JPH0959179A - Mouth hygienic agent effective for preventing cedar pollinosis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a mouth hygienic agent containing an antigenic protein extracted from cedar pollen as an active component, enabling easy prevention and/or treatment of cedar pollinosis and usable even by junior children. SOLUTION: The active component of this agent is an antigenic protein extracted from cedar pollen, a peptide having high homology to the protein at amino acid level or a peptide constituting a part of the protein. The antigenic protein is preferably Cry j I which is a major allergen existing in cedar pollen. The protein having high homology preferably has a homology of >=50% at amino acid level to the antigenic protein extracted from cedar pollen. The mouth hygienic agent is preferably administered at a daily dose of 0.4mg/kg body-weight in terms of the active component. The agent is preferably absorbed by sublingual absorption in the form of dentifrice, mouthwash, etc.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an oral hygiene agent for preventing and / or treating cedar pollinosis.

[0002]

2. Description of the Related Art Conventionally, cedar pollinosis has been prevented by physically wearing eyeglasses or a mask to prevent contact with cedar pollen, or treated by injection therapy called hyposensitization therapy. It was

However, in order to avoid the cedar pollen, the method of wearing a mask or spectacles exclusively for hay fever when going out is, as a practical problem, accompanied by pain and requires considerable labor. Furthermore, the scattering of pollen occurs not only outdoors but also indoors. This is because cedar pollen is being brought indoors due to ventilation and adhesion to hair, clothes or laundry. Therefore, there is a strong possibility that the cedar pollen may come into contact with the pollen indoors or outdoors during the period when it scatters.Therefore, in order to physically prevent contact with the pollen, strictly speaking Regardless, it is necessary to wear a mask and glasses all day long.

Further, the desensitization therapy for decreasing the sensitivity to the causative antigen is repeated by periodically and gradually increasing the amount of the antigen extract extracted from cedar pollen once or twice a week. It is a method of subcutaneous injection. The treatment is said to be effective when the amount of extract to be injected is increased, and when the maintenance amount is reached, the injection is continued at monthly intervals for at least one year, and in some cases for two years. There is. Thus, desensitization therapy is effective, takes considerable time, effort, and expense, and is not currently effective for all.

Since cedar pollinosis is an immune reaction, once it is sensitized, until it becomes old and all the immunity weakens,
The symptoms are not alleviated. On the other hand, the age at which cedar pollinosis develops has been decreasing year by year, and many cases have now been reported even in the lower grades of elementary school. People who have been sensitized in the lower grades of elementary school will suffer from cedar pollinosis every spring, for a long time before their immunity weakens. Therefore, it is desirable that the above-mentioned prevention and treatment of cedar pollinosis be performed before and after going to elementary school, but these methods, which are thought to be difficult to practice even in adults, will be more difficult at a young age. Is easy to imagine.

[0006]

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide an oral hygiene agent which can prevent and / or treat cedar pollinosis easily, particularly even in young children.

[0007]

As a result of diligent efforts to solve the above problems, the present inventors orally administered Cry j I, which is a major allergen of cedar pollen, to rats, and after a certain period of time, the antigen (Cry j I ), And then measuring the amount of antibody produced, the method of absorbing Cry j I under the tongue induces the suppression of anti-Cry j I antibody production most efficiently, and further, this effect It was found that there is an optimum intake amount to be exerted, and the present invention has been completed. That is, the present invention comprises a protein having antigenicity extracted from cedar pollen, a protein having high homology with the protein at the amino acid level, or a peptide consisting of a part thereof as an active ingredient,
An oral hygiene agent for preventing and / or treating cedar pollinosis is provided.

Although not limited to a particular theory, the use of the oral hygiene agent of the present invention allows the cedar pollen allergen to be absorbed into the body from the mucous membrane of the oral cavity and suppress the production of anti-cedar pollen allergen antibody. It is considered to be one.
If an oral hygiene agent containing a cedar pollen allergen is simply swallowed, it is easily hydrolyzed by pepsin, which is a gastric digestive enzyme, and loses its antigenicity as an allergen, resulting in loss of antibody production-lowering ability. Therefore, it is necessary to avoid hydrolysis by digestive enzymes and be absorbed into the body, and a form is preferred in which the cedar pollen allergen is retained in the oral cavity for a long period of time.

By the way, antibody production requires interaction between T lymphocytes and B lymphocytes, and this interaction produces antibodies against foreign substances that have entered the body. vice versa,
To suppress antibody production, it is required that either or both of these lymphocytes become tolerant. Generally, it is believed that B lymphocytes become more tolerant than T lymphocytes in a tolerant state, so that the cedar pollinosis of the present invention is prevented and / or treated. Oral hygiene agents to help put T lymphocytes in a tolerated state. Further, until now, Japanese cedar pollinosis was said to be mainly caused by type I allergy involving IgE antibodies corresponding to the Japanese cedar pollen allergen.
Allergic contact dermatitis, which is classified as type V allergy, has also become a problem. Since this type IV allergy is an allergic reaction involving T lymphocytes, it is considered possible to suppress type IV allergy if T lymphocytes can be made tolerant.

