KR102034881B1 - Polypeptide having Antimicrobial Activity and Use thereof - Google Patents

Abstract

The present invention relates to polypeptides having antimicrobial activities and uses thereof. The antimicrobial peptides of the present invention exhibit excellent antimicrobial activities against bacteria causing oral diseases, and are nontoxic to cells constituting the dental tissue, and thus can be useful for the prevention or treatment of oral diseases, in particular, the root canal treatment.

Description

Polypeptide having antimicrobial activity and its use {Polypeptide having Antimicrobial Activity and Use}

The present invention relates to polypeptides having antimicrobial activity and uses thereof.

The pulp and root disease are infectious diseases caused by bacteria that occur most frequently in the oral cavity along with dental caries and periodontal disease, and can be classified into acute and chronic pulpitis according to the duration of its development, and depending on the progress of the lesion, Periodontal periodontitis can be divided into. Because of the anatomical characteristics of teeth surrounded by periodontal tissue including alveolar bone, all bacteria present in saliva and dental plaque can be invaded by pulp by dental caries. In order to study the etiology of the pulp and root disease, it is necessary to study related bacteria.

Currently, researches on bacterial species related to pulp and root disease have been actively conducted. The results of these studies many are associated with mainly dimensions, and sometimes is bacterial paper distinguish appears mainly associated with periapical lesions, however, periodontal disease, such as Porphyromonas endodontalis Porphyromonas gingivalis, Prevotella nigrescens, Bacteroides forsythus, Prevotella intermedia, Fusobacterium nucleatum, Actinobacillus actinomycetemcomitans , Campylobacter rectus, etc. are also detected in the pulp and root root lesions. This is thought to be observed by the traffic of the lesion because the pulp and periodontal tissue are connected to each other.

 The ultimate goal of root canal treatment is to prevent the development of anterior periodontal periodontitis and to create a suitable environment for the healing of surrounding tissues. Because root canal periodontitis is an infectious disease, the basis for root canal treatment is to remove the infection that has occurred, and to prevent infection or re-infection of the root canal or surrounding tissue by the bacteria.

In order to treat pulp and root canal disease, a strong alkaline root canal agent having an antibacterial action is used along with the root canal treatment, but the effect is not obtained. Clinically, a diluent solution containing sodium hypochlorite and chlorohexidine is used, and a patch paste containing calcium hydroxide is used.

On the other hand, antimicrobial peptide (antimicrobial peptide) is a natural antimicrobial material that is responsible for the natural immunity (innate immunity) mechanism in vivo, and has an antimicrobial activity against a variety of microorganisms, including bacteria (fungi), viruses (viruses) It is known as a small molecule peptide having the characteristics of inducing local infection inhibition and systemic immune response. Antimicrobial peptides generally have an amphipathic structure and exhibit antimicrobial activity based on the principle that the cation site of the antimicrobial peptide binds to the anionic phospholipids of the microbial cell membrane and destroys the cell membrane of the microorganism.

In the meantime, peptide antibiotics have been tested in various organisms to find antibiotics that can replace conventional antibiotics. In practice, however, there are very few cases in which it is commercially produced, because of the problems of stability and human safety when peptide antibiotics are injected in vivo.

Accordingly, the present inventors completed the present invention by conducting a study for discovering antimicrobial peptides that can improve oral diseases without cytotoxicity.

Republic of Korea Patent Publication No. 10-2011-0137236

One object of the present invention is to provide an antimicrobial peptide consisting of the amino acid sequence of SEQ ID NO: 1.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of oral diseases comprising an antimicrobial peptide consisting of the amino acid sequence of SEQ ID NO: 1.

Still another object of the present invention is to provide a food composition for preventing or improving oral disease, comprising an antimicrobial peptide consisting of the amino acid sequence of SEQ ID NO: 1.

One aspect of the invention provides an antimicrobial peptide consisting of the amino acid sequence of SEQ ID NO: 1.

The antimicrobial peptide consisting of the amino acid sequence of SEQ ID NO: 1 of the present invention has an amino acid sequence of KPIFRLRSTLLAPRA (Lys-Pro-Ile-Phe-Arg-Leu-Arg-Ser-Thr-Leu-Leu-Ala-Pro-Arg-Ala) .

