JP2008195631A - IL-12 PRODUCTION PROMOTING METHOD AND Th1 INDUCING AGENT SUBJECTED TO THE TREATMENT - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new Th1 inducing agent having high safety, free from the problems of side actions, etc., and enabling the regulation of cytokine produced by helper T cell, based on the presumption attained by the recent advance in the immunological research that the balance of Th1 and Th2 has grave action on the onset and development of diseases, and to solve the recent problems of the increasing tendency of the promotion of Th2 from the living environment and, for example, the onset of cancer, immunodeficiency, asthma, dermatitis, allergic diseases, nephritis, infectious diseases, etc., caused by the function promotion of Th2. <P>SOLUTION: The problems can be solved by finely pulverizing an immunizing antigen substance to a size smaller than 1 μm, and applying a reagglomeration preventing treatment to the fine particles. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

The present invention provides a method for enhancing the ability to produce IL-12 from antigen-presenting cells by subjecting the immunoantigen substance to a particle size of less than 1 micron and subjecting the particles to a reaggregation prevention treatment, and the treatment. The Th1 inducer.

Helper T cells (hereinafter abbreviated as Th) are functionally divided into type I helper T cells (hereinafter abbreviated as Th1) and type II helper T cells (hereinafter abbreviated as Th2).

Th1 produces Th1-type cytokines such as IFN-γ and IL-12 (interleukin-12), attacks and destroys foreign substances such as bacteria and viruses to protect against infection, and also activates macrophages. IL-12 is a cytokine secreted also from antigen-presenting cells such as dendritic cells and macrophages. Natural killer cells (NK cells) that directly attack cancer cells, rack cells (LAK cells), killer T It is known as a very powerful immunologically active substance that activates cells (CTL cells) and enhances production of interferon γ (IFN-γ).

On the other hand, Th2 produces Th2-type cytokines such as IL-4, IL-5, and IL-10, and may be involved in humoral immunity that causes B cells to produce IgE antibodies in response to fungi and mites. Are known.

The Th1-type cytokine suppresses Th2, while the Th2-type cytokine suppresses Th1, and the two are related to each other in order to maintain the balance of the entire immunity.

In recent years, it has been speculated that the balance between Th1 and Th2 plays an important role in the onset and progression of disease as immune research progresses. Recently, there is a strong tendency for Th2 to increase from the living environment. For example, cancer, immunodeficiency, asthma, dermatitis, allergic disease, nephritis, infectious diseases, etc. are thought to develop due to the increased function of Th2. (Refer nonpatent literature 1).
Springer Seminars in Immunopathology Vol. 21 (3), 1999 and the latest medicine "Clinical 1998 of Autoimmune Disease", 32, 1998

In order to treat or prevent such diseases, it is considered that control of Th2 activation, and thus regulation of Th-producing cytokines, is very important.

As one of the methods, a method of regulating Th-producing cytokine by immune antigen stimulation to antigen-presenting cells is disclosed (see Patent Documents 1-3).
JP-A-Heisei 11-228425 JP 2006-016336 JP JP 2006-131623 A

For example, when lactic acid bacteria are used as an immunizing antigen substance, it is disclosed that a positive correlation exists between the phagocytosis rate of lactic acid bacteria by macrophages and the ability to induce IL-12 production (see Non-Patent Document 2). ).
Hideyuki Wakabayashi et al., Analysis of anti-allergic effect of L. paracasei KW3110 strain using mouse cells and human peripheral blood mononuclear cells, published on March 26, 2006

Furthermore, regarding the relationship between the phagocytosis of particles by antigen-presenting cells and their particle size, the total antigen weight taken into Peyer's patches by the study using latex beads increased until the particle size increased to 11 microns. It is disclosed that, when the particle size becomes 21 μm or more, it is not taken in at all (see Non-Patent Document 3).

However, it is disclosed that in the case of the total number of antigen particles, the smaller particle diameter is larger, and when the particle diameter is about 0.6 microns, the uptake amount is the largest (see Non-Patent Document 3). .

