WO1996027391A1 - Drug for treating immunity-related diseases and method of searching for the same - Google Patents

Abstract

A method of searching for chemicals capable of suppressing the production of cytokines in T cells, which closely relate to the establishment of immunity-related diseases, at the gene transcription level so as to enable the cure of these diseases through the clinical application of this chemical; and a drug for treating immunity-related diseases which comprises as the active ingredient a macrolide antibiotic capable of suppressing the production of at least one cytokine in human T cells.

Description

 Harm

 Agent for treating immune-related disease and method for searching for the same

[Technical Field of the Invention]

 The present invention relates to an agent for treating an immune-related disease and a method for searching for the same. It makes possible the treatment of immune-related diseases.

 In the present specification, “IL” indicates interleukin.

 [Prior art]

 Conventionally, for the treatment of immune-related diseases (allergic disease, eosinophilic inflammatory disease, collagen disease, autoimmune disease, rheumatoid arthritis, etc.), palliative treatment using exclusively antipyretic, analgesic, anti-inflammatory drugs, etc. Treatment has been performed with dohormon. On the other hand, for immune-related diseases, the involvement of various cytokines produced by T cells has been proven for each disease state. In particular, IL-2, IL-14, IL-15 and IL-18 are closely and importantly involved. Involvement has been suggested. Therefore, it is thought that suppressing the production of at least one of them will be effective in treating immune-related diseases.

By the way, the above-mentioned T-cell cytokine is originally a useful substance for living organisms if it is in an appropriate amount.Therefore, as long as its production is performed normally, it is necessary to suppress production. Rather, it can be said that suppression of production is not desirable. Therefore, it is desirable to suppress the production of cytokines whose production is abnormally enhanced, but not to suppress the production of cytokines whose production is performed normally, that is, it is desirable to perform selective suppression. Thus, it is understandable that the selective suppression of cytokine production is desirable. The fact that selective production of cytokines has been selectively performed has not been reported so far, and it is not clear whether such selective suppression of cytokine production is possible. Moreover, it was not known whether there were substances that would allow such selective suppression.

 For example, steroids are commonly used for cell cytokines such as IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-8, IL-12, 11-13, etc. Although it is known to suppress production universally, it is not known to suppress the production of specific cytokines. In addition, steroids have the disadvantage of causing a wide variety of powerful side effects, such as diabetes, hypertension, obesity, cataracts, and psychiatric disorders, when administered in overdose because they have general effects on organs and tissues throughout the body. is there. Therefore, we often encounter cases where the dosage is limited and we cannot administer a sufficient amount.

 Also, conventionally known immunosuppressive drugs such as cyclosporin FK506 are T cell-specific drugs and do not universally act on organs and tissues. Is limited. However, these substances also produce T-cell-produced sites, i.e., IL-2 IL-13, IL-4, IL-5. Production of IL-16, IL-18, IL-13, etc. It is a general and widespread control, which leads to a general suppression of immunity. Therefore, it is reasonable to assume that improving the condition with immunosuppressants is only one part of overall immunosuppression.

 [Problems to be solved by the invention]

The present invention is directed to IL-12, IL-14, IL-5 and IL-18, which have been suggested to be particularly closely related to immune-related diseases, among T cell cytokines, and at least one of them. Substances that substantially inhibit production and do not substantially inhibit at least one type of production, especially production inhibition The technical problem is to search for a substance that achieves the above by suppressing gene transcription, and to treat an immune-related disease using such a substance.

 [Means to solve the problem]

 In order to solve the above-mentioned problems, the present inventors have developed human T cells, such as human T cell clones, human T cell hybridomas, and human normal peripheral blood T cells, which have the potential to produce cytokines. Cells, that is, cytokine カ gene-expressing human T cells are grown and stimulated in the presence of the test substance to determine whether the desired cytokine is produced in the culture. We searched for a wide range of substances by the method of observation. As a result, they found that macrolide antibiotics (including their derivatives) contain a large number of drugs that selectively inhibit only specific cytokines.

 That is, the present invention suppresses the production of a specific cytokine (not limited to one species) among T cell cytokines, and produces another specific cytokine (not necessarily one species). It is possible to search for drugs that do not suppress the disease. Whether such selective production suppression at the gene level is actually feasible has not been known so far, and it is the first time that the present invention enables such selective production suppression. Confirmed. As a result, it was possible to proceed with the search in the future while expecting success, and in this respect, the technical significance of the present invention is extremely large.

The gist of the present invention is to suppress the production of at least one of the cytokines IL-12, IL-4, IL-15 and IL-18 to be produced therein in human T cells, and An agent for treating an immune-related disease, comprising as an active ingredient a substance that does not suppress the production of lipase. In this case, the suppression of cytokine production is achieved by suppressing the transcription of the cytokine gene (for example, m-RNA) in human T cells, and particularly by suppressing the abnormal increase in transcription. As described above, it is conventionally known whether selective suppression, in which transcription of a certain cytokine gene is suppressed but not transcription of another certain cytokine gene is actually possible, is possible. The present invention, which has never been confirmed and the possibility of which has been confirmed for the first time, can be said to be based on knowledge that could not be predicted at all in this respect.

 In the present invention, searching for a drug having a desired selective cytokine inhibitory activity is performed using cytokine gene-expressing τ cells. For example, the present inventors have previously succeeded in cloning and expanding τ cells that recognize allergens from peripheral blood lymphocytes from an allergic patient, but have obtained the T cell clone obtained here. Establish a T cell hybridoma by cell fusion with a T cell tumor line. A test substance is added to a medium in which these T cell clones, T cell hybridomas, and normal peripheral blood τ cells grow, and the cells are cultured with an activation stimulus. IL-12 in the culture (eg, supernatant) is added. Observe the production of IL-14, IL-15 and Z or IL-18. From the results, it is determined whether or not the test drug has the property of inhibiting the production of any one type of cytokine but not inhibiting the production of any one type of cytokine. In this way, for example, a relatively large number of macrolide antibiotics showing such selective suppression could be found.

