WO2002038179A1 - Preventives or remedies for endoplasmic reticulum stress-associated diseases - Google Patents
Preventives or remedies for endoplasmic reticulum stress-associated diseases Download PDFInfo
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- WO2002038179A1 WO2002038179A1 PCT/JP2001/009792 JP0109792W WO0238179A1 WO 2002038179 A1 WO2002038179 A1 WO 2002038179A1 JP 0109792 W JP0109792 W JP 0109792W WO 0238179 A1 WO0238179 A1 WO 0238179A1
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- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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Definitions
- the present invention relates to a disease caused by endoplasmic reticulum stress, which comprises an ASK1 (Apto os Sis Si gn a l-r e gu la ti ng Kinase 1) inhibitor.
- ASK1 Apto os Sis Si gn a l-r e gu la ti ng Kinase 1
- a method for preventing or treating ER stress-induced diseases including using an effective amount of an ASK1 inhibitor, and a formulation for preventing or treating ER stress-induced diseases
- ASK 1 inhibitors for preventing or treating ER stress-induced diseases
- Endoplasmic reticulum stress refers to a phenomenon in which abnormal proteins aggregate or accumulate in the endoplasmic reticulum.
- Endoplasmic reticulum stress is a phenomenon of polyglutamine disease, Alzheimer's disease, prion disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) ), FTDP-17 (anterotemporal dementia linked to chromosome 17), and other neurodegenerative diseases have been reported.
- abnormal proteins for example, polyglutamine is found in the brain or nerves in polyglutamine disease, and; 3 amyloid (Amy1oid ⁇ ) is found in brain in Alzheimer's disease.
- IRE1 which is understood to be a sensor molecule that recognizes abnormal proteins in the endoplasmic reticulum, activates MAP kinase JNK through TRAF2, an adapter protein. It is clear that this will happen.
- apoptosis caused by endoplasmic reticulum stress was suppressed in knockout mice of caspase 12, a cysteine protease. (Urano F et al., Science, Vol.287, pp.664-666 (2000); Atsushi Matsuzawa et al., Biological Science, Vol.51, No.4, pp.266-272 (2000))
- ASK1 is a kind of kinase belonging to the MAPKK kinase (MAPKKK) family.
- human ASK1 is composed of 1375 amino acids (protein of SEQ ID NO: 1 described in TO97 / 40143). 1 is composed of 1379 amino acids (Journal of the Oral Disease Society, No. 3, pp. 45 (1998), protein shown in Fig. 1), and is extremely high at 91.9% for human ASK 1 and mouse ASK 1 It is reported that homology is observed.
- M APKK kinase is a kinase belonging to the signal transduction cascade of the MAP (Mitogen-activated Protein) kinase family known as a conserved intracellular signaling pathway from mammals including humans to yeast. Downstream of the kinase, there are two kinds of kinases, MAPK kinase (MAPKK) and MAP kinase (MAPK). (Ichijo H.
- ASK1 is activated by TNF (tumor necrosis factor) via the TNF receptor, and the activated ASK1 is involved in the induction of apoptosis in cells via the signaling cascade. It was reported that when a dominant-negative human ASK1 mutant, Jurkat cells, was highly expressed in Jurkat cells, apoptosis induced by TNF- ⁇ was suppressed. I have. Therefore, it has been disclosed that ASK 1 is useful as a therapeutic agent for malignant tumors or a gene therapeutic for malignant tumors. However, no involvement of A SKI in ER stress has been reported. (Ichijo H. et al., Science, Vol.275, pp.90-94 (1997); WO97 / 40143)
- the present invention relates to an agent for preventing or treating a disease caused by endoplasmic reticulum stress, comprising an ASK1 inhibitor.
- the present invention also relates to a method for preventing or treating a disease caused by endoplasmic reticulum stress, comprising using an effective amount of an ASK1 inhibitor.
- the present invention relates to the use of an ASK1 inhibitor for producing a drug for preventing or treating a disease caused by ER stress.
- Figure 1 shows that apoptotic cells induced by endoplasmic reticulum stress by thapsigargin were converted into MESK cells from ASK1 knockout mice (ASK1 ⁇ / ⁇ ) and wild-type MEFS cells using the TUNEL method.
- ASK1 + / + This is a photo of comparative detection. Representative photographs 6 hours after induction of ER stress are shown. The upper row shows unstimulated control cells, and the lower row shows evening psigargin-treated cells. You.
- FIG. 2 shows the results of the TUNEL method for detecting the percentage of ER stress-induced apoptosis cells by thapsigargin shown in FIG. 1, and shows the results of MEFS cells of ASK1 knockout mice (ASK 1 ⁇ / ⁇ ; black bar). ) And wild type ME F s cells (ASK + / +; white bar) are compared and graphed over time (after 0, 2 and 6 hours). In the data, more than 100 cells were counted in each of three or more fields, and TUNEL-positive cells were regarded as apoptotic cells, and the average value of the ratio was shown. Error bars represent standard deviation. The vertical axis shows the percentage (%) of apoptotic cells, and the horizontal axis shows time (hour).
- Fig. 3 shows the resistance to cell death induced by endoplasmic reticulum stress induced by thapsigargin using the MTT method.
- the MESKs of ASK1 knockout mice (AS K1-/-; black circles) and wild-type MEFS cells ( ASK + Z +; open circles) are graphs showing the cell viability of each cell over time (after 0, 8, 16 and 24 hours). Data are shown as the average of five or more independent experiments. Error bars represent standard deviation. The vertical axis indicates cell viability (%), and the horizontal axis indicates time (hour).