Hereinafter, the present invention will be described in detail. The oral hygiene agent of the present invention contains, as an active ingredient, a protein having antigenicity extracted from cedar pollen, a protein highly homologous to the protein at the amino acid level, or a peptide consisting of a part thereof.

Examples of the protein having an antigenicity extracted from cedar pollen include a protein contained in cedar pollen capable of inducing production of IgE antibody specific to cedar pollen. These proteins consist of major and minor allergens. Among allergens contained in pollen, the major sensitizers of most patients are called major allergens, and most of them are the largest in pollen (J. Allergy Clin. Immun.
ol. (1983) 71, 77-86 and Allergy (1990) 45, 309-3.
12). In addition, a component that is sensitized to some patients in a small amount is called a minor allergen (new bioscience series "Science of pollinosis-exploring topical allergic diseases" by Yozo Saito, Takeshi Ide). The molecular weight of both allergens is approximately 5 to 50 kDa. Among these allergens, Cry j I, Cry j II (the above “science of hay fever”) and mixtures thereof are preferable, and Cry j I is particularly effective. The protein having antigenicity extracted from cedar pollen used in the oral hygiene agent of the present invention is J. Allergy Clin.
It can be obtained according to the method described in Immunol. (1983) 71, 77-86.

The protein having a high amino acid level homology with the protein having antigenicity extracted from the cedar pollen described above is 40% or more at the amino acid level with the protein having antigenicity extracted from the cedar pollen, preferably , A protein having a homology of 50% or more.

[0013] The protein having antigenicity extracted from the above-mentioned cedar pollen or a peptide consisting of a part of a protein having high homology with the protein at the amino acid level may be any peptide having antigenicity.

When the oral hygiene agent of the present invention is used in small animals such as rats, it is preferable to use allergens such as Cry j I which have been purified to increase the purity. This is because small animals have very small stomach and oral cavity volumes, and therefore the amount that can be administered is limited, so that it is necessary to orally administer a small amount of highly pure allergen. On the other hand, since the human oral cavity is large and it is possible to eat a large number of small amounts, it is considered that the effect is sufficiently exerted without increasing the purity of the allergen as in the rat. For example, a so-called crude antigen extracted from a cedar pollen protein with an organic solvent and then defatted with a weak alkaline solution may be sufficient, and if it is colored when added to an oral hygiene agent, it is purified by ion exchange. Is desirable.

The oral hygiene agent of the present invention is preferably 0.2 to 0.6 mg / kg, more preferably 0.3 to 0.5 mg / kg per day.
, Most preferably about 0.4 mg / kg (converted to the amount per body weight), which is an antigenic protein extracted from cedar pollen, a protein having high homology with the protein at the amino acid level, or one of them. It may be designed to ingest the multipartite peptide.

[0016] A protein having antigenicity extracted from cedar pollen in an amount sufficient for preventing and / or treating cedar pollinosis, a protein highly homologous to the protein at the amino acid level, or a part thereof. A peptide consisting of
It may be designed to be absorbed under the tongue. In order to absorb a sufficient amount of the above-mentioned allergen under the tongue, it is sufficient to take a formulation form in which the residence time of the allergen in the mouth is long, and as a formulation form that matches this purpose, toothpaste,
Examples include mouthwash. Preparations in these forms have the advantage that they can be used habitually and easily by anyone of any age.

As the raw material for the oral hygiene agent, all known raw materials used for the oral hygiene agent can be mentioned, and they are selected and used in a timely manner. Furthermore, since it is unlikely that other additives inhibit the hay fever preventive effect, it is possible to develop products for various age groups by combining various pigments, flavors and sweeteners. Examples of toothpaste include powder toothpaste, moisturizing toothpaste, toothpaste, and water toothpaste. Combining various pigments, fragrances, herbal extracts, and sweeteners makes it possible to develop products for various age groups. One or more flavors and herb extracts may be used as long as they are used in foods. It is preferable to use one or a plurality of materials that give a refreshing feeling. Such flavors and herbal extracts include mint, menthol, cineole, peppermint, spearmint, liquorice, cinnamon, hawthorn,
Perilla and the like.

