JPH0952822A - Composition for oral cavity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a composition for the oral cavity, excellent in practicality, having high effectiveness and safety and effective in preventing and treating periodontal diseases. SOLUTION: This composition for the oral cavity is obtained by blending an antibody in blood, an antibody in milk or an antibody in an egg produced after immunizing an animal (a mammal or a domestic fowl) with a synthetic peptide corresponding to a fragment derived from an amino acid sequence, represented by the formula and constituting a pilus of Actinobacillus actinomycetemcomitans or a polymerized lysine bonded thereto as an antigen. The daily dose of the antibody is preferably adjusted to 0.0001-50g/kg. The antibody is blended in an amount of preferably 0.0002-10wt.%, especially preferably 0.002-5wt.% based on the whole composition. The composition can be prepared as dentifrices such as a toothpaste, a tooth powder and a liquid dentifrice, liquid mouth refreshers such as a mouthwash, a troche, a pasta for the oral cavity, a massage cream for the gingiva, a solution for gargling, a chewing gum, a candy, a dairy product, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to Actinobacillus actinomycetemcomitans, which is one of the causative bacteria of periodontal disease.
The present invention relates to a composition for the oral cavity, which can prevent the colonization of omycetemcomitans) in the oral cavity and prevent periodontal disease.

[0002]

2. Description of the Related Art Actinobacillus actinomycetemcomitance is considered to be the most influential causative agent of juvenile periodontitis. It has been detected and isolated from the periodontal pockets with high frequency, and has been attracting more attention as a bacterium involved in the onset and progress of periodontal disease.

In order to prevent periodontal disease, it is effective to suppress the colonization of causative bacteria in the oral cavity. Conventionally, whole bacterial cells or bacterial cell components of Actinobacillus actinomycetemcomitans have been used in mammals or It is known that an antibody obtained by immunizing poultry is incorporated into a composition for the oral cavity to suppress the colonization of the bacterium in the oral cavity and prevent oral diseases (Japanese Patent Laid-Open Publication No. 6-58242).
0-146834 and JP-A-2-53716).

However, when whole bacteria or a fraction containing a plurality of proteins is used as an antigen, there is a problem in the homogeneity or safety of the obtained antibody. Further, when a specific protein of the bacterium is used as an antigen, it takes labor to prepare the antigen, and there is a problem in mass productivity, which is far from practical use.

The present invention has been made in view of the above circumstances.
By combining highly uniform antibodies that can be efficiently obtained by using synthetic peptide antigens that can be mass-produced,
It is an object of the present invention to provide a composition for oral cavity which is excellent in practicality, has high efficacy and safety, and is effective in preventing and treating periodontal disease.

[0006]

Means for Solving the Problems and Modes for Carrying Out the Invention As a result of intensive studies to meet the above-mentioned demands, the present inventor has found that a fragment derived from an amino acid sequence constituting Actinobacillus actinomycetemcomitans pili. When an animal (mammal or poultry) is immunized with a synthetic peptide corresponding to the above or a polymer lysine bound thereto as an antigen, the obtained blood antibody, milk antibody, and egg antibody are Actinobacillus actinomycetemcomi It was found that the oral colonization of the chest of drawers is significantly suppressed, and therefore, the incorporation of these antibodies into the oral composition enables effective prevention and treatment of periodontal disease. In particular, the present inventor deciphered the amino acid sequence from the N-terminal to the 41st position of the pilus protein obtained from the bacterium of Actinobacillus actinomycetemcomitans, and found that the amino acid sequence was as follows: Found.

[0007]

[Sequence Table 1]

In this case, of the above-mentioned sequences, the amino acid sequence from the N-terminal to the 18th to 38th amino acid, that is, the oligopeptide having the following amino acid sequence has strong antigenicity, and a synthetic peptide having such amino acid sequence is used as an antigen. By doing so, they have found that an antibody effective against periodontal disease can be obtained, and completed the present invention. [Amino acid sequence] Val Ala Val Phe Tyr Ser Asn Asn Gly Phe Ile Ala Asn Leu Gln Ser Lys Phe Phe Ser Leu

The present invention will be described in more detail below. The oral composition of the present invention comprises a synthetic peptide derived from the pilus protein of Actinobacillus actinomycetemcomitans, preferably this synthetic peptide is linked to polymer lysine. The obtained product is used as an antigen, and an antibody obtained by immunizing an animal with this is mixed.