- Fig. 4 shows that the MTT method was used to demonstrate the resistance to cell death induced by dithiothreitol and tunicamycin as various ER stress inducers, in addition to thapsigargin, using MEFs (AS Kl_
- AS Kl_ This is a graph comparing the comparison between wild type MEFS cells (ASK + Z +; white bar) and wild type MEFS cells (black bar). Data were expressed as the mean cell viability 8 hours after ER stress induction, performed in 5 or more independent experiments. Error bars represent standard deviation. The vertical axis represents the cell viability (3 ⁇ 4), and the horizontal axis represents the ER stress inducer used. Indicates the name.
- Fig. 5 shows the primary culture of ASK1 knockout mouse primary cultured neurons (ASK1-/-; black bars) using the MTT method for the resistance to neuronal cell death induced by the neurodegenerative protein Amy1oid-3.
- wild-type primary cultured neurons ASK + / +; open bar
- Error-number represents standard deviation.
- the vertical axis indicates neuronal cell viability (%), and the horizontal axis indicates time (days).
- the present inventors have conducted intensive studies to find a drug useful for the prevention or treatment of a disease caused by endoplasmic reticulum stress.As a result, ASK1 is involved in apoptosis caused by endoplasmic reticulum stress, and ASK1 inhibitor The present inventors have found that they are useful for endoplasmic reticulum stress-related diseases, and have accomplished the present invention.
- the present inventors first produced ASK1 knockout mice by the method described below. Fetal fibroblasts were collected from the prepared ASK1 knockout mice, and the occurrence of apoptosis was observed using various inducers that induce ER stress, e.g., evening psigargin and tunicamycin dithiothreitol. As a result, in all cases, apoptosis was significantly suppressed in fetal fibroblasts of ASK1 knockout mice as compared with fetal fibroblasts of wild-type (normal) mice. This indicates that ASK1 is closely involved in the induction of apoptosis by ER stress.
- the 709th lysine residue of ASK1 is an ATP binding site, and by replacing this with arginine or methionine, the kinase catalytic activity can be inactivated.
- This mutant ASK1 (K709R, K709M) functions as a dominant negative (dominantly suppressed) form.
- ASK1 dominant negative Integrates a base sequence encoding a mutant ASK1 (e.g., K709R, K709M) into an expression vector and transfects the vector into cells by lipofection or adenovirus infection. By doing so, it can be overexpressed in cells. Overexpressed cells can significantly suppress apoptosis due to ER stress.
- an ASK1 inhibitor as an active ingredient, a drug useful for preventing or treating a disease caused by endoplasmic reticulum stress can be provided.
- an effective amount of an ASK1 inhibitor it is possible to provide a method useful for preventing or treating a disease caused by endoplasmic reticulum stress.
- 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.
- ⁇ + / + '' means that two A SKI loci present on each of a pair of homologous chromosomes in a mouse or mouse-derived cells are both normal, that is, wild-type.
- ⁇ -Z- '' indicates 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 the ASK1 protein is not expressed, That is, it indicates that ASK1 is a knocked out mouse or a mouse-derived cell.
- the disease caused by endoplasmic reticulum stress refers to a disease in which endoplasmic reticulum stress is involved in the onset or progression of the pathological condition, such as polyglutamine disease, Alzheimer's disease, prion disease, Parkinson's disease, and muscle.
- the pathological condition such as polyglutamine disease, Alzheimer's disease, prion disease, Parkinson's disease, and muscle.
- neurodegenerative diseases such as amyotrophic lateral sclerosis and FTDP-17.
- the ASK1 inhibitor is not limited to a substance having an ASK1 inhibitory action itself, and may be an ASK1 inhibitor in vivo or in a culture system. Includes those that produce inhibitors, including, for example, adding, inserting, substituting and / or deleting one or more amino acids in the amino acid sequence (eg, deletion of the 960th alanine residue) (For example, Wang X Set al., J. Biol.
- ASK1 antisense oligonucleotide for human ASK1 hereinafter, ASK1 antisense oligonucleotide
- chemical substance having ASK1 inhibitory action eg, daltathione S
- Transferase Mul-l, etc.
- Nef Nef
- 1413-3 protein thioredoxin, etc.
- a recombinant vector capable of expressing the dominant negative or antisense oligonucleotide (hereinafter referred to as an ASK1 dominant negative expression recombinant vector or ASK1 antisense oligonucleotide expressing recombinant vector), ASK1 inhibition
- a recombinant vector capable of expressing a chemical substance having an action or its sense oligonucleotide in a target tissue or host cell hereinafter referred to as a chemical substance having an ASK1 inhibitory action.
- Recombinant expression vectors or sense oligonucleotide-expressing recombinant vectors of chemicals having ASK1 inhibitory activity), and host cells transformed with those recombinant vectors hereinafter ASK1 dominant-negative in some cases
- 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 substance expression vector having ASK1 inhibitory activity, or Recombinant expression of a sense oligonucleotide expressing a chemical substance having ASK 1 inhibitory activity or a host cell transformed with such a recombinant vector into a target tissue in a living body by an appropriate method to obtain a desired ASK 1 dominant Negative form, ASK1 antisense nucleotide, use as a genetic preventive or therapeutic agent by expressing a chemical substance having ASK1 inhibitory activity or its sense oligonucleotide, ASK1 dominant negative form, ASK1 antisense Oligonucleotides, ASK1 inhibitory chemicals, or ASK (1) Use of a preparation containing a sense oligonucleotide, which is a chemical substance having an inhibitory action, as an active ingredient orally or parent
- the vector examples include a plasmid vector, a virus vector (for example, a retrovirus vector, an adenovirus vector, a herpes virus vector, a Sendai virus vector, a vaccinia virus vector), and a ribosome vector (for example, Kachonic Ribosome vector).
