JP2009155204A - Medicinal composition for allergic disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for regulating or promoting the production of IL-4 and IgE by controlling the expression and function of synoviolin, and also a medicinal composition containing a substance for controlling the expression and activity of the synoviolin. <P>SOLUTION: This medicinal composition is provided by containing the substance for controlling the expression and function of the synoviolin, and a treating agent for allergic diseases is characterized by inhibiting the expression and function of the synoviolin. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for suppressing or inhibiting IL-4 and / or IgE production by regulating synoviolin expression and activity. The present invention also relates to a pharmaceutical composition comprising a substance that regulates IL-4 and / or IgE production by regulating the expression and activity of Synoviolin.

Synoviolin is a novel protein discovered as a membrane protein that is overexpressed in synovial cells derived from rheumatic patients (WO02 / 052007). Studies using genetically modified animals revealed that Synoviolin is an essential molecule for the development of rheumatoid arthritis.

  A protein structure prediction system suggests that Synoviolin has a RING finger motif. This motif is often found in the enzyme E3 ubiquitin ligase, which plays an important role in protein ubiquitination, but in fact, cioviolin has the self-ubiquitination activity that is one of the characteristics of E3 ubiquitin ligase. Proven (WO02 / 052007). However, the involvement of Synoviolin and the immune system has not been clarified.

  On the other hand, allergic diseases such as bronchial asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, anaphylactic shock, hay fever and food allergies have been increasing worldwide in recent years and have become major problems. Conventional antiallergic agents are inhibitors of the release of chemical mediators from mast cells, receptor inhibitors of released chemical mediators or inhibitors of allergic inflammatory reactions, but these are all symptomatic treatments It has not been a treatment for fundamental allergic diseases. Allergic reactions are classified into type I allergic reactions, type II allergic reactions, type III allergic reactions, and type IV allergic reactions depending on the mechanism of onset and the difference in symptoms. In the type I allergic reaction, IgE is caused by an antigen entering the living body, and this is caused by the reaction of the IgE antibody adhered to the cell membrane of tissue mast cells or blood basophil cells with the antigen. Releases histamine, serotonin and various other chemical mediators. These chemical mediators cause various tissue reactions such as smooth muscle contraction and increased vascular permeability.

  However, there are few chemical mediators that are known to specifically inhibit the release or production of chemical mediators or specific antagonists, and there are a wide variety of chemical mediators, which suppresses type I allergic reactions. In order to do so, it is currently necessary to analyze the interaction of various chemical mediators.

As therapeutic agents for fundamental allergic diseases, IgE antibody production inhibitors that are deeply involved in the development of allergic diseases are considered. One compound having an inhibitory effect on IgE production is suplatast tosylate (IPD-1151-T). This has been reported to act on type 2 T cells (Th2 cells) and suppress the differentiation of B cells into IgE antibody-producing cells by suppressing IL-4 production (Yanagihara Y et al., Jpn. Pharmacol. (1993) 61: 31-39) (non-patent document 1). Examples of the compound that directly acts on B cells and suppresses IgE antibody production include DSCG (interl), which is a mast cell degranulation inhibitor, or Negchromylsodium. These have been reported to inhibit class switching of B cells (J. Exp. Med. (1994) 180: 663-671 (Non-patent Document 2), J. Allergy Clin. Immunol. (1996) 97: 1141- 1150 (Non-Patent Document 3)). J. Med. Chem. (1997) 40: 395-407 also describes compounds that act directly on B cells to suppress IgE production. Attempts have also been made to suppress IgE production with non-specific immunosuppressive agents such as cyclosporin A to treat allergic diseases, but to suppress immunoglobulins other than IgE antibodies. Is not preferable, and development of a potent inhibitor with high IgE selectivity is desired.
Yanagihara Y et al., Jpn. Pharmacol. (1993) 61: 31-39 Loh RK et al., J. Exp. Med. (1994) 180, 663-671 Loh RK et al., J. Allergy Clin. Immunol. (1996) 97: 1141-1150

  An object of the present invention is to provide a pharmaceutical composition that regulates IL-4 and IgE production by regulating the expression and function of Synoviolin.

The present inventor has intensively studied to solve the above problems. As a result of detailed analysis of synoviolin hetero knockout (syno +/−) mice, it was found that IL-4 and IgE production was suppressed as compared with the wild type, and the present invention was completed.

That is, the present invention is as follows.
(1) A pharmaceutical composition comprising a substance that regulates the production of IL-4 and / or IgE.

  Examples of the substance that regulates the expression and function of Synoviolin include those that inhibit or promote the expression and / or function of Synoviolin. Specifically, it is selected from the group consisting of low molecular weight compounds, peptides, antibodies, decoy nucleic acids, antisense nucleic acids, siRNAs and shRNAs for synoviolin or a gene encoding synoviolin, using synoviolin expression and / or function as inhibitors. At least one can be exemplified. Moreover, as a substance which promotes the expression and / or function of Synoviolin, at least one selected from the group consisting of Synoviolin promoter activity promoter, Synoviolin polypeptide and Synoviolin polynucleotide can be exemplified.

  Here, the gene encoding Synoviolin includes, for example, the base sequence shown in SEQ ID NO: 1.

