JPH0640869A - Composition for oral cavity application - Google Patents

Abstract

PURPOSE:To obtain the subject composition effective in inhibiting the colonization of Actinobacillus actinomycetem comitans in the oral cavity, having excellent safety and activity and useful for the prevention and treatment of periodontosis, etc., by using a specific polysaccharide as an antigen, immunizing an animal with the antigen and using the obtained antibody as a component. CONSTITUTION:The composition contains preferably 0.002-5wt.% of an antibody obtained by immunizing an animal such as rabbit with an antigen selected from one or more kinds of polysaccharides of formula I to formula III (Ac is acetyl; (n) is >=1), preferably an antigen produced by bonding the polysaccharide to a polypeptide (e.g. bovine serum albumin). The antibody is preferably administered at a daily dose of 0.0001-50g/kg.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 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 oral cavity, which can prevent the colonization of the oral cavity of erythem citcomans) 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 cells or 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 prevent colonization of the bacterium in the oral cavity and prevent oral diseases (Japanese Patent Laid-Open No. 146834/1985). , JP-A-2-53716).

However, in general, 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 homogeneous antibodies that can be efficiently obtained using antigens that can be mass-produced and easily purified, it is highly practical, highly effective and safe, and effective in preventing and treating periodontal disease. The present invention aims to provide a composition for oral cavity.

[0006]

Means and Actions for Solving the Problems As a result of intensive studies to meet the above-mentioned demands, the present inventor has found that the bacterial cell surface polysaccharide of Actinobacillus actinomycetemcomitans (compound (a) represented by the following formula) , (B), (c)), which are used alone or in combination with a polypeptide as an antigen to immunize an animal (mammal or poultry), the obtained blood antibody, milk antibody, egg It was found that the antibodies remarkably suppress the oral colonization of Actinobacillus actinomycetemcomitans, and therefore, it is possible to effectively prevent and treat periodontal disease by incorporating these antibodies into the oral composition. The present invention has been completed.

[0007]

[Chemical 2]

Therefore, the present invention is based on the above general formula (a),
There is provided a composition for oral cavity obtained by immunizing an animal with one or more polysaccharides selected from (b) and (c) or a combination of the polysaccharide and a polypeptide as an antigen.

The present invention will be described in more detail below. The antigens used to obtain the antibody to be incorporated into the oral composition of the present invention are the polysaccharides represented by the following (a) to (c) or the polysaccharides and polysaccharides. It is a bond with a peptide.

[0010]

[Chemical 3]

In the above formula, Ac is an acetyl group, and n is an integer of 1 or more, preferably 10 to 1000.

Compounds (a), (b) represented by the above formula,
(C) As the antigen, a polysaccharide chemically synthesized from a monosaccharide or a polysaccharide synthesized by a sugar chain engineering method can be used, but is usually extracted from Actinobacillus actinomycetemcomitans cells. -Serotype-specific polysaccharide antigen obtained by purification is preferably used.

The above compounds (a), (b) and (c) can be prepared from the bacteria of serotypes a, b and c of Actinobacillus actinomycetemcomitans, respectively. Type a strains include Actinobacillus actinomycetemcomitans ATCC295
23, SUNYaB75, etc.
4, ATCC29522 and other strains of serotype c are N
CTC9710, SUNYaB67, etc. are mentioned.

On the other hand, the polypeptide to be bound to the above-mentioned polysaccharide is not particularly limited, but it is a polypeptide that is inexpensive and significantly increases the antibody titer against the polysaccharide when bound to the polysaccharide to form an immunogen. preferable. Polypeptides used for this purpose include BSA
(Bovine serum albumin), various albumins such as ovalbumin, γ-glogrin, casein, biotin, cholera toxin B subunit, ferritin, transferrin, cytochrome C, myosin and the like.

As the method for binding the polysaccharide to the polypeptide, a general method for binding the polysaccharide to the polypeptide is used, and it may be a method capable of binding the polysaccharide to the polypeptide and not changing the antigenicity of the polysaccharide. Any combination of methods can be used. Specifically, the binding can be achieved by either the adsorption method or the covalent binding method, but the covalent binding method is more preferable from the viewpoint of the strength of the binding force. There are many known methods for covalently linking a polysaccharide and a polypeptide, and any method can be used. For example, after activating a polysaccharide with cyanogen bromide, a method for binding a polypeptide, a method for introducing an aromatic amino group into a polysaccharide, and then a method for diazo coupling with an aromatic group of a polypeptide, and after introducing an amino group for a polysaccharide , A method of reacting with an amino group of a polypeptide to form a Schiff base, and a method of reacting a polysaccharide with tresyl chloride and then binding it with an amino group or a thiol group of the polypeptide.

As a method for immunizing animals with the above-mentioned antigen,
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. As the antigen to be immunized, one of the above-mentioned antigens may be used alone, or two or more of them may be used in combination.