The cytokines produced by T cells include interleukins (IL-1, IL-12, I-3, IL-4, IL-15, IL-16, IL-7, IL-8), interferon (IFN, IFN / 9, IFNa), granulocyte macrophage. Colony stimulating factor (GM—CSF), granulocyte 'colony stimulating factor (G—CSF), macrophage colony stimulating factor (M—CSF) , CSF-1 1), macrophage migration inhibitory factor (MIF), etc., are known. IL-12, IL-14, IL-15 and IL-8 that are deeply involved in c-protein.c It is preferable that the production of other cytokines does not particularly affect the production of cytokines. Whether or not to give is not important for the purpose of the present invention to provide a therapeutic agent for an immune-related disease.

 The major immune-related diseases that are generally recognized as involving IL-12, IL-14, IL-15 and IL-18, respectively, are:

 IL-2: collagen disease (systemic erythematosus, polymyositis, cutaneous I * myositis, scleroderma, MCTD), rheumatoid arthritis, etc .;

 I L-4: immediate hypersensitivity (hay fever, allergic rhinitis, allergic conjunctivitis), IgE mast cell-dependent hypersensitivity, etc .;

 I L-5: Allergic or eosinophilic diseases (bronchial asthma, atopic dermatitis, hay fever, allergic rhinitis, allergic conjunctivitis, hypereosinophilic syndrome, allergic vasculitis, eosinophils Fasciitis, eosinophilic pneumonitis, PIE syndrome), etc .;

 IL-8: Uveitis, Behcet's disease, etc.

 The present invention, which has been shown to suppress the production of any one of these cytokines, is useful for treating the above-mentioned immune-related diseases involving the cytokine. The agent for treating an immune-related disease found by the present invention, for example, suppresses the production of IL-12, but at least one of IL-14, IL-5 and IL-18 (for example, IL-4 and IL-15). Does not suppress the production of IL-15, or suppresses the production of IL-15. Does not suppress the production of at least one of IL-2, IL-14, and IL-18 (for example, IL-12 and IL-14). And the production of both IL-14 and IL-15 but not the production of at least one of IL-12 and IL-18 (eg, IL-2). I suppress the production of 5 and IL-8,

-0- Some do not suppress IL-12 production.

 In particular, taking drugs that suppress the production of IL-15 as examples, such drugs can be used to treat allergic diseases such as bronchial asthma, atopic dermatitis, hay fever, allergic rhinitis, allergic conjunctivitis, etc. It can be used for It can also be used to treat eosinophilic inflammatory diseases, such as hypereosinophilic syndrome, allergic vasculitis, eosinophilic fasciosis, eosinophilic pneumonitis, and PIE syndrome. I can do it.

 As an example of a preferable object of the search according to the present invention, the above-mentioned macrolide antibiotics can be mentioned. The ability of the macrolide antibiotics to inhibit cytokine production found by the present invention is based on the fact that the macrolide antibiotics suppress abnormal transcription of m-RNA, particularly m-RNA transcription, of the cytokine in T cells. It is by doing.

The term “macrolide antibiotics” in the present specification is used in its broadest sense, and refers to multi-membered lactones having many methyl side chains or 4- to 4-conjugated double lactones. (Including those having 4 to 7 conjugated double bonds are sometimes referred to as “polyene antibiotics”). _C Usually, another group, for example, “macrotetroid antibiotics” Substances classified as "substances" are also included in the category of "macrolide antibiotics" in this specification in that they are multi-membered lactones having many methyl side chains.

As a macroscopic antibiotic in a narrow sense, a lactone structure consisting of 10 to 40 ring atoms is known today, but most of them are 12-membered rings and 14-membered. Ring and 16-membered ring. Specific examples include 12-membered rings such as methymycin and neomethymycin, and 14-membered rings such as erythromycin, picokumycin, and narvomycin. And 16-membered rings such as leucomycin, spiromycin, rozamycin, sirlamycin, juvenimycin, delltamicin, carbomycin, angolamycin, Thai cinnacin and mycinamicin. Specific examples of those usually classified as “macrotetroid antibiotics” include nonactin (), monactin, dinactin, trinactin, and tetranactin. These “macrolide antibiotics” are generally produced by actinomycetes / micromonospora, but the present invention is not limited to those produced directly by these bacteria, and those obtained by appropriately modifying these And derivatives thereof.

 For macrolide antibiotics (including their derivatives), see U1 lmann's Encyclopedia of Industrial Chemistry ^ 5 fiR, Volume A2, pages 494-496 (1985), Biotechnology ^ A Comprehensive Treatise ^ 4, 359-363 (1986), published by Hirokawa Shoten, Kenji Maeda et al., “Development of Pharmaceuticals 5-Antibiotics and Antibacterials”, 10-11 (1990), University of Tokyo Press, Nobuo Tanaka A wide range of descriptions exist for various academic harms, such as “Analysis of Antibiotics and Chemistry and Biological Activities”, pages 128-159 and 250-251 (1992). And their descriptions are incorporated herein as a part of the present specification.

 The general relationship between individual macrolide antibiotics and the sites at which production is blocked is not known at this time, but this may be necessary, as described above and in more detail below. It can be easily confirmed by the search method. .

式中、 R,、 R₂、 R₃および R₄はそれぞれ独立して炭素数 1〜6のアル キル基であり、 これらは同一であってもそれぞれ異なっていてもよい。 好 ましくは R,、 R₂、 R₃および R₄がメチル基またはェチル基である。 具体 例としては、 ノナクチン、 モナクチン、 ジナクチン、 トリナクチン、 テト ラナクチンなどを挙げることが出来る。 For example, macrolide antibiotics of the formula (I) typically selectively inhibit IL-15 production but do not inhibit IL-2 or IL-14 production:

In the formula, R, R ₂ , R ₃ and R ₄ are each independently an alkyl group having 1 to 6 carbon atoms, which may be the same or different. Preferably, R, R ₂ , R ₃ and R ₄ are a methyl group or an ethyl group. Specific examples include nonactin, monactin, dinactin, trinactin, and tetranactin.