In the present invention, siRNA includes those targeting a part of the base sequence shown in SEQ ID NO: 1.

  The present invention also provides a pharmaceutical composition for preventing and / or treating allergic diseases, infectious diseases, autoimmune diseases or leukemia.

  Examples of the allergic disease include at least one selected from the group consisting of bronchial asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, anaphylactic shock, tick allergic disease, hay fever and food allergic disease Can do.

  Examples of infectious diseases include parasitic infections such as amoebiasis, roundworm, babesiosis, cryptosporidiosis, giardiasis, helminthiasis, malaria, helminthiasis, schistosomiasis, tapeworm infection, Toxocara Exemplifying at least one selected from the group consisting of infectious disease, toxoplasmosis, trichinosis, trichinosis, scabies, anisakiasis, echinococcosis, foodborne parasitic helminthiasis, lice disease, larva migrans and sarcoidosis it can.

  Examples of autoimmune diseases include rheumatoid arthritis, Sjogren's syndrome, systemic sclerosis, polymyositis, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, bullous pemphigoid, Graves' disease (Graves' disease), Hashimoto's thyroiditis, type 1 diabetes, systemic lupus erythematosus, myasthenia gravis, pemphigus, pernicious anemia, collagen disease, hypoIgemia and agammaglobulinemia One can be exemplified.

  Furthermore, the leukemia is exemplified by at least one selected from the group consisting of myelogenesis, multiple myeloma, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia and chronic myelogenous leukemia Can do.

(2) A food, beverage or feed for regulating cytokine production, comprising a substance that regulates the expression and / or function of Synoviolin.
(3) An antiallergic food, beverage or feed containing a substance that inhibits the expression and / or function of Synoviolin.
(4) A method for regulating IL-4 and IgE production, comprising regulating the expression and / or function of Synoviolin.

  The present invention provides a substance that regulates the expression and function of Synoviolin. Since this substance can regulate (suppress or promote) the production of IL-4 and / or IgE by regulating the expression and function of Synoviolin, the pharmaceutical composition for the treatment of allergic diseases when the production is inhibited When the production is promoted, it is useful as a pharmaceutical composition for treating infectious diseases, autoimmune diseases and leukemia.

  Hereinafter, the present invention will be described in detail.

  The present invention is characterized by treating a disease using a pharmaceutical composition containing a substance that regulates the expression and function of Synoviolin. Specifically, allergic diseases are treated by inhibiting the function of Synoviolin and suppressing the production of IL-4 and / or IgE. In addition, the present invention is characterized by treating infectious diseases, autoimmune diseases, and leukemias by promoting the function of Synoviolin to promote the production of IL-4 and / or IgE.

  In the present invention, in order to make synoviolin expression and activity inhibition one of the effective methods for treating allergic diseases, the function of synoviolin was focused. Synoviolin hetero knockout animals were prepared and analyzed in detail, and IL-4 and IgE production suppression was observed as compared to the wild type. That is, it has been found that inhibition of the function of Synoviolin suppresses the production of IL-4 and IgE that are deeply involved in allergic diseases, and the inhibition of Synoviolin function leads to treatment of allergic diseases. In addition, when the present inventors overexpress the Synoviolin gene with a viral vector for gene therapy, the production of IL-4 and IgE is promoted, leading to the treatment of infectious diseases, autoimmune diseases and leukemia. I found out.

  Accordingly, the present invention provides a pharmaceutical composition comprising a substance that modulates IL-4 production and / or IgE production. Here, “regulation” means to promote or suppress IL-4 production, IgE production or synoviolin expression, and is also referred to as positive regulation when promoted, or unregulated when inhibited. One skilled in the art can adjust to promote or inhibit IL-4 production, IgE production or synoviolin expression depending on the disease to be treated.

2. Synoviolin expression and / or activity regulation
In order to regulate IL-4 and IgE production, methods for inhibiting or promoting the expression and / or function of Synoviolin are employed.

(1) Synoviolin expression inhibition Synoviolin expression inhibition is not particularly limited. For example, RNA interference (RNAi) can be used. RNAi can be caused by designing and synthesizing siRNA (small interfering RNA) for the Synoviolin gene and introducing it into the cell. In the present invention, as a substance that inhibits the expression and / or function of Synoviolin, a low molecular compound, peptide, antibody, decoy nucleic acid, or antisense nucleic acid for Synoviolin or a gene encoding Synoviolin can also be used.

  RNAi is a phenomenon in which dsRNA (double-strand RNA) specifically and selectively binds to a target gene and efficiently cleaves the target gene by cleaving the target gene. For example, when dsRNA is introduced into a cell, expression of a gene having a homologous sequence with that RNA is suppressed (knocked down).

  siRNA can be designed as follows.

  (a) There is no particular limitation as long as it is a gene encoding Synoviolin, and any region can be used as a candidate. For example, in the case of a human, an arbitrary region of GenBank Accession number AB024690 (SEQ ID NO: 1) can be used as a candidate.