Here, the synthetic peptide having the above-mentioned [amino acid sequence] is suitable as the synthetic peptide.
In this case, the synthetic peptide is Pep according to a known method.
side synthesizer (430A, App
lied Biosystems Inc. , Fast
er City, CA).

[0012] Further, a method for binding the synthetic peptide with polymer lysine in order to enhance the antigenicity is described by Narde.
Polymer lysine method (J. Immunol.,
148, 912-920, 1992).

[0012] As a method of immunizing animals with the above antigen,
Known methods such as subcutaneous injection, intramuscular injection, buccal injection, intravenous injection, nasal administration, oral administration and oral cavity mucosal administration can be adopted. In addition, in order to increase the antibody titer, a method of immunizing with an adjuvant such as Freund's complete adjuvant, incomplete adjuvant, cholera toxin B subunit can be appropriately used. Examples of animals to be immunized include mammals such as rabbits, goats, sheep, cows and horses, and poultry such as chickens, ducks, ostriches, ducks and quails.

The oral composition of the present invention comprises the thus obtained blood antibody or milk antibody, or the egg antibody obtained by immunizing poultry. Antiserum containing such antibodies, milk,
Eggs may be blended as they are, or serum, milk, and purified antibody separated from eggs may be blended according to a conventional purification method. Here, as the antibody purification method, salting-out method, gel filtration method, ion exchange chromatography, affinity chromatography and the like can be used.

The dose of the above antibody is 0.0001 to 50 g.
/ Kg / day, in which case the antibody is 0.0002 to 10% (wt%, the same applies hereinafter) of the entire composition,
In particular, it is preferable to set the compounding amount to 0.002 to 5%.

The oral composition of the present invention comprises toothpaste, powdered toothpaste, liquid toothpaste and other dentifrices, liquid refreshing agents such as mouthwash, troches, oral pasta, gingival massage cream, gargle solution, chewing gum, It is used as a candy, a dairy product or the like, and as the other components of the oral composition of the present invention, appropriate components depending on the type of the oral composition and the like are used.

For example, in the case of toothpaste, dibasic calcium phosphate, calcium carbonate, calcium pyrophosphate,
Abrasives such as insoluble sodium metaphosphate, amorphous silica, crystalline silica, aluminosilicate, aluminum oxide, aluminum hydroxide, resin, carboxymethyl cellulose, hydroxyethyl cellulose, alginate, carrageenan, gum arabic, polyvinyl alcohol, etc. Agent, polyethylene glycol, sorbitol,
Glycerin, thickener such as propylene glycol, sodium lauryl sulfate, sodium dodecylbenzene sulfonate, hydrogenated coconut fatty acid monoglyceride, sodium monosulfate, sodium lauryl sulfoacetate, N-
Foaming agents such as sodium lauroyl sarcosinate, N-acyl glutamate, sucrose fatty acid ester, and essential oils such as peppermint and spearmint, 1-menthol,
Fragrances such as fragrance materials such as carvone, eugenol, and anethole, sodium saccharin, stevioside, neohesperidyl dihydrochalcone, sweeteners such as glycyrrhizin, perillartin, p-methoxycinnamic aldehyde, preservatives, etc. It is manufactured according to the law. Further, in mouthwashes such as mouthwash and the like, components suitable for the properties of the product are appropriately mixed.

In the present invention, dextranase, mutanase, sorbic acid, chlorhexidine, hinokitiol, cetylpyridinium chloride, alkylglycine, alkyldiaminoethylglycine salt, allantoin, ε-aminocaproic acid, tranexamic acid, azulene, vitamin E. , A water-soluble primary or secondary phosphate,
An active ingredient such as a quaternary ammonium compound, sodium chloride or a crude drug extract can also be added.

[0018]

EFFECTS OF THE INVENTION The oral composition of the present invention contains an antibody obtained by immunizing an animal with the above-mentioned antigen to effectively prevent the colonization of Actinobacillus actinomycetemcomitans in the oral cavity. However, periodontal disease can be effectively prevented or treated. Moreover, the antibody is highly safe because it is directed against a specific antigen.

[0019]

The present invention will be described below in more detail with reference to Examples, but the present invention is not limited to the following Examples.

