Such ingredients may be used independently or in mixtures. The ratio of such additives is not very important, but is generally 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.

[0074] The food composition of the present invention may contain the peptide of the present invention in various weight percentages as long as it exhibits the effect of preventing or improving dental caries. Specifically, the food composition may contain the peptide of the present invention in an amount of 0.00001 weight % to 100 weight %, or 0.01 weight % to 80 weight %, relative to the total weight of the food composition, but is not limited thereto. Effect of the Invention

[0075] The present invention can find use in improving or treating caries, including root caries, caries in baby teeth that are about to fall out, and caries in erupting teeth, and pain caused by caries.

[0076] Another object of the present invention is to provide an improving or treating effect on dental caries, which increases the healing ability of dental pulp damaged by dental caries and reduces the expression of inflammatory factors, thereby preventing the progression of the disease to irreversible pain.

[0077] The present invention can provide an application for improving or treating dental caries by increasing autophagy of odontoblasts in an environment where dental pulp has been damaged by dental caries, thereby protecting the dental pulp and reactivating odontoblasts.

[0078] The effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those having ordinary skill in the art to which the present invention pertains from the following description. [Brief description of the drawings]

[0079] [Figure 1a] FIG. 1a shows the results of observing the permeability and stability of a peptide (SEQ ID NO: 96) contained in a pharmaceutical composition according to an embodiment of the present invention in a caries environment. A peptide conjugated with the fluorescent dye rhodamine was applied to a tooth extracted from a human and showing dentin caries (stages 2-3 of caries). After about 1 minute, the tooth was fixed with 4% paraformaldehyde and sliced ​​in the sagittal direction using a digital low-speed diamond cutter to a thickness of 0.1 mm, and observed using a confocal microscope. [Figure 1b] Figure 1b shows the results of observing the permeability and stability of a peptide (sequence number 96) contained in a pharmaceutical composition according to an embodiment of the present invention in a caries environment, and confirms the stability of the peptide in the pharmaceutical composition in an environment where S. Mutans, a caries-causing bacterium, is present, by MALDI-TOF analysis. [Figure 2a] FIG. 2a shows the results of treatment of a pharmaceutical composition according to an embodiment of the present invention according to the stages of caries in a rat model of caries. [Figure 2b]FIG. 2b shows the results of comparing the number of inflammatory cells when a pharmaceutical composition according to an embodiment of the present invention was applied to a tooth affected by caries in a rat model of caries. [Diagram 3] FIG. 3 is a cell experiment mimicking inflammation of dental pulp caused by caries corresponding to stage 4, and is a graph showing the effect of treatment of human dental pulp cells with a pharmaceutical composition according to an embodiment of the present invention under an inflammatory environment on gene expression related to inflammatory factors. [Figure 4] Figure 4 is a cell experiment simulating the situation when dental pulp inflammation occurs due to caries, which corresponds to the fourth stage, and is a graph showing the effect of treating human dental pulp cells with a pharmaceutical composition according to an embodiment of the present invention under an inflammatory environment on the survival rate of dental pulp cells. [Diagram 5] FIG. 5 shows a cell experiment simulating the situation when dental pulp inflammation occurs due to caries, which corresponds to the fourth stage, and shows the effect of treating human dental pulp cells damaged under an inflammatory environment with a pharmaceutical composition according to an embodiment of the present invention on the repair of dental pulp cells. [Figure 6] Figure 6 shows a cell experiment that mimics the situation when pulp inflammation occurs due to caries, which corresponds to the fourth stage, and shows the results of observing the effects on the mineralization ability of human dental pulp cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0080] The objectives and effects of the present invention, as well as the technical configuration for achieving them, will become apparent by referring to the following detailed examples together with the accompanying drawings. In the description of the present invention, if it is determined that a detailed description of a known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description will be omitted. The terms described below are terms defined for the description of the embodiments of the present invention, and may vary depending on the intention or practice of the user or operator.