Furthermore, it is disclosed that a particle size of about 7 microns is most preferable for efficiently producing antibodies such as IgG and IgA induced by Th2-type cytokines (see Non-Patent Document 3).
Tabata Y, Inoue Y, Ikada Y. Vaccine. 1996; 14: 1677-1685

Anti-histamines, steroids, antiallergic agents, etc. are used to regulate Th-producing cytokines, but there are side effects such as diarrhea caused by destruction of intestinal microflora due to overloading of the patient and drugs However, the present situation is that a complete method has not yet been developed for both prevention and treatment.

In addition, in the method of regulating Th-producing cytokines by immune antigen stimulation to antigen-presenting cells, there is a problem that IL-12 production in antigen-presenting cells is suppressed when engulfment of antigenic substances is inhibited ( Non-Patent Document 3).
Tonoyuki Tano et al., Cancer and Chemotherapy, Vol. 31, No. 11, p. 1767-1789.

However, at present, no information is disclosed about what the IL-12 producing ability of antigen-presenting cells depends on.

Thus, there is a strong craving to provide a method for efficiently enhancing IL-12 production ability by antigen-presenting cells and a Th1 inducer.

Therefore, an object of the present invention is to solve the conventional problems and to provide a method for efficiently enhancing IL-12 production ability by antigen-presenting cells and a Th1 inducer.

In order to solve the above-mentioned problems, the present inventor diligently searched for what the IL-12 production ability of antigen-presenting cells depends on.

In the process, it was discovered that IL-12 production from antigen-presenting cells is dependent on the total number of immune antigen particles incorporated into the antigen-presenting cells, and the particle size of the immune antigen substance is reduced to less than 1 micron. It has been found that by applying a treatment to prevent reaggregation, it can be efficiently taken up by antigen-presenting cells, and as a result, the ability to produce IL-12 is enhanced.

This is due to a mechanism different from that in which the production of antibodies such as IgG and IgA induced by Th2-type cytokines correlates with the total weight of immune antigen particles incorporated into antigen-presenting cells. In the meantime, it was not yet known.

Thus, the present inventor has completed a method for enhancing the ability to produce IL-12 from antigen-presenting cells, and provides a Th1 inducer.

Hereinafter, the present invention will be described in detail. First, the present invention makes it possible to efficiently incorporate antigen-presenting cells by finely pulverizing the immunoantigen substance to a particle size of less than 1 micron, and subjecting the particles to a reaggregation prevention treatment. As a result, IL-12 is obtained. The present invention provides a method that can enhance productivity. The flow of the method is shown in FIG. 1 and will be described in detail below.

The “immunoantigen substance” of the present invention is specifically a substance that can be an immune antigen, regardless of whether it is known or unknown, origin of animals, plants, microorganisms, etc., forms of living bodies, dead bodies, dried bodies, etc. It is not intended to limit.

Examples include microorganisms such as bacteria, yeast, and filamentous fungi, mosses, plants, animals, and the like, and constituents such as proteins and glucans extracted from them by known methods, and substances secreted by them are also included. . Moreover, those combinations may be sufficient.

Bacteria that can be used for the immunogenic substance of the present invention include Lactobacillus acidphilus, Lactobacillus gasseri, L.mali, Lactobacillus plantarum (L. plantarum), Lactobacillus buchneri (L.buchneri), Lactobacillus casei (L.casei), Lactobacillus johnsonii (L.johnsonii), Lactobacillus gallinarum (L.gallinarum), Lactobacillus amylobolus (L .amylovorus), Lactobacillus brevis (L.brevis), Lactobacillus rhamnosus (L.rhamnosus), Lactobacillus kefir (L.kefir), Lactobacillus paracasei (L.paracasei), Lactobacillus crispatus (L . Lactobacillus genus bacteria, Streptcoccus thermophilus (Streptcoccus thermophilus) Streptococcus bacteria such as Lactococcus lactis, Bifidobacterium bifidum, B. longum, Bifidobacterium address Bifidobacterium such as B. adolescentis, B. infantis, B. breve, B. catenulatum, etc. Examples include bacteria belonging to the genus, Bacillus subtilis, Bacillus bacterium such as B. coagulans, and Clostridium bacterium such as Clostridium butilicum.

Examples of yeasts that can be used for the immunogenic substance of the present invention include Saccharomyces cerevisiae, Torulaspora delbrueckii, Candida kefir, and other Saccharomyces genera, Torlaspora genera, and Candida genera.