- a virus vector for example, a retrovirus vector, an adenovirus vector, a herpes virus vector, a Sendai virus vector, a vaccinia virus vector
- a ribosome vector for example, Kachonic Ribosome vector
- nucleotide sequences that control the expression to express antisense oligonucleotides eg, promoter sequence, terminator sequence, enhancer sequence
- oligonucleotides eg, promoter sequence, terminator sequence, enhancer sequence
- Gene markers for selecting microorganisms, insect cells, animal cultured cells, etc. Eg, a neomycin resistance gene, a kanamycin resistance gene
- host cells include Escherichia coli, yeast, insect cells, and animal cells such as CHO cells, COS cells, mink lung epithelial cells (eg, MvlLu), lymphocytes, fibroblasts, blood cells, and tumor cells. be able to.
- Methods for introducing a recombinant vector into a target tissue or host cell include the HV J liposome method (Kaneda, Experimental Medicine, Vol. 12, No. 2, p. 78 (1994); Morishita et al., Experimental Medicine , Vol. 12, No. 15, p. 158 (1994)), a method of directly administering an ASK1 inhibitor by injection, etc., a calcium phosphate method, a DEAE-dextran method, an electroporation method, and a method using a gene gun. (TM Klein et al., Bio / Technology 10, pp. 286-291 (1992)), a method of administration by the lipofection method (Nabel et al., Science, Vol. 244, p. 1285 (1990)) (For example, a retrovirus vector, an adenovirus vector, a herpes virus vector, a vaccinia virus vector, etc.).
- preparation forms are used depending on the usage.
- the formulation include tablets, capsules, granules, powders, pills, fine granules, troches, injections, rectal administration, suppositories, etc., and are administered orally or parenterally Is done.
- preparations containing an ASK1 inhibitor can be used as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents, etc., using the ASK1 inhibitor as an active ingredient. 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.
- pharmaceutical additives such as agents, preservatives, wetting agents, emulsifiers, dispersing agents, stabilizers, and solubilizing agents, and dispensing according to a conventional method.
- the dosage of the ASK1 inhibitor in the pharmacological prevention or treatment of a disease caused by endoplasmic reticulum stress is appropriately determined in consideration of the usage, patient age, gender, degree of symptoms, type of disease, etc.
- the dose is about 0.1 to 500 mg, preferably about 0.5 to 10 Omg per day for an adult, and it can be administered once or several times a day.
- AS K1 inhibitors cause ER stress.
- the dose for genetic prevention or treatment of a disease can be determined accordingly.
- an ASK1 dominant negative, an ASK1 antisense oligonucleotide, a chemical substance having an ASK1 inhibitory action or a nucleotide sequence encoding a sense oligonucleotide thereof, or a vector containing the same, or a host transformed with the vector By introducing cells into target tissues using cells, it is possible to suppress the development or progression of vesicles such as neurodegenerative diseases and diseases caused by body stress.
- oral administration or parenteral administration of an ASK1 dominant negative body, an ASK1 antisense oligonucleotide, a chemical substance having ASK1 inhibitory activity, or a preparation containing the sense oligonucleotide thereof may reduce the risk of neurodegenerative diseases. It can suppress the onset or progress of diseases caused by endoplasmic reticulum stress.
- Test example 1 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.
- chromosomal DNA fragments including the first exon of ASK1 were cloned from the chromosome (dienomic) DNA library of 129ZSv J 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 positive with GFP (green fluo rescencerotein).
- a targeting vector was constructed by substituting the neomycin resistance gene for selection.
- pBluescript SK Strate gene
- DT-A diphtheria toxin heavy chain
- the setting vector was introduced into J1 ES cells (embryon icst emce11) by an electoral poration method. After selecting a neomycin ffiH live ES cell colony, 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 C57BL / 6J 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 2
- ASK1 is involved in endoplasmic reticulum stress-induced apoptosis using fetal fibroblasts (m0 use embryon icfibr ob lasts; MEFs) derived from ASK1 knockout mice on day 5. It was examined whether or not it was. Incubate MEFs at a concentration of 1 x 10 5 ce 11 s / we 11 in a 24-well plate, convert to serum-free medium 2 hours before stimulation, and 2 ⁇ thapsigargin, 2.5 agZmL Endoplasmic reticulum stress was induced by adding tunicamycin or 1 OmM dithiothreitol to the medium.
- Thapsigargin is a drug that inhibits calcium uptake into the endoplasmic reticulum and induces endoplasmic reticulum stress by disrupting calcium homeostasis in the endoplasmic reticulum.
- tunicamycin is an agent that induces endoplasmic reticulum stress by inhibiting protein glycosylation and dithiothreitol by inhibiting disulfide bonds.
- Detection of cell death by apoptosis was performed using the TUNEL (Evening Minal Deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling) method and the MTT (3- [4,5-dimethylthiazole-2 1) 2,5 di-Fe2Lu 2 JJ—Tetrazoliu (Bromide) method.
- TUNEL method is a method that can specifically stain the fragmented DNA.
- Hoechst 33258 (Ing / mL) and propidiumiodide (10 g / mL), which can stain both live and dead cells, were used.
