WO2003063905A1

WO2003063905A1 - Preventives or remedies for immunological diseases

Info

Publication number: WO2003063905A1
Application number: PCT/JP2003/000826
Authority: WO
Inventors: Hidenori Ichijo; Koji Hashimoto; Atsushi Matsuzawa
Original assignee: Center For Advanced Science And Technology Incubation, Ltd.
Priority date: 2002-01-31
Filing date: 2003-01-29
Publication date: 2003-08-07
Also published as: JPWO2003063905A1

Abstract

It is intended to provide preventives or remedies for immunological diseases such as rheumatoid arthritis, type I diabetes, systemic lups erythematosus, psoriasis, inflammatory colon disease, multiple sclerosis, thrmobopenia, Sjoegren’s syndrome, bronchial asthma, atopic dermatitis and sepsis, which are characterized by containing a chemical having an ASK1 inhibitory effect (for example, an ASK1 dominant negative compound, an ASK1 antisense oligonucleotide, glutathione, an S-transferase (Mul-1, etc.), Nef, 14-3-3 protein or thioredoxin), a sense oligonucleotide thereof, an expression vector thereof or host cells transformed by the expression vector, and have an effect of inhibiting the production of various cytokines or chemokines.

Description

Description Preventive or therapeutic agent for immune diseases

The present invention relates to a preventive or therapeutic agent for an immune disease, comprising an ASKl (Apop tosis Signa l-regu lati ng Kinase 1) inhibitor, and a method for preventing an immune disease, comprising using an effective amount of an ASK1 inhibitor. Or a method of treatment, and the use of an ASK1 inhibitor for the manufacture of a preparation for the prevention or treatment of an immune disease. Background art

ASK 1 is a type of kinase belonging to the MAPKK kinase (MAPKKK) family. For example, human ASK 1 is composed of 1375 amino acids (see Reference 1 below), and mouse ASK 1 is composed of 1379 amino acids It has been reported that human ASK1 and mouse ASK1 have an extremely high homology of 91.9% (see Reference 2 below). Similarly, human ASK1 cDNA (see Reference 3 below) and mouse ASK1 cDNA (see Reference 2 below) are disclosed. MAPKK kinase is known to be a conserved intracellular signaling pathway from mammals, including humans, to yeast, MAP (

Mitogen-activated Protein) Kinase is a kinase that belongs to the signal transduction cascade by the kinase superfamily. Two types of kinases, MAPK kinase (MAPKK) and MAP kinase (MAPK), exist downstream of MAPKK kinase. (See references 4 to 6 below)

ASK1 is distributed in almost all organs of humans, including the heart, kidney, liver, ligament, large intestine, brain, and lung, and is activated by TNF (tumor necrosis factor) -α via the TNF receptor and activated It has been confirmed that the activated ASΚ1 is involved in the induction of apoptosis of “t cells” via the signaling cascade. It has been reported that when a dominant-negative mutant, which is a mutant of the present invention, was highly expressed in Jurkat cells, apoptosis induced by TNF-hi was suppressed. Therefore, it is disclosed that ASK1 is useful as a therapeutic agent for malignant tumor or a gene therapeutic agent for malignant tumor. (See References 4 and 5 below) Furthermore, it has been reported that ASK1 is highly expressed in skin keratinocytes, suggesting that it is involved in promoting its differentiation. However, there is no report that ASK1 is involved in the onset or progression of immune diseases.

Various reports have been reported that abnormal production of cytokines and chemokines is involved as one of the causes of immune diseases. For example, IL-1 is involved in the development of rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, inflammatory bowel disease, atopic dermatitis, psoriasis, transplant rejection, bronchial asthma, etc. It is involved in the development of immune diseases such as rheumatoid arthritis, systemic lupus erythema, death, inflammatory bowel disease, Caste eman disease, mesangial proliferative nephritis, and IL-8 is associated with rheumatoid arthritis, atopic dermatitis, It has been reported to be involved in the development of inflammatory bowel disease, psoriasis, bronchial asthma and the like. MIP-3 (also known as LARC (liver and activation-regulated chemokine)) is very highly expressed in psoriatic lesions, and a marked increase is observed in the skin of patients with atopic dermatitis. In addition, MlP-3 is involved in the invasion, migration and resident of immature dendritic cells, antigen-presenting cells, tissue-directed memory T cells, and Langerhans cells, which are important antigen-presenting cells of the skin. It is also involved in the development of inflammatory bowel disease, transplant rejection, and bronchial asthma. MIP-1α and MIP_1) 3 are involved in the migration and invasion of Th1 cells and macrophages, and are expressed in conditions such as rheumatoid arthritis, transplant rejection, graft-versus-host disease, and bronchial asthma Has been reported to play an important role in the formation of the disease state. (For example, see the following references 8-28)

The number of patients with immunological diseases including rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, psoriasis, inflammatory bowel disease, bronchial asthma, atopic dermatitis is increasing year by year. Currently, steroid therapy is frequently used for these patients, but the symptoms are often not improved, and if used for a long period of time, diabetes or high fat may occur. Hematosis, osteoporosis, high blood pressure, susceptibility to infection, etc. may be observed, and are still unsatisfactory for pharmacotherapy. Therefore, various researches are currently being conducted on immunological diseases, and early development of a preventive or therapeutic agent for the disease is highly demanded.