As the filamentous fungus that can be used for the immunogenic substance of the present invention, Aspergillius or Monascus, preferably A. awamori, A. oryzae, A. niger, A.M. sojae, A. usami, A. kawachii, A. Examples are gonococci such as saitoi. Moreover, the cocoon manufactured from these strains may be sufficient.

Examples of the moss that can be used for the immunogenic substance of the present invention include mycelia and fruiting bodies such as agaricus, maitake, agaricus, shiitake, enoki mushroom, and matsutake.

Examples of animal secretory substances that can be used for the immunogenic substance of the present invention include lactoferrin contained in mammalian breast milk, and nests (axillas) that swallows form their own saliva as a thread.

Bacterial secretory substances that can be used for the immunogenic substance of the present invention include Lactococcus lactis subsp. Examples include viscous substances produced extracellularly by microorganisms belonging to cremoris lactic acid bacteria and Aureobasidium sp.

Furthermore, the immunoantigen substance in the present invention may be an artificial antigen particle obtained by adding a base material such as starch, carrageenan, agar, gelatin, or protein to the above-mentioned material.

“Immunoantigen substance particle size is reduced to less than 1 micron” according to the present invention means that the immunoantigen substance has an average particle diameter of nanometer (nm) less than 1 micron (preferably about 0.6 microns). Refers to pulverization or dispersion until. The pulverization process and the dispersion process can be performed separately or simultaneously. Whether the particle size is less than 1 micron can be measured with a particle size distribution meter or an electron microscope.

As a method of “pulverizing the immunoantigen substance to a particle size of less than 1 micron” according to the present invention, known methods such as stirring, mixer, ball mill, bead mill, jet mill, homogenizer, and generator can be used regardless of wet or dry methods. Can be mentioned.

For example, in the case of bacteria, the bacterial culture can be microparticulated with a high-pressure homogenizer of about 150 kgf / cm ^{2 when} wet. On the other hand, in the case of a high molecular weight polymer immunostimulatory substance, when the substance is made into a fine particle by dry process, when water is added to the substance, the water is absorbed and the volume is expanded several times, so that the particle diameter is not nanometer size. Therefore, it is preferable to make fine particles with a high-pressure generator of about 1,000 to 2,500 kgf / cm ² when wet.

In the present invention, “the reaggregation prevention treatment is applied to the particles” means that a known dispersant or excipient is added to the pulverized / dispersed particles in order to prevent reaggregation of fine particles pulverized / dispersed to less than 1 micron. Is added and dispersed again with a mixer, homogenizer or the like. In this case, a dispersant or an excipient may be added in advance when pulverizing / dispersing.

In this case, the amount of the dispersant / excipient used varies depending on the properties of the immunizing antigen substance, but can be 1 to 100 times, preferably 4 to 10 times the weight.

In the case where the Th1 inducer of the present invention is finally obtained as a powder, it is dispersed by freeze-drying or spray-drying under reduced pressure after a treatment that prevents the particles from re-adhering with a known dispersant or excipient. A powder having excellent properties can be obtained.

Next, the present invention also provides a Th1 inducer.

“Th1 induction” in the present invention means that IL-12 is efficiently produced from antigen-presenting cells, thereby promoting differentiation of Th0 cells (naive Th cells) into Th1, and creating a Th1-dominant state in vivo. Refers to that.

The “Th1 inducer” of the present invention is characterized by comprising as an active ingredient an immunoantigen substance obtained by micronizing to less than 1 micron and subjecting the particles to a reaggregation prevention treatment.

The “Th1 inducer” of the present invention can be used as a product as it is, but in general, various ingredients are added and blended to increase the flavor and form the required shape, and further add flavor. And can be the final product.

Examples of the component added to and mixed with the “Th1 inducer” of the present invention include various sugars, emulsifiers, sweeteners, acidulants, fruit juices and the like. More specifically, sugars such as glucose, sucrose, fructose, and honey, sugar alcohols such as sorbitol, xylitol, erythritol, lactitol, and palatinit, emulsifiers such as sucrose fatty acid ester, glycerin sugar fatty acid ester, and lecithin are included. . In addition to this, even if various vitamins such as vitamin A, vitamin B, vitamin C, vitamin E and the like, herbal extracts, cereal ingredients, vegetable ingredients, milk ingredients, etc. are blended, an excellent flavor Th1 inducer is obtained. be able to.