(b) A sequence beginning with AA is selected from the selected region, and the length of the sequence is 19 to 25 bases, preferably 19 to 21 bases. The GC content of the sequence may be selected, for example, from 40 to 60%. Specifically, a DNA containing at least one sequence selected from the following base sequences among the base sequence shown in SEQ ID NO: 1 can be used as a target sequence of siRNA. In particular, it targets (i) (SEQ ID NO: 2), (ii) (SEQ ID NO: 3), (vi) (SEQ ID NO: 7), (vii) (SEQ ID NO: 8), (viii) (SEQ ID NO: 9) It is preferable.
(i) AA TGTCTGCATCATCTGCCGA GA (SEQ ID NO: 2)
(ii) AA GCTGTGACAGATGCCATCA TG (SEQ ID NO: 3)
(iii) AA AGCTGTGACAGATGCCATC AT (SEQ ID NO: 4)
(iv) AA GAAAGCTGTGACAGATGCC AT (SEQ ID NO: 5)
(v) AA GGTTCTGCTGTACATGGCC TT (SEQ ID NO: 6)
(vi) AA CAAGGCTGTGTACATGCTC TA (SEQ ID NO: 7)
(vii) AA ATGTTTCCACTGGCTGGCT GA (SEQ ID NO: 8)
(viii) AA GGTGTTCTTTGGGCAACTG AG (SEQ ID NO: 9)
(ix) AA CATCCACACACTGCTGGAC GC (SEQ ID NO: 10)
(x) AA CACCCTGTATCCAGATGCC AC (SEQ ID NO: 11)
(xi) AA GGTGCACACCTTCCCACTC TT (SEQ ID NO: 12)
(xii) AA TGTTTCCACTGGCTGGCTG AG (SEQ ID NO: 13)
(xiii) AA GAGACTGCCCTGCAACCAC AT (SEQ ID NO: 14)
(xiv) AA CGTTCCTGGTACGCCGTCA CA (SEQ ID NO: 15)
In order to introduce siRNA into a cell, a method in which siRNA synthesized in vitro is linked to plasmid DNA and introduced into the cell, a method in which two RNAs are annealed, or the like can be employed.

  The present invention can also use shRNA to produce RNAi effects. shRNA is an RNA molecule called a short hairpin RNA (short hairpin RNA) and has a stem-loop structure in which a part of a single strand forms a complementary strand with another region.

  shRNA can be designed such that part of it forms a stem loop. For example, if the sequence of a certain region is set as sequence A and the complementary strand to sequence A is set as sequence B, these sequences are present in one RNA strand so that the sequence A, spacer, and sequence B are in this order. Design to be 45-60 bases in length. Sequence A is a sequence of a partial region of the target Synoviolin gene (SEQ ID NO: 1), and the target region is not particularly limited, and any region can be a candidate. The length of the sequence A is 19 to 25 bases, preferably 19 to 21 bases.

  “Low molecular weight compound” means a compound that inhibits the expression and function of Synoviolin, and examples thereof include Synoviolin enzyme activity inhibitor and Synoviolin promoter activity inhibitor. Such low molecular weight compounds can be obtained by synthesis or by purchasing from a reagent company.

  “Peptide” means a peptide having an amino acid sequence similar to that of Synoviolin, which inhibits Synoviolin function by competitively binding to Synoviolin receptor. Such peptides can be synthesized by ordinary peptide synthesis methods. Examples of peptide synthesis methods include an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a DCC method, an active ester method, a carboimidazole method, and a redox method. In addition, the solid phase synthesis method and the liquid phase synthesis method can be applied to the synthesis. A commercially available peptide synthesizer may be used.

“Antibody” means an antibody that inhibits the function of Synoviolin by binding to Synoviolin, whether it is a polyclonal antibody or a monoclonal antibody. Antibodies also include Fab fragments, F (ab ′) ₂ , Fv fragments and the like.

  In order to prepare a polyclonal antibody, an animal (eg, rat, mouse, rabbit, etc.) is immunized using Synoviolin or a partial fragment thereof as an antigen, and the antibody titer is measured after the final immunization. Blood samples can be collected to obtain antiserum. The dose of the antigen per animal is 1 to 10 mg in the case of rabbits, and is appropriately adjusted depending on the presence or absence of an adjuvant. Examples of the adjuvant include Freund's complete adjuvant (FCA) and Freund's incomplete adjuvant (FIA). When purification of antibody from antiserum is required, it should be purified by selecting a known method such as ammonium sulfate salting-out method, ion exchange chromatography, gel filtration, affinity chromatography, or a combination thereof. Can do.

  Monoclonal antibodies can be produced by hybridoma methods well known in the art. For example, antibody-producing cells (spleen cells, lymph node cells, etc.) and myeloma cells (eg P3X63-Ag.8.U1, NS-I etc.) obtained after immunization of mammals using synoviolin or a partial fragment thereof as an antigen And then hybridomas that produce the desired monoclonal antibody after cell fusion may be screened. Cloning of the fused cells is performed by limiting dilution or the like, and finally a hybridoma that is a monoclonal antibody-producing cell is established. As a method for collecting the monoclonal antibody from the established hybridoma, a normal cell culture method or ascites formation method can be employed.