In order to exert the effect of preventing and / or treating hay fever, it is desirable to continuously take 0.2-0.6 mg / kg of allergen equivalent per day (converted into the amount per body weight). Therefore, it is desirable that these oral hygiene agents are designed so that the above-mentioned amount of allergen can be ingested per day.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described with reference to the following examples.
This will be described more specifically. These examples are illustrative and do not limit the scope of the invention. [Example 1] First, a large amount of polysaccharide components were removed from the crude antigen and the basic protein fraction was efficiently collected by the following method to obtain a major allergen of cedar pollen.
Got Cryj I.

The cedar pollen defatted with acetone was extracted with a weak alkaline solution, the extract was salted out with 80% saturated ammonium sulfate, and a sample of this ammonium sulfate fraction was dialyzed to pH 7.8 and the same. The mixture was mixed with DEAE-cellulose adjusted to pH and slowly stirred at 4 ° C for 1 hour. These were centrifuged and the non-adsorbed fraction of the supernatant was collected and redialyzed against an acetate buffer of pH 5.0. Furthermore, it was mixed with CM-cellulose adjusted to pH 5.0, and this was slowly stirred at 4 ° C. for 4 hours. These were centrifuged, and the precipitated CM-cellulose was washed with pH 5.0 acetate buffer, then dispersed in 0.3M NaCl-100 mM phosphate buffer (pH 7.0), and slowly stirred overnight at 4 ° C. . Then, centrifugation was performed to collect the supernatant, which was purified by an ion exchange method to obtain Cry j I. Further, Cry j I was dialyzed against 50 mM ammonium bicarbonate buffer (pH 7.8), concentrated, and then subjected to Sephadex G-150 at a flow rate of 20 ml.
/ 1 hour, fractionated and collected main peak (Fig. 1
(Fractions 60 to 75 / first). Purity test is SDS-
I did it on PAGE and got only Cry j I.

Extracted and purified from cedar pollen as described above
Cry j I was solubilized in physiological saline, adjusted to 70 μg / day / rat, and orally administered for 5 days, 2 days, and 5 days, for a total of 10 days. At that time, in order to see the effect of hydrolysis by pepsin, untreated control was started, Cry j I hydrolyzed with pepsin and undegraded Cry j I were administered sublingually, and undegraded Cry j
I was directly administered into the stomach using a sonde. Ten days after the final oral administration, the antigen was sensitized by intraperitoneally administering adjuvanted Cry j I. During this breeding period, no change in body weight was observed in any of the animal groups (Fig. 2). Therefore, it is suggested that this method does not have side effects that affect body weight. Furthermore, when the amount of anti-Cry j I antibody was measured with the ELISA method over time from the day of antigen sensitization, it was revealed that the sublingual administration group had a clearly lower antibody production amount than the control group. Further, it was shown that this tendency was stronger in the sublingual administration method than the intragastric administration method (Fig. 3).

Particularly, when the amount of anti-Cry j I antibody contained in plasma on the 46th day from the day of antigen sensitization was measured, the sublingual administration group was higher than that of the control group which was not orally administered in advance. It was shown that the antibody production amount was significantly low at a risk rate of 5% (Fig. 4). In addition, the amount of anti-Cryj I specific IgE antibody was adjusted to c
When the antibody amount was measured by fluorescence intensity using the apture-IgE antibody measurement method, the same tendency was observed (Fig. 5).

However, even when the sublingual administration method is used in the same manner, the decrease in the amount of antibody production expected when using Cry j I hydrolyzed with pepsin in advance is not observed.
In order to confirm this cause, the antigenicity when Cry j I was hydrolyzed with pepsin was examined. In other words, the antigenicity of Cry j I hydrolyzed over time was determined by three types of ELISA.
When the degree of hydrolysis was examined by the TNBS method as well as by the TNBS method, it was suggested that the antigenicity became lower as the TNBS value increased (FIG. 6). In addition, analysis by SDS-PAGE revealed that Cry j I was fragmented into small fragments so that the antigenicity disappeared immediately after the reaction. Thus, since Cry j I is easily hydrolyzed by pepsin, it is suggested that it is desirable to absorb Cry j I from the oral mucosa or enclose Cry j I in an enteric-coated capsule and ingest it.

Next, the sublingual dose of Cry j I was examined using the same experimental method. Since the oral volume of the rat is small, the maximum dose was 150 μg / rat · day. In the 100 μg-administered group and the 150 μg-administered group, suppression of antibody production was observed with a significant difference of 5% as compared with the control group. However, the difference in the amount of antibody produced between the 100 μg-administered group and the 150 μg-administered group was not as dramatic as the difference between the 50 μg-administered group and the 100 μg-administered group, and therefore this suppressive effect was 100 μg.
It is considered that saturation is almost reached in g / rat / day (Fig. 7).

After sensitizing once, the antigen was sensitized again for 3 months or more, and the antibody amount was measured with time from the second antigen sensitization. The amount of antibody produced was higher than that of the group. Nevertheless, it was revealed that the antibody production inhibitory effect continued in the 100 μg administration group (FIG. 8).

