Compositions for the prevention or treatment of periodontal disease and damage caused by periodontal disease, containing GV1001

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease. More specifically, the present invention relates to a pharmaceutical composition for the prevention or treatment of periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease, which contains a peptide having the amino acid sequence of SEQ ID NO: 1, is effective in suppressing osteoclast formation, suppressing colony formation of Porphyromonas gingivalis, and suppressing gingipain expression, is safe for the body, and has few side effects including abnormal reactions.

Description

Technical Field

[0001] The present invention relates to a composition for preventing or treating periodontal diseases including GV1001, as well as atherosclerosis and Alzheimer's disease caused by periodontal diseases.

Background Art

[0002] Periodontal diseases are diseases that occur in the surrounding tissues of teeth, such as gums, periodontal ligaments, and alveolar bones surrounding the teeth, and are classified into gingivitis and periodontitis according to the severity of the diseases. Periodontitis induces an immune-inflammatory reaction that causes irreversible changes in the periodontal tissues (periodontium), and as a result, it is a chronic oral and systemic inflammatory reaction in which the alveolar bone is gradually destroyed, leading to tooth loss. Periodontitis has been reported to be associated with the onset of various systemic diseases, such as rheumatoid arthritis, psoriasis, systemic sclerosis, Alzheimer's disease (AD), and cardiovascular diseases (CVD) including atherosclerosis.

[0003] Periodontal diseases are diseases with a high morbidity rate. In Korea, the morbidity rate of periodontal diseases in adults aged 19 or older is 29.8%, and the morbidity rate increases with age. In particular, the morbidity rate of periodontal diseases increases rapidly in those aged 50 or older.

[0004] According to a 2022 report by TechNavio, the size of the periodontal disease treatment drug market is predicted to grow by $1,061 million from 2022 to 2026, and the average annual growth rate (CAGR) during the prediction period is 9.75%.

[0005] Therefore, antibacterial and bacteriostatic effects against anaerobic Gram-negative bacteria, which are the underlying causative factors of periodontal disease, removal of bacterial toxic products, and restoration of lost periodontal tissue to its original state are important elements in the prevention and treatment of periodontal disease.

[0006] Treatment for periodontal disease involves improving the patient's oral hygiene, non-surgical treatments, and surgical treatments (scaling, root planing, gingival curettage, and periodontal tissue regeneration using reattachment). However, surgical treatment, the most effective method, has the limitation that it is usually performed after the disease has progressed to a certain extent, rather than for prevention, and as a result, most periodontal diseases progress to chronic conditions. Systemic antibiotics and topical sustained-release formulations have been used as supplementary treatments. However, these methods cause side effects due to excessive delivery of drugs to unnecessary sites, and recently, cases of periodontal bacteria resistant to antibiotics have been reported, highlighting the difficulty of preventing and treating periodontal disease.

[0007] Therefore, there is a growing need for periodontal disease prevention and treatment formulations that can overcome the limitations of surgical treatment and the problems associated with antibiotic use.

[0008] On the other hand, GV1001 (SEQ ID NO: 1), which consists of 16 amino acids selected from human telomerase reverse transcriptase (hTERT), has been reported to exhibit not only anti-cancer, anti-inflammatory, and antioxidant effects, but also effects that alleviate Alzheimer's symptoms. Furthermore, toxicity tests for GV1001 have been completed in numerous clinical trials targeting various diseases, and the safety of GV1001 in terms of side effects has been confirmed.

[0009] Based on this, the inventors diligently researched the preventive or therapeutic uses of GV1001 for periodontal disease and disorders caused by periodontal disease, such as atherosclerosis and Alzheimer's disease. As a result, the inventors confirmed that GV1001 exhibits preventive and / or therapeutic effects on periodontal disease, as well as atherosclerosis and Alzheimer's disease caused by periodontal disease, and thus completed the present invention. [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] The present invention aims to provide a pharmaceutical composition for the prevention or treatment of periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease.

[0011] The present invention aims to provide a pharmaceutical composition for the prevention or treatment of Porphyromonas gingivalis-induced periodontitis.

[0012] The present invention aims to provide a kit for the prevention or treatment of periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease.

[0013] The present invention aims to provide a health functional food for the prevention or improvement of periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease.

[0014] The present invention aims to provide a health functional food for the prevention or improvement of Porphyromonas gingivalis-induced periodontitis. [Means for solving the problem]

[0015] 1. A pharmaceutical composition for the prevention or treatment of periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease, comprising a peptide having the amino acid sequence of Sequence ID No. 1.

