Novel peptide and antibacterial composition comprising same

Abstract

The present invention relates to a novel peptide comprising the amino acid sequence of SEQ ID NO: 1, and an antibacterial composition comprising same. The peptide of the present invention, which is an Enterococcus lactis-derived antibacterial peptide APP101, has specific antibacterial activity against periodontitis-causing bacteria including Porphyromonas gingivalis, Fusobacterium nucleatum, Enterococcus faecalis, Streptococcus parasanguinis, and Streptococcus salivarius, and has stability against proteases, pH, and salt concentrations. The antimicrobial composition of the present invention can be usefully employed for the prevention or treatment of periodontitis.

Description

Novel peptide and antimicrobial composition containing the same

[0001] The present invention relates to APP101, a peptide antimicrobial substance derived from Enterococcus lactis that inhibits Porphyromonas gingivalis and Fusobacterium nucleatum, which cause various diseases including periodontitis, and its uses.

[0002]

[0003] Propyromonas gingivalis is known to primarily cause oral diseases such as gingivitis and periodontitis. This bacterium promotes inflammatory responses, destroying the tissue between the teeth and gums and ultimately leading to tooth loss. Furthermore, Propyromonas gingivalis is known to be associated with various diseases, including cardiovascular disease and diabetes. It is known to increase the risk of various chronic diseases by producing gingipain, a toxin that not only induces inflammation in the human body but also affects the entire body system. Recently, the link between Propyromonas gingivalis and Alzheimer's disease is also being investigated. Research suggests that the toxins from this bacterium can penetrate the brain, which may accelerate the progression of Alzheimer's disease by inducing neuroinflammation and causing brain cell damage. Additionally, Propyromonas gingivalis is known to induce the formation of amyloid beta protein, which is one of the major pathological characteristics of Alzheimer's disease.

[0004] Fusobacterium nucleatum is also primarily found in the oral cavity and, like Propyromonas gingivalis, is known to cause oral diseases such as periodontitis. In other words, this bacterium also induces inflammation and damages tooth and gum tissues. Similar to Propyromonas gingivalis, this bacterium is known to be linked to various diseases such as colorectal cancer, cardiovascular disease, and diabetes.

[0005] Therefore, technology to control Propyromonas kingivalis and Fusobacterium nucleatum is essential not only to prevent or treat various oral diseases but also to prevent various diseases associated with these bacteria.

[0006] The objective of the present invention is to provide APP101, a peptide antimicrobial substance derived from Enterococcus lactis that inhibits Porphyromonas gingivalis and Fusobacterium nucleatum, and the use thereof.

[0007]

[0008] The present invention relates to a peptide comprising the amino acid sequence of SEQ ID NO. 1.

[0009] The present invention relates to an antibacterial composition comprising the above-mentioned peptide.

[0010] The present invention relates to a pharmaceutical composition for the prevention or treatment of periodontitis comprising the above-mentioned peptide.

[0011] The present invention relates to a health functional food for improving oral health comprising the above-mentioned peptide.

[0012] The present invention relates to a quasi-drug for oral hygiene comprising the above-mentioned peptide.

[0013] The peptide of the present invention has specific antibacterial activity against periodontitis-causing bacteria.

[0014] The antimicrobial composition of the present invention protects beneficial bacteria in the oral cavity, minimizes side effects, and enables efficient management of periodontitis.

[0015] The pharmaceutical composition of the present invention can effectively prevent or treat periodontitis.

[0016] Figure 1 is a photograph showing the results of confirming the antibacterial activity of the antimicrobial peptide APP101 separated by electrophoresis.

[0017] Figure 2 shows the analysis results of the antimicrobial peptide APP101, where (a) is an HPLC chromatogram, (b) is a MALDI-ToF spectrum, and (c) is the bacterial endotoxin analysis result.

[0018] Figure 3 shows the results of confirming the antimicrobial activity of the antimicrobial peptide APP101 against Enterococcus faecalis, with (a) showing the results for the KCOM 1083 strain and (b) showing the results for the KCOM 2823 strain.

