JP3040744B2 - Antiallergic agent and method for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antiallergic agent comprising a cell belonging to the genus Enterococcus or a processed product thereof as a main component and a method for producing the same. In addition, the present invention relates to a drug capable of enhancing an anti-allergic effect by using in combination with a commercially available release inhibitor or antagonist of a chemical messenger such as histamine and leukotriene and reducing side effects of the therapeutic agent, and a method for producing the same. It is.

[0002]

2. Description of the Related Art In recent years, allergic diseases have been increasing. In particular, an increasing number of people are afflicted with diseases that are greatly related to immediate allergy, such as allergic rhinitis, hay fever, and allergic dermatitis. This is due to the fact that the ground is covered with asphalt, etc. and that flying antigens such as pollen are not adsorbed on the ground, environmental factors such as an increase in antigens due to an increase in chemical substances in the air, etc. It is considered that mites and fungi increase. Furthermore, it is presumed that an increase in allergic diseases is observed due to one or more causes such as a change in lifestyle, such as a change in diet and eating habits.

On the other hand, as the number of patients exhibiting allergic symptoms increases, sales of pharmaceuticals for suppressing allergic reactions have been increasing year by year (BioIndustry Vol.13 No.12 42-50).
(1996)). The mechanism of action that suppresses allergy is diversifying, including those that prevent release of chemical mediators such as histamine and those that exhibit antagonistic effects with chemical mediators. The market size is currently leveling off, but is expected to increase as new drugs with new mechanisms of action emerge. By the way, in 1985 when the development of antiallergic drugs began to flourish, the market was less than 25 billion yen.
A year later, in 1995, it grew nearly 6 times to 144 billion yen. Among them, antihistamines are currently the mainstream of antiallergic drugs, and currently account for 70% of the market.

[0004] However, since antihistamines not only suppress allergic reactions but also act on the central nervous system, in most cases side effects such as drowsiness and malaise occur. These side effects often hinder daytime activities and affect daily life. Drugs that exhibit other mechanisms of action also include those that damage the liver and those that damage the cardiovascular system.
Many have side effects that cause administration discontinuation.

[0005] Immunoglobulin E (IgE) is an antibody involved in the development of immediate allergy. Elevated levels of this antibody often indicate allergic symptoms. Japanese Patent Laid-Open No. 9-2959 is a patent focusing on this. This patent describes the addition of lactic acid bacteria to the spleen cell suspension of mice sensitized with ovalbumin in vitro,
This shows the effect of suppressing the amount of E production.

[0006] However, it is known that, in clinical practice, there is a person who does not show allergic symptoms even when the IgE level is increased, and conversely, there is a person who shows allergic symptoms even when the IgE level is in a normal range. It is also known that many of the asymptomatic humans are usually positive for the antigen when subjected to a skin test. Actually not showing any allergic symptoms 5
It has been found that close to 30% of 000 humans are positive for one or more normal antigens. From these facts, it can be said that complex factors as well as IgE levels are involved in causing allergic symptoms.

[0007]

It is a well-known fact that an immediate allergic reaction is not caused by only one factor such as an increase in IgE level or an increase in sensitizing antigen, but is caused by multiple factors. It is. Therefore, there is a need for a drug that can suppress not only one reaction but also a plurality of reactions related to an allergic reaction.

[0008] In addition, there are many prescription examples in which an antiallergic agent is continuously taken as a preventive drug before the onset of symptoms, rather than taken after the onset of allergic symptoms. If side effects such as drowsiness and malaise continue during the drinking period at that time, daily life will be greatly affected.

[0009] In order to solve these problems, an object of the present invention is to provide a drug which has an effect of preventing allergies or an effect of suppressing a plurality of reactions related to allergic reactions and has no side effect.

[0010]

It is known that lactic acid bacteria have various actions. In particular, bacteria belonging to the genus Enterococcus are expected to have some type of allergy-suppressing action because they are lactic acid bacteria resident in the intestine, which is the largest organ involved in mucosal immunity and digestion and absorption. Then, when the inhibitory action of the active cutaneous anaphylactic reaction was observed using dead cells of a bacterium belonging to the genus Enterococcus, a slight inhibition was observed.