Reference 1: Sequence Listing SEQ ID NO: 1 described in WO 97/40143

Reference 2: Journal of the Japanese Society of Stomatology, No.3, p.45 (1998) Figure 1

Reference 3: Sequence Listing SEQ ID NO: 2 described in WO 97/40143

Reference 4: Ichijo H. et al., Science, Vol.275, pp.90-94 (1997)

Reference 5: W097 / 40143

Reference 6: Kei Tome, Journal of Oral Disease Society, o.3, pp.42-52 (1998)

Bunnan Inu 7: Sayama K. et al., Journal of Biological Chemistry, Vol.276, No.2, pp.999-1004 (2001)

Reference 8: Kikuo Onozaki, Site Power Inn, The Japan Medical Museum, pp.95-105

Reference 9: Masataka Kuwana, Molecular Medicine, Vol.38, No.8, pp.900-907 (2001) Reference 10: Shuji Takei, Pediatrics, Vol.33, No.6, pp.861-865 (2001) Reference 11: Shinki Tokura, Journal of the Japanese Society of Skin Allergy, Vol.5, No.4, pp.117-

121 (1997)

Reference 12: Junichi Unohara, Clinical Pathology, Vol. 46, No. 6, pp. 587-592 (1998) Reference 13: Yasushi Tanaka et al., History of Medicine, Vol. 174, No. 14, pp. 1218- 1222 (1995) Article 14: Akihisa Harada et al., Inflammation and immunity, Vol.2, No.2, pp.211-220 (1994) Article 15: Masamichi Sugimoto et al., Molecular Medicine, Vol.38, No.4, pp. .410-417 (2001)

Reference 16: Akio Otani et al., Modern Medicine, Vol.33, No.6, pp.1527-1532 (2001) Reference 17: Kazuya Anezaki et al., History of Medicine, Vol.173, No.6, pp.588-592 (1995) Reference 18: Makoto Sada et al., Asthma, Vol.10, No.2, pp.31-35 (1997)

Reference 19: Kazuki Kurashima, Modern Medicine, Vol.33, No.6, pp.1517-1521 (2001) Article 20: Tsunaharu Matsushima et al., Infection, Inflammation, Immunity, Voshi 24, No.1, pp.9 -17 (1994) Reference 21: Naofumi Mukada, Clinical Pathology, Vol.40, No.4, pp.317-379 (1992) Reference 22 Noriko Suenobu et al., Molecular Medicine, Vol.38, No.2, pp.144-151

(2001)

Literature 23 Takashi Nakayama et al., Molecular Medicine, Vol.38, No.2, pp.126-134

(2001)

Reference 24 Masaru Ishikawa et al., Experimental Medicine, Vol.18, No.15, pp.150-156 (2000) Article 25 K. Hieshima, Cell Engineering, Vol.19, No.5, pp.708-716 ( 2000) Reference 26 Osamu Yoshie, Clinical Immunity, Vol.35, No.1, pp.86-94 (2001)

Reference 27 Masako Murai et al., Molecular Medicine, Vol.38, No.2, pp.168-174

(2001)

Reference 28 Suzuki Seiichi, Clinical Immunity, Vol.34, No.4, pp.504-511 (2000) Disclosure of the Invention

The present invention provides a preventive or therapeutic agent for an immune disease, which has an inhibitory effect on the production of various cytokines / chemokines that are deeply involved in the onset and progress of the immune disease. Detailed description of the drawings

Fig. 1 shows various cytokines (IL-1 and IL-1 / 3) in normal cultured human skin keratinocytes when activated ASK1 (ASK1-ΔΝ) was overexpressed by the adenovirus method. Increased mRNA expression of various chemokines (IL-8, MIP-3, MIP-1β) and GAPDH was increased by control (instead of untreated cells and activated ASK 1) 3-galactosidase (/ 3- FIG. 9 is a diagram quantified by using the RNase protection assay in comparison with (Cells expressing Gal)). The vertical axis shows the names of the detected genes, that is, the names of various cytokins and chemokines, and GAPDH as a negative control, and the horizontal axis shows the group names.

Fig. 2 shows the actual status of IL-18 when activated ASK1 (ASK1-ΔΝ) was overexpressed in cultured normal human skin keratinocytes by the adenovirus vector method. The expression level of the protein was compared with the control (untreated cells and cells expressing 3-galactosidase (] 3-Ga 1) instead of active ASK 1) in a graph quantified by ELISA. is there. The vertical axis indicates the concentration (pg / mL) of IL-8 secreted into the culture supernatant, and the horizontal axis indicates the group name. The error number indicates the standard deviation.