[0019] Figure 4 is a photograph showing the results of confirming the antimicrobial activity of the antimicrobial peptide APP101 against (a) Streptococcus salivarius and (b) Streptococcus parasanguinis.

[0020] Figure 5 is a schematic diagram illustrating the split-mouth design and clinical measurement items (BOP, PPD, CAL) applied to the clinical trial of the present invention.

[0021] Figure 6 is a graph comparing the reduction rate of bleeding (BOP) upon probing between the control group (minoclin) and the test group (APP101) in a clinical trial targeting patients with periodontitis.

[0022] Figure 7 is a graph comparing the number of sites where the periodontal pocket depth (PPD) exceeded 6 mm and improved to 4 mm or less after treatment with the control group (minoclin) and the test group (APP101) in a clinical trial on patients with periodontitis.

[0023] Figure 8 is a graph showing the improvement effect (number of site reductions) of periodontal pockets according to the control group (minoclin) and the test group (APP101) treatment for each individual patient participating in the clinical trial.

[0024] Figure 9 shows the oral clinical photographs and changes in periodontal indicators (BOP, PPD) before treatment and 3 months after APP101 treatment of clinical trial case 1 (female, 54 years old).

[0025] Figure 10 shows the oral clinical photographs and changes in periodontal indicators (BOP, PPD) before treatment and 3 months after APP101 treatment of clinical trial case 2 (42-year-old male).

[0026] The present invention will be described in detail below.

[0027]

[0028] The present invention relates to a novel peptide.

[0029] The peptide of the present invention comprises the amino acid sequence of SEQ ID NO. 1.

[0030] The amino acid sequence of SEQ ID NO. 1 is N-Lys-Tyr-Tyr-Gly-Asn-Gly-Val-Ser-Cys-Asn-Lys-Lys-Gly-Cys-Ser-Val-Asp-Trp-Gly-Lys-Ala-Ile-Gly-Ile-Ile-Gly-Asn-Asn-Ala-Ala-Ala-Asn-Leu-Ala-Thr-Gly-Gly-Lys-Ala-Gly-Trp-Lys-Gly-C.

[0031] The above peptide may have a molecular weight of 4.3 KDa.

[0032] The above peptide may be derived from Enterococcus lactis.

[0033] The above peptide can be prepared by known methods. For example, it can be obtained by culturing Enterococcus lactis and isolating and purifying the peptide from the culture medium. Alternatively, it can be prepared by a chemical synthesis method or an expression method using recombinant DNA technology.

[0034] The chemical synthesis of peptides can be carried out by known methods, such as Solid Phase Peptide Synthesis (SPPS) and liquid phase synthesis.

[0035] After synthesis, it can be purified using High Performance Liquid Chromatography (HPLC), etc. For example, it can be purified to a purity of 98% or higher.

[0036] The molecular weight of the synthesized peptide can be confirmed using Matrix-Assisted Laser Desorption / Ionization Time-of-Flight (MALDI-ToF) or similar methods.

[0037] The endotoxin content of the synthesized peptide may be, for example, 10 EU / mL or less, 7 EU / mL or less, or 5 to 6 EU / mL.

[0038] The peptide of the present invention has antibacterial activity against periodontitis-causing bacteria.

[0039] Bacteria that cause periodontitis include, for example, Porphyromonas gingivalis, Fusobacterium nucleatum, Treponema denticola, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Campylobacter rectus, Eikenella corrodens, Peptoanaerobacter stomatis, Streptococcus mutans, Actinomyces viscosus, and Bacteroides It could be *Bacteroides forsythus*, etc.

[0040] The peptide of the present invention may have antibacterial activity against Enterococcus faecalis, Streptococcus parasanguinis, and Streptococcus salivarius.

[0041] Enterococcus faecalis is recently found at a higher frequency in the oral cavity of patients with periodontitis than in healthy individuals and is known to be a factor that can accelerate the progression and worsening of periodontitis. In particular, this bacterium possesses multidrug resistance, which can make treatment difficult.