  “Decoy nucleic acid” refers to a decoy nucleic acid (decoy oligonucleotide) that binds to a transcription factor that binds to the promoter of synoviolin and can suppress the function of synoviolin, or directly binds to a synoviolin gene and suppresses the function of synoviolin. It is a decoy nucleic acid that can

Examples of preferred decoy nucleic acids that can be used in the present invention include, for example, nucleic acids that can bind to transcription factors that bind to EBS (Ets binding site) of the Synoviolin promoter, nucleic acids that can bind to transcription factors that bind to the ABS of the promoter, promoters Examples include nucleic acids that can bind to a transcription factor that binds to SBS (Sp1 binding site), oligonucleotides containing these complements, variants thereof, or nucleic acids containing these in the molecule. The decoy nucleic acid can be designed as a single strand or a double strand including a complementary strand based on the sequence of EBS, ABS (AML binding site) or SBS. The length is not particularly limited, and is 15 to 60 bases, preferably 20 to 30 bases. The nucleic acid may be DNA or RNA, or may include modified nucleic acids and / or pseudo-nucleic acids within the nucleic acid. Further, these nucleic acids, variants thereof, or nucleic acids containing these in the molecule may be single-stranded or double-stranded, and may be circular or linear. The decoy nucleic acid used in the present invention can be produced using a chemical synthesis method or a biochemical synthesis method known in the art. For example, a nucleic acid synthesis method using a DNA synthesizer generally used as a gene recombination technique can be used. In addition, after isolating or synthesizing a base sequence serving as a template, a PCR method or a gene amplification method using a cloning vector can also be used. Furthermore, the desired nucleic acid may be produced by cleaving the nucleic acid obtained by these methods using a restriction enzyme or the like and binding using a DNA ligase. Furthermore, in order to obtain a more stable decoy nucleic acid in a cell, chemical modification such as alkylation or acylation can be added to a base or the like. A method for producing a mutant of a decoy nucleic acid can also be synthesized by a method known in the art, for example, a site-directed mutagenesis method. Site-directed mutagenesis is well known in the art, and commercially available kits such as GeneTailor ^™ Site-Directed Mutagenesis System (Invitrogen), TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc.) (Manufactured by Takara Bio Inc.)).

  “Antisense nucleic acid” means a nucleic acid having a sequence complementary to a Synoviolin gene and capable of inhibiting the Synoviolin gene expression by hybridizing to the gene. An antisense nucleic acid can be prepared by a synthetic chemical method or the like by a nucleic acid compound complementary to a partial base sequence of a gene encoding synoviolin. In order to evaluate whether or not the nucleic acid compound efficiently inhibits synoviolin production, a screening test using the gene expression level as an index may be performed. As the antisense nucleic acid compound, for example, synoviolin expression can be suppressed to at least 50% or less as compared with the control.

(2) Expression promotion of Synoviolin On the other hand, although expression of Synoviolin is not particularly limited, for example, a transcription factor, an endoplasmic reticulum stress inducer, and the like can be used.

“Transcription factor” used to promote the expression of Synoviolin means a transcription promoter that regulates the transcriptional regulatory region of Synoviolin promoter gene. For example, GABPα / β (GA-binding protein α / β) , IRE1, ATF6 (activating transcription factor 6), etc. (WO05093067, M. Kaneko et al., FEBS Letters (2002) 532: 147-152). An ER stress inducer used to promote synoviolin expression means a substance that promotes transcription by activating the ER stress response elements (ERSE) of the synoviolin promoter gene. Examples thereof include tunicamycin and thapsigargin (M. Kaneko et al., FEBS Letters (2002) 532: 147-152).
3. Pharmaceutical composition The shRNA, siRNA, low molecular weight compound, peptide, antibody, decoy nucleic acid and antisense nucleic acid produced in the present invention are substances that suppress synoviolin expression, and a pharmaceutical composition that suppresses IL-4 and IgE production. It can be used as a product (particularly a gene therapy agent for allergic diseases). The transcription factor and endoplasmic reticulum stress inducer produced in the present invention is a substance that promotes the expression of Synoviolin, and a pharmaceutical composition that promotes IL-4 and IgE production (especially treatment of infectious diseases, autoimmune diseases, leukemia) Agent).

  When the pharmaceutical composition of the present invention (a substance that suppresses the expression of Synoviolin) is used as an allergy gene therapy agent, the application site is not particularly limited, and bronchial asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis , Anaphylaxis shock, tick allergic disease, hay fever, food allergic disease and the like.

  The above-mentioned diseases may be accompanied with other diseases.

  When the pharmaceutical composition of the present invention (substance that promotes the expression of Synoviolin) is used as a therapeutic agent for infectious diseases, autoimmune diseases, and leukemias, the application site is not limited.

  Examples of infectious diseases (mainly parasitic infections) include amebiasis, roundworm, babesiosis, cryptosporidiosis, giardiasis, helminthiasis, malaria, helminthiasis, schistosomiasis, tapeworm infection, toxocariasis , Toxoplasmosis, trichinosis, trichinosis, scabies, anisakiasis, echinococcosis, foodborne parasitic helminthiasis, lice disease, larva migrans, and sarcoidosis. Synoviolin expression to treat infections A substance that suppresses is used as a pharmaceutical composition.