- the stained cells were embedded with Mowio 1 (Wako Pure Chemical Industries, Ltd.), and dead cells were identified by fluorescence microscopy. %) Means at least 3 or more cells as a whole More than 100 cells were counted in each field of view, and the ratio was calculated as the ratio of the number of cells positive for TUNEL staining.
- the MTT method is an Atsey method for detecting the ratio of living cells using the change in absorbance due to reduction of methylth'iazolteletrazolium to formazan as an index.
- MTT assy was performed.
- MTT Atsushi solution stock solution (Dojindo) was added to the cell culture at a 10-fold dilution, allowed to stand at 37 ° C for 1 hour, and the absorbance in the culture was measured at 45 Onm. The percentage of viable cells () was calculated assuming the absorbance value in the untreated cell culture solution as 100%.
- FIG. 1 is a visualization of apoptotic cells by the TUNEL method. Apoptotic cells are stained with green fluorescence. The whole picture of the cells is stained with blue Hoechst 33258 and red pr0pidimuiodide.
- FIG. 1 is a representative visual field 6 hours after stimulation
- FIG. 2 is a graph summarizing the results of observations in three or more visual fields. Compared to wild-type ME F s (+ / +), fetal fibroblast ME F s (-/-) from ASK 1 knockout mice were more resistant to endoplasmic reticulum stress-induced cell death by evening pshigargin. , Markedly resistant. Similar results were confirmed by the MTT method (Fig. 3).
- ASK1-deficient MEFS cells are also resistant to apoptosis by tunicamycin dithiothreitol, a representative compound that induces endoplasmic reticulum stress by a mechanism different from evening psigargin. ( Figure 4).
- Amy 1 oid Apoptosis of primary cultured neurons by the neurodegenerative protein Embryos 14. Using primary cultured neurons derived from the cerebrum of the ASK 1 knockout mouse on day 5, ASK1 has been converted to the neurodegenerative protein Amy 1 oid —) We examined whether we are involved in apoptosis by (3). After isolating the mouse cerebral hemisphere and dislodging the tissue by pipetting in the medium, a concentration of approximately 1 x 10 5 ce 11 s / we 11 on a 24-L plate coated with poly-L-lysine and fibronectin And sowed. On day 1 and day 3 of culture 10118 1111 ⁇ ?
- Amy 1 oid— / 3 is Alzheimer's disease It is a denatured protein that aggregates in large amounts in the brain tissue of the elderly, has strong neurotoxicity, and is a causative protein of neurodegeneration in Alzheimer's disease.
- Amy 1 oid-/ 3 (25-35) (The amino acid sequence of the Amy 1 oid- / 3 protein from the 25th to the 35th peptide, which is said to be the most neurotoxic part)
- the viability after 3 days was added to the medium at a concentration of 25 M and the viability was calculated as the viable cell ratio (%) to the untreated primary cultured neurons by the same method as the MTT method described in Test Example 2.
- Figure 5 shows the survival rate against neurotoxicity caused by Amy 1 oid—; 3 in neurons derived from wild-type mouse (+ / +) and neurons derived from ASK 1 knockout mouse (1-/-). It is. While most wild-type neurons undergo Amy 1 oid_j3-induced cell death, neurons derived from ASK 1 knockout mice are significantly more prone to Amy 1 oid-induced neuronal death. In »showed a life.
- Amy1 oid—] 3 a neurodegenerative protein, has been shown to induce ER stress, and the results in Figure 5 show that ASK1 is actually responsible for ER stress-induced neuronal apoptosis. It is shown that it is done. In addition, the fact that ASK1 deficiency abolishes Amy1oid-3 / 3-induced apoptosis demonstrates the effect of ASK1 inhibitors on neurodegenerative diseases. [Industrial applicability]
- the present invention relates to suppression of ER stress-induced apoptosis by inhibiting ASK1.
- Prevention or treatment of diseases caused by endoplasmic reticulum stress in particular, neurodegenerative diseases such as polyglutamine disease, Alzheimer's disease, Parkinson's disease, prion disease, amyotrophic lateral sclerosis, and FTDP-17 according to the present invention.
- An agent or a prophylactic or therapeutic method can be provided.
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Abstract
Preventives or remedies for diseases caused by endoplasmic reticulum stress (for example, neurodegenerative diseases typified by poyglutamine disease, Alzheimer's disease, Parkinson's disease, prion disease, amyotrophic lateral sclerosis and FTDP-17) which contain chemicals having an effect of inhibiting endoplasmic reticulum stress-induced apoptosis and an ASK1 inhibitory effect (for example, ASK1 dominant negative compounds, ASK1 antisense oligonucleotides, glutathione, S-transferases (Mul-1, etc.), Nef, 14-3-3 protein, thioredoxin), sense oligonucleotides thereof, expression vectors thereof or host cells transformed by these expression vectors.
Description
小胞体ストレス関連疾患の予防又は治療剤 〔技術分野〕 Agent for preventing or treating ER stress-related diseases [Technical field]
本発明は、 ASK1 (Ap op t o s i s S i gn a l -r e gu l a t i ng K i na s e 1 ) 阻害物質を含む、 小胞体ストレスに起因する疾患 明 The present invention relates to a disease caused by endoplasmic reticulum stress, which comprises an ASK1 (Apto os Sis Si gn a l-r e gu la ti ng Kinase 1) inhibitor.