[0042] The peptide of the present invention may have stability against proteolytic enzymes, pH, and salt concentration. For example, it may maintain antibacterial activity against proteolytic enzymes such as trypsin, papain, and pepsin. In addition, it may maintain antibacterial activity even under conditions of pH 2.5 or a NaCl concentration of 140 mM.

[0043]

[0044] In addition, the present invention relates to an antibacterial composition.

[0045] The antibacterial composition of the present invention comprises the above peptide.

[0046] The above peptide may be included in the composition alone or in combination with other antimicrobial agents.

[0047] The above peptide may be included together with a delivery system. The delivery system may be intended to enhance the in vivo stability of the peptide and sustain the antimicrobial effect. For example, it may be manufactured in the form of nanoparticles, liposomes, microcapsules, etc.

[0048] Nanoparticles can be manufactured using polymers such as, for example, chitosan, alginate, and PLGA (poly(lactic-co-glycolic acid)). The particle size can be, for example, 50 to 500 nm.

[0049] Liposomes can be manufactured using, for example, phospholipids, cholesterol, etc. The particle size can be, for example, 100 to 1000 nm.

[0050] Microcapsules can be manufactured using, for example, alginate, chitosan, gelatin, etc. The particle size can be, for example, 1 to 100 μm.

[0051] The antibacterial composition of the present invention may be for antibacterial use against bacteria that cause periodontitis.

[0052] The antimicrobial composition of the present invention can be provided in various formulations. For example, it can be manufactured as a liquid formulation such as a mouthwash, spray, or detergent, and as a gel formulation used as a dental topical agent or a topical application agent. Additionally, it can be manufactured in the form of toothpaste for use in daily oral hygiene management, or manufactured in the form of an oral tablet or troche to be applied by slowly dissolving in the mouth to inhibit bacteria that cause periodontitis. Furthermore, it can be manufactured in the form of an antimicrobial film or patch to be attached to periodontal pockets or specific areas, and can also be utilized as an antimicrobial solution for oral irrigators.

[0053] The antimicrobial composition of the present invention possesses specific antimicrobial activity against bacteria that cause periodontitis. Accordingly, it protects beneficial bacteria in the oral cavity, minimizes side effects, and enables efficient management of periodontitis.

[0054]

[0055] In addition, the present invention relates to a pharmaceutical composition.

[0056] The pharmaceutical composition of the present invention may be used for the prevention or treatment of periodontitis. Since the peptide has specific antibacterial activity against bacteria that cause periodontitis, periodontitis can be effectively managed while preserving beneficial bacteria in the oral cavity.

[0057] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is not particularly limited as long as it is commonly used in the art. For example, excipients, diluents, disintegrants, binders, lubricants, absorption promoters, colorants, flavorings, preservatives, stabilizers, buffers, etc. may be used.

[0058] The pharmaceutical composition of the present invention can be prepared in various formulations, such as liquids, gels, sprays, tablets, troches, films, patches, and dental topicals.

[0059] Liquid preparations may take the form of, for example, mouthwash or oral rinse.

[0060] Gels may be in the form of, for example, dental coatings, topical applications, etc.

[0061] The tablet may be in a form that slowly dissolves in the oral cavity to exhibit an antibacterial effect, for example.

[0062] Troches may be in a form that dissolves slowly in the oral cavity and exhibits a sustained antibacterial effect, for example.

[0063] Film formulations may be in the form of adhering to the oral mucosa, for example, and releasing antibacterial components.

[0064] Patch formulations may be in the form of a sustained-release antibacterial agent that attaches to a periodontal pocket or gum, for example.

[0065]

[0066] In addition, the present invention relates to a health functional food.

[0067] The health functional food of the present invention may be for improving oral health. The health functional food may be manufactured using raw materials notified by the Ministry of Food and Drug Safety and may be manufactured in various formulations such as tablets, capsules, powders, granules, liquids, and gels.