Figure 3 shows the actual protein expression level of MIP-1 when activated ASK 1 (ASK 1 -ΔΝ) was overexpressed in cultured normal human skin keratinocytes by the adenovirus vector method. FIG. 4 is a graph quantified by an ELISA method as compared with a control (untreated cells and cells expressing i3-galactosidase (3-Gal) instead of active ASK1). The vertical axis indicates the concentration (pg / mL) of MlP-1α secreted into the culture supernatant, and the horizontal axis indicates the group name. The error bar represents the standard deviation.

Fig. 4 shows the actual protein expression level of MIP-1 / 3 when activated ASK1 (ASK1-ΔΝ) was overexpressed in cultured normal human skin keratinocytes by the adenovirus vector method. FIG. 4 is a graph quantified by ELISA in comparison with a control (untreated cells and cells expressing 3-galactosidase (instead of activated ASK1) 3-galactosidase). The vertical axis indicates the concentration (pgZmL) of MlP_l secreted into the culture supernatant, and the horizontal axis indicates the group name. Error bars indicate standard deviation.

Fig. 5 shows the actual protein expression level of MIP-3 when activated ASK1 (ASK1-ΔΝ) was overexpressed in normal human cultured skin keratinocytes by the adenovirus vector method. FIG. 4 is a graph quantified by an ELISA method as compared with a control (untreated cells and cells expressing / 3-galactosidase (/ 3-Gal) instead of active ASK1). The vertical axis indicates the concentration (pgZmL) of MlP-3 secreted into the culture supernatant, and the horizontal axis indicates the group name. The error bar indicates the standard deviation.

Fig. 6 shows the expression level of IL-16 protein induced by LPS using fetal fibroblasts (MEFs) as cells derived from ASK1 knockout mice. The time-dependent changes were determined by ELISA using MESK s cells (ASK1 − / −; open bar) and wild-type MEF s cells (ASK 1 + / +; black bar) derived from ASK1 knockout mice. It is a graph. The vertical axis shows the concentration (pgZmL) of IL-16 protein secreted into the culture supernatant, and the horizontal axis shows time (hour). The error number indicates the standard deviation.

Fig. 7 shows the dose dependence of LPS-stimulated IL-6 protein expression on LPS using fetal fibroblasts (MEFs) as cells derived from ASK1 knockout mice. FIG. 4 is a graph comparing and quantifying MEF s cells (ASK 1 − / −; white bars) and wild type MEF s cells (ASK 1 + / +; black bars) by the ELISA method. The vertical axis shows the concentration (pg / mL) of IL-6 protein secreted into the culture supernatant, and the horizontal axis shows the concentration of LPS (/ mL). The error number indicates the standard deviation.

FIG. 8 shows the temporal change in the expression level of IL_1] 3 protein induced by LPS using spleen cells (sp1e no cytes) derived from ASK1 knockout mice. This is a graph showing comparative quantification of sp 1 enocytes (ASK1-no-; open circle) derived from wild-type sp 1 enocytes (ASK 1 + / +; black circle) by ELISA. The vertical axis indicates the concentration (pg / mL) of IL-1 / 3 protein secreted into the culture supernatant, and the horizontal axis indicates time (hour). Error bars represent standard deviation.

Fig. 9 shows the time-dependent changes in the expression level of IL-6 protein induced by LPS using spleen cells (sp 1 e no cytes) derived from ASK1 knockout mice. This is a graph of comparative quantification of eno cytes (ASK1—Z_; open circle) and wild-type sp 1 enocytes (ASK 1 + / +; black circle) by the ELISA method. The vertical axis shows the concentration (pgZmL) of IL-6 protein secreted into the culture supernatant, and the horizontal axis shows time (hours). Error bars represent standard deviation.

FIG. 10 shows the expression level of MIP-1α protein stimulated by LPS using spleen cells (sp 1 enocytes) derived from ASK1 knockout mice. The temporal changes were determined by ELISA using sp1 enocytes (ASK1——; open circles) and wild-type sp1 enocytes (ASK1 +++; black circles) derived from ASK1 knockout mice. It is a graph. The vertical axis shows the concentration (pgZmL) of MIP-1α protein secreted into the culture supernatant, and the horizontal axis shows time (hours). Error bars represent standard deviation.

Fig. 11 is a graph comparing the survival rates of ASK1 knockout mice (ASK1-/-; open circles) and wild-type mice (ASKl + 10; black circles) with respect to the sepsis model over time. The vertical axis shows the survival rate (%), and the horizontal axis shows the group name.

Fig. 12 shows the blood TNF-α concentration 2 hours after LPS induction in a sepsis model in ASK1 knockout mice (ASK1 − / −; open circles) and wild-type mouse (ASK1 + Z +; black circles). It is the graph which compared. The vertical axis shows blood TNF-serum concentration (ngZmL), and the horizontal axis shows group names. The error bar represents the standard deviation. BEST MODE FOR CARRYING OUT THE INVENTION

The present inventors have conducted intensive studies to find drugs useful for the prevention or treatment of immune diseases.As a result, ASK 1 is involved in the development and progression of immune diseases, and ASK 1 inhibitors are useful for immune diseases. The knowledge was obtained, and the present invention was achieved.