Therefore, the present inventors consider that not only immune cells gathered near the intestinal tract, but also immune cells in the body may have some function related to allergy suppression by absorption into the body. Using a cell in which the cell wall of a lactic acid bacterium was broken so as to be easily absorbed into the body, the inhibitory effect of an active skin anaphylactic reaction was observed.
Then, by administering the cells, ovalbumin,
Regardless of the type of antigen that induces the anaphylactic reaction, such as cedar pollen, a strong inhibition of the reaction was observed as compared to the control. From these results, it was found that the processed product of the bacterium belonging to the genus Enterococcus has an antiallergic effect,
The present invention has been completed. Also, when administered in combination with ketotifen fumarate, which is used as an antiallergic agent, the action of the drug was enhanced.

The active cutaneous anaphylaxis reaction used as an index is an observation of the entire mechanism of the onset mechanism of type I allergy. Allergens from outside the body are recognized by T cells and B cells after being taken up by antigen presenting cells. Allergen-recognized cells are allergen-specific I
Promotes gE production. An experimental system reproduces a series of flows in which, when the same allergen enters the body again after the IgE has adhered to the mast cells, the mast cells react and release a chemical messenger to cause an anaphylactic reaction.

The fact that the anti-allergic effect was observed in the active cutaneous anaphylaxis reaction, which is an experimental system for type I allergy, means that the effect of suppressing a part of the process from antigen presenting cells to IgE production was confirmed. It is presumed to have any of the action of inhibiting cell binding, the action of inhibiting histamine release, and the action of a histamine blocker, and can be said to be used as an allergy preventive drug.

The microorganism belonging to the genus Enterococcus used in the present invention is a strain isolated from the stool of food or from healthy humans, and therefore has no risk of side effects. In addition, as shown in Example 3, it was confirmed that no toxicity was observed in acute, subacute, and chronic toxicity tests of the cell preparation obtained by enzymatically and heat-treating the cells.

To prepare the cells or a processed product thereof, carriers such as starch, lactose and soy protein, excipients, binders,
It is formulated into tablets or granules by a known method using additives such as a disintegrant, a lubricant, a stabilizer, and a flavoring agent.

The lytic enzyme used in the present invention may be any type commonly used for lysing bacteria, such as actinase, zymolyase, chitalase, lysozyme, mutanolicin, achromopeptidase, and the like. It is also possible to use a mixture of one or more enzymes. In addition, heat treatment is performed for the purpose of enhancing the effect of the lytic enzyme, decomposing the cell wall, and completely extracting the cell contents. The heat treatment may be performed at a temperature of 100 ° C. or higher, but a temperature range in which autoclaving can be performed is preferable in consideration of the extraction effect.

[0017] The amount used depends on symptoms, age, etc.
As an active ingredient, 0.002 to 0.1 g / kg body weight per day can be usually administered to an adult once or several times a day.

[0018]

【Example】

Embodiment 1 FIG. Enterococcus faecalis
us faecalis) NF-1011 (No. 1256
No. 4) was inoculated into a Rogosa liquid medium having the following composition (the number of bacteria: 10 ⁶ cells / ml), and cultured at 37 ° C. for 10 to 16 hours to obtain a culture solution with about 10 ⁹ cells / ml. Was. The obtained culture was centrifuged at 12,000 rpm for 20 minutes to collect cells, and the cells were washed twice with distilled water to obtain cells. The cells were suspended in distilled water, mutanolysin was added at a final concentration of 30 μg / ml, the mixture was treated at 37 ° C. for 4 hours, and then heated at 110 ° C. for 10 minutes. The cell suspension was dried by a freeze-drying method to obtain a dried cell sample (hereinafter referred to as a cell sample).