 In addition, for example, a macrolide antibiotic represented by the following formula (Π) usually suppresses the production of IL-12, but does not substantially suppress the production of IL-4 or IL-5 Or conversely, it inhibits the production of IL-4 and IL-5, but does not substantially inhibit the production of IL-2, etc .:

式中、 R₅は、 それが結合する炭素原子と共にォキシム化されていても よいカルボニル基、 R_eおよび R₇はヒ ドロキシ基またはそれぞれ一緒になつ て一 0(CH₂)₂0—、 一 0(C = 0)0—、 -NH (CH₂) ₂0—または一 NH (C = 0) 0—を形成する。 R₈は〉 C = 0または〉 CH (0-R) (た だし、 Rは糖残基またはァシル基） 、 R₉は糖残基、 R₁₀は （低級） アル キル化されていてもよいヒ ドロキシ基である。 ただし、 R₅がそれが結合 する炭素原子と共にォキシム化されたカルボニル基であって、 R_eと R₇が それぞれ一緒に一 NH (CH₂) ₂0—または一 NH (C = 0) O—を形成 する場合、 前者の中の窒素原子と後者の中の窒素原子が低級アルキレン鎖 を介して連結していてもよい。

Where R ₅ is oxidized with the carbon atom to which it is attached Good carbonyl group, R _e and R ₇ an 0 (CH ₂₎ Te summer with human Dorokishi group or respectively ₂ 0-, single 0 (C = 0) 0-, -NH (CH 2) 2 0- or a NH (C = 0) forms 0—. R ₈ may be> C = 0 or> CH (0-R) (where R is a sugar residue or an acyl group), R ₉ may be a sugar residue, and R ₁₀ may be (lower) alkylated It is a hydroxy group. Where R ₅ is a carbonyl group oxidized together with the carbon atom to which it is attached, and R _e and R ₇ are each taken together with one NH (CH ₂ ) ₂₀ — or one NH (C = 0) O — When forming the above, the nitrogen atom in the former and the nitrogen atom in the latter may be connected via a lower alkylene chain.

 The oxidized carbonyl group represents, for example, a group represented by the formula:> C = NOR '. In the formula, R ′ may be a hydrogen atom, a substituted or unsubstituted lower alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aryl lower alkyl group.

Examples of the acryl group represented by R include a substituted or unsubstituted lower alkanol group, an aryl group substituted or unsubstituted on an aryl group (lower) alkanol group, and an aryl group substituted or unsubstituted on an aryl group. Examples thereof include a carbonyl group. Sugar residue represented by R or R _e may be a sugar residue Makurorai de antibiotics has usually as a specific example, D- Dekosamin, D- mycaminose, D- angolosamine, D-Forosamine, L-Megosamine, D-Calcose, D-Aldgarose, 4, 6-Dideoxy D-Threohexos 1-3-Perose, D-Micinose, 6-Doxy D-aroose, 6-Doxy 2-O-methyl-D-alose, L-micarose, cladinose, L-orean drose, L-sinerulose VIII, L-alcoholic north, 3-methyl-2,3,6-tridoxy-1 L-threohexase 2-enobiranose and These derivatives and the like O

 In the present specification, the term "lower" usually indicates a group having no more than 8 carbon atoms, particularly a group having no more than 5, and the term "aryl group" means a heterocyclic aromatic group (for example, pyridyl group). , Pyrimidyl), but usually represents a carbocyclic aromatic group such as phenyl. Examples of the substituent which may be present in the alkyl group, aryl group, etc. include, for example, lower alkyl, halogen, hydroxy, lower alkyne, nitro, amino, lower alkylamino, di (lower) alkylamino, phenyl, fuunyl (lower) alkyl, Examples include phenyl lower alkenyl and the like, and one or more of them may be used. If there are substituents, the number is 5 or less, usually 1-3.

Among the compounds of the above formula (II), particularly those compounds in which R ⁵ is, for example, the formula:> C = NOR ”, substantially suppress the production of IL-14 and IL-15, such as ^ IL-2. In the formula, R "represents an aryl lower alkyl group which may be substituted on an aryl group (especially a phenyl group) or a phenyl lower alkenyl aryl group (eg, a styrylphenyl group). You.

 These substances may be produced by cultivation of actinomycetes or Micromonospora, and such products may be subjected to conventional oximation, ketalization, imino ketalization, etc. It can be manufactured by modifying it.

Hereinafter, the present invention will be described in detail mainly with reference to macrolide antibiotics of the formula (I) as typical examples. However, macrolide antibiotics of the formula (II) can be used in the same manner. The specific example that is presently preferred is nonactin, but monactin, dinactin, trinactin, tetranactin and the like corresponding to the modified form of the side chain are similar to nonactin for the purpose of the present invention. May be used.

 Cyclosporine and FK506, which are T cell-specific immunosuppressants known to date, are both macrolide antibiotics produced by actinomycetes. Nonactin, monactin, dinactin, trinactin, tetranactin and the like used as the treatment agent of the present invention are also macrolide antibiotics produced by actinomycetes.

 Of these, tetranactin is known to suppress the proliferation of human T cells and the induction of NK cells [Tanouchi et. Al., Immunopharmacology, 16, 25-32 (1988) 11). It also has an inhibitory effect on antibody production and has been reported to treat experimental autoimmune grape retinitis in rats in animal models [Tanouchi et.al., Jpn. J. Ophthalmol. 31, 218-229. (1987); Shichi et. Al.. U.S. Patent No. 4,843,092, Jun. 27, (1989); Tanouchi et. Al., Immunology, 63, 47 1-475 (1988)].

 Based on these findings, nonactin and its analogs can suppress autologous diseases such as suppression of rejection associated with organ transplantation, rheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis, uveitis and thyroid autoimmune diseases. Patents have been filed for the treatment of immune diseases [see Shichi et. Al., US Pat. No. 4,843,092, Jun. 27, (1989)]. However, the above documents do not disclose or suggest the use of the same as a therapeutic agent for so-called allergic diseases or eosinophilic diseases. Prior to the filing of the present invention, the fact that nonactin and its analogs suppressed IL-15 telegene transcription, mRNA expression and protein production in human T cells was not known.