Specifically, first, to investigate the physiological function of ASK1, the present inventors established normal cultured human skin keratinocytes overexpressing activated ASK1, and established various types of cytokines, chemokines. The expression level was measured. As a result, in activated ASK1 overexpressing cells, IL-1 and IL-6 cytodynamics, IL-8, MIP-1α, MIΡ-1) 3 and MIP_3α chemokines were highly expressed. I confirmed that These facts indicate that ASK1 is involved in enhancing the expression of these cytokines, chemokines, which are deeply involved in the development and progression of immune diseases.

In addition, the present inventors produced ASK1 knockout mice by the method described below. Made. Fetal fibroblasts and spleen cells were collected from the prepared ASK1 knockout mice, and the amounts of production of IL-1, IL-6 and MIP-1α induced by LPS (lipopolysaccharide) stimulation were measured. As a result, fetal fibroblasts and spleen cells of ASK1 knockout mice were significantly different from those of wild type (normal) mouse fetal fibroblasts and spleen cells. The production of spikes was suppressed. From these facts, it was revealed that reducing the activity of ASK1 can actually suppress the production of the cytokine ゃ chemokine. Therefore, the above experimental results indicate that ASK1 inhibitors are extremely useful for prevention and treatment of various immune diseases.

Furthermore, the present inventors have found that in an in vivo test using a sepsis model, the survival rate of ASK1 knockout mice is significantly improved as compared with wild-type mice, and that the ASK1 knockout mice Observed that there was little increase in blood TNF concentration. From these facts, it was found that reducing the activity of ASK1 can remarkably improve the pathology of various immune diseases such as sepsis. These are nothing less than further supporting that ASK1 inhibitors are extremely useful for prevention and treatment of various immune diseases.

That is, by containing an ASK1 inhibitor as an active ingredient, a drug useful for prevention or treatment of an immune disease can be provided. Use of an effective amount of an ASK1 inhibitor can provide a useful method for preventing or treating an immune disease.

As used herein, `` ++ '' indicates that two ASK1 loci present on a pair of homologous chromosomes in a mouse or mouse-derived cells are both normal, that is, wild-type, “One Z—” is a homozygote in which a mutation has been introduced into both of the two ASK1 loci present on each of a pair of homologous chromosomes and has no ASK1 protein expression, ie, ASK1 Indicates that the mouse or mouse-derived cell has been knocked out.

In the present invention, the term “immune disease” refers to a disease that develops or develops due to the breakdown of the immune system due to abnormal functions of various cells, such as rheumatoid arthritis, Surin dependence) Diabetes, systemic lupus erythematosus (SLE), psoriasis, inflammatory bowel disease, multiple sclerosis, systemic sclerosis, hemolytic anemia, thrombocytopenia, neutropenia, siedalen syndrome, IgA kidney Disease, lupus nephritis, mesangial proliferative nephritis, polymyositis, Hashimoto's disease, Graves' disease, Addison's disease, primary biliary cirrhosis (PBC), ankylosing myelitis, autoimmune hepatitis, pemphigus vulgaris, severe Autoimmune diseases such as myasthenia, anti-glomerular basement membrane disease, Casteman disease, celiac disease, and Graves disease, bronchial asthma, allergic rhinitis, atopic dermatitis, robbery measles, anaphylactic disease And allergic diseases such as sepsis, transplant rejection, graft host disease (GVHD) and the like. The pharmaceutical composition of the present invention is particularly useful for treating rheumatoid arthritis, type I (insulin-dependent) diabetes, systemic lupus erythematosus (SLE), psoriasis, inflammatory bowel disease, multiple sclerosis, thrombocytopenia, siedalen syndrome, etc. It is suitable for allergic diseases such as autoimmune disease, bronchial asthma, atopic dermatitis, and sepsis.

In the present invention, the ASK1 inhibitor is not limited to a substance having an ASK1 inhibitory action per se, but produces an ASK1 inhibitory substance in vivo or in a culture system. And, for example, a derivative in which one or more amino acids are added, inserted, substituted, and Z or deleted (for example, deletion of the alanine residue at position 960) in the amino acid sequence (see, for example, Reference 29 below) ), Dominant negative for human ASK1 (eg, K709R, K709M; hereinafter, referred to as ASK1 dominant negative), antisense oligonucleotide for human ASK1 (hereinafter, referred to as ASK1 antisense oligonucleotide), AS Chemicals with K1 inhibitory activity (eg, Daryuthion S-transferase (Mu 1 -1 etc.), Nef (Ne f), 14—3—3 White matter, thioredoxin, heat shock protein Hsp72, protein phosphatase 5 (PP5), Akt (Akt3, etc.), CDC25A, etc. (for example, see references 30 to 37 below) and their sense oligonucleotides (such as And pharmacologically acceptable salts thereof), and the nucleotide sequence encoding ASK1 dominant negative or ASK1 antisense oligonucleotide. A recombinant vector capable of replicating and expressing the dominant negative or antisense oligonucleotide in a target tissue or host cell (hereinafter, referred to as a “recombinant vector”).