[0016] 2. In item 1 above, the periodontal disease is Porphyromonas gingivalis-induced periodontitis, and the pharmaceutical composition is as described above.

[0017] 3. In item 1 above, the pharmaceutical composition is a pharmaceutical composition that prevents or treats periodontal disease by suppressing osteoclast formation.

[0018] 4. In item 1 above, the pharmaceutical composition is a pharmaceutical composition that prevents or treats periodontal disease by suppressing the expression of gingipain.

[0019] 5. A kit for the prevention or treatment of periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease, comprising a pharmaceutical composition described in any of items 1 to 4 above, and instructions describing a method for preventing or treating periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease.

[0020] 6. In item 5 above, the method of prevention or treatment is a kit comprising the step of administering a pharmaceutical composition to an individual who has developed or is at risk of developing periodontal disease, or atherosclerosis or Alzheimer's disease due to periodontal disease.

[0021] 7. A health functional food containing a peptide having the amino acid sequence of SEQ ID NO: 1, for the prevention or improvement of periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease.

[0022] 8. In item 7 above, periodontal disease is Porphyromonas gingivalis-induced periodontitis, a health functional food.

[0023] 9. In item 7 above, a health functional food is a health functional food that prevents or improves periodontal disease by suppressing osteoclast formation.

[0024] 10. In item 7 above, the health functional food is a health functional food that prevents or improves periodontal disease by suppressing the expression of gingerpin.

Effects of the Invention

[0025] The pharmaceutical composition of the present invention is effective in preventing or treating periodontal disease by suppressing osteoclast formation.

[0026] The pharmaceutical composition of the present invention is effective in preventing or treating atherosclerosis induced by periodontitis by reducing inflammatory cytokines in the blood, lipid deposition in the arterial wall, and the expression of CD47 at the root of the artery.

[0027] The pharmaceutical composition of the present invention is effective in preventing or treating Alzheimer's disease induced by periodontitis by reducing inflammatory cytokines and Alzheimer's disease-related biomarkers in the brain tissue.

[0028] The health functional food of the present invention is expected to be effective in preventing or improving periodontal disease by suppressing osteoclast formation.

[0029] The health functional food of the present invention is expected to be effective in preventing or improving atherosclerosis induced by periodontitis by reducing inflammatory cytokines in the blood, lipid deposition in the arterial wall, and the expression of CD47 at the root of the artery.

[0030] The health functional food of the present invention is expected to be effective in preventing or improving Alzheimer's disease induced by periodontitis by reducing inflammatory cytokines and Alzheimer's disease-related biomarkers in the brain tissue.

[0031] The pharmaceutical composition, kit, and health functional food of the present invention are expected to provide economic or healthcare support to patients suffering from periodontal disease and their families.

[0032] Understanding the pathophysiology of periodontal disease development according to this invention is expected to be a driving force for the future development of new periodontal disease treatments. [Brief explanation of the drawing]