From the above results, Cry j I added to an oral hygiene agent for preventing and / or treating cedar pollinosis
Was found to be optimal for rats, 100 μg.
Converting this to the amount per weight, it corresponds to 0.4 mg / kg.

Example 2 Production of Toothpaste Powder Base Calcium Phosphate (Calcium Carbonate) 35% Silicic Acid 5% Binder and Diluent 56% Foaming Agent 2% Allergen 0.4% Sweetener Appropriate Coloring Agent Proper amount Perfume Proper amount The powder base material was swollen, deaerated, and kneading was started with a kneader. In advance, a binder solution dissolved in a diluent was added to the powder base material, and after uniformly mixing, a foaming agent, a sweetener,
The allergen Cry j I and the like obtained in Example 1 were sequentially added. The final fragrance was added, and when the mixture was uniformly mixed, the kneading was stopped, the kneading was sent to a filling machine, the tube was loaded and sealed, and a toothpaste was manufactured.

When using, it is preferable to use toothpaste so that the amount of the tooth is about 3 g at a time, and it is desirable to brush the tooth once to three times a day.

Example 3 Production of Mouthwash Menthol was dissolved in 15% of the total amount of ethanol, and 1
A solvent system solution was prepared by adding 0% glycerin and a fragrance and thoroughly mixing them with stirring. Citric acid, chlorhexidine gluconate and sodium hydrogenphosphate were dissolved in water, and the dye and 3% of the total amount of Epan were added and stirred to prepare an aqueous solvent.

The solvent-based solution was added little by little to the water-based solvent in the agitated state, and the allergen Cry j I obtained in Example 1 was further added so as to be 6% of the total amount, and this was stirred and mixed to obtain a product. did. This mouthwash is designed so that 100 μl can be sprayed at one time, and it is desirable to spray this into the oral cavity about 2 to 6 times a day.

[0031]

The oral hygiene agent of the present invention can prevent and / or treat cedar pollinosis.

[Brief description of drawings]

FIG. 1 is a diagram showing a fraction number and a colorimetric value. Fractions with high colorimetric values indicate high protein content.

FIG. 2 is a diagram showing changes in body weight of rats during the breeding period.

FIG. 3 is a diagram showing a time course of the relative value of the amount of anti-Cry j I antibody starting from the day of antigen sensitization.

FIG. 4: Anti-Cr contained in plasma 46 days after antigen sensitization
It is a figure which shows the amount of yj I antibody. * Indicates that there is a significant difference in antibody production amount at a risk rate of 5%.

FIG. 5: Anti-Cr contained in plasma 46 days after antigen sensitization
It is a figure which shows the amount of yj I specific IgE antibody.

FIG. 6 is a diagram showing the relationship between the relative value of antigenicity and TNBS value measured by three types of immunoassays and the hydrolysis time.

[Fig. 7] Fig. 7 is a view showing a change over time in the relative value of the amount of anti-Cry j I antibody. * Indicates that the risk rate is 5% and the antibody production amount is lower than that of the control group.

[Fig. 8] Fig. 8 is a graph showing the change over time in the relative value of the amount of anti-Cry j I antibody starting from the day of sensitization once again after 3 months or more after sensitization.

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Claims (7)

[Claims] 1. A method for preventing and / or preventing cedar pollinosis, which comprises an antigenic protein extracted from cedar pollen, a protein highly homologous to the protein at the amino acid level, or a peptide consisting of a part thereof as an active ingredient. Or an oral hygiene agent to treat. 2. The oral hygiene agent according to claim 1, wherein the protein having antigenicity extracted from cedar pollen is capable of inducing production of IgE antibody specific to cedar pollen. 3. The oral hygiene agent according to claim 1, wherein the protein having antigenicity extracted from cedar pollen is a mixture of a major allergen and a minor allergen. 4. The major allergens are Cry j I and Cry j.
The oral hygiene agent according to claim 3, which is selected from the group consisting of II and mixtures thereof. 5. A protein having an antigenicity, which is extracted from cedar pollen in an amount of 0.2 to 0.6 mg / kg (calculated per body weight) per day, and a protein having high homology with the protein at the amino acid level,
The oral hygiene agent according to claim 1, which is designed so that a peptide consisting of them can be ingested. 6. A protein having antigenicity extracted from a sufficient amount of cedar pollen for preventing and / or treating cedar pollinosis, a protein having high homology at the amino acid level with the protein, or a part thereof. The oral hygiene agent according to claim 1, wherein the peptide consisting of is designed to be absorbed under the tongue. 7. The oral hygiene agent according to claim 6, wherein the formulation form is toothpaste or mouthwash.