ASK1 dominant negative body expression recombinant vector or ASK1 antisense oligonucleotide expression recombinant vector), which expresses a chemical substance having ASK1 inhibitory activity or its sense oligonucleotide in target tissues or host cells (Hereinafter referred to as a recombinant vector expressing a chemical substance having an ASK1 inhibitory action or a recombinant oligonucleotide vector expressing a sense oligonucleotide having a chemical substance having an ASK1 inhibitory action). (C) a host cell transformed with an ASK1 dominant-negative expression recombinant vector, a host cell transformed with an ASK1 antisense oligonucleotide-expressing recombinant vector , ASK1 inhibitory chemical expression vector And a host cell transformed with a recombinant vector expressing a sense oligonucleotide which expresses a chemical substance having an ASK1 inhibitory action).

The method of using the ASK1 inhibitor of the present invention includes ASK1 dominant negative expression recombinant vector, ASK1 antisense oligonucleotide expression recombinant vector, chemical expression recombinant vector having ASK1 inhibitory activity, and Introduces a recombinant vector expressing senseoligonucleotide of a chemical substance having ASK1 inhibitory activity or a host cell transformed by the recombinant vector into a target tissue in a living body by an appropriate method, and ASK1 dominant negative, ASK1 antisense nucleotide, use as a genetic preventive or therapeutic agent by expressing a chemical substance having ASK1 inhibitory activity or its sense oligonucleotide, or ASK1 dominant negative, ASK1 antisense oligonucleotide, ASK1 inhibitory chemical, or ASK (1) The use of a preparation containing an inhibitory chemical substance, senseoligonucleotide, as an active ingredient orally or parenterally, as a drug preventive or therapeutic agent can be mentioned. The lysine residue at position 709 of ASK 1 is an ATP-binding site, which can be inactivated by substituting it with arginine or methionine. Therefore, dominant negative ( A dominant-negative type) ASK1 (K709R, K709M), which functions as a mutant, is inserted into an expression vector, and the vector is transfected or infused into cells by the lipofection method or adenovirus infection method. Thus, overexpression in cells can be exemplified. The ASK1 overexpressing cells transformed in this way can remarkably suppress the production of cytokinetic chemokines.

In the present specification, when a dominant negative mutant, which is a specific mutant, is expressed, the original amino acid residue (one-letter code) is first, the position number is second, and the amino acid residue after substitution. (Single-letter notation) is the third one, and “K709 R” and “K709M” indicate that the 709th amino acid residue K (Lys: lysine) is R (Arg: arginine) or M (Met: methionine) Represents that it has been replaced by

Examples of the vector include a plasmid vector, a virus vector (for example, a retrovirus vector, an adenovirus vector, a herpes virus vector, a Sendai virus vector, and a vaccinia virus vector), and a ribosome vector (for example, a click). Ribosome vector). In a recombinant vector, in addition to the nucleotide sequence encoding the ASK1 dominant negative and the ASK1 antisense oligonucleotide, this is actually introduced into a target tissue or host cell to produce the desired dominant negative In order to express the sense oligonucleotide, a nucleotide sequence that controls its expression (eg, a promoter sequence, a terminator sequence, an enhancer sequence) 遺伝子 a gene marker for selecting a microorganism, insect cell, animal cultured cell, etc. (for example, , Neomycin resistance gene, kanamycin resistance gene) and the like.

Host cells include, for example, E. coli, yeast, insect cells, and CH〇 cells, Examples include animal cells such as COS cells, mink lung epithelial cells (eg, MvI Lu), lymphocytes, fibroblasts, blood cells, and tumor cells.

Methods for introducing a recombinant vector into a target tissue or host cell include the HVJ liposome method (see References 38 and 39 below), the method of directly administering an ASK1 inhibitor by injection, the calcium phosphate method, and the DEAE-dextran method. A method using a gene gun (see Reference 40 below), a method using a ribofection method (see Reference 41 below), a vector (for example, a retrovirus vector, an adenovirus vector, a herpes virus vector, Vaccinia virus vector).

When a preparation containing an ASK1 inhibitor as an active ingredient is actually used in the prevention or treatment of an immune disease, various preparation forms are used depending on the usage. Examples of the formulation form include tablets, capsules, granules, powders, pills, fine granules, troches, injections, rectal administration, suppositories, and the like. Is administered.

These preparations containing an ASK1 inhibitor contain the above-mentioned ASK1 inhibitor as an active ingredient, and are commonly used as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents. It can be produced by appropriately mixing, diluting or dissolving with pharmaceutical additives such as agents, preservatives, wetting agents, emulsifiers, dispersing agents, stabilizers, and solubilizing agents, and dispensing according to a conventional method.

The dose of an ASK1 inhibitor for pharmacological prophylaxis or treatment of immune disease is determined as appropriate, taking into account usage, patient age, gender, severity of symptoms, type of disease, etc. The dose may be 0.1 to 50 Omg, preferably about 0.5 to 10 Omg, and may be administered once or several times a day. In addition, the dose of the ASK1 inhibitor in the genetic prevention or treatment of an immune disease can also be appropriately determined according to the above.