[0068]

[0069] In addition, the present invention relates to quasi-drugs.

[0070] The quasi-drug of the present invention can be used as an oral hygiene product. For example, it can be manufactured in the form of toothpaste, mouthwash, mouthwash, etc. The quasi-drug can help maintain oral health by inhibiting the growth of periodontitis-causing bacteria in the oral cavity.

[0071] Toothpaste can be manufactured by including the above peptide together with, for example, abrasives, surfactants, moisturizers, fragrances, etc.

[0072] A mouthwash can be manufactured by including the above peptide together with, for example, ethanol, glycerin, fragrances, coloring agents, etc.

[0073] A mouthwash can be prepared by including the above peptide together with, for example, purified water, glycerin, ethanol, fragrance, etc.

[0074]

[0075] The pharmaceutical composition, health functional food, and quasi-drug of the present invention may include an appropriate delivery system to increase the delivery efficiency of the peptide. For example, it may be manufactured in the form of nanoparticles, liposomes, microcapsules, etc., to improve the stability and bioavailability of the peptide.

[0076]

[0077] Example 1. Isolation of APP101, an antimicrobial peptide derived from Enterococcus lactis, having antimicrobial activity against Propyromonas gingivalis

[0078] To isolate peptides derived from Enterococcus lactis possessing antimicrobial activity, the strains were cultured in the minimal medium shown in Table 1 for 24 hours, followed by centrifugation and removal of the cells using a 0.2 µm filter. After concentrating the supernatant, Pur-A-Lyzer TMAfter dialysis using a Mega 3500 Dialysis kit, electrophoresis was performed using a 4–20% Tis-Glycine gel. The electrophoresed gel was placed on a BHI agar medium supplemented with 5% FBS inoculated with Propyromonas kingivalis KCTC 5352 and incubated in an anaerobic chamber for 24 hours to confirm the presence of a clear band indicating inhibited bacterial growth (Fig. 1).

[0079] Minimal medium for isolating antimicrobial substances

[0080]

[0081] Example 2. Amino Acid Sequence Analysis and Synthesis of Antimicrobial Peptide APP101

[0082] The band in which inhibitory activity was confirmed in Example 1 was cut and submitted for LC-MS / MS analysis. As a result, the amino acid sequence of the antimicrobial peptide APP101 was as follows, and the exact molecular weight was calculated to be 4.3 KDa.

[0083] N-Lys-Tyr-Tyr-Gly-Asn-Gly-Val-Ser-Cys-Asn-Lys-Lys-Gly-Cys-Ser-Val-Asp-Trp-Gly-Lys-Ala-I le-Gly-Ile-Ile-Gly-Asn-Asn-Ala-Ala-Ala-Asn-Leu-Ala-Thr-Gly-Gly-Lys-Ala-Gly-Trp-Lys-Gly-C

[0084] APP101, whose amino acid sequence was confirmed, was synthesized using a chemical synthesis method. A purification rate of over 98% was confirmed via high-performance liquid chromatography (HPLC) (Fig. 2a), and mass analysis via matrix-assisted laser desorption / ionization mass spectrometry (MALDI-ToF) confirmed that it has a molecular weight of 4.3 kDa, identical to that of an antimicrobial peptide derived from Enterococcus lactis (Fig. 2b). Additionally, a bacterial endotoxin test confirmed a value of 5.394 EU / mg (Fig. 2c).

[0085] [Example 3] Effect of Antimicrobial Peptide APP101 on Protease, pH, and Salt Concentration

[0086] The isolated antimicrobial peptide APP101 was incubated at 37°C for 2 hours with proteolytic enzymes derived from Bacillus licheniformis, Chymotrypsin, Trypsin, Papain, and Pepsin at a final concentration of 1 mg / mL, and its antimicrobial activity was measured. As a result, it lost antimicrobial activity against the proteolytic enzyme derived from Bacillus licheniformis and Chymotrypsin, but maintained antimicrobial activity against other proteolytic enzymes. In addition, when the antimicrobial peptide APP101 was incubated at 37°C for 2 hours under conditions of pH 2.5, it maintained antimicrobial activity, and it also maintained antimicrobial activity at a concentration of 140 mM NaCl (Table 2).