[0033] [Figure 1] Figure 1 shows the results of comparing cytokine mRNA levels to confirm changes in inflammatory cytokines in periodontal tissue between the control group and the GV1001-treated group. [Figure 2a] Figure 2 shows the results of measuring cytokine expression levels by fluorescence staining to confirm changes in inflammatory cytokines in the periodontal tissue of the control group and the GV1001-treated group. [Figure 2b] Same as above [Figure 3a] Figure 3 shows the results of examining the number of Porphyromonas gingivalis colonies and Porphyromonas gingivalis LPS in the gingival tissue of the control group and the GV1001-treated group, as well as the results of examining changes in gingipain (Kgp, RgpB) aggregates. [Figure 3b] Same as above [Figure 4a] Figure 4 shows the results of confirming alveolar bone loss using μCT in the control group and the GV1001-treated group. [Figure 4b] Same as above [Figure 4c] Same as above [Figure 5a] Figure 5 shows the results of confirming the number of osteoclasts in the alveolar bone by TRAP staining in the control group and the GV1001-treated group. [Figure 5b] Same as above [Figure 6a] Figure 6 shows the results of measuring the degree of osteoclast formation at the in vitro level. [Figure 6b] Same as above [Figure 6c] Same as above [Figure 6d] Same as above [Figure 6e] Same as above [Figure 7a]Figure 7 shows the results of measuring serum levels of inflammatory cytokines to confirm the progression level of systemic inflammation due to periodontitis in the control group and the GV1001-treated group. [Figure 7b] Same as above [Figure 8] Figure 8 shows the results of measuring mRNA levels of inflammatory cytokines in arterial tissue to confirm the possibility of progression to atherosclerosis due to periodontitis in the control group and the GV1001-treated group. [Figure 9a] Figure 9 shows the results of measuring the expression levels of inflammatory cytokines in arterial tissue by fluorescence staining to confirm the possibility of progression to atherosclerosis due to periodontitis in the control group and the GV1001-treated group. [Figure 9b] Same as above [Figure 10a] Figure 10 shows the results of examining the number of Porphyromonas gingivalis colonies and changes in Porphyromonas gingivalis LPS and gingipain (Kgp, RgpB) aggregates in the arterial wall of the control group and the GV1001-treated group. [Figure 10b] Same as above [Figure 11a] Figure 11 shows the results of examining serum triglycerides, total cholesterol, high-density lipoprotein, low-density lipoprotein, and very low-density lipoprotein levels in the control group and the GV1001-treated group to confirm changes in cholesterol due to chronic inflammation. [Figure 11b] Same as above [Figure 12a] Figure 12 shows the results of examining lipid deposition levels in the walls of the aorta-iliac bifurcation to verify the induction of atherosclerosis by periodontitis in the control group and the GV1001-treated group. [Figure 12b] Same as above [Figure 13a] Figure 13 shows the results of an experiment to determine whether HUVECs were transformed into EndMT by TNF-α and Porphyromonas gingivalis LPS in the control group and the GV1001-treated group. [Figure 13b] Same as above [Figure 14a] Figure 14 shows the results of an experiment to determine whether ox-LDL is taken up by TNF-α and Porphyromonas gingivalis LPS in the control group and the GV1001-treated group. [Figure 14b] Same as above [Figure 15a] Figure 15 shows the results of measuring CD47 expression levels at the base of the aorta in the control group and the GV1001-treated group. [Figure 15b] Same as above [Figure 16a] Figure 16 shows the results of confirming CD47 expression levels using human coronary artery smooth muscle cells (HCASMCs) at the in vitro level. [Figure 16b] Same as above [Figure 16c] Same as above [Figure 16d] Same as above [Figure 16e] Same as above [Figure 16f] Same as above [Figure 17] Figure 17 shows the results of examining mRNA levels of inflammatory cytokines in brain tissue in the control group and the GV1001-treated group to confirm the possibility of progression to Alzheimer's disease due to periodontitis. [Figure 18a] Figure 18 shows the results of examining cytokine expression levels by fluorescence staining in the control group and the GV1001-treated group to confirm the possibility of progression to Alzheimer's disease due to periodontitis. [Figure 18b] Same as above [Figure 19a] Figure 19 shows the results of examining cytokine expression levels by fluorescence staining in the control group and the GV1001-treated group to confirm the possibility of progression to Alzheimer's disease due to periodontitis. [Figure 19b] Same as above [Figure 20a]Figure 20 shows the results of examining the number of Porphyromonas gingivalis colonies and changes in Porphyromonas gingivalis LPS and gingipain (Kgp, RgpB) aggregates in the cerebral cortex of the control group and the GV1001-treated group. [Figure 20b] Same as above [Figure 21a] Figure 21 shows the results of examining the number of Porphyromonas gingivalis colonies and changes in Porphyromonas gingivalis LPS and gingipain (Kgp, RgpB) aggregates in the hippocampus of the control group and the GV1001-treated group. [Figure 21b] Same as above [Figure 22a] Figure 22 shows the results of confirming the accumulation of Aβ42 and p-Tau, biomarkers for Alzheimer's disease, in the brains of the control group and the GV1001-treated group. [Figure 22b] Same as above [Figure 23] Figure 23 shows the results of confirming the effect of GV1001 on the expression of gingipain (RgpA, RgpB, Kgp). [Modes for carrying out the invention]

[0034] The present invention provides a pharmaceutical composition for the prevention or treatment of periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease, comprising a peptide having the amino acid sequence of Sequence ID No. 1.

[0035] In the present invention, the peptide having the amino acid sequence of SEQ ID NO: 1 includes its functional equivalent. A "functional equivalent" refers to a peptide that, as a result of the addition, substitution, or deletion of amino acids, has at least 70%, 80%, 90%, or 95% sequence homology to the amino acid sequence of SEQ ID NO: 1 and exhibits substantially the same physiological activity as the peptide having the amino acid sequence of SEQ ID NO: 1. "Substantially the same physiological activity" refers to activity involved in the prevention, improvement, or treatment of periodontal disease.

[0036] In this invention, "periodontal disease" includes gingivitis and periodontitis.

[0037] In one embodiment, the periodontal disease may be gingivitis.

[0038] In one embodiment, the periodontal disease may be periodontitis.