[0081] However, the present invention is not limited to the embodiments disclosed below, and may be realized in various different forms. However, the embodiments are provided to make the disclosure of the present invention complete and to fully inform those skilled in the art of the invention of the category of the invention, and the present invention is defined only by the claims. Therefore, the definition should be based on the contents of the entire specification.

[0082] The present invention will be described in more detail below with reference to examples. However, these examples are merely for illustrative purposes and the scope of the present invention is not limited to these examples.

[0083] Example 1: Synthesis of peptides The inventors synthesized a peptide (SEQ ID NO: 1) exhibiting the effect of promoting the regeneration of dentin or dental pulp tissue using 9-fluorenylmethyloxycarbonyl (Fmoc), and synthesized each group of peptides by substituting the amino acids of the synthesized peptide (Tables 1 to 12).

[0084] N-KYQRRKKNKY-C (SEQ ID NO: 1) First, the peptides of Group 1 were synthesized by substituting the peptide of SEQ ID NO: 1 or the 5th to 7th amino acids of the peptide of SEQ ID NO: 1 with lysine or arginine (Table 1).

[0085] [Table 1]

[0086] Next, peptides in group 2 were synthesized by substituting the 5th to 7th amino acids of the peptide of SEQ ID NO:1 with lysine or arginine, and substituting the 8th amino acid with serine (Table 2).

[0087] [Table 2]

[0088] Next, peptides in group 3 were synthesized by substituting the 5th to 7th amino acids of the peptide of SEQ ID NO:1 with lysine or arginine, and substituting the 9th amino acid with tyrosine (Table 3).

[0089] [Table 3]

[0090] Next, peptides in group 4 were synthesized by substituting the 5th to 7th amino acids of the peptide of sequence number 1 with lysine or arginine, the 8th amino acid with serine, the 9th amino acid with tyrosine, and the 10th amino acid with lysine (Table 4).

[0091] [Table 4]

[0092] Next, peptides in group 5 were synthesized by substituting the third amino acid of the peptide of sequence number 1 with arginine, the fourth amino acid with glutamine, and the fifth to seventh amino acids with lysine or arginine (Table 5).

[0093] [Table 5]

[0094] Next, the peptides of group 6 were synthesized by replacing the third amino acid of the peptide of sequence number 1 with arginine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, and the eighth amino acid with serine (Table 6).

[0095] [Table 6]

[0096] Next, the peptides of group 7 were synthesized by replacing the third amino acid of the peptide of SEQ ID NO:1 with arginine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, the ninth amino acid with tyrosine, and the tenth amino acid with lysine (Table 7).

[0097] [Table 7]

[0098] Next, the peptides of group 8 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO:1 with arginine acid, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, the eighth amino acid with serine, the ninth amino acid with tyrosine, and the tenth amino acid with lysine (Table 8).

[0099] [Table 8]

[0100] Next, the peptides of group 9 were synthesized by substituting the third amino acid of the peptide of sequence number 1 with lysine acid, the fourth amino acid with glutamine, and the fifth to seventh amino acids with lysine or arginine (Table 9).

[0101] [Table 9]

[0102] Next, the peptides in group 10 were synthesized by replacing the third amino acid of the peptide of sequence number 1 with lysine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, and the eighth amino acid with serine (Table 10).

[0103] [Table 10]

[0104] Next, the peptides of group 11 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO:1 with lysine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, the ninth amino acid with tyrosine, and the tenth amino acid with lysine (Table 11).

[0105] [Table 11]

[0106] Finally, the peptides of group 12 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO:1 with lysine, the fourth amino acid with glutamine, the fifth through seventh amino acids with lysine or arginine, the eighth amino acid with serine, the ninth amino acid with tyrosine, and the tenth amino acid with lysine (Table 12).