The composition of Rogosa liquid medium is shown. Trypticase 10 g Yeast extract 5 g Tryptose 3 g Monopotassium phosphate 3 g Dipotassium phosphate 3 g Triammonium citrate 2 g Tween 80 (surfactant) 1 g Glucose 20 g Cysteine hydrochloride 0.2 g Salt solution (as per 1) 5 ml Distilled water 1000 ml (adjusted to pH 7.0, 121 ° C. 15 minutes heat-sterilized in) (1) _{saline: MgSO 4 · 7H 2 O 11.5g} FeSO 4 · 7H 2 O 0.68g MnSO 4 · 2H 2 O 2.4g distilled 100 ml of water

Embodiment 2 FIG. Active skin anaphylaxis reaction 2-1 Reaction using ovalbumin as antigen 5 week-old BALB / c female mouse (Japan SLC)
The cells were divided into groups, and one group contained 5 specimens of the bacterial cell prepared in Example 1.
% Powder CE-2 (Clear Japan)
Groups received only powdered CE-2 until the end of the experiment (28 days).

On the 14th day after feeding various foods, ovalbumin (hereinafter referred to as OVA) (Wako Pure Chemical Industries) was dissolved in physiological saline, and muscles were added to both thighs of mice so that the amount of 1 mg per mouse was obtained. It was sensitized by internal administration. The two groups divided according to the type of food were divided into three groups of ten each on the 28th day from the start of the experiment, thereby dividing the group into a total of six groups. To these, ketotifen fumarate (hereinafter, ketotifen) (Sigma) suspended in physiological saline was orally administered to each concentration. The group to which ketotifen was not administered received only saline. Table 1 shows the group composition.

Table 1 ────────────────────────────────── Group name Ketotifen (mg / kg) Product 対 照 control group − − vs. 0.2 group 0.2 − vs. 1 0.0 group 1.0-bacteria administration group-+ bacteria 0.2 group 0.2 + bacteria 1.0 group 1.0 + ─────────────────── ───────────────

One hour after the administration of ketotifen, 0.06 ml of 1% Evans blue (Sigma) was administered from the tail vein of the mouse. Immediately after that, 0.05 ml of physiological saline or OVA adjusted to 600 μg / ml with physiological saline was intradermally administered to each of the left and right sides under ether anesthesia, and the solution was induced.

Thirty minutes after the induction, the mice were killed with carbon dioxide gas, and the skin on the back was peeled off. Pigment spots at each of the elicited sites were cut out and cut into small pieces. This is acetone:
The dye was added to 3 ml of a solution mixed at a ratio of 0.5% sodium sulfate = 7: 3 and shaken at room temperature for 24 hours to extract a dye. The absorbance at a wavelength of 620 nm of the supernatant obtained by centrifuging them (3000 rpm, 10 minutes) was measured.

FIG. 1 shows the results of the induction with OVA. Looking at the value of each group when the value of the absorbance of the control group was set to 1, the bacterial cell-administered group was significantly (p <0.0) compared to the control group.
The anaphylaxis-suppressing effect was obtained in 5), which was equivalent to that of the group administered with 0.2 mg / kg of ketotifen (vs. 0.2 group). In addition, as compared with ketotifen alone administration, significant (p <0.0
5) The anaphylaxis inhibitory effect was enhanced.

2-2 Reaction Using Cedar Pollen Extract as an Antigen A 5-week-old BALB / c female mouse (Japan SLC) was
The cells were divided into groups, and one group contained the bacterial sample 60 prepared in Example 1.
mg / animal / day by gavage (hereinafter referred to as the group to which the cells were administered).
Each group received only physiological saline (hereinafter referred to as a control group) until the end of the experiment (28 days).

On the 14th day after the start of the administration of the bacterial cell preparation, the cedar pollen extract (Torii Pharmaceutical Co., Ltd.) was diluted 5-fold with physiological saline to each group, and the concentration was adjusted to 0.1 ml per mouse.
The mice were sensitized by intramuscular administration to both thighs.

On the 28th day from the start of the experiment, all mice were administered with 1% Evans blue (Sigma) in a dose of 0.06 ml via the tail vein. Immediately thereafter, under ether anesthesia, the cedar pollen extract diluted twice with physiological saline was added to each of the right and left sides in an amount of 0.1.
It was induced by intradermal administration of 05 ml each.