[0039] In one embodiment, the periodontal disease may be Porphyromonas gingivalis-induced periodontitis.

[0040] Porphyromonas gingivalis is a representative bacterium that causes periodontal disease, and it uses a protease called gingipain to invade tissues and cause other problems.

[0041] In this invention, "prevention" means all actions that suppress or delay periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease.

[0042] In the present invention, "treatment" means all actions that improve or beneficially modify the symptoms of an individual suspected of having periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease, or who has actually developed such disease.

[0043] In one embodiment, the composition of the present invention can prevent or improve periodontal disease by suppressing osteoclast formation.

[0044] In one embodiment, the composition of the present invention can prevent or improve periodontal disease by suppressing the expression of gingipain, thereby reducing the colony formation of Porphyromonas gingivalis, Porphyromonas gingivalis LPS, and gingipain in gingival tissue.

[0045] In the present invention, "individual" means all animals, such as livestock and mice, that have developed or are likely to develop periodontal disease, or atherosclerosis or Alzheimer's disease due to periodontal disease, and may include mammals, such as humans.

[0046] The pharmaceutical composition of the present invention may be provided as a pharmaceutical composition comprising an active ingredient alone, or as a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, excipients, or diluents.

[0047] Examples of carriers, excipients, or diluents that may be included in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, dextrin, maltodextrin, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil.

[0048] The administration route of the pharmaceutical composition of the present invention may be, but is not limited to, the oral cavity, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual, or rectal.

[0049] The composition of the present invention may be administered orally or parenterally.

[0050] When administering the composition of the present invention parenterally, it is preferable to select an injection method such as topical application to the skin, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection, but is not limited to these.

[0051] The pharmaceutical composition of the present invention may be a solid dosage form for oral administration, such as a tablet, pill, powder, granule, or capsule.

[0052] The pharmaceutical composition of the present invention may be a liquid formulation for oral administration, such as a suspension, an oral solution, an emulsion, or a syrup.

[0053] The pharmaceutical composition of the present invention may be a formulation for parenteral administration, such as a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized formulation, or a suppository.

[0054] The present invention provides a kit for the prevention or treatment of periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease, comprising a pharmaceutical composition for the prevention or treatment of periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease, and instructions describing a method for preventing or treating these diseases.

[0055] In one embodiment, a method for preventing or treating periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease, may include the step of administering the pharmaceutical composition of the present invention to an individual who has developed or is at risk of developing periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease.

[0056] In this invention, "administration" means introducing a predetermined substance into an individual using an appropriate method.

[0057] In one embodiment, a method for preventing or treating periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease, may include the step of administering a pharmaceutically effective amount of the pharmaceutical composition of the present invention to an individual who has developed or is at risk of developing periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease.

[0058] In this invention, "pharmaceutically effective amount" means an amount sufficient to treat periodontal disease with a reasonable benefit / risk ratio applicable to medical treatment.

[0059] The pharmaceutically effective amount of the pharmaceutical composition of the present invention may be determined according to factors including the severity of the disease, the activity of the drug, the patient's sensitivity to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment and concomitant drugs, and other factors well known in the medical field, and may be appropriately selected by those skilled in the art.

[0060] The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents, in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, or as single or multiple doses, which can be easily determined by a person of ordinary skill.

[0061] The present invention provides a health functional food for preventing or improving periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease, comprising a peptide having the amino acid sequence of SEQ ID NO: 1.

[0062] The health functional foods of the present invention refer to foods manufactured and / or processed in various forms to provide functions useful to the human body.

[0063] The health functional foods of the present invention may be included in various foods or pharmaceuticals known in the field.

[0064] The types of foods that may contain the health functional foods of the present invention are not particularly limited. For example, the health functional foods of the present invention may be contained in meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes.

[0065] The health functional foods of the present invention encompass all forms, including functional foods, nutritional supplements, health foods, and food additives, and these types of foods can be manufactured in various forms according to common methods known in the art. For example, health foods can be manufactured and consumed in the form of liquid drinks, or ingested in the form of granules, capsules, spherical tablets (such as balls), and powders, and can also be manufactured and ingested in the form of powders, capsules, soft capsules, tablets, gums, and sticky liquid compositions. Functional foods also include beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruit, bottled fruit, jam, marmalade, etc.), fish, meats and their processed foods (e.g., ham, sausage, corned beef, etc.), breads and noodles (e.g., udon, soba, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, candies, dairy products (e.g., butter, cheese, etc.), edible vegetable oils and fats, margarine, vegetable protein, retort foods, frozen foods, herbal decoctions, and various seasonings (e.g., miso, soy sauce, sauces, etc.).