[0107] [Table 12]

[0108] Example 2: Preparation of pharmaceutical compositions The pharmaceutical composition (KH002) according to the embodiment of the present invention was prepared as follows. The pharmaceutical composition (KH002) was prepared by mixing with about 1.5% carboxymethyl cellulose (Sigma-Aldrich, C5678). The carboxymethyl cellulose was mixed with distilled water at about 3%, boiled at 100°C, and sterilized under pressure, and was adjusted so that the final concentration would be about 1.5% when mixed with the peptide (KH001) of SEQ ID NO:96 among the peptides prepared in Example 1.

[0109] Example 3: Permeability and stability of pharmaceutical compositions in a carious environment [Example 3-1: Permeability of pharmaceutical composition in caries environment] A pharmaceutical composition (KH002) containing a peptide conjugated with the fluorescent dye rhodamine (KH001) was applied to extracted human teeth with dentin caries (stages 2-3) and fixed with 4% paraformaldehyde after 1 min. Slices of the teeth were then cut in the sagittal direction with a digital low-speed diamond cutter to a thickness of approximately 0.1 mm and observed under a confocal microscope (Figure 1a).

[0110] As shown in FIG. 1a, the red staining can be observed, and it was confirmed that the peptide (KH001) component contained in the pharmaceutical composition of Example 2 successfully penetrated the caries-affected tooth tissue and reached the dental pulp.

[0111] [Example 3-2: Stability analysis of pharmaceutical compositions in a caries environment] A. Stability analysis of peptides in pharmaceutical compositions provided in Example 2 The standard strain S. mutans (Streptococcus mutans, KCOM 1054) used in this experiment was provided by the Korean Collection of Oral Microbiology (KCOM). The bacteria was smeared on an agar plate containing about 20% sucrose (MSB, bacitracin concentration 0.5 μg / mL) and cultured in a candle jar at about 37° C. for about 48 hours. Single colonies were cultured in Tryptic Soy Broth (TSB; BD DIFCO®, USA) for about 24 hours and used in the experiment. The S. mutans strain was cultured until the absorbance (OD600) value reached 1.0, and 100 μl of the cultured strain was mixed with 100 μg of pharmaceutical composition (KH002) in 1.5% carboxymethylcellulose, and the stability of the peptide (KH001) contained in the pharmaceutical composition (KH002) depending on the reaction time with the strain was analyzed by MALDI-TOF analysis (UltraflexIII TOF / TOF (Bruker Daltonics)).

[0112] The compositions (a) and (b) were collected at intervals of 1 minute, 5 minutes, 15 minutes, and 30 minutes, respectively, and centrifuged at about 15,000 rpm for about 10 minutes, and the supernatant was collected.

[0113] 2 μl of each supernatant collected from the halo and loco was mixed with 2 μl of substrate solution (10 mg / mL α-cyano-4-hydroxycinnamic acid (CHCA) in 0.1% TFA / ACN (1:1, v / v)) for MALDI-TOF analysis (Figure 1b).

[0114] FIG. 1b confirms that the peptide (KH001) in the pharmaceutical composition (KH002) provided in Example 2 was stably maintained in the environment of S. Mutans, a bacterium that causes dental caries.

[0115] Example 4: Analysis of the results of treatment with pharmaceutical compositions for dental caries at each stage (Example 4-1: Preparation of rat models of caries at different stages) Figure 2a shows the stages of dentin caries in a rat caries model, divided into early, moderate, and advanced stages for each third of the dentin thickness, with the most severe stage being when the dentin above the pulp is completely infected with caries and the caries has invaded the pulp (n=4). This classification is almost the same as the human caries stage, where early is caries stage 2, moderate and advanced are caries stage 3, and severe is caries stage 4.

[0116] (Example 4-2: Observation of the results of treatment with pharmaceutical compositions in rat models) The control group was a group suffering from dental caries but not treated with the pharmaceutical composition (KH002) prepared in Example 2. The tissue photographs below show that the pharmaceutical composition (KH002) was applied at 3 μg per tooth at the early, middle, late and severe stages, and the rats were sacrificed after 4 weeks, fixed and decalcified, and tissue slides were prepared and stained with hematoxylin and eosin.