Thirty minutes after the induction, the mice were killed with carbon dioxide gas, and the skin on the back was peeled off. Pigment spots at each of the elicited sites were cut out and cut into small pieces. This is acetone:
The dye was added to 3 ml of a solution mixed at a ratio of 0.5% sodium sulfate = 7: 3 and shaken at room temperature for 24 hours to extract a dye. The absorbance at a wavelength of 620 nm of the supernatant obtained by centrifuging them (3000 rpm, 10 minutes) was measured.

FIG. 2 shows the results. When the value of the absorbance of the control group was 1, the value of the group to which the cells were administered was 0.7, which significantly (p <0.05) suppressed the anaphylactic reaction.
This indicates that not only food-derived antigens but also flying antigens such as pollen have an anaphylactic suppression effect.

Embodiment 3 FIG. Toxicity Test 3-1 Acute Toxicity Test 2500 mg / kg amount of the bacterial cell preparation prepared in Example 1 was administered to male and female ICR mice (Charles River Japan) and SD rats (Charles River Japan) using a gastric probe. No abnormalities were found in the general condition.

3-2 Subacute Toxicity Test Female ICR mice (Charles River Japan) were divided into 10 females, and the bacterial sample prepared in Example 1 was used for 125, 5
00 or 2000 mg / kg, 6 with gastric sonde
Administration was performed for 0 days. No abnormalities were observed in the general condition and weight at the time of the experiment, and no abnormalities were observed in the general blood test and anatomical findings after the experiment.

3-3 Chronic Toxicity Test The male and female SD rats (Charles River Japan) were divided into 10 males and 10 females, and the bacterial cell preparation prepared in Example 1 was added to powder CE-2 in an amount of 1% (500 mg / kg). Conversion) or 5%
The mixture (in terms of 2500 mg / kg amount) was administered to each group for 190 days. No abnormalities were observed in the general condition and body weight during the administration period, and no abnormalities were observed in general blood tests and anatomical findings after the administration.

[0034]

According to the present invention, it can be used as an antiallergic agent having no side effects. Also,
When allergic symptoms are strong, the combined use with a small amount of an antiallergic agent enhances the allergy suppressing effect and also has an effect of reducing side effects due to the antiallergic agent. In addition, since the allergy can be suppressed without side effects by taking it before the allergen sensitization time, it can be used as a preventive drug for allergic reactions, such as hay fever, whose illness time is known.

[Brief description of the drawings]

FIG. 1 is a diagram showing the intensity of an active skin anaphylactic reaction in the case of using OVA as an antigen in terms of absorbance (relative value).

FIG. 2 is a diagram showing the intensity of an active skin anaphylactic reaction when a cedar pollen extract is used as an antigen in terms of absorbance (relative value).

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Tetsuro Yamamoto 1406-69 Entokuin, Ayama-cho, Ayama-gun, Mie Examiner Naoko Tsukanaka (56) References JP-A-7-265064 (JP, A) Hei 9-2959 (JP, A) JP-A-56-158717 (JP, A) Nanzando Medical Dictionary, Nanzando, Inc., (1990), p. 31 Chikako Tanaka, Ryuichi Kato, “New Pharmacology”, Nanzando Co., Ltd., (1989), pp. 467-469, 475-478 (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 35 / 74 A61P 27/16 A61P 37/08 WPIDS (STN)

Claims (5)

(57) [Claims] 1. An anti-allergic agent comprising an enzyme-treated bacterium belonging to the genus Enterococcus as a main component. 2. An anti-allergic agent comprising a substance belonging to the genus Enterococcus treated with a lytic enzyme and heat-treated. 3. The antiallergic agent according to claim 1, wherein the antiallergic effect suppresses an anaphylactic reaction. 4. The antiallergic agent according to claim 1, wherein the antiallergic effect is to suppress hay fever. 5. A method for producing an anti-allergic agent, comprising treating cells belonging to the genus Enterococcus with one or more lytic enzymes, followed by heat treatment.