[0066] The health functional food of the present invention may further contain ingredients that are typically added during the manufacture of food, without departing from the ultimate objective of the present invention, such as proteins, carbohydrates, fats, other nutrients, seasonings, and flavorings.

[0067] The health functional food of the present invention may further contain various nutritional supplements, vitamins, electrolytes, flavorings, colorings, pectin acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like.

[0068] The health functional food of the present invention may also contain fruit pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages. These components can be used independently or in combination.

[0069] The present invention will be described in detail below with reference to examples. However, the following examples are provided to aid in understanding the present invention, and the content of the present invention is not limited to these examples. [Examples]

[0070] 1. Experimental Method 1.1. Synthesis of GV1001 A peptide consisting of 16 amino acids, having the structural formula shown in Chemical Formula 1 below and possessing the following Sequence ID No. 1 (GV1001), was synthesized from human telomerase.

[0071] [ka]

[0072] Peptide GV1001, sequence number 1, was prepared according to a conventional solid-phase peptide synthesis method. Specifically, the peptide was synthesized using ASP48S (Peptron, Inc., Daejeon, Korea) by coupling amino acids one by one from the C-terminus using the Fmoc solid-phase peptide synthesis (SPPS) method. The peptide used had the first amino acid of the C-terminus attached to the resin, as shown below. For example: NH2-Lys(Boc)-2-chloro-Trityl Resin NH2-Ala-2-chloro-Trityl Resin NH2-Arg(Pbf)-2-chloro-Trityl Resin

[0073] All amino acid starting materials used in peptide synthesis were protected with Fmoc at the N-terminus and all residues removed by acid, such as Trt, Boc, t-Bu (t-butylester), and Pbf (2,2,4,6,7-pentamethyl dihydro-benzofuran-5-sulfonyl). For example: Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH , Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ahx-OH, Trt-Mercaptoacetic acid.

[0074] The coupling reagent used was HBTU[2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetamethylaminium hexafluorophosphate] / HOBt[N-Hydroxxybenzotriazole] / NMM[4-Methylmorpholine]. Piperidine in 20% DMF was used to remove Fmoc. The synthesized peptide was separated from the resin, and a cleavage cocktail [TFA(trifluoroacetic acid) / TIS(triisopropylsilane) / EDT(ethanedithiol) / H2O=92.5 / 2.5 / 2.5 / 2.5] was used to remove the protecting groups from the residues.

[0075] Using a solid support to which amino acid protecting groups were attached as starting amino acids, each amino acid was reacted with the support, washed with a solvent, and then deprotected. This process was repeated to synthesize each peptide. After the synthesized peptides were cleaved from the resin, they were purified by HPLC, their synthesis was confirmed by MS, and they were freeze-dried.

[0076] The specific synthesis process for GV1001 is as follows: 1) Coupling NH2-Lys(Boc)-2-chloro-Trityl Resin-protected amino acids (8 equivalents) and the coupling reagents HBTU (8 equivalents) / HOBt (8 equivalents) / NMM (16 equivalents) were dissolved in DMF and added. The mixture was then reacted at room temperature for 2 hours, and washed in the following order: DMF, MeOH, and DMF. 2) Fmoc deprotection Piperidine in 20% DMF was added, and the mixture was reacted twice at room temperature for 5 minutes each time. The mixture was then washed in the following order: DMF, MeOH, and DMF. 3) The basic peptide skeleton was constructed by repeating reactions 1 and 2. 4) Cleavage: The cleavage cocktail was added to the synthesized peptide resin, and the peptide was separated from the resin. 5) Cooling diethyl ether was added to the resulting mixture, and the obtained peptide was precipitated by centrifugation. 6) After purification by Prep-HPLC, the molecular weight was confirmed by LC / MS, and the product was frozen to produce a powder.

[0077] 1.2. Animal Model Preparation and Experimental Protocol An animal model was created using ApoE- / - mice (C57BL / 6 background, male, 4 weeks old).

[0078] Animal models were fed a high-fat diet (HFD), and using the Placement of Ligatures induction method, 6-0 silk ligature was applied for one week to form gingival pockets around the maxillary second molar. These pockets were then treated with Porphyromonas gingivalis (Pg) or PBS for five weeks. The animal models used in the experiment are shown in Table 1 below.