The antimicrobial peptides of the present invention are not cytotoxic and exhibit excellent antimicrobial activity against bacteria that cause oral disease without affecting proliferation of pulp stem cells.

According to one embodiment of the present invention, the peptide may have antimicrobial activity against Porphyromonas gingivalis .

The antimicrobial peptide of the present invention is effective in inhibiting quorum sensing of prophylomonas gingivalis with excellent antibacterial activity against bacteria causing oral diseases such as pulp and root disease, in particular, propiromonas gingivalis. can do. Because the inhibition of quorum sensing effectively inhibits the formation of biofilm by propiromonas gingivalis, the antimicrobial peptide of the present invention prevents, treats or ameliorates oral diseases caused by propiromonas gingivalis. This can be useful for.

As used herein, the term 'quorum sensing' is a phenomenon in which microorganisms belonging to a species monitor the density of microorganisms that are homologous to each other, and when a concentration of a signal substance secreted by each microorganism reaches a certain limit, it is determined from a microbial population. It refers to a phenomenon in which a gene (for example, a gene encoding a disease agent) is expressed.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating oral disease, comprising an antimicrobial peptide consisting of the amino acid sequence of SEQ ID NO: 1.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in the manufacture of a medicament, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin , Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. It is not limited. The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (22th ed., 2013).

The pharmaceutical composition of the present invention may be administered with a substance that exhibits one or more prophylactic or therapeutic activities for oral disease.

The pharmaceutical compositions of the present invention may be used alone or in combination with methods using procedures, hormone therapy, drug therapy and / or biological response modifiers for the prevention or treatment of oral disease.

The pharmaceutical composition of the present invention may include various bases and / or additives necessary and appropriate for the formulation of the formulation, and nonionic surfactants, silicone polymers, extender pigments, fragrances, and preservatives without departing from the effect thereof. , Fungicides, oxidation stabilizers, organic solvents, ionic or nonionic thickeners, softeners, antioxidants, free radical destroyers, opacifiers, stabilizers, emollients, silicones, α-hydroxy acids, defoamers, humectants It may be prepared further comprising known compounds such as vitamins, insect repellents, flavoring agents, preservatives, surfactants, anti-inflammatory agents, substance P antagonists, fillers, polymers, propellants, basicizing or acidifying agents, or coloring agents.

Suitable dosages of the pharmaceutical compositions of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and response to response of the patient. Can be. The dosage of the pharmaceutical composition of the present invention may be 0.001 to 1000 mg / kg on an adult basis, it may be administered orally.

The antimicrobial peptide included in the pharmaceutical composition of the present invention is present in the root canal and has excellent antibacterial activity against bacteria that cause oral diseases, particularly Porphyromonas gingivalis , and thus, the root canal is cleaned during neurotherapy. It can be particularly useful as a cleaning agent for.

According to one embodiment of the present invention, the oral disease may be caused by infection of Porphyromonas gingivalis .

According to one embodiment of the invention, the oral disease may be periodontitis, or pulp and root disease.

According to an embodiment of the present invention, the pulp and root disease may be pulp or periodontal periodontitis.

The chemical preparation (eg, NaOCl, etc.) used as a root canal cleaning solution in the root canal treatment of teeth causes a fatal chemical burn on the tissues around the teeth when used in high concentrations and excessive amounts, and thus is restricted in use. On the other hand, the antimicrobial peptide of the present invention exhibits excellent antimicrobial activity against porphyromonas gingivalis, which is present in the root canal without being toxic to cells. Thus, the root canal is washed and periodontitis or pulp and root disease, specifically, pulpitis Or it can be usefully used to prevent or treat the periodontal periodontitis.

Another aspect of the present invention provides a food composition for preventing or improving oral disease, comprising an antimicrobial peptide consisting of the amino acid sequence of SEQ ID NO: 1.

In the food composition of the present invention, natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.), disaccharides (eg, maltose, sucrose, etc.), and polysaccharides (eg, dextrins). Conventional sugars such as cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Such flavoring agents may include natural flavoring agents (tauumatin), stevia extracts (eg, rebaudioside A, glycyrrhizin, etc.) and / or synthetic flavoring agents (saccharin, aspartame, etc.).