[0117] In the control group not treated with the pharmaceutical composition (KH002) prepared in Example 2, liquefaction necrosis was observed inside the dental pulp, the surrounding state of odontoblasts was damaged below the part affected by caries, and infiltration of inflammatory cells into the dental pulp was observed. In addition, in the group treated with the pharmaceutical composition (KH002), the lower part of the caries-affected odontoblasts showed a surrounding state, and in the dental pulp as a whole, astrocytes, which are characteristic of healthy dental pulp, were observed in the early and middle stages, and it was confirmed that healthy dental pulp was maintained in the root part even in a severe state where all the dentin above the dental pulp was affected by caries.

[0118] When treated with the pharmaceutical composition (KH002), it was observed that dentin was regenerated in all stages of caries, and even in a model of a severely damaged tooth, a dentin bridge, which is formed as one of the defensive mechanisms against caries, was formed above the preserved dental pulp.

[0119] (Example 4-3: Observation of inflammatory cells caused by dental caries) The number of inflammatory cells relative to the total number of pulp cells was compared as a percentage (number of individuals n = 4, number of teeth n = 6). Three sites inside the pulp were randomly designated, and readings were performed by three researchers who were blinded to ensure accuracy of readings.

[0120] Figure 2a shows the results of treatment of a pharmaceutical composition according to an embodiment of the present invention according to the caries stage in a rat caries model. Referring to Figure 2a, it was confirmed that the percentage of inflammatory cells stained with ED1 was significantly reduced in the experimental group treated with pharmaceutical composition (KH002) compared to the control group not treated with pharmaceutical composition (KH002). Based on these results, it can be predicted that treatment with pharmaceutical composition (KH002) is effective in relieving inflammation caused by caries.

[0121] Figure 2b shows the results of treating a rat caries model with a depth equivalent to stage 3 human caries with a pharmaceutical composition (KH002). In order to quantify the degree of inflammation in caries, the number of inflammatory cells that had infiltrated into the dental pulp was confirmed, and the results were compared using immunohistochemical techniques with ED1, a representative inflammatory cell marker for dental pulp.

[0122] (Example 5: Analysis of the effect of dental caries on the expression of inflammatory factors) To confirm the preventive and therapeutic effects of the pharmaceutical composition (KH002) in the fourth stage of caries, when inflammation occurs after caries, human dental pulp cells were treated with LPS (Lipopolysaccharide) and the pharmaceutical composition (KH002) and then the expression of inflammatory factors was compared.

[0123] All experiments using human dental pulp cells were approved by the Institutional Review Board (IRB; S-D20140007) of Seoul National University Dental Hospital and were performed in accordance with the guidelines and regulations of the institution. Dental pulp cells were isolated by extracting the third molar (wisdom tooth) of patients who had signed a consent form at Seoul National University Dental Hospital, using a previously reported method (Choung et al., 2016). The tooth was cut and the internal pulp was isolated with forceps, and the pulp tissue was finely chopped into a 60 mm diameter cell culture dish. The finely chopped pulp tissue was then covered with a cover glass and cultured in cell culture medium to isolate human dental pulp cells. Human dental pulp cells were cultured in a 6-well dish at 1 × 10 5 After seeding the cells at 1000x1000 cells / well, the negative control group was treated with culture medium alone, the positive control group was treated with 10μg / mL LPS and 10μg / mL pharmaceutical composition (KH002) alone, and the experimental group was treated with 10μg / mL LPS and 10μg / mL pharmaceutical composition (KH002) simultaneously to determine the preventive effect. In the experimental group, the cells were pretreated with LPS for 24 hours and then treated with pharmaceutical composition (KH002) to confirm the therapeutic effect.

[0124] RT-PCR was performed to compare the expression of inflammatory factors, and the ratio to the expression level of GAPDH was confirmed to quantitatively compare the expression level of each factor (Figure 3).