[0079] [Table 1]

[0080] 1.3. Evaluation Items 1) Evaluation of the effectiveness against periodontitis We examined the depth of periodontal pockets, the expression levels of inflammatory cytokines in gingival tissue (mRNA level, IF staining), the aggregation of Porphyromonas gingivalis, Porphyromonas gingivalis LPS, Kgp, and RgpB in gingival tissue, the amount of alveolar bone loss, and the number of osteoclasts in the alveolar bone. We also examined the effects on osteoclast formation in an in vitro study.

[0081] 2) Evaluation of the effect on atherosclerosis We examined the expression levels of inflammatory cytokines in serum and arterial tissue, the aggregation of Porphyromonas gingivalis, Porphyromonas gingivalis LPS, Kgp, and RgpB in arterial tissue, changes in serum cholesterol levels, and lipid deposition (atherosclerosis) in the arterial wall. In vitro studies confirmed inhibition of EndMT and macrophage uptake of dil-ox-LDL. CD47 expression levels were examined in the aortic root in in vivo studies and in vitro studies.

[0082] 3) Evaluation of the effects on AD Expression levels of inflammatory cytokines in brain tissue, aggregation of Porphyromonas gingivalis, Porphyromonas gingivalis LPS, Kgp, RgpB, and Aβ 42 We also confirmed the accumulation of p-Tau.

[0083] 2. Experimental Results 2.1. Effects of GV1001 in periodontitis 1) Inflammation To examine changes in inflammatory cytokines (IL-1β, TNF-α, IL-6) in periodontal tissue, cytokine mRNA levels (Figure 1) and cytokine expression levels (Figure 2) were measured using fluorescence staining. -Control group: In the periodontitis induction model (control group), the amount of cytokines increased. -GV1001 administration group: Administration of GV1001 significantly reduced cytokine mRNA levels, and fluorescence staining confirmed a decrease in cytokine expression.

[0084] 2) Colonies and gingipains of Porphyromonas gingivalis The number of Porphyromonas gingivalis colonies and Porphyromonas gingivalis LPS were examined in gingival tissue (Figure 3), and changes in gingipain (Kgp, RgpB) aggregates were observed. -Control group: In the periodontitis induction model (control group), the number of Porphyromonas gingivalis colonies, Porphyromonas gingivalis LPS, and gingipain increased in the gingival tissue. -GV1001 administration group: The number of Porphyromonas gingivalis colonies, Porphyromonas gingivalis LPS, and gingipain in gingival tissue were significantly reduced.

[0085] 3) Osteoclasts To confirm the effect of GV1001 on alveolar bone loss, alveolar bone loss was confirmed using μCT in an animal model of periodontitis (Figure 4), and the number of osteoclasts in the alveolar bone was confirmed by TRAP staining (Figure 5). Furthermore, osteoclast formation, i.e., the degree to which mononuclear cells differentiate into osteoclasts, was measured at the in vitro level (Figure 6). -Control group: In the periodontitis induction model (control group), increased osteoclast formation in the alveolar bone and alveolar bone loss were observed. -GV1001 administration group: GV1001 administration significantly suppressed osteoclast formation and significantly reduced alveolar bone loss. After inducing macrophage cell line (Raw264.7) into osteoclasts, GV1001 significantly suppressed osteoclast formation.

[0086] 4) Systemic inflammation To assess the progression of systemic inflammation caused by periodontitis, the levels of inflammatory cytokines (IL-1β, TNF-α, IL-6, GM-CSF, VEGF, IL-17, KC) in the serum of a periodontitis-induced model were measured (Figure 7). -Control group: In the serum of the periodontitis induction model (control group), all cytokine levels increased. -GV1001 administration group: GV1001 administration significantly suppressed all cytokine levels in the serum.

[0087] 2.2. Effects of GV1001 in periodontitis-related atherosclerosis 1) Vascular inflammation Since it is well known that vascular inflammation can induce atherosclerosis, we measured changes in the levels of inflammatory cytokines (IL-1β, TNF-α, IL-6) in arterial tissue to confirm the possibility of periodontitis progressing to atherosclerosis. For this purpose, we measured cytokine mRNA levels (Figure 8) and cytokine expression levels by fluorescence staining (Figure 9). -Control group: In the periodontitis induction model (control group), the amount of cytokines in the arteries increased. -GV1001 administration group: Administration of GV1001 significantly reduced cytokine mRNA levels, and fluorescence staining confirmed a decrease in cytokine expression.

[0088] 2) Colonies and gingipains of Porphyromonas gingivalis We examined the number of Porphyromonas gingivalis colonies in the arterial wall, as well as changes in the aggregates of Porphyromonas gingivalis LPS and gingipain (Kgp, RgpB) (Figure 10). -Control group: In the periodontitis induction model (control group), the number of Porphyromonas gingivalis colonies, Porphyromonas gingivalis LPS, and gingipain increased in the arterial wall. -GV1001 administration group: A significant decrease in the number of Porphyromonas gingivalis colonies in the arterial wall, as well as Porphyromonas gingivalis LPS and gingipain, was confirmed.

