JPH0768110B2 - Oral composition - Google Patents

Description

TECHNICAL FIELD The present invention relates to an oral composition for preventing and treating periodontal disease,
Treponema denticola, which is a kind of bacteria especially involved in the onset and progression of periodontal disease
The present invention relates to an oral composition capable of suppressing oral colonization and preventing periodontal disease.

Conventional technology and problems Spirochetes that live in the oral cavity of human adults have been difficult to separate and culture because they are obligate anaerobic bacteria and their cell structures are very easily destroyed. Recent advances have led to the isolation of various oral spirochetes species. Treponema denticola is the most frequently found bacterial species of spirochete in subgingival plaque, and has recently attracted attention as a bacterium involved in the onset and progression of periodontal disease. In particular, examples of pathogenicity of the bacterium in relation to the onset and progression of periodontal disease include invasion ability of periodontal tissue, production of tissue-destructive enzymes such as trypsin-like enzyme activity, and lymph of host Examples include damage to system cells.

To prevent or treat periodontal disease caused by these spirochetes, which is characterized by a marked numerical increase in inflamed gingival pockets, it is thought to prevent colonization of spirochetes in the oral cavity or suppress proliferation. To be

In general, as a method of preventing colonization of bacteria or suppressing proliferation, a so-called immunization method that uses a bactericidal agent and a vaccine that is expected to produce an antibody with high specificity against the bacteria can be considered. Immunization methods are roughly classified into active immunity in which whole bacterial cells are used as antigens, and active immunity in which blood cells obtained by immunizing mammals and the like are administered, and passive immunity.

In order to utilize passive immunity, those containing an antibody having high agglutinating activity against a target bacterium are preferable, and it is considered preferable from the viewpoint of rapid-acting. In that case, from the viewpoint of safety, it is better to use only a specific antigen of the cells involved in aggregation as an immunogen and to use serum containing only antibodies against this antigen, rather than immunizing animals with whole cells. it is conceivable that. From the above point of view, no method has been established for Treponema denticola against the colonization or growth of bacteria.

In view of such circumstances, the present inventors have conducted earnest research on a highly safe method capable of effectively suppressing oral colonization of Treponema denticola, and more reliably preventing / treating periodontal disease. , Proteins of Treponema denticola, especially proteins present on the surface of the film (molecular weight 50,000 = 50K, 5
3,000 = 53K, 56,000 = 56K, etc.) as an antigen, and antibodies in blood or milk obtained by immunizing mammals with this or antibodies in eggs obtained by immunizing birds, and further antibodies against protein antigens A monoclonal antibody against the protein antigen produced by a hybridoma obtained by cell fusion of a producing cell and a myeloma cell, for example, the monoclonal antibody of Japanese Patent Application No. 63-312348 significantly suppresses the oral colonization of Treponema denticola, and thus The present inventors have completed the present invention by finding that by incorporating these antibodies into a composition for oral cavity, effective prevention and treatment of periodontal disease can be achieved.

Means for Solving the Problems The oral composition of the present invention has a coating of Treponema denticola, in particular, a protein present in the coating surface layer as an antigen, and a blood antibody or milk obtained by immunizing a mammal with this. The antibody comprises an antibody, an egg antibody obtained by immunizing a bird, and a monoclonal antibody against the coat protein antigen.

Here, the Treponema denticola used for antigen production may be any strain as long as it belongs to this strain,
Strains available from American Type Culture Collection (ATCC) in Rockville, Maryland, USA, eg Treponema denticola ATCC 33520
A strain, or a strain isolated from the focal site of periodontitis is used. The culture of Treponema denticola is not particularly limited, and any culture can be used, for example, TYGVS medium or the like is used.

The antigen used for immunization is prepared, for example, as follows.

Treponema denticola cells anaerobically cultured in TYGVS medium are suspended in 0.01 M phosphate buffer (pH 7.2), sonicated, centrifuged and the supernatant is dialyzed against the same buffer. . Then, the protein component is subjected to reduction treatment to be solubilized, subjected to slab-type sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis, the peptide is developed and separated, and the 20K to 75K protein band portion is cut out.

Immunization of mammals or birds with this antibody can be performed according to a conventional method, and well-immunized mammals include rabbits, goats, sheep, cows, horses, and the like.
Examples of birds include chickens, ducks and ostriches.

Further, a monoclonal antibody against the coat protein may be prepared by a conventional method. For example, BALB / c mice and the like are immunized with the antigen, and the resulting antibody-producing cells against the coat protein are fused with myeloma cells, and the resulting hybridoma is cultured. Can be obtained.

The antibody can be purified from the antiserum or milk according to a conventional purification method. As an antibody purification method, a salting-out method,
Gel filtration, ion exchange chromatography, affinity chromatography and the like can be used.