Thus, the oral composition of the present invention uses the bacterial cell surface polysaccharide of Actinobacillus actinomycetemcomitans (compounds (a), (b), (c)) as an antigen,
Alternatively, an antigen obtained by binding these polysaccharides and a polypeptide is used, and a blood antibody or milk antibody obtained by immunizing a mammal with this, or an egg antibody obtained by immunizing poultry is contained. However, the antiserum, milk, or egg containing such an antibody may be blended as it is, or the purified antibody separated from serum, milk or egg may be blended in accordance with a usual purification method. May be. Here, as the antibody purification method, salting-out method, gel filtration method, ion exchange chromatography, affinity chromatography and the like can be used.

In the oral composition of the present invention, one kind of the above-mentioned antibodies may be blended alone, or two or more kinds thereof may be used in combination. The dose of the antibody is 0.0001 to 50.
The amount is preferably g / kg / day, and in this case, the amount of the antibody is preferably 0.0002 to 10% (% by weight, the same applies hereinafter) of the entire composition, and more preferably 0.002 to 5%.

The oral composition of the present invention is a toothpaste such as toothpaste, powder toothpaste, liquid toothpaste, liquid refreshing agent such as mouthwash, troche, pasta for oral cavity, massage cream for gums, mouthwash 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, essential oils such as peppermint and spearmint, 1-menthol,
Fragrances such as fragrance materials such as carvone, eugenol and anethole, saccharin sodium, stevioside, neohesperidyl dihydrochalcone, sweeteners such as glycyrrhizin, perillartin, p-methoxycinnamic aldehyde, preservatives and the like are mixed with water, 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.

[0022]

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.

[0023]

[Experimental example]

(1) Preparation of Polysaccharide Antigen Actinobacillus actinomycetemcomitans strain (ATCC 29523, Y4 and NCTC 9710 strain)
Todd hebit broth containing 1% yeast extract was inoculated and incubated at 37 ° C. for 3 hours in an incubator containing 5% CO _2.
Cultured for a day. After collecting the cells, the cells were washed 3 times with physiological saline, suspended in physiological saline, and autoclaved at 121 ° C. for 15 minutes. After cooling, centrifugation was performed at 10,000 × g for 20 minutes to separate the supernatant, and physiological saline was added again to the precipitate, and the above extraction operation was repeated. The obtained supernatant was collected, dialyzed thoroughly against distilled water, and freeze-dried. This standard is 0.
100 mg / in 01M Tris-HCl buffer (pH 8.2)
It was dissolved so that the amount became ml and dialyzed against the same buffer solution at 4 ° C for 2 days. DEAE prepared by equilibrating the same solution with the above buffer
Ion exchange chromatography was performed using a Sephadex A-25 column. Fractional sugar peak fractions eluted with the same buffer were collected, concentrated with a rotary evaporator, and then dialyzed sufficiently against distilled water. This is Sep
Gel filtration was performed using a hacryl S-300 column. Single sugar peak fractions were collected and lyophilized. This was used as a polysaccharide antigen.

(2) Binding of Polysaccharide Antigen to Polypeptide The polysaccharide antigen prepared above was activated with cyanogen bromide and then reacted with adipic acid dihydrazide to give an amino group. Further, it was bound to bovine serum albumin (BSA) by the carbodiimide method. This was designated as polysaccharide-BSA antigen.

(3) Preparation of antibody Rabbits were immunized with the prepared polysaccharide antigen or polysaccharide-BSA antigen to obtain antibodies. The method of immunization was carried out according to a commonly used method. That is, for the primary immunization, 1 mg of the polysaccharide antigen was subcutaneously injected together with Freund's complete adjuvant. Then, every 7 days, 2 mg of the polysaccharide antigen was administered 4 times in total into the ear vein. The obtained antiserum was salted out twice with 50% ammonium sulfate,
It was dialyzed against physiological saline to obtain an antibody preparation.

(4) Effect of Actinobacillus actinomycetemcomitans on colonization of hamsters in the oral cavity of rabbit serum antibodies. Actinobacillus actinomycetemcomitans cultured for 3 days had a turbidity of OD _{550 nm} in 1 mM phosphate buffer. =
The suspension was adjusted to 1.0, mixed with various antibodies at a volume ratio of 1: 1 and reacted at 37 ° C. for 30 minutes. As a control group, a mixture with a phosphate buffer solution was used instead of the antibody solution.

Hamsters of 8 animals in each group were inoculated with 0.1 ml each of the above treated bacterial solution for 5 consecutive days. One week later, the gingival margin was swabbed with a sterilized cotton ball, and the number of Actinobacillus actinomycetemcomitans and the total number of bacteria were measured. The ratio of the number of bacteria of Actinobacillus actinomycetemcomitans to the total number of bacteria was obtained and used as the colonization rate of Actinobacillus actinomycetemcomitans. The experiment was carried out three times by changing the strain to be inoculated (Experiments A, B, C). The results are shown in Tables 1 to 3.