Experimental Example (1) Preparation of Antigen Actinobacillus actinomycetemcomitans 31
The 0-a strain was cultured in Todd-Hebit medium under CO ₂ conditions at 37 ° C. for 72 hours. Next, the method of Inoue et al. (FEMS Microbiol. Lett., 6
9, 13-17, 1990) to prepare pili antigens. Growing bacteria attached to the glass wall was scraped off with a policeman, and phosphate buffered saline (PBS, pH
It was suspended in 7.2) and centrifuged at 12,000 × g. The collected bacteria were suspended in 0.15 M ethanolamine-hydrochloric acid buffer (pH 10.5), and homogenized for 30 minutes on ice with a blender. 20 at 10,000 xg
Centrifuge for 30 minutes at 25,000 xg to remove bacterial debris and add 4 mL of saturated ammonium sulfate solution to the supernatant.
Dropwise at 0 C until a final concentration of 10% was reached. The precipitate (coarse pili) was resuspended in ethanolamine-hydrochloric acid buffer,
The components adhering to the cell membrane were removed by centrifugation at 25,000 xg. This operation of salting out the supernatant was repeated under the same conditions.
20 mM of precipitate containing 5% sodium deoxycholate
It was dissolved in Tris-HCl buffer (pH 8.2) and homogenized in a rolling stirrer for 30 minutes at room temperature. 1
The insoluble matter in the precipitate was removed by centrifugation at 30,000 xg for 30 minutes. The same operation was repeated using 20 mM Tris-hydrochloric acid buffer containing 0.7% n-octyl-β-glucopyranoside, and finally the precipitate was suspended in Tris-hydrochloric acid buffer.
This suspension was used as a pili preparation. It was confirmed by SDS-PAGE that the prepared pili were single-stranded.

After removing the Tris-HCl buffer from the pilus preparation by filtration and freeze-drying, the purified pilus was subjected to a protein sequencer (PSQ-10, Shimadzu Corporation).
Edman reaction (J. Biol. C) in which the amino acid sequence from the N-terminal to the 41st position was improved by Hewick et al.
hem. , 256, 7990-7997, 1981). The molecular weight of the pili was about 54,000.

[0022]

[Sequence Table 2]

Next, based on the above amino acid sequence, several kinds of oligopeptides were selected from Peptide Synthesize.
r (430A, Applied Biosystems
Inc. , Foster City, CA). As a result of examining their antigenicity, the following sequences were found to have strong antigenicity. [Amino acid sequence] Val Ala Val Phe Tyr Ser Asn Asn Gly Phe Ile Ala Asn Leu Gln Ser Lys Phe Phe Ser Leu

The molecular weight of the synthetic peptide having this amino acid sequence is Nardelli for the purpose of enhancing the antigenicity.
Polymer lysine method (J. Immunol., 1
48, 912-920, 1992).
The branched lysine polymer resin was treated with a deprotecting reagent to remove the resin. The augmented polypeptide was extracted with 2N sodium acetate, dialyzed against distilled water and lyophilized.

(2) Preparation of antibody The antibody was prepared according to a commonly used immunization method. 50 mg of lysine-binding oligopeptide for the first immunization
Was subcutaneously injected into a New Zealand white rabbit (male) together with Freund's complete adjuvant. Two weeks and three weeks after the initial immunization, the same antigen was subcutaneously injected, and the final immunization was carried out by ear vein injection by suspending the antigen in phosphate buffer, and then preparing an antibody according to a conventional method (hereinafter, this was synthesized. Called peptide antibody).

The obtained antibody was not only pili of Actinobacillus actinomycetemcomitans 310-a strain but also other test strains, that is, AS9 collected from periodontal disease patients.
0017 strain, AS900070 strain, AS9000322
It was confirmed to react with the pili of the strain Km-1.

(3) Adhesion Inhibitory Effect of Actinobacillus actinomycetemcomitans on Human Buccal Mucosal Epithelium by Rabbit Serum Antibody The experiment was conducted according to the method of Gibbons et al. That is,
Human buccal mucosal cells were collected and suspended in phosphate buffer,
After treatment with μm Millipore, target cells were obtained. Finally, the cells were prepared so that the number of cells was 10 ⁵ in 1 ml of phosphate buffer (solution A). On the other hand, in 1 ml of a phosphate buffer containing a diluted synthetic peptide antibody or control serum, Actinobacillus actinomycetemcomitans 310-
It was prepared so that the strain a would be 10 ⁸ (solution B).

The solutions A and B were mixed and reacted at 37 ° C. for 30 minutes. The number of bacteria attached to the buccal mucosal cells was stained with 2% crystal violet and then counted with a microscope. The results are shown in FIG.