の予防または治療剤、 ASK 1阻害物質を有効量使用することを含む、 小胞体 ストレスに起因する疾患の予防または治療方法、 および小胞体ストレスに起因 する疾患の予防または治療用の製剤を製造するための ASK 1阻害物質の使用 に関するものである。 A method for preventing or treating ER stress-induced diseases, including using an effective amount of an ASK1 inhibitor, and a formulation for preventing or treating ER stress-induced diseases The use of ASK 1 inhibitors for
〔背景技術〕 (Background technology)
小胞体ストレスとは、異常蛋白質が小胞体内に凝集又は蓄積する現象をいい、 小胞体ストレスがポリグルタミン病、 アルツハイマー病、 プリオン病、 パ一キ ンソン病、 筋萎縮性側索硬化症（ALS)、 FTDP- 17 (17番染色体にリ ンクする前側頭葉性痴呆症） 等の神経変性疾患に深く関与していることが報告 されている。 異常蛋白質については、 例えば、 ポリグルタミン病においては脳 内又は神経にポリグルタミンの沈着が認められ、 アルツハイマー病においては 脳内に ;3アミロイド （Amy 1 o i d β) の沈着が認められている。同様に、 プリオン病においては異常型プリオン （P r P 、 パーキンソン病において は α—シヌクレイン（a— Synuc l e i n)、筋萎縮性側索硬化症において は変異型 SOD 1遺伝子産物、 FTDP— 17においてはタウ （Tau) の沈 着がそれぞれ認められている。 そして、 この小胞体ストレスが過剰な場合ある いは長時間持続した場合、細胞がアポトーシス様の形態変化を起こして死滅し、 上記神経変性疾患は小胞体を起源とするアポ卜一シスであることが指摘されて いる。 このように、 小胞体ストレスは、 これらの神経変性疾患の発症における
由々しき危険因子であると考えられている。 （Niwa N. et al.， Cell, Vol.99, pp.691-702 (1999)； Katayama T. et al., Nature Cell Biol., Vol.1, pp.479-702 (1999)； Nakagawa T. et al" Nature, Vol.403, pp.98-103 (2000)； 垣塚彰，細胞 工学， Vol.18, Ν0·6,ρρ.782-788 (1999); 今泉和則， 実験医学， Vol.l8， Νο·13, ρρ.1781-1786 (2000)) Endoplasmic reticulum stress refers to a phenomenon in which abnormal proteins aggregate or accumulate in the endoplasmic reticulum. Endoplasmic reticulum stress is a phenomenon of polyglutamine disease, Alzheimer's disease, prion disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS) ), FTDP-17 (anterotemporal dementia linked to chromosome 17), and other neurodegenerative diseases have been reported. As for abnormal proteins, for example, polyglutamine is found in the brain or nerves in polyglutamine disease, and; 3 amyloid (Amy1oid β) is found in brain in Alzheimer's disease. Similarly, abnormal prions (PrP in prion diseases, α-synuclein (a-Synuclein) in Parkinson's disease, mutant SOD1 gene product in amyotrophic lateral sclerosis, and FTDP-17 Tau deposits have been observed, and when this ER stress is excessive or long-lasting, cells undergo apoptotic-like morphological changes and die, resulting in the above neurodegenerative diseases. It has been pointed out that ER is an apoptosis originating in the endoplasmic reticulum. It is considered a serious risk factor. (Niwa N. et al., Cell, Vol. 99, pp. 691-702 (1999); Katayama T. et al., Nature Cell Biol., Vol. 1, pp. 479-702 (1999); Nakagawa T et al "Nature, Vol.403, pp.98-103 (2000); Akira Kakizuka, Cell Engineering, Vol.18, Ν0.6, ρρ782-788 (1999); Kazunori Imaizumi, Experimental Medicine, Vol. l8, Νο · 13, ρρ.1781-1786 (2000))
小胞体ストレスの伝達経路やその活性化のメカニズムとしては、 小胞体内の 異常蛋白質を認識するセンサー分子と解される I RE 1が、 アダプター蛋白で ある TRAF2を介して MAPキナーゼである J N Kを活性化することが明ら かになつている。 また、 システィンプロテア一ゼであるカスパーゼ 12のノッ クアウトマウスでは小胞体ストレスによるアポトーシスが抑制されたことが報 告されている。 （Urano F et al., Science, Vol.287, pp.664-666 (2000)； 松沢厚 ら， 生体の科学， Vol.51, No.4, pp.266-272 (2000)) Regarding the endoplasmic reticulum stress transmission pathway and its activation mechanism, IRE1, which is understood to be a sensor molecule that recognizes abnormal proteins in the endoplasmic reticulum, activates MAP kinase JNK through TRAF2, an adapter protein. It is clear that this will happen. In addition, it has been reported that apoptosis caused by endoplasmic reticulum stress was suppressed in knockout mice of caspase 12, a cysteine protease. (Urano F et al., Science, Vol.287, pp.664-666 (2000); Atsushi Matsuzawa et al., Biological Science, Vol.51, No.4, pp.266-272 (2000))
ASK1は MAPKKキナーゼ （MAPKKK) ファミリーに属するキナー ゼの一種であり、 例えば、 ヒト ASK 1は 1375個のアミノ酸により構成さ れ （TO97/40143号公報記載の配列表配列番号 1の蛋白質）、 マウス ASK 1は 1379個のアミノ酸から構成されており （口腔病学会雑誌， No.3,pp.45 (1998) 図 1記載の蛋白質）、 ヒト ASK 1とマウス ASK 1には 91. 9%の 極めて高い相同性が認められることが報告されている。 同様に、 ヒト ASK1 の c D N A (WO97/40143号公報記載の配列表配列番号 2) やマウス ASK 1 の cDNA (口腔病学会雑誌， No.3， pp.45 (1998) 図 1) が開示されている。 M APKKキナーゼは、 ヒトを含む哺乳類から酵母に至るまで保存された細胞内 シグナル伝達経路として知られている MAP (Mitogen-activated Protein) キ ナーゼスーパ一ファミリーによるシグナル伝達力スケードに属するキナーゼで あり、 MAPKKキナーゼの下流には、 MAPKキナーゼ （MAPKK) およ び MAPキナーゼ（MAP K)の 2種類のキナーゼが存在する。 （Ichijo H. et al.， Science, Vol.275,pp.90-94 (1997)； WO97/40143号公報； 飛梅圭， 口腔病学会 雑誌， No.3, pp.42-52 (1998))