  Examples of autoimmune diseases include rheumatoid arthritis, Sjogren's syndrome, systemic sclerosis, polymyositis, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, bullous pemphigoid, Graves' disease (Graves' disease), Hashimoto's thyroiditis, type 1 diabetes, systemic lupus erythematosus (SLE), myasthenia gravis, pemphigus, pernicious anemia, collagen disease, hypo-IgE and a-globulinemia, etc. A substance that suppresses the expression of is used as a pharmaceutical composition.

  Examples of leukemia include myelogenesis, multiple myeloma, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, etc., and pharmaceutical compositions containing substances that promote the expression of Synoviolin Use as

  The above-mentioned diseases may be accompanied with other diseases.

  The pharmaceutical composition of the present invention can be systemically or locally administered orally or parenterally. When the medicament of the present invention is orally administered, it may be any of tablets, capsules, granules, powders, pills, troches, liquids for internal use, suspensions, emulsions, syrups, etc. It may be a dry product that is redissolved when used. In addition, when the therapeutic agent of the present invention is administered parenterally, it can be selected from pharmaceutical forms such as intravenous injection (including infusion), intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, etc. In the case of a preparation, it is provided in a unit dose ampoule or a multi-dose container.

  These various preparations include excipients, extenders, binders, wetting agents, disintegrants, lubricants, surfactants, dispersants, buffers, preservatives, solubilizers, preservatives, A flavoring agent, a soothing agent, a stabilizer, a tonicity agent and the like can be appropriately selected and produced by a conventional method.

  The above various preparations may contain a pharmaceutically acceptable carrier or additive. Examples of such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin , Propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose and the like. The additive to be used is selected appropriately or in combination from the above according to the dosage form of the present invention.

  The dosage of the therapeutic agent of the present invention varies depending on the age of the administration subject, the administration route, and the number of administrations, and can vary widely. The effective amount administered as a combination of an effective amount of the peptide of the present invention and an appropriate diluent and a pharmacologically usable carrier is 0.1 μg to 100 mg / body, preferably 1 to 10 μg / body at a time. The dose can be selected in the range of 1 to several times, and the dose is administered for 1 day or more.

  When the pharmaceutical composition of the present invention is used as a gene therapy agent, a method of administering a vector in which a nucleic acid is incorporated may be mentioned in addition to a method of directly administering the pharmaceutical composition of the present invention by injection. Examples of the vector include an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a vaccinia virus vector, a retrovirus vector, a lentivirus vector, and the like, and can be efficiently administered by using these virus vectors.

  It is also possible to introduce the pharmaceutical composition of the present invention into a phospholipid vesicle such as a liposome and administer the vesicle. The endoplasmic reticulum holding siRNA or shRNA is introduced into a predetermined cell by the lipofection method. Then, the obtained cells are administered systemically, for example, intravenously or intraarterially. It can also be administered locally to the brain or the like.

The dosage of the pharmaceutical composition of the present invention varies depending on age, sex, symptoms, administration route, administration frequency, dosage form, for example, the dosage in the case of adenovirus is about 10 ⁶ to 10 ¹³ per day And are administered at 1-8 week intervals.

In order to introduce siRNA or shRNA into a target tissue or organ, a commercially available gene introduction kit (for example, Adeno Express: Clontech) can also be used.
4). TECHNICAL FIELD The present invention provides a food, beverage or feed containing a substance that regulates the expression and / or function of Synoviolin. These foods, beverages, feeds and cosmetics are used for regulation of cytokine production, and when they contain substances that inhibit the expression and / or function of Synoviolin, they are used for antiallergic foods.

 Here, “regulation of cytokine production” means to regulate (promote or suppress) production of IL-4. Examples of cytokines that are subject to production regulation include IL-4.

  In the foods, beverages, feeds and cosmetics of the present invention, the content ratio of the substance that regulates the expression and / or function of Synoviolin is not particularly limited, and should be appropriately set according to various applications (and the types). Can do. Moreover, it does not limit about the method of obtaining the foodstuff, drink, feed, and cosmetics of this invention, The well-known manufacturing method according to various uses (and the kind) can be employ | adopted.

  The food of the present invention is not limited, for example, confectionery such as edible breads, biscuits, chocolate, jelly, ice cream, noodles such as udon, buckwheat, Chinese soba, various pasta, hamburger, ham, bacon, Examples include processed meats such as wiener, dairy products such as cheese and milk, health foods, various seasonings, and various instant foods.

  Although it does not limit as a drink of the present invention, For example, coffee, black tea, green tea, vegetable juice, fruit juice, various sports drinks, etc. are mentioned.

  The feed of the present invention is not limited, and examples thereof include livestock feed and pet food.

  Although it does not limit as cosmetics of this invention, For example, a lotion, emulsion, foundation, essence, cream, sunscreen lotion etc. are mentioned.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

  In this example, synoviolin expression analysis was performed in each immune system cell of a wild-type mouse. As each immune system cell, peripheral blood mononuclear cells, liver mononuclear cells, spleen cells (B cells and T cells) and intraperitoneal cells were used.

  Peripheral blood mononuclear cells were collected by the following method. Specifically, whole blood was collected from a wild-type mouse, and 5 mL of RPMI1640 medium (hereinafter abbreviated as “medium”) containing 10% FCS was added to the whole blood, and the mixture layered on 5 mL of Lympholyte-M (CEDARLANE) was centrifuged at 1500 rpm for 20 minutes. Went. The intermediate layer was recovered, washed twice with medium, and suspended in 1 mL of medium.