According to the present invention, it has been found that nonactin and analogs thereof suppress the production of human IL-15 in human T cells. Furthermore, the mechanism is that these substances are involved in human IL-15 gene transcription and transcription in human T cells. It has been shown that the production of protein is suppressed by suppressing the expression of niRNA.Taking nonactin as an example, T cell activity can be determined by isolating and culturing peripheral blood mononuclear cells from peripheral blood of allergic patients. When stimulated with concanavalin A, a conjugating agent, and simultaneously adding nonactin, nonactin suppressed the production of IL-15 in a dose-dependent manner (Fig. 1). Concanavalin A has a function of activating T cells via T cell antigen receptors. It has been demonstrated by the present inventors that when peripheral blood mononuclear cells are stimulated with concanapalin A, IL-15 is almost entirely produced from CD4 ⁺ T cells. Therefore, it is considered that nonactin suppressed the production of IL-15 from CD4 + T cells. To clarify that nonactin acts directly on T cells, an allergen-specific T cell clone was established and IL-2 was immobilized using an immobilized anti-CD3 monoclonal antibody, a T cell receptor-mediated stimulator. When nonactin was added to 15 high-producing T cell clones, it was observed that IL-15 production was completely suppressed at a concentration of 10 OnM or more (Fig. 2).

To determine whether the inhibitory effect of nonactin on IL-15 production is through suppression of IL-15 gene expression (mRNA expression), the same experiment as in Fig. 1 was performed, and RT-PCR ( Reverse Transcription-Polymerase Chain Reaction & {From this, we demonstrated that non-actin suppresses the expression of IL-5¾fc in a site-dependent manner (Fig. 3). Inhibiting IL-15 raRNA expression by directly acting on cells suppresses IL-15 raRNA expression induced in clones of T cells stimulated with phorbol ester and CA ++ ionophore. (Figure 4). When T cells are activated in vivo by antigens, which are physiological activators, protein kinase C (PKC) is intracellularly activated by an activation signal via a cell receptor. It is known that activation and simultaneous Ca ⁺⁺ influx occur. Therefore, a phorbol ester having functions to directly activate ^{P KC (PMA), Ca +} + causes influx Ca ^{+ +} Ionofoa, e.g. Ionomaishin: by both the (I onomyc i I 0M), T cell antigen It can give the same level of activation as when stimulated with, and is commonly used in many immunological experimental systems.

Furthermore, in order to examine the effect of IL-15 on gene transcription, the above T cell clone was fused with a human T cell tumor cell line to produce a human IL-15 gene-expressing T cell hybridoma. did. Pi L-5 Luc (Fig. 6) in which the DNA of the promoter Z enhancer region located 5 'upstream of the transcription start point of the human IL-15 gene was connected to the luciferase gene, a reporter gene. Using a gene transcription experiment system obtained by transient transfection into T cell hybridomas, a nonactin gene transcription suppression experiment was performed. pi L-5 Luc is temporarily transduced into the cells of the IL-15 gene-expressing T cell hybridoma, and is stimulated into the cell by the stimulation of PMA + I0M. (Transcription factor) recognizes the IL-15 promoter Z enhancer region, binds it and transcribes the luciferase gene located downstream, so that luciferase mRNA appears in the cell and luciferase protein is expressed. Produced quickly. Luciferase mRNA, unlike mRNA of cytokine, has a constant stability and a constant rate of protein synthesis from mRNA, so that nonactin acts on the IL-15 gene promoter noenhancer. Can be determined. In this experimental system, 100 nM of nonactin and 1 M of human IL-15 gene It was observed that it inhibits the expression of the function of the oral motor enhancer and represses the transcription of the IL-15 gene (Fig. 7).

 Therefore, nonactin and its analogous compounds are useful as remedies for allergic diseases and eosinophilic diseases, like FK506, cyclosporin A, etc., which have IL-15 inhibitory activity. It is.

 On the other hand, when the effect of nonactin on IL-2 and IL-4 was examined using a similar experimental system, nonactin did not suppress the production of IL-12 and IL-14 (Fig. 5). Did not suppress gene transcription at all (Figs. 8 and 9). Therefore, nonactin has a small effect on other immune systems such as infectious immunity, that is, it can avoid the side effects of cyclosporin FK506 such as susceptibility to infection due to reduced immunity and the possibility of developing malignant tumors. It is expected. In addition, it has the advantage that it can be used as a safe clinical treatment, avoiding the aforementioned various side effects of corticosteroid drugs.

 Furthermore, in an in vivo test using BDF1 mice sensitized with ovalbumin, the effect of nonactin on eosinophil accumulation was tested, and it was confirmed that nonactin blocks eosinophil accumulation (Fig. 1). 0). This indicates that the compound of the present invention suppresses eosinophilic inflammation in vivo, and demonstrates the usefulness of using the compound of the present invention as a therapeutic agent for allergic diseases and eosinophilic diseases. It is shown.

Drugs such as the compounds of formulas (I) and (II) which have been shown by the present invention to inhibit the production of at least one cytokine and not to inhibit the production of at least one cytokine are It can be used for the treatment of various immune-related diseases described above, such as allergic diseases and eosinophilic diseases. To administer such a treatment, one of these drugs may be used alone or in combination of two or more. At that time, administer those drugs as they are Or a solid or liquid pharmaceutical carrier into granules, tablets, hard capsules, capsules, granules, powders, fine granules, emulsions, suspensions or liquids. Orally, or as an injection, intravenous, intramuscular, intradermal or subcutaneous. Injectables can also be prepared from powders at the time of use. On the other hand, it is used as a suppository, nasal drop, eye drop or inhalant in the form of topical preparation for rectal, nasal, ocular and pulmonary administration. In the case of inhalants, it is used as an aerosol in the form of an aqueous solution or a partial aqueous solution. Oral preparations may also be administered as enteric agents.

 The ratio of active ingredient drug to carrier component, in solid form, varies from 0.01% to 90% by weight. Suitable solid carriers include lactose, dextrin, starch, calcium carbonate and the like. Preparation of drug product according to ft'Ά

 In liquid form preparations, ie, emulsions, suspensions, syrups, solutions and the like, water, alcohols and their derivatives and oils (such as fractionated coconut oil and peanut oil) are used as carriers. In the case of parenteral administration, carriers are also used in the form of oily esters such as ethyl oleate and isopropyl myristate. Preparation of pharmaceutical preparations is carried out by a conventional method.

 The drug can be administered topically as a solution, descendant, cream, or lotion containing the required amount of the drug in an acceptable excipient to achieve its clinical effect.