ASK1については TNF受容体を介して TNF (腫瘍壊死因子） 一ひによ り活性化され、 活性化された A S K 1が当該シグナル伝達カスケ一ドを介して 細胞のアポト一シス誘導に関与していることが確認されており、 ヒト ASK 1 の変異体であるドミナントネガティブ体を J u r k a t細胞に高発現させた場 合、 TNF— αにより誘導されるアポト一シスが抑制されたことが報告されて いる。 それ故、 ASK 1が悪性腫瘍治療剤または悪性腫瘍遺伝子治療剤として 有用であることが開示されている。 しかしながら、 小胞体ストレスにおける A SKIの関与については何ら報告されていない。 （Ichijo H. et al.， Science, Vol.275, pp.90-94 (1997)； WO97/40143号公報） ASK1 is a kind of kinase belonging to the MAPKK kinase (MAPKKK) family. For example, human ASK1 is composed of 1375 amino acids (protein of SEQ ID NO: 1 described in TO97 / 40143). 1 is composed of 1379 amino acids (Journal of the Oral Disease Society, No. 3, pp. 45 (1998), protein shown in Fig. 1), and is extremely high at 91.9% for human ASK 1 and mouse ASK 1 It is reported that homology is observed. Similarly, the cDNA of human ASK1 (SEQ ID NO: 2 in WO97 / 40143) and the cDNA of mouse ASK1 (Journal of the Society of Oral Diseases, No.3, pp.45 (1998), FIG. 1) are disclosed. ing. M APKK kinase is a kinase belonging to the signal transduction cascade of the MAP (Mitogen-activated Protein) kinase family known as a conserved intracellular signaling pathway from mammals including humans to yeast. Downstream of the kinase, there are two kinds of kinases, MAPK kinase (MAPKK) and MAP kinase (MAPK). (Ichijo H. et al., Science, Vol.275, pp.90-94 (1997); WO97 / 40143; Kei Tobiume, Journal of Oral Disease Society, No.3, pp.42-52 (1998) ) ASK1 is activated by TNF (tumor necrosis factor) via the TNF receptor, and the activated ASK1 is involved in the induction of apoptosis in cells via the signaling cascade. It was reported that when a dominant-negative human ASK1 mutant, Jurkat cells, was highly expressed in Jurkat cells, apoptosis induced by TNF-α was suppressed. I have. Therefore, it has been disclosed that ASK 1 is useful as a therapeutic agent for malignant tumors or a gene therapeutic for malignant tumors. However, no involvement of A SKI in ER stress has been reported. (Ichijo H. et al., Science, Vol.275, pp.90-94 (1997); WO97 / 40143)
現在、 小胞体ストレスによる神経変性疾患に有用な予防または治療剤は全く 知られておらず、 目下これらの神経変性疾患の予防または治療剤を早期に開発 すべく鋭意研究が行われている。 At present, no useful preventive or therapeutic agent for neurodegenerative diseases caused by endoplasmic reticulum stress is known at all, and intensive research is currently being conducted to develop early preventive or therapeutic agents for these neurodegenerative diseases.
〔発明の開示〕 [Disclosure of the Invention]
本発明は、 ASK 1阻害物質を含む、 小胞体ストレスに起因する疾患の予防 または治療剤に関するものである。 The present invention relates to an agent for preventing or treating a disease caused by endoplasmic reticulum stress, comprising an ASK1 inhibitor.
また、 本発明は、 AS K1阻害物質を有効量使用することを含む、 小胞体ス トレスに起因する疾患の予防または治療方法に関するものである。 The present invention also relates to a method for preventing or treating a disease caused by endoplasmic reticulum stress, comprising using an effective amount of an ASK1 inhibitor.
更には、 本発明は、 小胞体ストレスに起因する疾患の予防または治療用の製 剤を製造するための ASK 1阻害物質の使用に関するものである。 Furthermore, the present invention relates to the use of an ASK1 inhibitor for producing a drug for preventing or treating a disease caused by ER stress.
〔図面の簡単な説明〕 [Brief description of drawings]
第 1図は、 TUNE L法を用いて、 タプシガルギンによる小胞体ストレスに よって誘導されたアポトーシス細胞を ASK 1ノックァゥトマウス由来の ME F s細胞 （ASK1—/—) と野生型 ME F s細胞 （ASK1+/ + ) にっき 比較検出した写真である。 小胞体ストレス誘導後 6時間目の代表的な写真を示 した。 上段は無刺激のコントロール細胞、 下段は夕プシガルギン処理細胞であ
る。 4枚ずつの写真のうち、 左側の Ho e c h s t 33258 (青色） と p r o p i d i urn i od i d e (赤色） での染色は、 細胞の全体像を示し、 それらのうちアポトーシスを引き起こしている細胞の核は、 右側の写真で TU NEL染色法により緑色を呈している。 TUNE L染色写真の右下の数字は、 アポトーシス細胞の割合 （％) である。 Figure 1 shows that apoptotic cells induced by endoplasmic reticulum stress by thapsigargin were converted into MESK cells from ASK1 knockout mice (ASK1 − / −) and wild-type MEFS cells using the TUNEL method. ASK1 + / +) This is a photo of comparative detection. Representative photographs 6 hours after induction of ER stress are shown. The upper row shows unstimulated control cells, and the lower row shows evening psigargin-treated cells. You. In each of the four photographs, staining with Ho echst 33258 (blue) and propidi urn iodide (red) on the left shows a complete picture of the cells, of which the nuclei of cells that are causing apoptosis are: The picture on the right shows a green color by TUNEL staining. The numbers at the bottom right of the TUNEL stained photographs are the percentage of apoptotic cells (%).