[0087] Effects of APP101 on proteolytic enzymes, pH, and salt concentration

[0088]

[0089] (+: Maintains antibacterial activity, -: Loses antibacterial activity)

[0090] Example 4. Evaluation of the antimicrobial activity of antimicrobial peptide APP101 against various strains

[0091] To evaluate antimicrobial activity, first, Propyromonas kingivalis KCTC 5352 was cultured for 18 hours at 37°C under anaerobic conditions in TSB medium supplemented with Hemin and Menadione. Fusobacterium nucleatum KCTC 2640 was cultured for 48 hours at 37°C under anaerobic conditions in BHI medium containing 1% Yeast extract, 0.05% L-cysteine, 1% Hemin, and 0.02% Menadione.

[0092] To evaluate antimicrobial activity, a culture of pathogens was applied to a cotton swab sterilized in an anaerobic chamber and inoculated onto the entire surface of a BHI agar medium supplemented with 5% FBS. After drying the surface of the medium for 30 minutes, a disk with a diameter of 6 mm was placed on top, and 20 liters of the antimicrobial peptide APP101 at a concentration of 1 mg / mL were dropped onto it. The medium was then incubated under anaerobic conditions at 37°C for 24 hours. Experiments on other strains, such as Escherichia coli, were conducted under aerobic conditions. As a result, APP101 was found to inhibit Propyromonas gingivalis and Fusobacterium nucleatum (Table 3).

[0093] Evaluation of the antimicrobial activity of antimicrobial peptide APP101 against various strains

[0094]

[0095] (+: Antibacterial activity present, -: Antibacterial activity absent)

[0096] Example 5. Evaluation of antimicrobial activity against Enterococcus faecalis

[0097] Recent studies are increasingly reporting that Enterococcus faecalis is found at a higher frequency in the oral cavities of patients with periodontitis than in healthy individuals. This suggests that E. faecalis may be a significant factor in accelerating the progression and worsening of periodontitis. In particular, this bacterium exhibits multidrug resistance, showing resistance to various types of antibiotics, which can make treatment more difficult.

[0098] Antimicrobial activity against Enterococcus faecalis KCOM 1083 (Fig. 3a) and KCOM 2823 (Fig. 3b) was confirmed using BHI medium in the same manner as in Example 4. As a result, APP101 exhibited antimicrobial activity against both strains by forming a distinct zone of inhibition. In contrast, no zone of inhibition was observed in the PBS control group.

[0099] Example 6. Evaluation of antimicrobial activity against Streptococcus parasanguinis and Streptococcus salivarius

[0100] Antimicrobial activity against Streptococcus salivarius (Fig. 4a) and Streptococcus parasanguinis (Fig. 4b) was confirmed using BHI medium in the same manner as in Example 4. As a result, APP101 exhibited antimicrobial activity against both strains by forming a zone of inhibition. In contrast, no zone of inhibition was observed in the PBS control group.

[0101] Example 7. Evaluation of clinical efficacy in patients with periodontitis

[0102] 7-1. Test Method

[0103] A human clinical trial was conducted to verify the efficacy of the peptide APP101 of the present invention in treating periodontitis in an actual clinical setting. The oral cavity of the subjects was divided into a total of six segments, including the left and right anterior and posterior regions of the upper and lower jaws. A split-mouth design was applied in which one side of the subjects' oral cavity (one anterior segment + two posterior segments) was assigned to the control group, Minocline, and the opposite side was assigned to the test group, APP101 (Fig. 5). All subjects received standard periodontal treatment followed by additional treatment with either Minocline or APP101, and major clinical indicators were measured 12 weeks (3 months) after treatment. Probing Pocket Depth (PPD), Bleeding on Probing (BOP), and Clinical Attachment Level (CAL) were measured and compared as evaluation indicators.