According to the present invention, an ASK1 dominant negative, an ASK1 antisense oligonucleotide, a chemical substance having ASK1 inhibitory activity, a nucleotide sequence encoding the sense oligonucleotide thereof, a vector containing the same, or a By introducing into a target tissue using a host cell transformed by a vector, the onset or progression of an immune disease can be suppressed. In addition, the administration of ASK1 dominant negative, ASK1 antisense oligonucleotide, a chemical substance having ASK1 inhibitory activity, or a preparation containing its senseoligonucleotide, orally or parenterally, causes the onset or development of an immune disease. Progress can be restrained.

Reference 29: Wang X. S. et al., Journal of Biological Chemistry, Vol.271, pp.31607-31611 (1996)

Reference 30: Ssang-Goo Cho et al., Journal of Biological Chemistry, Vol.276, No.16, pp.12749-12755 (2001)

Reference 31: Romas Geleziunas et al., Ature, Vol. 410, pp. 834-838 (2001) Reference 32: FASEB Journal, Vol. 15, No. 4, pp. A235 (2001)

Reference 33: EMB0 Journal, Vol.17, No.9, pp.2596-2606 (1998)

Reference 34: Hee-Sae Park et al., Molecular and Cellular Biology, Vol.22, No.22, pp.7721-7730 (2002)

Reference 35: EMB0 Journal, Vol.20, No.21, p.6028-6036 (2001)

Reference 36: Albert H. Kim et al., Molecular and Cellular Biology, Vol.21, No.3, pp.893-901 (2001)

Bunnan Inu 37: Xianghong Zou et al., Molecular and Cellular Biology, Vol. 21, No. 14, pp. 4818-4828 (2001)

Reference 38: Kaneda, Experimental Medicine, Vol.12, No.2, p.78 (1994)

Reference 39: Morishita et al., Experimental Medicine, Vol.12, No.15, p.158 (1994)

Reference 40: T. M. Klein et al., Bio / Technology 10, pp.286-291 (1992) Reference 41: Nabel et al., Science, Vol.244, p.1285 (1990)

The content of the present invention will be described in more detail by the following test examples, but the present invention is not limited to the content. Test example 1

Induction of cytokines and chemokines in cultured normal human skin keratinocytes by expression of activated ASK1

Activated ASK1 (ASK1-ΔΝ; see Reference 42 below), a molecule that is permanently activated by deleting the N-terminal activity suppression region of the ASK1 molecule, is overexpressed in cells, and the physiology of ASK1 The target function was amplified and detected by the following method. First, an adenovirus vector (Ad-ASK1-ΔΝ) incorporating this active ASK1 was prepared, and the active human ASK1 was overexpressed in cultured normal human skin keratinocytes by the adenovirus method. (Multiplicity of infection) The recombinant virus is infected with 3 x 10 ⁶ eel 1 s / we 11 normal human cultured skin keratinocytes at an efficiency of about 5 and the cells and culture supernatant 5 mL was collected. As described below, the increase in mRNA expression levels of various cytokins and chemokines was quantified using the RNase protection assay, and the protein concentration released into the culture supernatant as the actual protein expression level was determined by ELISA. It was quantified using. As controls, untreated cells and cells expressing) 3-galactosidase instead of active ASK1 were compared. GAPDH, a gene whose mRNA level did not change with stimulation, was used as a negative control.

1) RNase protection method

Using a radioisotope-labeled probe specific for the target mRNA, leaving only the complementary double-stranded oligonucleotide resistant to endonuclease in the sample, separating it by polyacrylamide electrophoresis, and expressing mRNA The amount was detected with high sensitivity (can be detected as a band), and the increase was quantified.

2) ELISA method

Using an antibody specific to the target protein (two types of antibodies were used, and one side used a biotin-labeled antibody), the expression level of the protein was trace-quantified by the biotin-avidin method. The results are shown in Fig. 1 for the mRNA expression levels of various cytokines and chemokines, and for the protein expression levels in Fig. 2 (IL-18), Fig. 3 (MIP-la), and Fig. 3 respectively. This is shown in Fig. 4 (Μ Ι Ρ_1; 3) and Fig. 5 (Μ Ι Ρ—3a). These figures show that activation of ASK1 causes cytokines such as IL-1 and IL-1 / 3, as well as IL-8, Μ Ρ Ρ_1 (¾, Μ Ι Ρ— 1/3, MI Ρ-3 This indicates that the expression of mRNA or various proteins of chemokines such as a is induced.