The flavors that can be added to the “Th1 inducer” of the present invention include yogurt, berry, orange, quince, perilla, citrus, apple, mint, grape, pair, custard. Examples include flavors such as cream, peach, melon, banana, tropical, herbal, tea, and coffee, and these can be used alone or in combination. The addition amount of the flavor is not particularly limited, but is preferably 0.05 to 0.5% by mass, particularly preferably about 0.1 to 0.3% by mass from the flavor side.

The “Th1 inducer” described above can be a product in any form such as solid or liquid.

The “Th1 inducer” of the present invention includes beverages, granules, tablets, pharmaceutically acceptable salts, excipients, preservatives, coloring agents, corrigents and the like by methods known in the field of pharmaceutical or food production. It can be used in various forms such as capsules.

In addition, the “Th1 inducer” in the present invention can be used for health food. Health food means a food that is more active than ordinary food, and is intended for health, health maintenance and promotion, for example, liquid or semi-solid, solid products such as cookies, Examples include confectionery such as rice crackers, jelly, yokan, yogurt and manju, soft drinks, nutritional drinks, soups and the like.

The amount of Th1 inducer used in the present invention varies depending on the type and quality of the used immune antigen substance, age, symptoms, and the like. For example, for prevention, about 0.01 to 10 g in terms of solid content per adult can be mentioned, and it is desirable to take 3 times a day about 30 minutes before meals. In addition, when used as a health food, it is appropriate to use it in an amount that does not adversely affect the taste and appearance of the food, for example, in the range of about 0.1 to 100 g in terms of solid content with respect to 1 kg of the target food. .

Further, the Th1 inducer in the present invention is a lotion (skin lotion), cosmetic cream, emulsion, lotion, pack, skin milk (emulsion), gel, powder, lip balm, lipstick, under makeup, foundation. , Suncare, bath preparations, body shampoos, body rinses, soaps, cleansing foams, ointments, patches, jellies, aerosols, and the like, and can also be used for external preparations for skin.

In addition, the Th1 inducer of the present invention can be appropriately mixed with various components and additives usually used in cosmetics, quasi-drugs, and pharmaceuticals as shown below, as necessary.

That is, humectants such as glycerin, petrolatum, urea, hyaluronic acid, heparin; PABA derivatives (paraaminobenzoic acid, Escalol 507, etc.), cinnamic acid derivatives (neoheliopan, pulsol MCX, sungard B, etc.), salicylic acid derivatives (octyl salicy) Rate), benzophenone derivatives (ASL-24, ASL-24S, etc.), dibenzoylmethane derivatives (Parsol A, Parsole DAM, etc.), heterocyclic derivatives (tinuvin type, etc.), ultraviolet absorbers / scattering agents such as titanium oxide; Metal sequestering agents such as disodium edetate, trisodium edetate, citric acid, sodium citrate, tartaric acid, sodium tartrate, lactic acid, malic acid, sodium polyphosphate, sodium metaphosphate, gluconic acid; salicylic acid, sulfur, caffeine, Sebum inhibitor such as tannin; salt Bactericides and disinfectants such as benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate; anti-inflammatory agents such as diphenhydramine hydrochloride, tranexamic acid, guaiazulene, azulene, allantoin, hinokitiol, glycyrrhizic acid and salts thereof, glycyrrhizic acid derivatives, glycyrrhetinic acid; vitamins A, vitamin B group (B1, B2, B6, B12, B15), folic acid, nicotinic acids, pantothenic acids, biotin, vitamin C, vitamin D group (D2, D3), vitamin E, ubiquinones, vitamin K (K1, Vitamins such as K2, K3, K4); amino acids and derivatives thereof such as aspartic acid, glutamic acid, alanine, lysine, glycine, glutamine, serine, cysteine, cystine, tyrosine, proline, arginine, pyrrolidone carboxylic acid; retinol, acetic acid Whitening agents such as coferol, magnesium ascorbate phosphate, glucoside ascorbate, arbutin, kojic acid, ellagic acid, placenta extract; antioxidants such as butylhydroxytoluene, butylhydroxyanisole, propyl gallate; zinc chloride, zinc sulfate Astringents such as zinc carbonate, zinc oxide, and aluminum potassium sulfate; sugars such as glucose, fructose, maltose, sucrose, trehalose, erythritol, mannitol, xylitol, lactitol; licorice, chamomile, maronier, yukinoshita, glaze, karin, ogon In addition to various plant extracts, such as agron, auren, jujube, and ginkgo biloba, oily components, surfactants, thickeners, alcohols, powder components, pigments, and the like can be appropriately blended.