The food composition of the present invention is a vitamin mixture consisting of vitamin A acetate, vitamin E, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, biotin, nicotinic acid amide, folic acid, calcium pantothenate, and ferrous sulfate, zinc oxide , Magnesium carbonate, potassium phosphate dibasic, potassium phosphate dibasic, potassium citrate, calcium carbonate and magnesium chloride, and the like, and one or more minerals conventionally added in the art.

 These ingredients may be used independently or in combination, and the ratio of such additives may be selected from 0 to about 20 parts by weight per 100 parts by weight of the food composition of the present invention, but is not limited thereto.

Since the food composition of the present invention is excellent in antibacterial activity, it can be usefully used for the prevention or improvement of oral diseases.

According to the polypeptide having an antimicrobial activity, since it shows excellent antimicrobial activity against bacteria causing oral disease, it can be usefully used for the prevention or treatment of oral disease, in particular, the root canal treatment.

1 is a graph showing the results of pulp cell survival analysis of the antimicrobial peptide consisting of the amino acid sequence of SEQ ID NO: 1.
Figure 2 is a graph showing the results of MTT proliferation analysis for crown pulp of the antimicrobial peptide consisting of the amino acid sequence of SEQ ID NO: 1.
Figure 3 is a graph showing the results of MTT proliferation analysis for root pulp of the antimicrobial peptide consisting of the amino acid sequence of SEQ ID NO: 1.
Figure 4 is a graph showing the results of the MTT proliferation analysis for root root papilla stem cells (SCAP) of the antimicrobial peptide consisting of the amino acid sequence of SEQ ID NO: 1.
Figure 5 is a graph showing the results of the sensitivity test of the antimicrobial peptide consisting of the amino acid sequence of SEQ ID NO: 1.

Hereinafter, the present invention will be described in more detail with reference to one or more embodiments. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1 Confirmation of Cytotoxicity of New Peptides by MTT Assay

KPIFRLRSTLLAPRA (Lys-Pro-Ile-Phe-Arg-Leu-Arg-Ser-Thr-Leu-Leu-Ala-Pro-Arg-Ala) (SEQ ID NO: 1) A new peptide consisting of the amino acid sequence prevents or treats oral diseases In order to confirm whether it can be used for the purpose, it was confirmed whether the new peptide cytotoxicity.

Specifically, 100 μl of 1 × 10 ⁵ cells / ml MDA-MB-231 breast cancer cell line was dispensed in 24-well plates, and cultured daily in RPMI 1640 medium at 37 ° C. and 5% CO ₂ , followed by new peptide or As a control, 0.9% sodium chloride was treated in each well at a concentration of 1, 2, 5, 7, 10, 15, 20, 25, 30, 40 or 50 μM and then incubated for 24 hours. Then, the medium was removed, and MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium bromide) (Sigma) solution was added to each well so that the final concentration was 2 mg / ml. Next, the reaction was incubated for 4 hours, the MTT solution was removed, and then 100 μl of DMSO was added thereto and stirred. Once thoroughly stirred, UV absorbance was measured at 570 nm using a microplate reader (Molecular Device, Sunnyvale, Cam USA). Cell viability (%) is expressed as a percentage of the absorbance values of the normal control group not treated with the new peptide and 0.9% sodium chloride.

As a result, it was confirmed that the novel peptide does not affect cell viability in the range of 1 to 50 μM concentration, thus showing no cytotoxicity (FIG. 1).

Example 2. Confirmation of proliferation inhibition of pulp stem cells of novel peptides by MTT proliferation assay

In order to determine whether the new peptide can be used for the prevention or treatment of oral diseases, it was confirmed whether the new peptide is toxic to the dental pulp stem cell (DPSC).

