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WO1996027391A1 - Medicament pour le traitement de maladies liees a l'immunite et leur technique d'elaboration - Google Patents

Medicament pour le traitement de maladies liees a l'immunite et leur technique d'elaboration Download PDF

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Publication number
WO1996027391A1
WO1996027391A1 PCT/JP1996/000553 JP9600553W WO9627391A1 WO 1996027391 A1 WO1996027391 A1 WO 1996027391A1 JP 9600553 W JP9600553 W JP 9600553W WO 9627391 A1 WO9627391 A1 WO 9627391A1
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production
agent according
nonactin
cells
suppressed
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Japanese (ja)
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Hirokazu Okudaira
Akio Mori
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Environmental Research Institute Inc
Environmental Research Institute of Michigan
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Environmental Research Institute Inc
Environmental Research Institute of Michigan
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/424—Oxazoles condensed with heterocyclic ring systems, e.g. clavulanic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom

Definitions

  • 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.
  • IL indicates interleukin.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ 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.
  • macrolide antibiotics including their derivatives
  • 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.
  • 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.
  • cytokine gene for example, m-RNA
  • 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.
  • searching for a drug having a desired selective cytokine inhibitory activity is performed using cytokine gene-expressing ⁇ cells.
  • the present inventors have previously succeeded in cloning and expanding ⁇ cells that recognize allergens from peripheral blood lymphocytes from an allergic patient, but have obtained the T cell clone obtained here.
  • a test substance is added to a medium in which these T cell clones, T cell hybridomas, and normal peripheral blood ⁇ cells grow, and the cells are cultured with an activation stimulus.
  • IL-12 in the culture eg, supernatant
  • 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,
  • drugs that suppress the production of IL-15 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.
  • 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.
  • 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.
  • 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.
  • macrotetroid antibiotics include nonactin (), monactin, dinactin, trinactin, and tetranactin.
  • macromonospora 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.
  • macrolide antibiotics of the formula (I) typically selectively inhibit IL-15 production but do not inhibit IL-2 or IL-14 production:
  • 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.
  • 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.
  • a macrolide antibiotic represented by the following formula ( ⁇ ) 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 .:
  • R 5 is oxidized with the carbon atom to which it is attached Good carbonyl group
  • the nitrogen atom in the former and the nitrogen atom in the latter may be connected via a lower alkylene chain.
  • 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
  • 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.
  • lower alkyl examples 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.
  • R 5 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).
  • 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.
  • macrolide antibiotics of the formula (I) 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.
  • Nonactin, monactin, dinactin, trinactin, tetranactin and the like used as the treatment agent of the present invention are also macrolide antibiotics produced by actinomycetes.
  • 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)].
  • 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)].
  • 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.
  • the fact that nonactin and its analogs suppressed IL-15 telegene transcription, mRNA expression and protein production in human T cells was not known.
  • 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.
  • 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.
  • PKC protein kinase C
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • one of these drugs may be used alone or in combination of two or more.
  • 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.
  • 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.
  • 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
  • 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 1 Nonactin suppression of IL-15 production
  • PBMC peripheral blood mononuclear cells
  • Example 2 Nonactin suppresses expression of IL-15 telegene
  • IL-5niRNA expression was performed by the reverse transcription polymerase chain reaction (RT-PCR) [see Mori A. et al. International Immunology: 7].
  • PBMCs peripheral blood of allergic patients as described above, cultured in 24-well plates (manufactured by Corning) at a concentration of 2 ⁇ 10 ⁇ - 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.
  • ConA 10 / g / ml
  • 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 ⁇ . 3M sodium acetate solution ( ⁇ 5.3) 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.
  • Actin was used as a positive control.
  • 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.
  • PBMC peripheral blood of an allergic patient was collected, and PBMC was isolated in the same manner as in Example 1.
  • 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
  • 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 ⁇ T cell hybridomas were suspended in 0.5 ml 1 RPM I medium (Gibco BRL), and pi L-5 Luc 10 ⁇ g was subjected to electroporation under the conditions of 250 V and 800 zF. However, transient transfection was performed.
  • 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.
  • 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 ⁇ and 10 ⁇ for 48 hours, and the induced luciferase activity was measured. As evident in FIG. 8, nonactin 10 ⁇ did not inhibit IL-12 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
  • mice 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.
  • OA ovalbumin
  • 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.
  • 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.
  • test drug compound 1
  • the test drug 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).
  • 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:
  • Example 7 Inhibition of IL-2 production of compound 2
  • 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:
  • IL-12, IL-14, and IL-15 production were performed by the specific sandwich ELISA method (G Enzyme kit), respectively.
  • 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:
  • mice 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.
  • 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:
  • IL-12, IL-14 and IL-5 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).
  • 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:
  • BDFi mice 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.
  • Example 12 Inhibition of production of IL-14 and I'L-15 of compound 7
  • Example 13 Inhibition of production of IL-14 and IL-5 of compound 8
  • Example 14 Inhibition of production of IL-9 and IL-15 of compound 9
  • the substances represented by the formulas (I) and ( ⁇ ) 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.
  • 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.
  • FIG. 2 shows the suppression of IL-5 production of T cell clones by nonactin. Each number in the figure indicates the following.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • FIG. 10 shows in vivo suppression of eosinophil accumulation of nonactin. Each number in the figure indicates the following.
  • 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. .
  • 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.

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Abstract

Technique d'élaboration de produits chimiques capables d'empêcher la production de cytokines dans des cellules T lesquelles sont étroitement liées à l'apparition de maladies liées à l'immunité au niveau de la transcription des gènes et dont l'utilisation clinique permet de traiter lesdites maladies; et médicament pour le traitement de maladies liées à l'immunité comportant comme principe actif un macrolide capable d'empêcher chez l'homme la production d'au moins une cytokine dans les cellules T.
PCT/JP1996/000553 1995-03-08 1996-03-07 Medicament pour le traitement de maladies liees a l'immunite et leur technique d'elaboration Ceased WO1996027391A1 (fr)

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JP7742995 1995-03-08
JP7/77429 1995-03-08
JP7/101696 1995-04-03
JP10169695 1995-04-03
JP7/258961 1995-10-05
JP25896195 1995-10-05

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002534531A (ja) * 1999-01-15 2002-10-15 ザンボン グループ エス．ピー．エー． 抗炎症活性を有するマクロライド

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
INTERNATIONAL ARCHIVES OF ALLERGY AND IMMUNOLOGY, 1994, Vol. 105, No. 3, KONNO S., pages 308-316. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2002534531A (ja) * 1999-01-15 2002-10-15 ザンボン グループ エス．ピー．エー． 抗炎症活性を有するマクロライド

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