Dosage may vary depending on the particular drug employed, the route of administration, the severity of the symptoms encountered and the particular patient being treated. Generally, it is preferred to administer the treatment of the present invention in the range of 0.1 to 10 OmgZkg per day. Clinical safety doses will be determined in future clinical safety studies (Phase I). Furthermore, the optimal dose and blood concentration are determined by conducting an effective concentration test (Phase II). can do,

【Example】

 Hereinafter, the present invention will be described more specifically with reference to examples.

 Example 1: Nonactin suppression of IL-15 production

 (1) Suppression of IL-15 production of peripheral blood mononuclear cells in allergic patients

 The present inventors have already reported that IL-15 production of peripheral T cells in allergic patients has been enhanced [Mori A. et al. International Immunology 7). In this experiment, blood was collected with consent from several allergic patients with markedly enhanced IL-15 production and used in the following studies.

That is, venous blood of an allergic patient was collected, and peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Paque specific gravity centrifugation. At 2 × 10 ⁶ cells / well, the cells were suspended in A11 ^!-¥ medium (Gibco BRL) and cultured in a 24-well plate (Corning). 10 zzgZml of concanapalin A (hereinafter referred to as ConA) was added and stimulated, and simultaneously, nonactin (manufactured by Sigma) was added. After culturing for 48 hours, the culture supernatant was collected and measured by a specific IL-15 ELISA method. The results obtained are shown in FIG. As is clear from the figure, nonactin suppressed the production of IL-15 in a dose-dependent manner.

 (2) Inhibition of IL-15 production of allergen-specific T cell clones

Allergic patients peripheral blood mononuclear cells were stimulated with Der f II protein, one of the major allergens contained in house dust, and T cell clones were established by limiting dilution (llori A). et al. International 1 Archives of Allergy and Immunology. 1995]. A clone producing IL-15 (1 x 10 ⁵ cells / well) upon antigen stimulation was cultured in a 96 / well round bottom culture plate. Apply 10 / zgZinl anti-CD3 monoclonal antibody (OKT3: Janssen Kyowa Co., Ltd.) Stimulation via the D3 molecule produced IL-15 from the cell clone. Nonactin completely suppressed IL-15 production at concentrations of 10 OnM or higher (Fig. 2).

 Example 2: Nonactin suppresses expression of IL-15 telegene

 (1) Allergic patients Inhibition of IL-15 mRNA expression in PBMC

 The study of IL-5niRNA expression was performed by the reverse transcription polymerase chain reaction (RT-PCR) [see Mori A. et al. International Immunology: 7].

PBMCs were isolated from peripheral blood of allergic patients as described above, cultured in 24-well plates (manufactured by Corning) at a concentration of 2 × 10 ^β- cell Nowell, and stimulated with ConA (10 / g / ml). Nonactin was added simultaneously with the start of the culture. Eight hours later, the cells were collected, and 1 ml of isogen (Isogeiu Nippon Gene) was added to the pellet to lyse the cells. The mixture was homogenized 10 times with a 5 ml injection cylinder with a 25 G needle. After addition of a black mouth form 200 // 1, the mixture was thoroughly mixed and centrifuged at 13000 rpm for 15 minutes at 4 ° C. The aqueous layer was collected, and isopropyl alcohol 5001 was added. After the stirring, the mixture was cooled for one hour or more at one end. After centrifugation at 13000 rpm for 15 minutes at 4 ° C, decantation was performed, and the pellet was washed by adding 80% ethanol. After centrifugation at 1300 Orpm. For 15 minutes at 4 ° C, decant the pellet, and add TE buffer-(lOmll Tris-HC1, pH 8.0, 5nH EDTA) 400 ΐ. 3M sodium acetate solution (ΡΗ5.3) 40 ^ 1, 100% ethanol (lm1) was added, and the mixture was cooled at 120 ° C for 1 hour or more. After centrifugation at 13000 rpm for 15 minutes at 4 ° C, the solution was decanted, dried at room temperature, and the pellet was dissolved in distilled water at 10 / z1.

After incubating for 70 minutes and 4 minutes, it was rapidly cooled on ice. Next, 9.5 1 of the RT reaction solution shown in Table 1 below was added, and reacted at 42 ° C for 3 hours to synthesize cDNA. (Reverse transcription: RT) ₀

 【table 1】

 5 First Buffer 4 βΐ

 0.1M DTT 1 ul

 RNasin 40U / / 1 1 \

 dNTPmixd OmM) 1 β \

 Random 'primer 1.5 ^ 1 reverse transcriptase

 After the reaction was completed, PCR reaction solutions shown in Table 2 below were prepared, and 451 PCR reaction solutions were added to cDNA (51).

 [Table 2]

 1 Ox PCR reaction buffer 5 1

2.5mM MgCl ₂ 3 l sterile water 32.6 1 dNTPmixCl OmM) 2 ΐ

20 jc M 5'—IL-5 primer 1 a

 20 jt / M 3'—IL-15 primer 1 zl 20 βΜ5 '— / Sactin primer 1 ϊ20 Μ3' — ^ actin primer 1 \ Taq polymerase (5UZ zl) 0.4 fil Further, 331 light mineral oil (Light mineral oil: manufactured by Sigma) was added, and the PCR method was performed according to the protocol shown in Table 3 below.

 [Table 3]

Process 1

2: 30 95 ° C 1:30 72

 2:00 60. C

Step 2—30 cycle iterations

 1: 00 95 ° C

 1:30 72. C

 2:00 60. C

 After completion of the PCR method, add 2/1 6X Typelll dye (manufactured by Nippon Gene) to each sample 10 1 and electrophorese on a 0.5XTBE 1% agarose gel.

O performed o

 Actin was used as a positive control.

 As a result, nonactin significantly suppressed IL-15 mRNA expression at a concentration of 10 OnM or more (FIG. 3).

 (2) Inhibition of IL-15 mRNA expression of allergen-specific T cell clones The Der f II-specific T cell clones described in Example 1 and (2) were replaced with phorbol 12-myristate 13- The cells were stimulated with acetate (PMA) 2 OnM and ionomycin (10M) 1, and the cells were collected 6 hours later. Nonactin was added simultaneously with the start of culture.