  Liver mononuclear cells were prepared by the following method. That is, the liver refluxed with the phosphate buffer was crushed with a mesh and suspended in 5 mL of 45% percoll solution. This suspension was layered on 3 mL of 67.5% Percoll solution and centrifuged at 2000 rpm for 20 minutes. The intermediate layer after centrifugation was collected, washed twice with medium, and suspended in 1 mL of medium.

  As spleen cells, organs were subdivided using a slide glass, those that passed through a steel mesh were collected, and those that were suspended in a medium after removing red blood cells were prepared.

Spleen B cells and T cells were collected by the following method. 5 μg of biotin-labeled mouse anti-CD45R antibody (RA3-6B2) was added to 250 μL of washed magnetic beads (Dynabeads) and incubated at 4 ° C. for 30 minutes to prepare a magnetic bead-labeled anti-mouse CD45R antibody. After adding 5 × 10 ⁷ spleen cells and incubating at 4 ° C. for 30 minutes, splenic CD45R positive cells were recovered using a magnet and used as spleen B cells. The remaining cells from which CD45R positive cells were removed were used as spleen T cells in the following experiments.

  As the intraperitoneal cells, a solution obtained by washing the intraperitoneal area of a mouse with 5 mL of medium was collected and resuspended in 1 mL of medium.

Each isolated cell was lysed by adding 1 mL of ISOGEN (WAKO), and total RNA was extracted according to a conventional method. DNaseI treatment and phenol chloroform treatment were followed by ethanol precipitation and recovery.
RNA was dissolved in 10 μL of DEPC water. Add 1 μL of total RNA to a mixture containing Synoviolin-specific TaqMan probe (ABI) or human18S (ABI) 0.5 μL, TaqMan Mixture (ABI) 5 μL, DEPC water 3.5 μL, and follow the 7500 Real-Time PCR System (ABI) program. RT-PCR was performed to determine the ratio of Synoviolin / 18S.

  As a result, synoviolin expression was observed in all the analyzed cells, but particularly high in B cells (FIG. 1).

  In this example, the production of antigen-specific antibodies during allergy induction in hetero knockout mice (syno +/-) of Synoviolin gene was examined. Syno +/− mice were prepared by the present inventors according to techniques well known to those skilled in the art (WO 02/052007, Genes Dev. 2003 Vol. 17: 2436-49).

  Aluminum hydroxide gel (alum) per animal for syno +/- mice and wild type mice (male: 5-9 weeks old, syno +/- 4, 2 wild type) with DBA / 1J mice as background Alum-OVA suspended in 0.2 mL of 0.1 M phosphate buffer (pH 7.5) (4 mg and ovalbumin (OVA) 10 μg) was intraperitoneally administered, and 14 days later, the same amount of alum-OVA was re-administered intraperitoneally. . On day 28, an autopsy was performed, and whole blood was collected from the heart of each mouse. Serum was collected from the obtained whole blood by centrifugation. Antigen-specific antibody production in serum was detected by ELISA.

  A 96-well plastic plate (Sumilon) was coated with OVA dissolved in phosphate buffer at 25 ng / well, and each mouse serum collected thereon was added and left at room temperature for 1 hour. After washing each well, 50 μL of 1.0 μg / mL HRP-labeled anti-mouse IgG1 antibody (LO-MG1-2) was added to each well to detect OVA-specific IgG1. For detection, a TMB substrate solution set (BD Pharmingen) was used, and after the reaction was stopped, measurement was performed at 450 nm using a microplate reader. OVA-specific IgG2a was detected using HRP-labeled anti-mouse IgG2a antibody (LO-MG2a-7) coated with 100 ng / well of OVA in the same manner as IgG1 detection.

  OVA-specific IgE was measured by the following method. Two-fold serially diluted serum was added to a 96-well plastic plate coated with 100 ng / well of anti-mouse IgE antibody (LO-ME-3) and allowed to stand at room temperature for 1 hour. After washing the wells, biotin-labeled OVA at a concentration of 5 μg / mL was added and left at room temperature for 1 hour. Biotin-labeled OVA was prepared using EZ-LinkSulfo-NHS-Biotin reagent (PIERCE). After washing each well, 1000-fold diluted ECL streptavidin HRP (Amersham) was added and left at room temperature for an additional hour. After washing each well, TMB substrate solution was added, and after stopping the reaction, measurement was performed at 450 nm using a microplate reader.

  As a result, the production of IgE specific to antigen (OVA) was decreased in syno +/−. On the other hand, no difference was observed between antigen-specific IgG1 and IgG2a (FIG. 2).

  In this example, production of total IgE and total IgM at the time of allergy induction in syno +/− mice in DBA / 1J background was examined. The serum total IgE concentration was measured using a total EIA IgE measurement kit (Yamasa), and the total IgM concentration was measured using a Mouse IgM ELISA Quantitation Kit kit (BETHYL). After washing each well, TMB substrate solution was added, and after stopping the reaction, measurement was performed at 450 nm using a microplate reader.

  As a result, total IgE production tended to decrease in syno +/− mice (FIG. 3).