In the composition for oral cavity of the present invention, one of the above-mentioned antibodies may be blended alone, or two or more thereof may be used in combination. The amount of the antibody compounded in the composition is 0.0001% to 10% (% by weight,
The same applies hereinafter), especially 0.001% to 5%, and the amount of the composition applied to the oral cavity is preferably 0.00001 g to 10 g / kg / day in terms of antibody amount.

The oral composition of the present invention is a toothpaste, a powdered toothpaste, and
Toothpaste such as liquid toothpaste, mouthwash, troche, oral pasta, gum massage cream, gargle solution,
It may be an irrigation solution, chewing gum, dairy product, or the like, and other appropriate components are used depending on the type of oral composition. For example, in the case of toothpaste, a binder, a thickener, a foaming agent, a stabilizer, a flavor, a sweetener,
Well-known ingredients such as antiseptics are used. Further, also in other shapes such as mouthwash, the components according to the properties of the product are appropriately mixed.

In addition, in the present invention, active ingredients such as chlorhexidine, cetylpyridinium chloride, sodium chloride, azulene, protease, lytic enzyme, mutanase, and herbal medicine extract may be blended.

Thus, the composition for oral cavity according to the present invention uses the protein present in the coat of Treponema denticola particularly as the coat surface layer as an antigen, and the antibody in blood or the antibody in milk or the bird obtained by immunizing a mammal with this. The egg antibody obtained by immunization, and further by mixing a monoclonal antibody against the 20K to 75K protein antigen, effectively blocks colonization of Treponema denticola in the oral cavity and effectively prevents periodontal disease or Can be treated. Moreover, the antibody is highly safe because it is against a specific antigen.

Hereinafter, the effects of the present invention will be specifically shown by experiments.

[Experiment]

(1) Preparation of antigen Treponema denticola strain (eg ATCC33520, A
TCC35404 or ATCC35405) inoculated into TYGVS medium at 37 ℃
Were cultured under anaerobic conditions until the logarithmic growth phase. After collecting the bacteria, 0.
The cells were washed 3 times with 01M phosphate buffer (pH 7.2). After ultrasonically destroying the washed cells, the supernatant obtained by centrifugation at 15000 g for 30 minutes was dialyzed against PBS. This was subjected to gel filtration with a Sephadex G-75 column, the first peak fraction of the measurement pattern of OD ₂₈₀ was concentrated and dialyzed with PBS to obtain a precipitate of 1000 g,
Centrifuge for 10 minutes to remove, and then add the supernatant again to 45000g, 60
Centrifugation was performed for a minute, and the sediment was used as a film fraction. Furthermore, this was subjected to SDS-polyacrylamide electrophoresis,
% Of the protein bands separated on a% acrylamide gel and positively stained with Coomassie Brilliant Blue stain, protein bands such as 50K, 53K, 56K, which are proteins on the surface layer of the capsule, were cut out and pooled.

(2) Preparation of antibody Rabbits were immunized with the prepared various antigens to obtain antibodies. The method of immunization was carried out according to a commonly used method. That is, 100 μg of each of the prepared various antigens
It was administered into the ear vein every 4 days for a total of 10 times. The obtained antiserum is
After salting out with 50% ammonium sulfate, it was dialyzed against distilled water to obtain an antibody sample. Table 1 shows the microbial cell aggregation activity of each antibody preparation against Treponema denticola ATCC 33520 strain.

(3) Preparation of monoclonal antibody 53K of Treponema pallidum ATCC 33520 strain prepared above
The protein band was disrupted in phosphate buffer, and a mixture of this and the same volume of Freund's complete adjuvant (protein mass: 10 μg / ml) was used as the immunogen. The antigen was administered three times at 14 ml intervals. The antibody titer in the blood of the mouse was measured by agglutination reaction 1:
An aggregated antibody titer of 256 or more was obtained. Three days after the final antigen administration, mouse spleens were aseptically collected, splenocyte suspensions were prepared in cold RPMI-1640 medium and washed twice in the same medium (85%).
The above-mentioned viable cell ratio) was subjected to fusion.

On the other hand, RPMI-1640 medium containing 15% fetal bovine serum (NS-
NS-1 myeloma cultured for 3 days in 1 medium) was suspended in RPMI-1640 medium immediately before fusion. The viable cell rate of NS-1 myeloma cells was 85% or more.