RNA was extracted in exactly the same manner as in (1), and IL-5 mRNA expression was examined by RT-PCR. As is evident from FIG. 4, nonactin did not affect the gene expression of yS-actin used as a positive control and efficiently suppressed the mRNA expression of IL-15.

Example 3 Effect of Nonactin on IL-12 and IL-14 Production Peripheral blood of an allergic patient was collected, and PBMC was isolated in the same manner as in Example 1. In 24 © El plates were cultured at a concentration of 2 X 10 ^beta cells / Ueru were stimulated with Con A 10 g / ml. Add nonactin at the same time did. After culturing for 48 hours, the culture supernatant was collected and measured with 1-2 11 14 ELI SA KIT (R & D System). Nonactin did not suppress the production of IL-12 and IL-14 in the range of 10 On M-1 // M (FIG. 5).

 Example 4: Nonactin suppresses transcription of the human cytokine gene

 (1) Effect on 1L-5 gene transcription

The T cell clone was fused with a human T cell tumor cell line to obtain a human IL-15 gene-expressing T cell hybridoma (Mori A. et al. International Archives of Allergy and Immunology, 1995] ₀ human IL-15 gene promoter About 50 Obp DNA of the enhancer region was connected to luciferase gene as a reporter gene to produce pi L-5 Luc (FIG. 6). 5 × 10 ^β T cell hybridomas were suspended in 0.5 ml 1 RPM I medium (Gibco BRL), and pi L-5 Luc 10 βg was subjected to electroporation under the conditions of 250 V and 800 zF. However, transient transfection was performed. After washing the cells, the cells were stimulated with 20 nM and 10 M of PMA, and after 24 hours, the cells were collected. The induced luciferase activity was measured using a luciferase 'Atsusy System (Toyo Ink). As shown in FIG. 7, luciferase activity derived from Nonactin 100 nM, l! Vn3, and pIL-5 Luc was completely suppressed. In other words, it exhibited the effect of inhibiting the functional expression of the human IL-15 gene promoter no enhancer and suppressing the transcription of the IL-15 gene.

 (2) Effect on 1L-1 2 telegene transcription

A vector pi L-2 Luc in which a DNA of human IL-12 gene promoter / enhancer region 275 bp was linked to a luciferase gene was prepared in the same manner as in the above section (1). the pi L- 2 Luc 1 0 _8, said of the human The T cell hybridoma was transduced by electroporation, stimulated with ΡΜΑ and 10Μ for 48 hours, and the induced luciferase activity was measured. As evident in FIG. 8, nonactin 10 {η} did not inhibit IL-12 gene transcription.

(3) Effect on 1 L-1 4 gene transcription

 A vector pi L-4 Luc was prepared in which the DNA of the human IL-14 gene promoter / enhancer region 27 Obp was connected to the luciferase gene. 10 g of pi L-4 Luc was transfected into human T cell hybridomas and stimulated with PMA and 10M for 48 hours. As is evident in FIG. 9, Nonactin 10 OnM did not suppress IL-14 gene transcription at all. From the above (1), (2) and (3), nonactin is induced by the activation of allergen-specific human T cell hybridomas and affects the transcription of IL-12 and IL-14 genes. , It was found that the transcription of IL-15 gene was efficiently suppressed without giving any effect.

 Example 5: In vivo suppression of eosinophil accumulation of nonactin

 Six weeks of BDF 1 mice were sensitized by administration of ovalbumin (OA) l / g + aluminum hydroxide gel (Alun lmg twice at 2 week intervals. Two weeks later, nonactin or control Vehicle (physiological saline) 2 mg / animal (1 OOmgZkg) was administered by subcutaneous injection for 3 consecutive days on day 3. 30 minutes after administration of nonactin or vehicle, the antigen (egg albumin in saline) was administered. 10 OmgZinl) was inhaled for 10 minutes.

 Twenty-four hours later, bronchoalveolar lavage was performed, cells were collected, the total number of cells was counted, and the fractions were analyzed by cell staining.

Fig. 10 shows the obtained fruits. As evident from Figure 10, nonactin blocks IL-15-dependent eosinophil accumulation in vivo and reduces eosinophil infiltration. It was confirmed that it was suppressed.

 Example 6: Inhibition of IL-1 production of compound 1

 A 6-week-old BDF mouse is immunized by intraperitoneal injection of 100 g of Ascaris (parasite extract, manufactured by Sigma). Two weeks later, booster immunization was performed again using the same immunization method. Two weeks later, spleen cells were obtained.

 Stimulate with Con A (10 ^ g / ml) and collect the supernatant 24 hours later. The test drug (compound 1) was added to the culture from the start of stimulation, and the effect on IL-12, IL-14, and IL-15 production was examined. The production of IL-2, IL-14 and IL-5 is carried out by the specific sandwich ELISA method (G enzyme kit).

 As shown in FIG. 11, compound 1 suppresses the production of IL-12, but does not substantially inhibit the production of IL-14 or IL-5. The chemical formula of compound 1 is as follows:

Compound 1

 Example 7: Inhibition of IL-2 production of compound 2

 Six-week-old BDF mice are immunized by intraperitoneal injection of Ascarisl 00 μg. Two weeks later, the same immunization method was used to boost the immunity again, and two weeks later, spleen cells were obtained.

Stimulate with Con A (10 g / ml) and collect the supernatant 24 hours later. Trial The test drug (compound 2) was added during the culture from the start of stimulation, and the effect on IL-2, IL-14, and IL-5 production was examined. The production of IL-2, IL-14, and IL-15 is carried out by specific sandwich ELISA (Genzyme kit).

 As shown in FIG. 12, compound 2 suppresses the production of IL-12, but does not substantially inhibit the production of IL-4 or IL-15. The chemical formula of compound 2 is as follows:

実施例 8 ：化合物 3の I L一 2産生抑制 Compound 2

Example 8: Inhibition of IL-2 production of compound 3

 Six weeks old BDF mice are immunized by intraperitoneal injection of Ascarisl 00 g. Two weeks later, the same immunization method was used to boost the immunity again, and two weeks later, spleen cells were obtained.

 Stimulate with Con A (10 fig / ml) and collect the supernatant 24 hours later. The test drug (compound 3) was added during the culture from the start of stimulation, and the effect on IL-12, IL-14 and IL-15 production was examined. IL-12, IL-14, and IL-15 production were performed by the specific sandwich ELISA method (G Enzyme kit), respectively.