第 2図は、 第 1図に示したタプシガルギンによる小胞体ストレス誘導性アポ トーシス細胞の割合について TUNE L法により検出した結果を、 ASK1ノ ックアウトマウスの ME F s細胞 (ASK 1 -/-；黒棒） と野生型 ME F s 細胞 （ASK + /+；白棒） にっき比較し、 経時的に （0、 2及び 6時間後） グラフ化したものである。 データは、 3つ以上の視野で、 それぞれ 100個以 上の細胞数を数えて、そのうちの TUNE L陽性細胞をアポトーシス細胞とし、 その割合の平均値を示した。 エラ一·バーは標準偏差を表す。 縦軸はアポトー シス細胞の割合 (%) を、 横軸は時間 （時間） を示す。 FIG. 2 shows the results of the TUNEL method for detecting the percentage of ER stress-induced apoptosis cells by thapsigargin shown in FIG. 1, and shows the results of MEFS cells of ASK1 knockout mice (ASK 1 − / −; black bar). ) And wild type ME F s cells (ASK + / +; white bar) are compared and graphed over time (after 0, 2 and 6 hours). In the data, more than 100 cells were counted in each of three or more fields, and TUNEL-positive cells were regarded as apoptotic cells, and the average value of the ratio was shown. Error bars represent standard deviation. The vertical axis shows the percentage (%) of apoptotic cells, and the horizontal axis shows time (hour).
第 3図は、 MTT法を用い、 タプシガルギンによる小胞体ストレスによって 誘導される細胞死に対する耐性について、 ASK 1ノックアウトマウスの ME F s (AS K1—/一；黒丸） と野生型 ME F s細胞 （ASK + Z+;白丸） にっき比較し、 経時的に （0、 8、 16及び 24時間後） それぞれの細胞生存 率を示したグラフである。 データは、 5回以上の独立した実験を行い、 その平 均値として示した。 エラ一 ·バ一は標準偏差を表す。 縦軸は細胞生存率 (%) を、 横軸は時間 （時間） を示す。 Fig. 3 shows the resistance to cell death induced by endoplasmic reticulum stress induced by thapsigargin using the MTT method. The MESKs of ASK1 knockout mice (AS K1-/-; black circles) and wild-type MEFS cells ( ASK + Z +; open circles) are graphs showing the cell viability of each cell over time (after 0, 8, 16 and 24 hours). Data are shown as the average of five or more independent experiments. Error bars represent standard deviation. The vertical axis indicates cell viability (%), and the horizontal axis indicates time (hour).
第 4図は、 MTT法を用い、 タプシガルギンの他に、 様々な小胞体ストレス 誘発剤として、 ジチオスレィトールおよびッニカマイシンによって誘導される 細胞死に対する耐性について、 ASK 1ノックアウトマウスの MEF s (AS Kl_/一；黒棒） と野生型 ME F s細胞 （ASK + Z+ ;白棒） にっき比較 したグラフである。 データは、 5回以上の独立した実験を行い、 小胞体ストレ ス誘導後 8時間目の細胞生存率の平均値として示した。 エラー ·バーは標準偏 差を表す。 縦軸は細胞生存率 (¾) を、 横軸は用いた小胞体ストレス誘発剤の
名称を示す。 Fig. 4 shows that the MTT method was used to demonstrate the resistance to cell death induced by dithiothreitol and tunicamycin as various ER stress inducers, in addition to thapsigargin, using MEFs (AS Kl_ This is a graph comparing the comparison between wild type MEFS cells (ASK + Z +; white bar) and wild type MEFS cells (black bar). Data were expressed as the mean cell viability 8 hours after ER stress induction, performed in 5 or more independent experiments. Error bars represent standard deviation. The vertical axis represents the cell viability (¾), and the horizontal axis represents the ER stress inducer used. Indicates the name.
第 5図は、 MTT法を用い、 神経変性蛋白質 Amy 1 o i d— /3によって誘 導される神経細胞死に対する耐性について、 ASK 1ノックァゥトマウスの初 代培養神経細胞 （ASK1 - /—；黒棒） と野生型初代培養神経細胞 （ASK + /+；白棒） にっき比較し、 Amy 1 o i d— j3を添加した当日 （0日）、 お よび 3日後におけるそれぞれの神経細胞の生存率を示したグラフである。 デー 夕は、 5回以上の独立した実験を行い、 その平均値として示した。 エラー -バ 一は標準偏差を表す。 縦軸は神経細胞生存率 (%) を、 横軸は時間 （日） を示 す。 Fig. 5 shows the primary culture of ASK1 knockout mouse primary cultured neurons (ASK1-/-; black bars) using the MTT method for the resistance to neuronal cell death induced by the neurodegenerative protein Amy1oid-3. ) And wild-type primary cultured neurons (ASK + / +; open bar) compared to each other, showing the survival rates of the respective neurons on the day (0 day) and 3 days after the addition of Amy 1 oid—j3 It is a graph. In the evening, five or more independent experiments were performed and the average value was shown. Error-number represents standard deviation. The vertical axis indicates neuronal cell viability (%), and the horizontal axis indicates time (days).