Reference 42: EMBO Journal, Vol.17, No.9, pp.2596-2606 (1998)

Test example 2

Production of ASK 1 knockout mouse

Several chromosomal DNA fragments, including the first exon of ASK1, were cloned from the chromosome (genomic) DNA library of 129ZS VJ mice (Strategene). Using one of the clones, 10 kb upstream and 2 kb downstream of the first exon were used as the homologous recombination region, and the first exon and the adjacent intron region were converted to GFP (greenflourescence protein) and neomycin for positive selection. A targeting vector replaced with a resistance gene was constructed. As a targeting vector, pBluescript SK (Strategene) containing DT-A (diphtheria toxin α chain) for negative selection was used. The evening vector was introduced into J1 ES cells (embryon icst emce11) by the electoporation method. After selecting neomycin-resistant ES cell colonies, homologous recombination cells were further confirmed by Southern blotting. Heterozygous mutant ES cells were introduced into C57BL / 6J blastocysts by microinjection. Backcrossing of the born chimeric mice to C57BLZ6J mice resulted in F1 (first generation) heterozygous mice. After confirming whether or not the gene mutation was introduced into germ cells by Southern blotting, ASK1 knockout mice, which are homozygotes of ASK1, were established from the crossing of these F1 heterozygous mice. . Test example 3

Decreased expression of various cytokines and chemokines in ASK1 knockout mouse-derived cells

Inflammatory cytokine IL-1β stimulated by LPS using fetal fibroblasts (mouse embryon icfibrob 1 asts; ME F s) and spleen cells (sp 1 e no cytes) from ASK 1 knockout mice Then, it was examined whether ASK1 is involved in the induction of the expression of IL-16 and the chemokine MIΡ-1α. For fetal fibroblasts (MEFs), 10 g / mL LPS was added to cells seeded at a concentration of 1 × 10 ⁵ ce 11 s / we 11 The temporal change in the secretion amount of IL-16 protein into the culture supernatant at 12, 24 hours was measured. At the same time, the dose dependence on LPS (0.1, 1, 10 βg / mL) after 24 hours was also measured. For spleen cells (sple no cytes), ILs were added to cells seeded at a concentration of 1 × 10 ⁵ ce 11 s Zwe 11 by adding 10 Z gZmL of LPS to about 0.5 mL of medium. 1) Induce the expression of 3, IL-6 and MIP-1α, and 1-1,3, IL-6 and MIP in the culture supernatant at 6, 12, and 24 hours after LPS stimulation. -The amount of secreted protein was measured. The expression levels of each of the cytokines and chemokines were determined using the ELISA method, as in Test Example 1.

'Figures 6 and 7 show the results for fetal fibroblasts (MEFs), and Figures 8, 9 and 10 show the results for spleen cells (sple no cytes). A In fetal fibroblasts (MEFs) derived from SKI knockout mice, the expression level of IL-6 protein was dramatically increased both temporally (Fig. 6) and dose-dependently with LPS (Fig. 7). Was confirmed to have decreased. In spleen cells derived from ASK1 knockout mice (splenenocytes), IL-13 (Fig. 8), IL-6 (Fig. 9), and MIP-1α (Fig. 10) It has been confirmed that the expression levels of all have dramatically decreased. In other words, in ASK1 knockout mouse-derived cells, It was found that the expression levels of IL_lj3, IL-6, and MIP-1 as chemokines, which were stimulated by LPS, decreased dramatically. These experimental results clearly demonstrate that inhibition of ASK1 activity actually suppresses cytokine and chemokine production, and that ASK1 inhibitors are useful for various immune diseases.

Test example 4

Resistance of ASK1 knockout mice to sepsis model

The survival rates of the ASK1 knockout mouse and the wild-type mouse in the sepsis model were compared. Sepsis was induced by injecting LPS into mice intraperitoneally at a dose of 18.7 mg Zkg body weight. In sepsis, a sharp rise in cytokines has been observed, which has been shown to cause subsequent shock death. Therefore, the TNF-α concentration in the blood 2 hours after the induction was measured, and thereafter the survival rate of the individual was observed over time. In addition, 10 knockout mice and 10 wild-type mice were used.

FIG. 11 shows the change over time in the survival rate of the individual, and FIG. 12 shows the TNF-α concentration in the blood 2 hours after the induction in the ASK1 knockout mouse and the wild type mouse, respectively. In the ASK1 knockout mice, there was almost no increase in blood TNF-α concentration (FIG. 12), and the survival rate was also significantly improved (FIG. 11). These results indicate that inhibition of ASK1 activity is remarkably effective in improving pathological conditions such as sepsis. Industrial applicability

The present invention relates to the prevention or treatment of an immune disease using an ASK1 inhibitor as an active ingredient. According to the present invention, various immune diseases such as rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, psoriasis, inflammatory bowel disease, multiple sclerosis, thrombocytopenia, siedalen syndrome, bronchial asthma, atopic dermatitis, and sepsis Of the present invention can be provided.

Claims

The scope of the claims

1. A preventive or therapeutic agent for an immune disease, including an ASK1 inhibitor.

2. The ASK 1 inhibitor is an ASK 1 dominant negative, a recombinant vector expressing ASK 1 dominant negative, or a host cell transformed by the recombinant vector expressing ASK 1 dominant negative. The described prophylactic or therapeutic agent.