As shown in FIG. 13, compound 3 suppresses the production of IL-12, but does not substantially suppress the production of IL-14 or IL-15. The chemical formula of compound 3 is Is:

実施例 9 ：化合物 4の I L一 2産生抑制 Compound 3

Example 9: Inhibition of IL-12 production by compound 4

 Six-week-old BDF! Mice are immunized by intraperitoneal injection of Ascarisl 00 / ig. Two weeks later, the same immunization method was used to boost the immunity again, and two weeks later, spleen cells were obtained.

 Stimulate with Con A (10 i / g / ml) and collect the supernatant 24 hours later. The test drug (compound 4) was added during the culture from the start of stimulation, and the effect on IL-12, IL-4 and IL-15 production was examined. The production of IL-12, IL-14 and IL-5 is carried out by the specific sandwich ELISA method (kit manufactured by Genzyme).

As shown in FIG. 14, compound 4 suppresses the production of IL-12, but does not substantially suppress the production of IL-14 or IL-5. The chemical formula of compound 4 is as follows:

実施例 10 ：化合物 5の I L一 2産生抑制 Compound 4

Example 10: Inhibition of IL-12 production by compound 5

 Six weeks old BDF mice are immunized by intraperitoneal injection of AscalisOO / ig. Two weeks later, the same immunization method was used to re-boost, and two weeks later, spleen cells were obtained.

 Stimulate with Con A (10 / zg / ml) and collect the supernatant 24 hours later. The test drug (compound 5) was added to the culture from the start of the stimulation, and the effect on IL-12 and IL-4 IL-15 production was examined. The production of IL-12, IL-14 and IL-5 is carried out by the specific sandwich ELISA method (G enzyme kit).

 As shown in FIG. 15, Compound 5 does not substantially suppress the production of IL-14 or IL-15, which suppresses the production of IL-12. The chemical formula of compound 5 is as follows:

実施例 11:化合物 6の I L一 4および I L一 5の産生抑制 Compound 5

Example 11: Inhibition of production of IL-14 and IL-15 of compound 6

 Six-week-old BDFi mice are immunized by intraperitoneal injection of Ascarisl 00 / g. Two weeks later, the same immunization method was used to boost the immunity again, and two weeks later, spleen cells were obtained.

 Stimulate with Con A (10 g / ml) and collect the supernatant 24 hours later. The test drug (compound 6) was added during the cultivation from the start of stimulation, and suppression of IL-12, IL-4, and IL-15 production was examined. The production of IL-12, IL-4 and IL-5 is carried out by a specific sandwich method (kit by Genzyme). As shown in FIG. 16, Compound 6 (Compound No .: TE-323) inhibits the production of IL-4 and IL-5, but does not substantially inhibit the production of IL-12. The chemical formula of compound 6 is as follows:

Compound 6

 Example 12: Inhibition of production of IL-14 and I'L-15 of compound 7

 In the same manner as in Example 11, suppression on IL-12, IL-14, and IL-15 production was examined.

However, compound 7 (TE-479) represented by the following chemical formula was used as a test drug. Compound 7

 As shown in FIG. 17, it was found that Compound 7 suppressed the production of IL-4 and IL-5, but did not substantially suppress the production of IL-12.

 Example 13: Inhibition of production of IL-14 and IL-5 of compound 8

 In the same manner as in Example 11, suppression on IL-12, IL-14, and IL-15 production was examined.

 However, compound 8 (TE-760) represented by the following chemical formula was used as the test drug.

Compound ⁸

 As shown in FIG. 18, it was found that Compound 7 suppressed the production of IL-4 and IL-5, but did not substantially suppress the production of IL-12.

 Example 14: Inhibition of production of IL-9 and IL-15 of compound 9

 In the same manner as in Example 11, suppression on IL-12, IL-14, and IL-15 production was examined.

However, as a test drug, compound 9 represented by the following chemical formula (TE-34 5) use

Compound 9

 As shown in FIG. 19, it was found that Compound 7 suppressed the production of IL-4 and IL-5, but did not substantially suppress the production of IL-12.

 【The invention's effect】

 According to the present invention, the substances represented by the formulas (I) and (Π) suppress the gene transcription of interleukin such as human IL-15, the expression of niRNA and the production of protein, and therefore, so-called allergic diseases It has been shown to be a useful therapeutic agent for and eosinophilic diseases. The therapeutic agent of the present invention is expected to be a useful therapeutic agent that has the same or better clinical effects as the corticosteroid drug and has no side effects.

 [Brief description of the drawings]

 FIG. 1 shows suppression of IL-15 production of peripheral blood mononuclear cells by allergic patients by nonactin. Each number in the figure indicates the following.

 1 No stimulation

 2 Con A 10

 3 2 + Nonactin InM

 4 2 + nonactin 10 nM

5 2 + nonactin 100 nM 6 2+ nonactin

 FIG. 2 shows the suppression of IL-5 production of T cell clones by nonactin. Each number in the figure indicates the following.

 1 No stimulation

 2 Anti-CD3 antibody stimulation

 3 2 + Nonactin InM

 4 2 + nonactin Ι ΟηΜ

 5 2 + nonactin 100 nM

 6 2+ Nonactin l ^ M

 FIG. 3 is a photograph of electrophoresis showing suppression of IL-15 gene transcription in peripheral blood mononuclear cells of an allergic patient by nonactin. Each lane number in the figure indicates the following.

 1 No stimulation

 3 2 + Nonactin 1 M

 4 2 + nonactin 100 nM

 5 2 + nonactin 10 nM

 FIG. 4 is a photograph of electrophoresis showing suppression of IL-5 gene transcription of a T cell clone by nonactin. Each lane number in the figure indicates the following.

 1 No stimulation

 2 PMA 20nM + 10M 1

 3 2 + nonactin 100 nM

FIG. 5 shows the effect of nonactin on IL-12 and IL-14 production. Each number in the figure indicates the following. 1 No stimulation

 2 Con A 10 ug / ml

 3 2 + nonactin l ^ M

 4 2 + nonactin 100 nM

 5 2 + nonactin 10 0 nM

 FIG. 6 is a schematic diagram showing the structure of plasmid pIL5Luc.