[0125] When human dental pulp cells were treated with LPS alone, it was confirmed that the expression of inflammatory factors TNF-α, IL-1α, IL-1β, IL-6, CXCL10, iNOS, and COX2 was significantly increased. In contrast, when the pharmaceutical composition (KH002) was applied after LPS pretreatment, and when LPS and the pharmaceutical composition (KH002) were treated together, the expression patterns were similar to those of the negative control group that received no treatment. These results indicate that when inflammation occurs after caries, the pharmaceutical composition (KH002) has the effect of preventively and therapeutically alleviating caries-related inflammation.

[0126] The RT-PCR primer sequences for each factor are as follows (Table 13).

[0127] [Table 13]

[0128] (Example 6: Analysis of the effect on the viability of dental pulp cells) The effect of a pharmaceutical composition (KH002) on cell viability in an inflammatory environment of human dental pulp cells was confirmed for four stages of caries in which inflammation occurred after tooth decay.

[0129] To measure cell viability, 5 × 10 human dental pulp cells were 3 After seeding cells / well into a 96-well plate, the cells were treated with 150 μl of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide solution, Medifab) for approximately 2 hours on the 1st, 3rd, and 5th days, respectively, and then dissolved in DMSO, and the absorbance at 570 nm was measured.

[0130] All experiments using human dental pulp cells were approved by the Institutional Bioethics Board (IRB) of Seoul National University Dental Hospital and were performed in accordance with the institution's guidelines and regulations. Dental pulp cells were isolated by extracting the third molar (wisdom tooth) of patients who had signed a consent form at Seoul National University Dental Hospital, using a previously reported method. The tooth was cut and the internal pulp was isolated with forceps, and the pulp tissue was finely chopped into a 60 mm diameter cell culture dish. The finely chopped pulp tissue was then covered with a cover glass and cultured in cell culture medium to isolate human dental pulp cells. Human dental pulp cells were cultured in a 6-well dish at 1 × 10 5 After seeding the cells at 1000x1000 cells / well, the negative control group was treated with culture medium alone, the positive control group was treated with 10μg / mL LPS and 10μg / mL pharmaceutical composition (KH002) alone, and the experimental group was treated with 10μg / mL LPS and 10μg / mL pharmaceutical composition (KH002) simultaneously to determine the preventive effect. In the experimental group, the cells were pretreated with LPS for 24 hours and then treated with pharmaceutical composition (KH002) to confirm the therapeutic effect.

[0131] Figure 4 is a cell experiment mimicking the situation when inflammation of the dental pulp occurs due to caries, which corresponds to stage 4, and is a graph showing the effect of treating human dental pulp cells with a pharmaceutical composition (KH002) under an inflammatory environment on the survival rate of dental pulp cells.

[0132] The experimental group treated with pharmaceutical composition (KH002) at a concentration of 10μg / mL showed no significant difference from the negative control group. The group treated with 10μg / mL LPS showed a significant decrease in the viability of dental pulp cells compared to the negative control group, while the group treated with 10μg / mL LPS and pharmaceutical composition (KH002) at 10μg / mL showed a recovery in the viability of dental pulp cells to a similar level to the negative control group.

[0133] These results indicate that when inflammation occurs after dental caries, the pharmaceutical composition (KH002) can increase the survival rate of human dental pulp cells, protect the dental pulp, and alleviate inflammation.

[0134] (Example 7: Analysis of the effect on dental pulp cell recovery) In a wound healing model using human dental pulp cells, the effects of the inflammatory environment and pharmaceutical composition (KH002) treatment on wound healing ability were examined.

[0135] Human dental pulp cells were cultured in a 6-well plate at 1 × 10 5 The day after seeding at 100 cells / well, the center was scraped off with the tip of a 200 μL pipette, and the rate at which the cells grew after 24 hours was compared.