From these results, even when the antibody was diluted 100-fold, the adhesion of Actinobacillus actinomycetemcomitans to the buccal mucosal cells was about 8 as compared with the control serum.
It was confirmed to suppress by 5%.

(4) Adhesion-inhibiting effect of Actinobacillus actinomycetemcomitance on rabbit saliva-treated hydroxyapatite beads by rabbit serum antibody Saliva-treated hydroxyapatite beads (hereinafter referred to as S-H)
A) is prepared by the method of Clark et al.
t. Immun. 19, 846-853, 1978). That is, the mixed saliva after centrifugation (12,000 × g, 15 minutes) was treated at 60 ° C. for 1 hour to inactivate oxygen. Then, centrifugation (3,000 xg, 5 minutes) was performed to obtain a supernatant (experimental saliva). 10 ml of saliva was added to 5 mg of hydroxyapatite beads and incubated at room temperature for 24 hours to obtain S-HA. Actinobacillus actinomycetemcomitance was labeled with a radioactive substance, tritium thymidine, and was finally adjusted to 2 × 10 ⁸ / ml with a KCl buffer solution (5 mg / ml) containing bovine serum albumin.

The adhesion inhibition experiment was carried out by incubating S-HA with radiolabeled actinobacillus actinomycetemcomitans and a synthetic peptide antibody at 37 ° C. for 1 hour, and then culturing bacteria attached to S-HA with a scintillation counter. It was done by measuring. The results are shown in FIG.

From the results shown in FIG. 2, it was confirmed that the synthetic peptide antibody specifically and in a concentration-dependent manner inhibits the adhesion of Actinobacillus actinomycetemcomitans to S-HA as compared with the control serum. .

Next, examples will be shown. In the following examples, for the preparation of antibodies and antisera, synthetic peptides having the [amino acid sequence] of (1) above were used, and various animals were immunized and prepared in the same manner as in (2) above according to a conventional method. What was done was used. In the following examples,% means% by weight.

[Example 1] Toothpaste dicalcium phosphate dihydrate 50.0% Glycerin 20.0 Carboxymethyl cellulose 1.0 Sodium lauryl sulfate 1.5 Sodium lauroyl sarcosinate 0.5 Perfume 1. 0 saccharin 0.1 dextranase 0.01 equine anti-synthetic peptide serum 0.1 water total 100.0%

Example 2 Toothpaste Dicalcium Phosphate Dihydrate 47.0% Sorbit 10.0 Glycerin 10.0 Carboxymethyl Cellulose 1.0 Sodium Lauryl Sulfate 2.0 Perfume 1.0 Saccharin 0.1 Ethanol 2.0 Chlorhexidine gluconate 0.01 Mutanase 0.1 Chicken egg anti-synthetic peptide antibody 0.1 Water balance 100.0%

[Example 3] Toothpaste Calcium carbonate 46.0% Glycerin 20.0 Carrageenan 0.5 Carboxymethyl cellulose 1.0 Lauryl diethanolamide 1.0 Sucrose monolaurate 2.0 Perfume 1.0 Tranexamic acid 0 .05 saccharin 0.1 bovine anti-synthetic peptide breast milk 0.05 water balance 100.0%

[Example 4] Toothpaste Aluminum hydroxide 50.0% Glycerin 20.0 Carboxymethyl cellulose 2.0 Sodium lauryl sulfate 2.0 Fragrance 1.0 Saccharin 0.1 Goat anti-synthetic peptide serum 0.1 Water Balance 100.0%

Example 5 Toothpaste Dicalcium Phosphate Dihydrate 50.0% Calcium Carbonate 30.0 Glycerin 10.0 α-Olefin Sulfonate 1.0 Perfume 1.0 Saccharin 0.1 Dextran 0 .5 Rabbit anti-synthetic peptide serum 0.05 Water balance 100.0%

[Example 6] Liquid toothpaste Sodium polyacrylate 50.0% Glycerin 30.0 Fragrance 0.9 Saccharin 0.1 Ethanol 3.0 Cetylpyridinium chloride 0.05 Linoleic acid 0.05 Goat anti-synthetic peptide breast milk 0.01 Water balance 100.0%

Example 7 Mouthwash Ethanol 20.0% Fragrance 1.0 Saccharin 0.05 Lauryldiethanolamide 0.3 Goat anti-synthetic peptide serum 0.1 Water Residual 100.0%