[0089] 3) Cholesterol To assess changes in cholesterol levels due to chronic inflammation, serum levels of triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL) were examined (Figure 11). -Control group: In the periodontitis induction model (control group), serum levels of TG, TC, HDL, LDL, and VLDL increased. -GV1001 administration group: Administration of GV1001 significantly reduced serum levels of TC and LDL.

[0090] 4) Lipid deposition To investigate the induction of atherosclerosis by periodontitis, the level of lipid deposition in the wall of the aorta-iliac bifurcation was examined using Sudan IV staining and Oil Red O staining (Figure 12). -Control group: In the periodontitis induction model (control group), intravascular lipid deposition increased. -GV1001 administration group: Administration of GV1001 significantly suppressed intravascular lipid deposition.

[0091] 5) Changes in the phenotype of endothelial cells It is known that when endothelial cells (ECs) undergo endothelial-to-mesenchymal transition (EndMT), their phenotype changes to that of mesenchymal cells (MCs), inducing various vascular diseases (such as atherosclerosis, pulmonary hypertension, and fibrosis). TNF-α is known to be involved in this process. In this invention, it was confirmed that when human umbilical vein endothelial cells (HUVECs) were stimulated with TNF-α and Porphyromonas gingivalis LPS, EndMT reduced ECs (marker; CD31) and increased MCs (marker; FSP-1) (Figure 13). -Control group: EndMT of HUVECs was confirmed to progress with TNF-α and Porphyromonas gingivalis LPS. -GV1001 administration group: It was confirmed that GV1001 suppressed the progression of EndMT in HUVECs. As a result, it was confirmed that GV1001 can suppress the development of atherosclerosis due to periodontitis by suppressing phenotypic changes in ECs.

[0092] 6) LDL uptake and foam cell formation The formation of foam cells by macrophages using LDL and TNF-α is known to be an important process in atherosclerosis. In this invention, we evaluated the uptake of ox-LDL by TNF-α and Porphyromonas gingivalis LPS in macrophages differentiated from THP-1 (human monocyte cell line) cells (Figure 14). -Control group: TNF-α and Porphyromonas gingivalis LPS increased ox-LDL uptake. -GV1001 administration group: GV1001 significantly suppressed ox-LDL uptake. This result suggests that the suppression of atheroma formation by GV1001 is partly due to its inhibitory effect on foam cell formation.

[0093] 7)CD47(Cluster of differentiation 47) CD47, a representative antiphagocytic factor, is known to inhibit the removal of cancer cells by macrophages, and overexpression of CD47 is known to cause atherosclerosis. In this invention, the expression level of CD47 at the root of the aorta was confirmed in an animal model of periodontitis (Figure 15). Furthermore, the expression level of CD47 was confirmed in vitro using human coronary artery smooth muscle cells (HCASMC) (Figure 16). -Control group: In the periodontitis induction model (control group), an increase in CD47 was observed at the root of the aorta, and it was confirmed that CD47 increased in HCASMC cells upon treatment with TNF-α and Porphyromonas gingivalis LPS. -GV1001 administration group: In an animal model of periodontitis, administration of GV1001 significantly reduced CD47 expression at the root of the aorta. In addition, in vitro HCASMC cell line studies confirmed that TNF-α and Porphyromonas gingivalis LPS treatment significantly reduced CD47 expression.

[0094] 2.3. Effects of GV1001 on periodontitis-related Alzheimer's disease 1) Inflammation Since Alzheimer's disease patients exhibit neuroinflammation characterized by changes in inflammatory cytokines in the brain, to confirm the possibility that periodontitis may progress to Alzheimer's disease, cytokine mRNA levels were checked to measure changes in the amount of inflammatory cytokines (IL-1β, TNF-α, IL-6) in brain tissue (Figure 17), and cytokine expression levels were measured by fluorescence staining (Figures 18 and 19). -Control group: In the periodontitis induction model (control group), the amount of cytokines in brain tissue increased. -GV1001 administration group: Administration of GV1001 significantly reduced cytokine mRNA levels, and fluorescence staining confirmed a decrease in cytokine expression.