3. The preventive or therapeutic agent according to claim 2, wherein the ASK1 dominant negative is K709R or K709M.

4. The ASK1 inhibitor according to claim 1, wherein the ASK1 inhibitor is a host cell transformed with the ASK1 antisense oligonucleotide, the ASK1 antisense oligonucleotide expression recombinant vector or the ASK1 antisense oligonucleotide expression recombinant vector. Prophylactic or therapeutic agent.

5. ASK1 inhibitory chemical substance whose ASK1 inhibitory substance is selected from daryuthion S-transferase, nef, 14-13_3 protein and thioredoxin, and sense oligo of the chemical substance having the ASK1 inhibitory action A nucleotide, a recombinant vector expressing a chemical substance having an ASK1 inhibitory action, a recombinant vector expressing a sense oligonucleotide having a chemical substance having an ASK1 inhibitory action, or a vector characterized by the recombinant vector 3. The preventive or therapeutic agent according to claim 1, which is a transformed host cell.

6. The preventive or therapeutic agent according to any one of claims 1 to 5, wherein the immune disease is an autoimmune disease.

7. The preventive or therapeutic agent according to any one of claims 1 to 5, wherein the immune disease is an allergic disease.

8. The autoimmune disease is a disease selected from the group consisting of rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, psoriasis, inflammatory bowel disease, multiple sclerosis, thrombocytopenia and siedalen syndrome. The prophylactic or therapeutic agent according to the above.

9. The preventive or therapeutic agent according to claim 7, wherein the allergic disease is bronchial asthma, atopic dermatitis or sepsis.

10. A method for preventing or treating an immune disease, comprising using an effective amount of an ASK1 inhibitor.

11.The ASK1 inhibitor according to claim 10, wherein the ASK1 inhibitor is an ASK1 dominant negative, a ASK1 dominant negative expression recombinant vector or a host cell transformed with an ASK1 dominant negative expression recombinant vector. Method of treatment.

12. The method according to claim 11, wherein the ASK1 dominant negative body is K709R or K709M.

13. The ASK1 inhibitor is an ASK1 antisense oligonucleotide, an ASK1 antisense oligonucleotide-expressing recombinant vector or a host cell transformed by an ASK1 antisense oligonucleotide-expressing recombinant vector. 10. The prevention or treatment method according to 10.

14. ASK1 inhibitory substance selected from daryuthion S-transferase, Nef, 14-133 protein and thioredoxin, which has ASK1 inhibitory action, and sense of ASK1 inhibitory chemical substance Oligonucleotides, recombinant vector expressing a chemical substance having ASK1 inhibitory activity, recombinant vector expressing sense oligonucleotide having a chemical substance having ASK1 inhibitory action, or host transformed with such a recombinant vector 11. The method according to claim 10, which is a cell.

15. The method according to any one of claims 10 to 14, wherein the immune disease is an autoimmune disease.

16. The method according to any one of claims 10 to 14, wherein the immune disease is an allergic disease.

17. The autoimmune disease according to claim 15, wherein the autoimmune disease is a disease selected from rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, psoriasis, inflammatory bowel disease, multiple sclerosis, thrombocytopenia and siedalen syndrome. Prevention or treatment method.

18. The method according to claim 16, wherein the allergic disease is bronchial asthma, atopic dermatitis or sepsis.

19. Use of an ASK1 inhibitor for the manufacture of a formulation for the prevention or treatment of an immune disease.

20. The ASK1 inhibitor is a host cell transformed by an ASK1 dominant negative body, an ASK1 dominant negative body expression recombinant vector or an ASK1 dominant negative body expression recombinant vector. Use of the description.

21. Use according to claim 20, wherein the ASK1 dominant negative body is K709R or K709M.

22. The ASK 1 inhibitor is a host cell transformed with an ASK 1 antisense oligonucleotide, an ASK 1 antisense oligonucleotide expression recombinant vector or an ASK 1 antisense oligonucleotide expression recombinant vector. Use according to claim 19.

23. ASK1 inhibitory substance selected from daryuthion S-transferase, Nef, 14-3-13 protein and thioredoxin, which has ASK1 inhibitory action, and sense of ASK1 inhibitory chemical substance An oligonucleotide, a recombinant vector expressing a chemical substance having an ASK1 inhibitory action, a recombinant vector expressing a sense oligonucleotide having a chemical substance having an ASK1 inhibitory action, or a recombinant vector thereof. 20. The use according to claim 19, wherein the host cell is an isolated host cell.

24. The use according to any one of claims 19 to 23, wherein the immune disease is an autoimmune disease.

25. The use according to any of claims 19 to 23, wherein the immune disease is an allergic disease.

26. The autoimmune disease according to claim 24, wherein the autoimmune disease is a disease selected from rheumatoid arthritis, type I diabetes, systemic lupus erythematosus, psoriasis, inflammatory bowel disease, multiple sclerosis, thrombocytopenia and siedalen syndrome. use.

27. The use according to claim 25, wherein the allergic disease is bronchial asthma, atopic dermatitis or sepsis.