EXAMPLES Next, although an Example is given and this invention is demonstrated in more detail, this invention is not restrict | limited at all by these Examples.

[Preparation of Th1 inducer]
The lactic acid bacterium Lactobacillus brevis strain FERM BP-4693 was cultured by a known method. Prepare heat-treated cells from the culture solution, and 0 times (no addition), 0.5 times, 1 times, 2 times, 4 times, 9 times the amount of the cells in terms of weight. Dextrin was added as an excipient, and a sample (numbers A to F) to be lyophilized as it was and a sample (numbers G to L) to be lyophilized after being dispersed with a mixer were prepared. Purified water was added to each of these freeze-dried samples so as to give a dry cell equivalent of 10 mg / ml to prepare a cell dispersion. Shake well by hand and stir in the refrigerator for 18 hours. The results are shown in Fig. 2-I.

Further, 0.1 ml of a supernatant 15 mm below the surface of each sample was taken, added to 0.9 ml of purified water, and stirred well, and then the absorbance at OD 660 nm was measured. The results are shown in Figure 2-II.

The floating rate of lactic acid bacteria was the smallest in the sample K in which the amount of dextrin was added four times as an excipient in terms of weight with respect to the bacteria, and further dispersed with a mixer and then freeze-dried (Fig. 2-I). ), The floating rate was the highest at 88.9% (FIG. 2-II), and it was found that the synergistic effect of the pulverization / dispersion treatment and the re-aggregation prevention treatment appeared.

In addition, since there is a relationship in which the floating ratio increases as the proportion of particles having a small particle size increases, it indicates that the proportion of particles having a small particle size in Sample K is the highest. Therefore, the particle size distribution of the bacterial cell particles in all the solutions of Samples A and K was measured. The result is shown in FIG.

As shown in FIG. 3, in sample A, there are two particle size peaks with an average value of the particle size weight conversion distribution of 106.3 ± 73.7 nm and 2,607.4 ± 126.5 nm. The average value of was 1,728.2 ± 786.3 nm. On the other hand, in sample K, the average value of the particle size weight conversion distribution was 555.8 ± 40.6 nm, indicating that the particles were distributed only in a very narrow range near 0.6 microns. This is probably because the particles near 0.6 microns in the sample K correspond to primary particles of lactic acid bacteria, and in the sample A, they are diploid and tetraploid.

[Comparison of IL-12 production ability]
The measurement of IL-12 induction activity was performed by the following method.
(1) Preparation of reagents PBS: NaCl 80 g, Na ₂ HPO ₄ · 12H ₂ O 29 g, KCl 2 g, KH ₂ PO ₄ 2 g were dissolved in distilled water to make 1 L.
FCS (Fetal Calf Serum): Frozen FCS is deactivated by treatment in a water bath at 56 ° C. for 40 minutes and cooled on ice. The sample was passed through a 70 μm cell strainer, placed in a sterilized container, stored refrigerated, and used in about 1 to 2 months.
ET (−) RPMI 1640 Medium: RPMI 1640 31.2 g, NaHCO ₃ 3.75 g, penicillin G-K (Million units of Ariyu Pharmaceutical) 0.18 g, kanamycin 0.18 g was dissolved in 3 L of water for injection, and CO ₂ gas was blown into the solution. Thereafter, it was filtered with a 0.45 μm filter.
After Turc staining: 0.01% gentian violet aqueous solution: acetic acid: water = 1: 1: 98, and stored refrigerated.