[0136] All experiments using human dental pulp cells were approved by the Institutional Bioethics Board (IRB) of Seoul National University Dental Hospital and were performed in accordance with the institution's guidelines and regulations. Dental pulp cells were isolated by extracting the third molar (wisdom tooth) of patients who had signed a consent form at Seoul National University Dental Hospital, using a previously reported method. The tooth was cut and the internal pulp was isolated with forceps, and the pulp tissue was finely chopped into a 60 mm diameter cell culture dish. The finely chopped pulp tissue was then covered with a cover glass and cultured in cell culture medium to isolate human dental pulp cells. Human dental pulp cells were cultured in a 6-well dish at 1 × 10 5 After seeding the cells at 1000x1000 cells / well, the negative control group was treated with culture medium alone, the positive control group with 10μg / mL LPS and 10μg / mL pharmaceutical composition (KH002) alone, and the experimental group was treated with 10μg / mL LPS and 10μg / mL pharmaceutical composition (KH002) simultaneously to determine the preventive effect. In the experimental group, LPS was pretreated for 24 hours, and then the pharmaceutical composition (KH002) was treated to confirm the effect on the recovery of dental pulp cells.

[0137] Photographs of the cells were taken under a light microscope using a 10x objective lens, and the photographs were analyzed using the ImageJ program.

[0138] Figure 5 shows a cell experiment simulating the situation when inflammation of the dental pulp occurs due to caries, which corresponds to the fourth stage, and shows the effect of treating human dental pulp cells damaged under an inflammatory environment with a pharmaceutical composition according to an embodiment of the present invention on the recovery of the dental pulp cells.

[0139] The wound healing ability of the experimental group was quantified in comparison with the control group. The group treated with only the pharmaceutical composition (KH002) showed no significant difference from the control group, but the group treated with LPS showed a significant decrease in wound healing ability, at about 50% of the control group. In the group treated with LPS and the pharmaceutical composition (KH002) together, the wound healing ability recovered to about 90%, confirming that the pharmaceutical composition (KH002) restores the wound healing ability of human dental pulp cells even in an inflammatory environment caused by caries.

[0140] (Example 8: Analysis of the effect on mineralization ability of dental pulp cells) When inflammation occurs after dental caries, the effect of treatment with a pharmaceutical composition (KH002) on the mineralization ability of human dental pulp cells was confirmed.

[0141] To do this, 1 × 10 human dental pulp cells were added to each 6-well dish. 5 The cells were seeded at 100 cells / well, and from the next day, they were cultured in differentiation medium containing ascorbic acid (50 μg / mL) and β-glycerophosphate (10 mM) for 7, 14, or 21 days.

[0142] All experiments using human dental pulp cells were approved by the Institutional Bioethics Board (IRB) of Seoul National University Dental Hospital and were performed in accordance with the institution's guidelines and regulations. Dental pulp cells were isolated by extracting the third molar (wisdom tooth) of patients who had signed a consent form at Seoul National University Dental Hospital, using a previously reported method. The tooth was cut and the internal pulp was isolated with forceps, and the pulp tissue was finely chopped into a 60 mm diameter cell culture dish. The finely chopped pulp tissue was then covered with a cover glass and cultured in cell culture medium to isolate human dental pulp cells. Human dental pulp cells were cultured in a 6-well dish at 1 × 10 5After seeding the cells at 1000x1000 cells / well, the negative control group was treated with culture medium alone, the positive control group was treated with 10μg / mL LPS and 10μg / mL pharmaceutical composition (KH002) alone, and the experimental group was treated with 10μg / mL LPS and 10μg / mL pharmaceutical composition (KH002) simultaneously to determine the preventive effect. In the experimental group, LPS was pretreated for 24 hours, and then the pharmaceutical composition (KH002) was treated to confirm the effect on mineralization.

[0143] To compare the amount of mineralized matrix, the tissue was fixed with 4% PFA overnight at 4°C, and then treated with 40 mM alizarin red S adjusted to pH 4.2 for 30 minutes at 25°C. Then, 0.5 mL of sodium dodecyl sulfate dissolved in 0.5 N hydrochloric acid was applied and the tissue was shaken for 30 minutes, after which the absorbance was measured at 405 nm.