 FIG. 7 shows the effect of nonactin on IL-15 gene transcription by measuring luciferase activity derived from pI L5 Luc. Each number in the figure indicates the following.

 1 No stimulation

 2 PMA + 10M

 3 2 + nonactin

 4 2 + Nonactin 0 OnM

 5 2+ Nonactin OnM

 FIG. 8 shows the effect of nonactin on IL-12 gene transcription by measuring luciferase activity derived from pIL2Luc. Each number in the figure indicates the following.

 1 No stimulation

 2 PMA + 10M

 3 2 + nonactin 100 nM

 FIG. 9 shows the effect of nonactin on IL-14 gene transcription by measuring luciferase activity derived from pIL2Luc. Each number in the figure indicates the following.

 1 No stimulation

2 PMA + 10M 3 2 + nonactin 100 nM

 FIG. 10 shows in vivo suppression of eosinophil accumulation of nonactin. Each number in the figure indicates the following.

 1 No antigen

 2 Vehicle

 3 Nonactin

 FIG. 11 shows that Compound 1 suppresses the production of IL-12 from T cells in vivo, but does not substantially suppress the production of IL4 and IL5.

 FIG. 12 shows that compound 2 suppresses the production of IL-12 from T cells in vivo, but does not substantially suppress the production of IL4 and IL5.

 FIG. 13 shows that Compound 3 suppresses the production of IL-2 from T cells in vivo, but does not substantially suppress the production of IL4 or IL5.

 FIG. 14 shows that compound 4 suppresses the production of IL-2 from T cells in vivo, but does not substantially suppress the production of IL4 or IL5.

 FIG. 15 shows that compound 5 suppresses the production of IL-2 from T cells in vivo, but does not substantially suppress the production of IL4 or IL5.

 FIG. 16 is a graph showing that compound 6 suppresses the production of IL-14 and IL-15 from T cells in vivo, and that it does not substantially suppress the production of IL-12. .

FIG. 17. Compound 7 demonstrates IL-14 and T cells from T cells in vivo. 3 is a graph showing that production of IL-2 and IL-5 is suppressed, but production of IL-2 is not substantially suppressed.

 FIG. 18 is a graph showing that compound 8 suppresses the production of IL-14 and IL-15 from T cells in vivo, but does not substantially suppress the production of IL-12.

 FIG. 19 is a graph showing that compound 9 suppresses the production of IL-4 and IL-15 from T cells in vivo, but does not substantially suppress the production of IL-12.

Claims

The scope of the claims 1. A substance, which suppresses the production of at least one of the cytokines IL-12, IL-14, IL-15 and IL-8 in T cells and does not inhibit the production of at least one of them, as an active ingredient. For treating immune-related diseases.  2. The treatment agent according to claim 1, wherein the production is suppressed by suppressing the transcription of a cytokine gene whose production is to be suppressed.  3. The therapeutic agent according to claim 1 or 2, wherein the cytokine whose production is to be suppressed is IL-12.  4. The treatment agent according to claim 3, wherein the site power-in of which production is not suppressed is at least one of IL14, 1-5 and 1-18.  5. The therapeutic agent according to claim 1 or 2, wherein the site force in which production is to be suppressed is at least one of IL-14, IL-5 and IL-8.  6. The therapeutic agent according to claim 5, wherein the cytokine whose production is not suppressed is IL-12.  7. The treatment according to any one of claims 1 to 6, wherein the substance is a macrolide antibiotic.  8. The therapeutic agent according to claim 7, wherein the macrolide antibiotic has a 10- to 40-membered lactone ring structure.  9. The therapeutic agent according to claim 8, wherein the macrolide antibiotic has a 12-membered lactone ring structure.  10. The treatment agent according to claim 8, wherein the macrolide antibiotic has a 14-membered lactone ring structure. 11. The treatment agent according to claim 8, wherein the macrolide antibiotic has a 16-membered lactone Hunch structure. 2. The macrolide antibiotic has the following formula (I)

[Wherein, R ₂ , R ₃ and R ₄ are each independently a Ci-C ₆ alkyl group. ] The treatment agent according to claim 7, which is represented by: 13. The treatment agent according to claim 12, wherein R !, R ₂ , R ₃ and R ₄ are a methyl group or an ethyl group.  14. The treatment agent according to claim 13, wherein the macrolide antibiotic is nonactin, monactin, dinactin, trinactin or tetranactin.  15. The macrolide antibiotic has the following formula (II):

Wherein R ₅ is a carbonyl group which may be oxidized together with the carbon atom to which it is attached, Re and R ₇ together form one 0 (CH ₂ ) ₂₀ —, -0 (C = 0) 0 —, -NH (CH ₂ ) ₂₀ — or one NH (C = 0) 0 — You may have R ₈ is> C = 0 or> CH (0—R) (where R is a sugar residue or an acyl group), R ₉ is a sugar residue, R ₁₀ is an optionally alkylated hydroquine, Where R ₅ is a carbonyl group oxidized together with the carbon atom to which it is attached, and R ₆ and R ₇ are each _NH (CH ₂ ) ₂₀ — or one NH (C = 0) 0 — When forming the above, the nitrogen atom in the former and the nitrogen atom in the latter may be linked via a lower alkylene chain. ] The treatment agent according to claim 7, which is represented by: 16. Makurorai de antibiotic is, R ₅ has the formula:> C = NOR "  16. The method according to claim 15, wherein R "is a compound of the above formula (II), which means a substituted or unsubstituted aryl lower alkyl group or a fuenyl lower alkenyl aryl group on the aryl group. Treatment agent.  17. The therapeutic agent according to claim 1, which is useful for treating collagen diseases, autoimmune diseases, organ transplantation, and the like by suppressing the production of IL-2.  18. The therapeutic agent according to claim 1, which is useful for treating IgE mast cell dependence hypersensitivity or the like by suppressing the production of IL-4.  19. The therapeutic agent according to claim 1, which is useful for treating allergic or eosinophilic diseases by suppressing the production of IL-5. 20. Suppressing IL-18 production is useful for treatment of neutrophilic inflammation The treatment agent _{c according to} claim 1