  In this example, syno +/− mice and wild type mice (female: 7-13 weeks old, syno +/− 4, wild type 3) with C57BL / 6 as the background were used. The experiment was conducted. Measurement of serum-specific OVA-specific IgE, IgG1, and IgG2a was performed by the method described in Example 2. The method for measuring OVA-specific IgM in serum is described below.

  Serially diluted serum was added to a plate coated with 50 ng / well of OVA and left at room temperature for 1 hour. After washing each well, 20 ng / mL HRP-labeled IgM was added, and the mixture was further allowed to stand at room temperature for 1 hour. After washing the well, TMB substrate solution was added, and after stopping the reaction, measurement was performed at 450 nm using a microplate reader.

  As a result, the antigen-specific IgE production during alum-OVA immunization was significantly reduced in C57BL / 6 background syno +/− mice as in the case of DBA / 1J background (FIG. 4).

  In this example, the production of total IgE and total IgM at the time of allergy induction in C57BL / 6 background syno +/− mice was examined. The total IgM concentration was measured by the method described in Example 3.

  The serum total IgE concentration was measured by adding serum to a 96-well plastic plate coated with 100 ng / well of anti-mouse IgE antibody (LO-ME-3) and left at room temperature for 1 hour. After washing the wells, measurement was performed using a total EIA IgE measurement kit (Yamasa).

  The total IgG1 concentration was measured by the following method.

  Serially diluted serum was added to a plate coated with 200-fold diluted purified anti-mouse IgG1 antibody (A85-3) and allowed to stand at room temperature for 1 hour. Anti-mouse IgG1 antibody (MOPC-31C) was used as a standard. After washing each well, 50 μL of 0.1 μg / mL HRP-labeled anti-mouse IgG1 antibody was added to each well and allowed to stand at room temperature for 1 hour. After washing each well, TMB substrate solution was added, and after stopping the reaction, measurement was performed at 450 nm using a microplate reader.

  As a result, in the C57BL / 6 background syno +/− mice, as in the case of the DBA / 1J background, total IgE production during alum-OVA immunization tended to decrease in the syno +/− mice (FIG. 5).

  In this example, cytokine production during allergy induction in syno +/− mice was examined.

Spleens were extracted from mice that had been immunized with alum-OVA according to the schedule described in Example 2, and were subdivided using a slide glass and suspended in 5 mL of medium. After erythrocyte removal, cells were pooled for each of the syno +/− group and the wild type group, and each was suspended in 5 mL of medium. Cells were seeded in a 96-well culture plate so that the number of cells was 2.5 × 10 ⁵ cells / well after measurement. To each well, only the medium or OVA was added to a final concentration of 0.1 mg / mL, and cultured for 90 hours at 37 ° C. with a CO ₂ concentration of 5%. After completion of the culture, the culture plate was centrifuged at 1200 rpm for 5 minutes, and the culture supernatant was collected on a new plate. The concentrations of IFN-γ and IL-4 in the culture supernatant were detected using OptEIA Set Mouse IFN-γ (BD Pharmingen) and OptEIA Set Mouse IL-4 (BD Pharmingen). For color development, TMB substrate solution was used, and after stopping the reaction, measurement was performed at 450 nm using a microplate reader.

  As a result, when OVA stimulation was applied to the spleen cells at the time of immunization with alum-OVA, IL-4 production decreased in the syno +/− mouse group under a low dose of OVA stimulation (FIG. 6).

  In this example, the expression of IL-4 receptor in syno +/− mouse spleen cells was examined.

Spleens were extracted from whole blood collected from syno +/− mice and wild type mice (female: 9-16 weeks old, syno +/− 3, 3 wild type) with DBA / 1J as background. Spleen cells were prepared by subdividing an organ using a slide glass, collecting those that passed through a steel mesh, removing red blood cells, and then suspending them in a medium. Anti-mouse CD16 / CD32 antibody (2.4G2) was added as a blocking solution to the spleen cells adjusted to 1 × 10 ⁶ / tube, incubated at 4 ° C. for 30 minutes, and then the excess blocking solution was removed. 1 μg each of FITC-labeled anti-mouse TCRβ antibody (H57-597), PE-labeled anti-mouse IL-4 receptor antibody (mIL4R-M1) as a T cell marker, and biotin-labeled anti-CD45R antibody as a B cell marker were added at 4 ° C. Incubated for 30 minutes. After washing twice with a staining buffer (phosphate buffer containing 1% BSA), 0.25 μg of streptavidin PerCP-Cy5.5 (BD) was added, and further incubated at 4 ° C. for 30 minutes. A sample suspended in 1 mL of staining buffer after washing three times was taken in with FACS Callibar (BD) and analyzed using CellQuest (BD).

  As a result, there was no difference in the expression ratio of IL-4 receptor in spleen cells between syno +/− mouse cells and wild type mice (FIG. 7).

  In this example, the expression of ICOS, which is a receptor involved in IL-4 production, was examined using syno +/− mouse spleen cells. The following experiment was performed using spleen cells of syno +/− mice and wild type mice prepared by the method described in Example 7.