Example 8 Mouthwash Tablet Sodium Bicarbonate 54.0% Dibasic Sodium Phosphate 10.0 Polyethylene Glycol 3.0 Citric Acid 17.0 Sodium Sulfate (Anhydrous) 13.6 Perfume 2.0 Chlorhexidine Glucon Acid salt 0.05 Oleic acid 0.1 Rabbit anti-synthetic peptide serum 0.1 Water balance 100.0%

Example 9 Gingival Massage Cream White Vaseline 8.0% Propylene Glycol 4.0 Stearyl Alcohol 8.0 Polyethylene Glycol 4000 25.0 Polyethylene Glycol 400 37.0 Tocopherol Acetate 0.1 Sucrose Stearate 0 .5 Bovine anti-synthetic peptide serum 0.5 water Residual total 100.0%

[Example 10] Chewing gum Gum base 43.85% Calcium carbonate 2.0 Water candy 15.0 Sugar 30.0 Sucrose palmitate 1.0 Fructose 4.0 Maltose 1.0 Flavor 1.0 Chicken egg anti-synthetic peptide Antibody 0.1 Total 100.0%

Example 11 Lozenge gum arabic 6.0% Glucose 72.0 Gelatin 3.0 Fragrance 0.2 l-menthol 0.1 Spearmint oil 0.1 Sodium ascorbate 0.1 Goat anti-synthetic peptide-casein Breast milk 0.05 water Residual total 100.0%

Example 12 Oral Pasta Polyoxyethylene Monostearate 2.0% Sorbitan Monooleate 2.0 Cetyl Alcohol 2.0 Palmityl Alcohol 3.0 Propylene Glycol 15.0 Carboxymethyl Cellulose 5.0 Gelatin 1.0 Saccharin 0.2 Pehermint oil 0.5 Spearmint oil 0.5 Horse anti-synthetic peptide serum 0.05 Lysozyme chloride 5000 units / g Remaining water 100.0% in total

Example 13 Oral Pasta Glyceryl Monolaurate 3.0% Oleyl Alcohol 5.0 Polyethylene Glycol 15.0 White Vaseline 3.0 N-Palmitoyl Glutamate Monosodium 0.5 Hydroxyethyl Cellulose 5.0 Tocopherol Acetate 0.1 Saccharin sodium 0.2 Japanese peppermint oil 0.7 Carvone 0.5 Anethole 0.3 Eugenol 0.1 Chicken egg anti-synthetic peptide-ovalbumin antibody 0.05 Water balance 100.0%

Example 14 Ice Cream Cream (Fat ratio 50%) 16.84% Milk (Fat ratio 3.7%) * 42.65 Sugar-free skimmed condensed milk 24.24 Sugar 11.25 Corn syrup 4.65 Stabilizer 0.35 Total 100.0% * Contains 0.5% bovine anti-synthetic peptide breast milk

Example 15 Yogurt Skim milk 320 kg Sweetened condensed milk ** 56 Sugar 23 The above components are fermented by a conventional method. ** Contains 10% bovine anti-synthetic peptide breast milk

Example 16 Bavarois Dough Milk *** 375 cc Sugar 60 g Vanilla Saya 1 Egg Yolk 5 Gelatin 20 g Fresh Cream 75 cc *** Contains 5% bovine anti-synthetic peptide breast milk

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the dilution ratio of a synthetic peptide antibody and the number of adherent bacteria.

FIG. 2 is a graph showing the relationship between the synthetic peptide antibody dilution rate and the bacterial adhesion inhibition rate.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication location A61K 39/395 A61K 39/395 DR C07K 14/195 ZNA 8517-4H C07K 14/195 ZNA

Claims (3)

[Claims] 1. A method comprising an antibody obtained by immunizing an animal with a synthetic peptide corresponding to a fragment derived from the amino acid sequence constituting the pili of Actinobacillus actinomycetemcomitans as an antigen. An oral composition comprising: 2. The oral composition according to claim 1, wherein the synthetic peptide bound to polymer lysine is used as an antigen. 3. The composition for oral cavity according to claim 1, wherein the synthetic peptide has the following amino acid sequence. [Amino acid sequence] Val Ala Val Phe Tyr Ser Asn Asn Gly Phe Ile Ala Asn Leu Gln Ser Lys Phe Phe Ser Leu