[0028]

[Table 1]

[0029]

[Table 2]

[0030]

[Table 3]

As is clear from the results shown in Tables 1 to 3, it was confirmed that the various antibodies significantly inhibited the oral colonization of Actinobacillus actinomycetemcomitans as compared with the control group. Moreover, when the BSA-bound polysaccharide was used as an antigen, it showed a much higher colonization-inhibiting effect than the case of using the polysaccharide alone.

Examples will be shown below. In addition, all% show weight%. [Example 1] Toothpaste dibasic calcium phosphate dihydrate 50.0% Glycerin 20.0 Carboxymethyl cellulose 1.0 Sodium lauryl sulphate 1.5 Sodium lauroyl sarcosinate 0.5 Perfume 1.0 Saccharin 0 .1 dextranase 0.01 horse anti-polysaccharide (b) serum 0.1 water balance 100.0%

Example 2 Toothpaste Dicalcium Phosphate Dihydrate 47.0% Solbit 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-polysaccharide (b) -BSA antibody 0.1 Water balance 100.0%

[Example 3] Toothpaste Calcium carbonate 48.0% 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-polysaccharide (a, b, c) 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-polysaccharide (c) -BSA serum 0.1 Water balance 100.0%

[Example 5] Toothpaste Silicic anhydride 30.0% Glycerin 30.0 Sorbit 20.0 Carboxymethyl cellulose 1.0 Sodium lauryl sulfate 2.0 Fragrance 1.0 Saccharin 0.1 Sodium fluoride 0. 1 Ethanol 2.0 Egg anti-polysaccharide (a) antibody 0.05 Egg anti-polysaccharide (b) antibody 0.05 Egg anti-polysaccharide (c) antibody 0.05 Water balance 100.0%

Example 6 Toothpaste Dicalcium phosphate dihydrate 50.0% Calcium carbonate 30.0 Glycerin 10.0 α-Olefin sulfonate 1.0 Fragrance 1.0 Saccharin 0.1 Dextran 0 .5 Rabbit anti-polysaccharide (a) -BSA serum 0.05 Water balance 100.0%

[Example 7] 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-polysaccharide (b ) -BSA Breast Milk 0.01 Water Residual 100.0%

[Example 8] Mouthwash Ethanol 20.0% Perfume 1.0 Saccharin 0.05 Lauryl diethanolamide 0.3 Goat anti-polysaccharide (c) serum 0.1 Water balance 100.0%

Example 9 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-polysaccharide (a, c) serum 0.1 Water balance 100.0%

Example 10 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-polysaccharide (b) -BSA serum 0.5 water residual 100.0%

[Example 11] 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-polysaccharide ( a, b, c) antibody 0.1 total 100.0%

Example 12 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-polysaccharide (a) -Casein breast milk 0.05 water Residual total 100.0%

Example 13 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-polysaccharide (b) -BSA serum 0.05 Lysozyme chloride 5000 units / g Leftovers 100.0% in total

Example 14 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 Sodium saccharin 0.2 Japanese peppermint oil 0.7 Carvone 0.5 Anethole 0.3 Eugenol 0.1 Chicken egg anti-polysaccharide (c) -ovalbumin antibody 0.05 Water balance 100.0%

[Example 15] Ice 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-polysaccharide (a, b, c) breast milk

Example 16 Yogurt Skim milk 320 kg Sweetened condensed milk ** 56 Sugar 23 The above components are fermented by a conventional method. ** Contains 10% bovine anti-polysaccharide (a, b, c) breast milk

Example 17 Bavarois Dough Milk *** 375 cc Sugar 60 g Vanilla Saya 1 egg yolk 5 gelatin 20 g fresh cream 75 cc *** Bovine anti-polysaccharide (a, b, c) containing 5% breast milk

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[Procedure amendment]

[Submission date] July 27, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0014

[Correction method] Change

[Correction content]

On the other hand, the polypeptide to be bound to the above-mentioned polysaccharide is not particularly limited, but it is a polypeptide that is inexpensive and significantly increases the antibody titer against the polysaccharide when bound to the polysaccharide to form an immunogen. preferable. Polypeptides used for this purpose include BSA
(Bovine serum albumin), various albumins such as ovalbumin, γ-glogrin, casein, avidin , cholera toxin B subunit, ferritin, transferrin, cytochrome C, myosin , gelatin, collagen and the like.

Claims (2)

[Claims] 1. An antibody obtained by immunizing an animal with one or more polysaccharides selected from the following general formulas (a), (b) and (c) as an antigen: A composition for oral cavity. [Chemical 1] 2. The general formulas (a), (b) according to claim 1,
An oral composition comprising an antibody obtained by immunizing an animal with an antigen in which a polysaccharide selected from (c) is bound to a polypeptide.