[0144] FIG. 6 shows the results of a cell experiment simulating the situation when dental pulp inflammation due to caries occurs, which corresponds to the fourth stage, and the effect on the mineralization ability of human dental pulp cells. Referring to FIG. 6, the group treated with pharmaceutical composition (KH002) showed an increase in the size of mineral nodules on the 21st day compared to the control group, and the amount of mineralized matrix also increased significantly on the 7th, 14th, and 21st days. In contrast, the group treated with LPS showed a significant decrease in the size of mineral nodules on the 21st day and the amount of mineralized matrix on the 7th, 14th, and 21st days compared to the control group. The group treated with LPS and pharmaceutical composition (KH002) showed an increase in the size of mineral nodules on the 21st day compared to the group treated with LPS, and the amount of mineralized matrix recovered to almost the same level as the negative control group on the 7th and 14th days. In the case of the experimental group, the recovery did not reach the same level as the control group after 21 days, but showed a statistically significant increase compared to the group treated with LPS alone. These results confirmed that the pharmaceutical composition (KH002) can restore and improve the dentin secretion ability of odontoblasts in human dental pulp cells under an inflammatory environment caused by caries, and can provide the effect of repairing the thickness of damaged dentin.

[0145] The pharmaceutical composition according to the embodiment of the present invention may be used for the prevention and treatment of various caries diseases, such as preventing or preventing root caries in elderly people to whom general caries treatment methods are difficult to apply, and preserving carious baby teeth of infant patients until they fall out.

[0146] In addition, in the case of the pharmaceutical composition according to the embodiment of the present invention, even when inflammation occurs in a severe stage of caries where general caries treatment cannot restore the condition of natural teeth, it can protect odontoblasts and provide autophagy of newly formed odontoblasts, thereby providing the effect of treating caries by protecting dental pulp cells.

[0147] In the present specification and drawings, preferred embodiments of the present invention are disclosed, and even if 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 the understanding of the invention, and are not intended to limit the scope of the present invention. It is obvious to those having ordinary knowledge in the technical field 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 pharmaceutical composition for preventing or treating dental caries, comprising as an active ingredient a peptide having an amino acid sequence represented by the following general formula 1: KY-R1-R2-R3-R4-R5-R6-R7-R8 (general formula 1) In the general formula 1, R1 is arginine (R), lysine (K) or glutamine (Q); R2 is arginine (R) or glutamine (Q); R3, R4 and R5 are each arginine (R) or lysine (K); R6 is asparagine (N) or serine (S), and R7 and R8 are lysine (K) or tyrosine (Y).

2. The pharmaceutical composition for preventing or treating dental caries according to claim 1, characterized in that the peptide consists of any one of the amino acid sequences of SEQ ID NOs: 1 to 96.

3. The pharmaceutical composition for preventing or treating dental caries according to claim 1, characterized in that the pharmaceutical composition comprises a polypeptide in which the peptides are linked in repeated fashion.

4. The pharmaceutical composition for preventing or treating dental caries according to claim 1, characterized in that the pharmaceutical composition comprises a complex in which the peptide is bound to a drug having an antibiotic effect against oral bacteria.

5. The pharmaceutical composition for preventing or treating dental caries according to claim 1, further comprising a pharma- ceutically acceptable carrier, excipient or diluent.

6. A quasi-drug composition for preventing or ameliorating dental caries, comprising the peptide according to claim 1 or 2.

7. A health functional food composition for preventing or improving dental caries, comprising the peptide according to claim 1 or 2.

8. A method for preventing or treating dental caries, comprising administering to an individual other than humans a pharmaceutical composition for preventing or treating dental caries, the pharmaceutical composition comprising a peptide having an amino acid sequence represented by the following general formula 1 as an active ingredient: KY-R1-R2-R3-R4-R5-R6-R7-R8 (general formula 1) In the general formula 1, R1 is arginine (R), lysine (K) or glutamine (Q); R2 is arginine (R) or glutamine (Q); R3, R4 and R5 are each arginine (R) or lysine (K); R6 is asparagine (N) or serine (S), and R7 and R8 are lysine (K) or tyrosine (Y).

Images