(2) Preparation of macrophages Male ICR mice (4-8W) were intraperitoneally injected with 0.4 ml of a 1 g / 100 ml glycerin solution and reared overnight. After the mouse was sacrificed by cervical dislocation, 5 ml of cooled PBS was injected into the abdominal cavity and the abdominal cavity was swallowed. The intraperitoneal solution was placed in a silicon-coated Spitz tube, and centrifuged at 1200 rpm for 5 minutes in a cooling centrifuge to remove the supernatant and red blood cells on the wall surface. Cold PBS was added and dispersed by pipetting, followed by centrifugation at 800 rpm for 5 minutes. The supernatant and wall red blood cells were removed. This operation was further repeated once, and finally 1 ml / mouse of 1% FCS-added ET-RPMI 1640 Medium (manufactured by Invitro Jane) was added. 50 μl of this solution and 50 μl of Cherk staining solution were mixed, and the number of viable cells was counted using a hemocytometer. From this value, the number of cells was adjusted to 1 × 10 ⁶ / ml using 1% FCS-added ET-RPMI 1640 Medium (macrophage fluid). 200 μl of macrophage solution was placed in a 96-well flat bottom plate and cultured in a CO ₂ incubator for 1.5 to 5 hours.

(3) After adjusting the IL-12 derived sample with 5% FCS-added ET-RPMI1640 Medium to 1 mg / ml in terms of bacterial powder, the cells were dispersed with ultrasound. This solution was diluted 100-fold with ET-RPMI 1640 Medium containing 5% FCS, placed in a 96-well U-shaped plate at 250 μl, and incubated for 1 hour in a CO ₂ incubator.

The supernatant of the macrophage fluid obtained in (2) above is removed and washed twice with PBS kept at 37 ° C. 200 μl of the above diluted bacterial powder was added thereto, and cultured in a CO ₂ incubator for 22 hours to induce IL-12.

(4) Measurement of IL-12 The measurement was performed using a mouse IL-12 measurement kit “Cytoscreen” manufactured by BIO SOURCE INTERNATIONAL. In addition, in order to adjust the lot difference of macrophages, OK432 having the same concentration as the sample was measured and expressed as a specific activity with the measured value. OK432 is an antineoplastic agent marketed as “Pisibanil” (trade name, manufactured by Chugai Pharmaceutical Co., Ltd.). Streptococcus pyogenes (group A type 3) Su strain is fixed in the presence of penicillin G. It is a cell preparation obtained by treatment under conditions and lyophilization. OK432 is widely used in the art as a substance that serves as an index for measuring immune activity.

The present inventor compared the IL-12 inducing ability of samples A and K prepared in Example 1. The result is shown in FIG.

From FIG. 4, it was found that Sample K has an IL-12 inducing ability 3.5 times that of Sample A. This means that the antigen uptake rate to macrophages increased 3.5 times as the cell size of the cells decreased.

From these results, it is possible to obtain a powder with excellent dispersibility by adding about 4 times the amount of excipient in terms of weight to the lactic acid bacteria and then lyophilizing the powder. It was confirmed that IL-12 can be efficiently produced from macrophages by the body.

Thus, a Th1 inducer comprising an immunoantigen substance, which is micronized to less than 1 micron and subjected to reaggregation prevention treatment to the particle, is very useful in creating a Th1-dominant state in vivo. .

It is the figure which showed the manufacturing method of Th1 inducer. It is the figure which showed the influence which the micronization and re-aggregation prevention process has on the particle floating rate. It is the figure which compared the particle size weight conversion distribution of the samples A and K. FIG. It is the figure which compared the IL-12 inducibility of the samples A and K.

Explanation of symbols

In FIG. 1-I, the numbers shown in the sample bottles in the figure are the samples (numbers A to F) added with dextrin as an excipient and lyophilized as they are, and the samples (numbered A to F) that were crushed with a mixer and then lyophilized ( Numbers G to L). As for the amount of dextrin added, in terms of weight with respect to the cells, numbers A and G are 0 times (no addition), numbers B and H are 0.5 times, and numbers C and I are 1 time. , Numbers D and J are double amounts, numbers E and K are four times amounts, and numbers F and L are nine times amounts.

Claims (2)

A method for enhancing the ability to produce IL-12 from antigen-presenting cells by micronizing the immunoantigen substance to a particle size of less than 1 micron and subjecting the particles to re-aggregation prevention treatment. A Th1 inducer comprising as an active ingredient an immunoantigen substance subjected to the treatment according to claim 1.