Add 1 μg each of FITC-labeled anti-mouse TCRβ antibody (H57-597) and biotin-labeled anti-mouse ICOS antibody (7E.17G9) as T cell markers to 1x10 ⁶ spleen cells prepared for flow cytometry at 4 ° C. And incubated for 30 minutes. After washing twice with a staining buffer (phosphate buffer containing 1% BSA), 0.25 μg of streptavidin PerCP-Cy5.5 (BD) was added, and further incubated at 4 ° C. for 30 minutes. A sample suspended in 1 mL of staining buffer after washing three times was taken in with FACS Callibar (BD) and analyzed using CellQuest (BD).

  As a result, the ICOS expression ratio in spleen T cells was not different between the syno +/− mouse cells and the wild type mice (FIG. 8).

  In this example, cell proliferation reaction was examined when IL-4 stimulation was applied to spleen cells.

Spleen cells were collected from syno +/− mice and wild type mice (3 females each) and seeded in 96-well plates at 5 × 10 ⁵ cells / well. Recombinant mouse IL-4 (PeproTech EC) was added at a final concentration of 1 and 10 ng / mL, and LPS was added as a positive control to a final concentration of 1 μg / mL, and 24 hours at 37 ° C in the presence of 5% CO _2. Then, 20 μL of WST-8 (Dojindo) was added, and 4 hours later, measurement was performed at a wavelength of 450 nm using a microplate reader.

  As a result, no significant difference was observed between the syno +/− group and the wild type group (FIG. 9).

  According to the present invention, there are provided a pharmaceutical composition containing a substance that regulates the expression and function of Synoviolin, and a food, beverage, feed or cosmetic containing the substance. In the present invention, substances that suppress the expression and function of Synoviolin are useful as pharmaceutical compositions such as antiallergic agents.

The figure which shows the expression analysis of Synoviolin in each immune system cell of a wild type mouse. The figure which shows the antigen-specific antibody production at the time of allergy induction in syno +/- mouse | mouth (DBA / 1J). The figure which shows the antibody production (total) at the time of allergy induction in syno +/- mouse | mouth (DBA / 1J). The figure which shows the antigen specific antibody production at the time of allergy induction in syno +/- mouse | mouth (C57BL / 6). The figure which shows the antibody production (total) at the time of allergy induction in syno +/- mouse (C57BL / 6). The figure which shows the cytokine production at the time of allergy induction in a syno +/- mouse | mouth (DBA / 1J). The figure which shows the expression of IL-4R in a syno +/- mouse | mouth spleen cell. The figure which shows the expression of ICOS in a syno +/- mouse | mouth spleen cell. The figure which shows the proliferation response with respect to IL-4 stimulation of a syno +/- mouse | mouth spleen cell.

Claims (13)

  A pharmaceutical composition comprising a substance that regulates IL-4 production and / or IgE production.   The pharmaceutical composition according to claim 1, wherein the IL-4 production and / or IgE production regulator inhibits or promotes synoviolin expression and / or function.   The substance that inhibits the expression and / or function of Synoviolin is at least one selected from the group consisting of Synoviolin or a small molecule compound for Synoviolin-encoding gene, peptide, antibody, decoy nucleic acid, antisense nucleic acid, siRNA and shRNA. The pharmaceutical composition according to claim 2.   The pharmaceutical composition according to claim 3, wherein the gene encoding synoviolin comprises the base sequence represented by SEQ ID NO: 1.   The pharmaceutical composition according to claim 3, wherein the siRNA targets a part of the nucleotide sequence shown in SEQ ID NO: 1.   A pharmaceutical composition for preventing and / or treating an allergic disease, infectious disease, autoimmune disease or leukemia, comprising a substance that regulates the production of IL-4 and / or IgE.   The allergic disease is at least one selected from the group consisting of bronchial asthma, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, anaphylactic shock, mite allergic disease, hay fever and food allergic disease. Pharmaceutical composition.   Infectious diseases are amebiasis, roundworm, babesiosis, cryptosporidiosis, giardiasis, helminthiasis, malaria, helminthiasis, schistosomiasis, tapeworm infection, toxocariasis, toxoplasmosis, trichinosis, trichinosis The pharmaceutical composition according to claim 6, which is at least one selected from the group consisting of: scabies, anisakiasis, echinococcosis, food-borne parasitic helminthiasis, lice disease, larva migrans, and sarcoidosis.   Autoimmune diseases include rheumatoid arthritis, Sjogren's syndrome, systemic sclerosis, polymyositis, Guillain-Barre syndrome, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, bullous pemphigoid, Graves' disease (Basedow) Disease), Hashimoto's thyroiditis, type 1 diabetes, systemic lupus erythematosus, myasthenia gravis, pemphigus, pernicious anemia, collagen disease, hypo-Igemia and agammaglobulinemia The pharmaceutical composition according to claim 6.   The leukemia is at least one selected from the group consisting of myelogenesis, multiple myeloma, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic white blood and chronic myelogenous leukemia. Pharmaceutical composition.   A food, beverage or feed for regulating cytokine production, comprising a substance that regulates the expression and / or function of synoviolin.   An antiallergic food, beverage or feed comprising a substance that inhibits the expression and / or function of Synoviolin. A method for regulating IL-4 and IgE production, which comprises regulating the expression and / or function of Synoviolin.

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