[0095] 2) Colonies and gingipains of Porphyromonas gingivalis In the cerebral cortex of an animal model of periodontitis, changes in the number of Porphyromonas gingivalis colonies and aggregates of Porphyromonas gingivalis LPS and gingipain (Kgp, RgpB) were observed (Figure 20). Similarly, changes in the number of Porphyromonas gingivalis colonies and aggregates of Porphyromonas gingivalis LPS and gingipain (Kgp, RgpB) were observed in the hippocampus (Figure 21). -Control group: In the periodontitis induction model (control group), the number of Porphyromonas gingivalis colonies, Porphyromonas gingivalis LPS, and gingipain increased in the cerebral cortex and hippocampus. -GV1001 administration group: A significant decrease in the number of Porphyromonas gingivalis colonies, Porphyromonas gingivalis LPS, and gingipain was confirmed in the cerebral cortex and hippocampus.

[0096] 3) Aβ 42 and p-Tau In the brains of animal models of periodontitis, Aβ, a biomarker for Alzheimer's disease, was discovered. 42 Accumulation of p-Tau was also confirmed (Figure 22). -Control group: In the periodontitis induction model (control group), Aβ in the cerebral cortex and hippocampus 42 And the accumulation of p-Tau increased. -GV1001 administration group: Aβ in the cerebral cortex and hippocampus 42 Furthermore, a significant decrease in p-Tau accumulation was confirmed.

[0097] 2.4. Elucidation of the mechanism of the inhibitory effect of GV1001 on Porphyromonas gingivalis expression. To understand the underlying mechanisms of Porphyromonas gingivalis colonization, Porphyromonas gingivalis LPS and gingipain reduction induced by GV1001 in gingival tissue, arterial walls, and the brain, we investigated the effect of GV1001 on gingipain expression, an enzyme crucial for Porphyromonas gingivalis proliferation and tissue invasion. The results showed that GV1001 significantly suppressed gingipain expression in in vitro studies. Very low doses (5-30 μM) of GV1001 reduced RgpA and RgpB expression by approximately 5-fold and Kgp expression by approximately 3-fold compared to the control group (Porphyromonas gingivalis 16S rRNA) (Figure 23).

[0098] 3. Conclusion A periodontitis model was established in a periodontitis induction model treated with Porphyromonas gingivalis, due to a significant increase in alveolar bone loss. Administration of GV1001 was verified to prevent and treat periodontitis by reducing inflammatory cytokines and suppressing osteoclast formation.

[0099] Furthermore, in atherosclerosis induced by increased lipid deposition in arterial blood vessels due to periodontitis, GV1001 was confirmed to suppress the progression of atherosclerosis by reducing inflammatory cytokines in the blood, lipid deposition in the arterial wall, and CD47 expression at the base of the artery.

[0100] Furthermore, in Alzheimer's disease induced by periodontitis, administration of GV1001 reduces inflammatory cytokines in brain tissue and lowers the Alzheimer's disease-related biomarker (Aβ). 42 It was confirmed that it reduces p-Tau.

[0101] In vitro studies confirmed that GV1001 suppresses the expression of gingipain, a protease used by Porphyromonas gingivalis for tissue invasion. This demonstrates that GV1001 reduces Porphyromonas gingivalis colonization, Porphyromonas gingivalis LPS, and gingipain in gingival tissue, arterial walls, and the brain.

Claims

1. A pharmaceutical composition for the prevention or treatment of periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease, comprising a peptide having the amino acid sequence of Sequence ID No.

1.

2. The pharmaceutical composition according to claim 1, wherein the periodontal disease is Porphyromonas gingivalis-induced periodontitis.

3. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition prevents or treats periodontal disease by inhibiting osteoclast formation.

4. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition prevents or treats periodontal disease by suppressing the expression of gingipain.

5. A pharmaceutical composition according to any one of claims 1 to 4, A kit for the prevention or treatment of periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease, including instructions describing methods for the prevention or treatment of periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease.

6. The kit according to claim 5, wherein the preventive or therapeutic method comprises the step of administering the pharmaceutical composition to an individual who has developed or is at risk of developing periodontal disease, or atherosclerosis or Alzheimer's disease due to periodontal disease.

7. A health functional food containing a peptide having the amino acid sequence of SEQ ID NO: 1, for the prevention or improvement of periodontal disease, or atherosclerosis or Alzheimer's disease caused by periodontal disease.

8. The health functional food according to claim 7, wherein the periodontal disease is Porphyromonas gingivalis-induced periodontitis.

9. The health functional food according to claim 7, wherein the health functional food prevents or improves periodontal disease by suppressing osteoclast formation.

10. The health functional food according to claim 7, wherein the health functional food prevents or improves periodontal disease by suppressing the expression of gingipain.

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