For fusion, the spleen cells and myeloma cells were mixed at a ratio of 5, and centrifuged at 1,000 rpm for 5 minutes. After completely removing the supernatant, the precipitate was thoroughly loosened, and 1 ml of 50% polyethylene glycol 4000 (Merck No. 9727) solution kept at 37 ° C. was added for 1 minute while vigorously rotating and vibrating the container. Continue the rotation vibration for the next 1 minute,
10 ml of RPMI-1640 medium kept at 37 ° C was added over 5 minutes. Immediately after centrifugation at 1000 rpm for 5 minutes,
The supernatant was removed. 5x10 ⁶ cells / spleen cells /
Add HAT medium kept at 37 ° C to make a uniform cell suspension to give a uniform cell suspension, and dispense 0.1 ml each into the wells of 96-well flat-bottom microplates treated with NS-1 medium from the previous day. Vibridomas were formed in a 6% carbon dioxide incubator at 0 ° C. 50 μl of HAT medium was added to each well at 3 day intervals. Cell proliferation was confirmed from around 9 days, and hybridoma proliferation was confirmed from 12 days onward, and the presence or absence of antibody production was screened by the ELISA method. Overall 61
Hybridoma formation was observed in% of the wells, and 16% of the wells were positive for antibody production. Each of the antibody-producing positive hybridomas screened was transferred to a 24-well microplate, cultured in HT medium for 3 days, and then cloned twice by the limiting dilution method. Monoclonal antibodies were similarly prepared for coat proteins of other strains.

The desired hybridoma thus obtained could be cultured in the following medium at 37 ° C. in the presence of 5% carbon dioxide gas.

RPMI-1640 containing 7 to 15% fetal bovine serum HT medium containing 7 to 15% fetal bovine serum HAT medium containing 7 to 15% fetal bovine serum Intraperitoneal of BALB / c mouse pretreated with pristane Could also be cultured. This hybridoma could be cryopreserved in growth medium containing 10% dimethylsulfoxide. This hybridoma is named P1-11C, and has been deposited on November 2, 1988 by the Institute of Industrial Science and Technology, Institute for Microbial Technology, as Microorganism Research Institute No. 10381.

The 50% saturated ammonium sulfate fraction of the culture supernatants of the thus-obtained 5 kinds of hybridomas was used as a monoclonal antibody sample, and the agglutination activity of each monoclonal antibody against Treponema denticola ATCC 33520 strain was examined by Western blotting. Table 2 shows the recognition protein antigens of each monoclonal antibody and the subclasses of each monoclonal antibody examined by the Octelronie method.

As is clear from this result, the subclass of the obtained monoclonal antibody was IgG ₁ or IgG ₃ , and the cell aggregation activity was shown although the activity was different.

(4) Macrophage phagocytosis enhancing effect of rabbit serum antibody and monoclonal antibody Using guinea pig peritoneal macrophages,
The enhancing effect of the above-mentioned antibody on the phagocytic ability against Denticola was examined.

Guinea pig peritoneal macrophages on the 4th day of glycogen stimulation, Treponema denticola ATCC 33520 strain cultured for 7 days and various antibodies (final concentration 10%) were added to Hank's solution, and the mixture was anaerobically cultivated at 37 ° C under shaking, 0, 0.5, 1, The viable cell count was measured after 2 hours. The number of added macrophages is 5.0
× 10 ⁵ cells / ml, and the number of test bacteria was 1.0 × 10 ⁶ cells / ml. The results are shown in Table 3.

As shown in Table 3, the target group, phosphate buffer (0.1
M, pH7.2), macrophage phagocytosis-enhancing effect was observed in various antibodies, but the effect was higher than that in anti-whole cell body antibody and anti-sonication fraction antibody. The 53K protein antibody and the monoclonal antibody were stronger.

(5) Inhibitory effect of Treponema denticola on rat oral colonization by rabbit serum antibody and monoclonal antibody Treponema denticola ATCC35404 strain, which had been cultured for 7 days, was suspended in 10 mM PBS at pH 7.2 so that the turbidity was OD ₆₆₀ = 1.0. Various antibodies were mixed at a volume ratio of 1: 1 and reacted at 37 ° C for 30 minutes. As a control group, the antibody solution was mixed with a phosphate buffer solution.

6 rats in each group were ligated with cotton thread for 7 days in advance, and 0.1 ml of each treated bacterial solution was inoculated to the first molar of the lower jaw for 5 consecutive days. One week after that, the ligature was taken, and the ratio of Treponema denticola to the total number of bacteria in the ligature was calculated by fluorescent antibody microscopy.

The results are shown in Table 4.

As shown in Table 4, it was confirmed that any of the tested antibodies inhibited the oral colonization of Treponema denticola with or without. Among the test antibodies, the anti-whole cell body antibody and the anti-sonication fraction antibody had lower colonization inhibition effects than the other antibodies.

EXAMPLES Next, the present invention will be described in more detail with reference to Examples. Here,% means% by weight.

[Example 1] Toothpaste dicalcium phosphate dihydrate 50.0% Sorbitol 20.0% Carboxymethyl cellulose 1.5% Sodium lauryl sulfate 1.0% Fragrance 1.0% Saccharin 0.1% Water remaining 100.0% Goat anti-capsule serum 0.1% to 0.5% or goat anti-20K to 75K protein serum 0.1% to 0.5% is added.