Specifically, 1 × 10 ⁵ cells / ml of crown pulp, root pulp, or root cells from the apical papilla (SCAP), respectively, in a 24-well plate. Dispense 100 μl, incubate in low glucose DMEM medium at 37 ° C. and 5% CO ₂ per day, and then treat the new peptides to each well at a concentration of 1, 10 or 20 μM, and then 1, 3, 5 Or incubated for 7 days. Then, MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium bromide) (Sigma) solution was added to each well so that the final concentration was 2 mg / ml. After the reaction, the MTT solution was removed, and 100 µl of DMSO was added thereto and stirred. Once thoroughly stirred, UV absorbance was measured at 570 nm using a microplate reader (Molecular Device, Sunnyvale, Cam USA). Cell growth rate (%) is expressed as a percentage of the original (day 0) absorbance value.

As a result, it was confirmed that the novel peptide does not affect the proliferation of stem cells existing in different parts of each tooth in the range of 1 to 20 μM concentration (FIGS. 2 to 4).

Example 3. Confirmation of antimicrobial activity of novel peptides by susceptibility test

In order to confirm whether the novel peptide can be used for the prevention or treatment of oral diseases, the antimicrobial activity of the novel peptide against Porphyromonas gingivalis strain was confirmed.

Specifically, the new peptide was dissolved in PBS (phosphate buffered saline) (pH 7.2) and incubated at 4 ° C. for 12 hours to form a tertiary structure. Antimicrobial susceptibility testing of the novel peptides against Porphyromonas gingivalis ATCC 33277 strain was performed according to the guidelines of the Clinical and Laboratory Standards Institute. Porphyromonas gingivalis was incubated in an exponential phase and harvested by centrifugation at 4 ° C. for 10 minutes at 4,000 × g. Porphyromonas gingivalis was then washed three times and resuspended in PBS. Porphyromonas gingivalis, counted with a Petroff-Hasser counting chamber (Hausser Scientific, Horsham, PA, USA), was prepared in 96-well polystyrene plates containing 10 μl of PBS, a series of two-fold dilutions of new peptides against PBS (SPL). Lifescience, Gyunggi). Plates containing porphyromonas gingivalis were incubated for 1 hour at 37 ° C. under anaerobic conditions, and oxygen-containing BHI broth containing vitamin K and hemin was added to each well. Porphyromonas gingivalis was then incubated for 3 days in an anaerobic chamber at 37 ° C. and measured spectrophotometrically at 660 nm to measure the growth of propiromonas gingivalis.

As a result, it was confirmed that the novel peptides exhibited antimicrobial activity against Pyromonas gingivalis from 6 to 12 μM concentration (FIG. 5).

Through the above results, the novel peptides can inhibit quorum sensing of prophylomonas gingivalis to inhibit the formation of biofilm, thus preventing, treating or ameliorating oral diseases, in particular, root canal treatment. It was confirmed that it can be usefully used for.

So far, the present invention has been described with reference to the embodiments. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown not in the above description but in the claims, and all differences within the scope will be construed as being included in the present invention.

<110> SOGANG UNIVERSITY RESEARCH FOUNDATION          SEOUL NATIONAL UNIVERSITY DENTAL HOSPITAL <120> Polypeptide having Antimicrobial Activity and Use <130> PN180291 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Antimicrobial Polypeptide <400> 1 Lys Pro Ile Phe Arg Leu Arg Ser Thr Leu Leu Ala Pro Arg Ala   1 5 10 15

Claims (7)

Antimicrobial peptide consisting of the amino acid sequence of SEQ ID NO: 1.
The antimicrobial peptide of claim 1, wherein the peptide has antimicrobial activity against Porphyromonas gingivalis .
In the pharmaceutical composition for preventing or treating oral diseases comprising the antimicrobial peptide of claim 1,
The oral disease is,
Oral diseases caused by infection with Porphyromonas gingivalis ; or
Periodontitis, or pulp and root disease
Pharmaceutical composition for preventing or treating oral disease.
delete delete According to claim 3, wherein the pulp and root disease is dental pulp or periodontal periodontitis is a pharmaceutical composition for preventing or treating oral disease.
In the food composition for preventing or improving oral disease comprising the antimicrobial peptide of claim 1,
The oral disease is,
Oral diseases caused by infection with Porphyromonas gingivalis ; or
Periodontitis, or pulp and root disease
Food composition for preventing or improving oral disease.

Images