[0104] 7-2. Test Results (Quantitative Evaluation)

[0105] As a result of clinical trials, APP101 demonstrated a superior effect in improving periodontitis compared to the existing treatment, minoclin. First, when measuring the reduction rate of bleeding on probing (BOP), a key indicator of inflammation, the APP101-treated group showed a statistically significantly higher reduction rate of bleeding compared to the minoclin-treated group (***p<0.001). This suggests that APP101 has an excellent effect in suppressing gum inflammation and promoting hemostasis (Fig. 6).

[0106] Second, the effect of improving periodontal pocket depth (PPD), which indicates the degree of destruction of periodontal tissue, was evaluated. When measuring the number of severe periodontitis sites with a pocket depth exceeding 6 mm that recovered to a normal or mild level of 4 mm or less after treatment, the APP101 treatment group showed approximately twice as many improved pocket sites compared to the minoclin treatment group (Fig. 7).

[0107] Third, as a result of analyzing the responses of individual patients (Fig. 8), the improvement effect on periodontal pockets was greater in the areas treated with APP101 (blue bars) than in the areas treated with minoclin (orange bars) in the majority of patients. In particular, for some patients (e.g., patients 8 and 11), the number of areas improved by APP101 treatment was more than three times higher compared to minoclin, confirming that it showed consistently superior efficacy despite individual differences.

[0108] 7-3. Case Report

[0109] The visual and numerical improvement effects of APP101 were confirmed through actual patient oral photographs and measurements.

[0110] (1) Case 1 (54-year-old female, Periodontitis Class III / Grade B) Before treatment with APP101, the patient had severe redness and swelling of the gums and deep periodontal pockets. However, in the clinical photograph (Fig. 9) taken after 3 months of treatment with APP101, it was observed that the swelling of the gums had significantly subsided and the healthy, bright red gum tone had been restored. Numerically, the incidence of bleeding (BOP) decreased sharply from 32.1% before treatment to 13.1% after treatment, and showed significant improvement with the number of deep pockets exceeding 6 mm decreasing from 7 to 3 and the number of mild pockets exceeding 4 mm decreasing from 20 to 13.

[0111] (2) Case 2 (42-year-old male, Periodontitis Class III / Grade B) The excellent therapeutic effect of APP101 was also confirmed in another patient with severe periodontitis (Fig. 10). The severe inflammatory findings before treatment were visibly improved 3 months after APP101 treatment. BOP decreased from 28.5% to 13.1%, less than half, and in particular, the number of periodontal pockets exceeding 4 mm decreased significantly from 26 to 12, confirming that extensive periodontal tissue recovery had occurred.

[0112] When synthesizing the above results, it has been proven that the peptide APP101 of the present invention possesses excellent therapeutic efficacy in suppressing bleeding in patients with periodontitis and restoring destroyed periodontal tissue in actual clinical practice, going beyond a simple antibacterial effect.

Claims

1. A peptide containing the amino acid sequence of Sequence No.

1.

2. An antimicrobial composition comprising the peptide of Claim 1.

3. An antimicrobial composition according to claim 2, wherein the antimicrobial agent is for one or more bacteria selected from the group consisting of Porphyromonas gingivalis, Fusobacterium nucleatum, Enterococcus faecalis, Streptococcus parasanguinis, and Streptococcus salivarius.

4. A pharmaceutical composition for the prevention or treatment of periodontitis comprising the peptide of Claim 1.

5. The pharmaceutical composition of claim 4, wherein the composition is a formulation selected from the group consisting of liquids, gels, sprays, tablets, troches, films, patches, and dental topicals.

6. A health functional food for improving oral health comprising the peptide of Claim 1.

7. An oral hygiene quasi-drug comprising the peptide of Claim 1.

8. In claim 7, the quasi-drug is a toothpaste, mouthwash, or gargle.

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