[Example 2] Toothpaste dicalcium phosphate dihydrate 50.0% Glycerin 10.0% Sorbitol 10.0% Sodium lauryl sulfate 1.5% Fragrance 1.0% Saccharin 0.1% Citric acid 0.5% Water Residual 100.0% or more bovine anti-coating Fraction serum 0.2% is mixed with sodium monofluorophosphate 0.2% or cetylpyridinium chloride 0.1%.

[Example 3] Toothpaste Silica dihydrate 40.0% Sorbitol 20.0% Titanium dioxide 5.0% Carboxymethyl cellulose 2.0% Sucrose monolaurate 2.0% Fragrance 1.0% Saccharin 0.1% Water Residue 100.0% or more against bovine 20K-75K Protein serum 0.01% and chlorhexidine gluconate 0.05% or lytic enzyme 0.05% are mixed.

[Example 4] Toothpaste dicalcium phosphate dihydrate 50.0% Calcium carbonate 30.0% Sorbitol 10.0% Dextran 0.5% Perfume 1.0% Palatinose 0.1% Water remaining 100.0% Sheep anti-53K protein serum 0.05% and chloride Cetylpyridinium 0.05% or lytic enzyme 0.05% is added.

[Example 5] Liquid dentifrice Sodium polyacrate 50.0% Glycerin 30.0% Fragrance 1.0% Palatinose 0.1% Ethanol 3.0% Water 100.0% Bovine anti-coating fraction breast milk 0.01% or 0.02%
And 0.1% zinc chloride or 100 units / g protease.

[Example 6] Mouthwash Ethanol 20.0% Fragrance 1.0% Cacaline 0.05% Dye 0.5% Water 100.0% 0.01% of chicken egg anti-20K-75K protein antibody
Add 0.02% and cetylpyridinium chloride 0.05% or chlorhexidine gluconate 0.05%.

[Example 7] Lozenge gum arabic 10.0% Lactose 60.0% Gelatin 3.0% Perfume 0.5% l-Menthol 0.1% Peppermint oil 0.1% Tocopherol 0.1% Water balance 100.0% Anti-53K protein monoclonal antibody 0.05% or 0.1% to the above components. Add 0.01% cetylpyridinium chloride or 0.01% azulene.

Example 8 Gingival Massage Cream White Vaseline 10.0% Propylene Glycol 3.0% Stearyl Alcohol 4.0% Polyethylene Glycol 6000 20.0% Polyethylene Glycol 600 40.0% Sucrose Oleate 0.5% Water Residue 100.0% bovine anti-coating fraction Serum 0.01% or 0.02% and lysozyme chloride 0.05% or crude drug extract 0.05% or tranexamic acid 0.1% are mixed.

[Example 9] Pasta for oral use Polyoxyethylene monostearate 2.0% Sorbitan monooleate 3.0% Olein alcohol 4.0% Polyethylene glycol 10.0% Carboxymethylcellulose 5.0% Gelatin 1.0% Peppermint oil 0.5% Water Residue 100.0% or more 50K protein monoclonal antibody 01% or 0.2% and eugenol 0.1% or lytic enzyme 0.05% or crude drug extract 0.05% are mixed.

[Example 10] Yogurt skim milk 200 kg Sweetened condensed milk ^** 50 kg Palatinose 10 kg The above components are fermented by a conventional method. ^** Contains 10% bovine anti-capsulated milk, 0.01% bacteriocin or 0.02 lytic enzyme
% Or 1000 units / g of mutanase.

EFFECTS OF THE INVENTION According to the present invention, it is possible to obtain an oral composition that significantly suppresses the oral colonization of Treponema denticola and is effective in the prevention and treatment of periodontal disease.

Claims (4)

[Claims] 1. An oral cavity for suppressing oral colonization of Treponema denticola, which comprises a blood antibody or a milk antibody obtained by immunizing a mammal with the Treponema denticola capsule as an antigen. Composition. 2. An oral cavity of Treponema denticola, which comprises a protein existing in the surface layer of Treponema denticola coating as an antigen, and a blood antibody or a milk antibody obtained by immunizing a mammal with these proteins. An oral composition for suppressing internal fixation. 3. An oral cavity of Treponema denticola, which comprises the antigen existing in the coat of Treponema denticola or the surface layer of the coat, immunized a bird with the protein, and containing the antibody contained in the egg. An oral composition for suppressing internal fixation. 4. A Treponema denticola comprising a monoclonal antibody against a protein antigen produced by a hybridoma obtained by cell fusion of an antibody-producing cell against a coat protein antigen of Treponema denticola and a myeloma cell. An oral composition for suppressing oral fixation.