〔発明を実施するための最良の形態〕 [Best mode for carrying out the invention]
本発明者らは、 小胞体ストレスに起因する疾患の予防または治療に有用な薬 剤を見出すベく鋭意検討した結果、 A S K 1が小胞体ストレスによるアポトー シスに関与し、 ASK 1阻害物質が小胞体ストレス関連疾患に有用であるとい う知見を得、 本発明を成すに至った。 The present inventors have conducted intensive studies to find a drug useful for the prevention or treatment of a disease caused by endoplasmic reticulum stress.As a result, ASK1 is involved in apoptosis caused by endoplasmic reticulum stress, and ASK1 inhibitor The present inventors have found that they are useful for endoplasmic reticulum stress-related diseases, and have accomplished the present invention.
詳細に述べれば、 本発明者らは、 第一に ASK1ノックアウトマウスを後述 した方法により作製した。 作製した A S K 1ノックアウトマウスより胎児線維 芽細胞を採取し、 小胞体ストレスを誘導する種々の誘発剤である夕プシガルギ ン、 ッニカマイシンゃジチオスレィトールを用いてアポトーシスの発生を観察 した。 その結果、 すべてにおいて、 ASK 1ノックアウトマウスの胎児線維芽 細胞においては野生型 （正常） マウスの胎児線維芽細胞の場合と比較して有意 にアポト一シスが抑制されていた。 この事から A S K 1は小胞体ストレスによ るアポトーシス誘導に密接に関与していることが認められる。 Specifically, the present inventors first produced ASK1 knockout mice by the method described below. Fetal fibroblasts were collected from the prepared ASK1 knockout mice, and the occurrence of apoptosis was observed using various inducers that induce ER stress, e.g., evening psigargin and tunicamycin dithiothreitol. As a result, in all cases, apoptosis was significantly suppressed in fetal fibroblasts of ASK1 knockout mice as compared with fetal fibroblasts of wild-type (normal) mice. This indicates that ASK1 is closely involved in the induction of apoptosis by ER stress.
また、 ASK 1の 709番目のリジン残基は ATP結合サイトであり、 これ をアルギニンまたはメチォニンに置換することによってキナーゼ触媒活性を不 活化できる。 この変異体 ASK 1 (K709 R、 K709M) はドミナントネ ガティブ （優性抑制型） 体として機能する。 ASK1 ドミナントネガティブ体
は、 変異体 ASK 1 (例えば、 K709 R、 K 709 M) をコードする塩基配 列を発現べクタ一に組み込み、 そのベクターをリポフエクチン法やアデノウィ ルス感染法などにより細胞にトランスフエクシヨンまたはインフエクシヨンす ることで、 細胞内に過剰発現させることができる。 過剰発現した細胞は、 小胞 体ストレスによるアポト一シスを顕著に抑制することができる。 The 709th lysine residue of ASK1 is an ATP binding site, and by replacing this with arginine or methionine, the kinase catalytic activity can be inactivated. This mutant ASK1 (K709R, K709M) functions as a dominant negative (dominantly suppressed) form. ASK1 dominant negative Integrates a base sequence encoding a mutant ASK1 (e.g., K709R, K709M) into an expression vector and transfects the vector into cells by lipofection or adenovirus infection. By doing so, it can be overexpressed in cells. Overexpressed cells can significantly suppress apoptosis due to ER stress.
即ち、 ASK1阻害物質を有効成分として含有させることにより、 小胞体ス トレスに起因する疾患の予防または治療に有用な薬剤を提供することができる。 また、 ASK 1阻害物質を有効量使用することにより、 小胞体ストレスに起因 する疾患の予防または治療として有用な方法を提供することができる。 That is, by containing an ASK1 inhibitor as an active ingredient, a drug useful for preventing or treating a disease caused by endoplasmic reticulum stress can be provided. In addition, by using an effective amount of an ASK1 inhibitor, it is possible to provide a method useful for preventing or treating a disease caused by endoplasmic reticulum stress.
尚、 本明細書において、 特定の変異体であるドミナントネガティブ体を表す 場合には、 本来のアミノ酸残基（一文字表記） を最初に、位置番号を二番目に、 置換された後のアミノ酸残基（一文字表記） を三番目に示し、 「K709 R」や 「K709M」 は 709番目のアミノ酸残基である K (Ly s ： リジン） が R (A r g：アルギニン） または M (Me t :メチォニン） で置換されているこ とを表す。 In the present specification, when a specific mutant, a dominant negative form, is expressed, the original amino acid residue (single-letter code) is first, the position number is second, and the amino acid residue after substitution. "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.
また、本明細書において、 「+/ +」 は、 マウスまたはマウス由来細胞におい て 1対の相同染色体それぞれに存在する 2ケ所の A SKI遺伝子座が共に正常 であること、 即ち野生型であることを示し、 「一 Z―」 は、 1対の相同染色体そ れぞれに存在する 2ケ所の A S K 1遺伝子座の両方に変異が導入されており、 ASK 1蛋白の発現がないホモ接合体、 即ち ASK 1がノックアウトされてい るマウスまたはマウス由来細胞であることを示す。 In addition, in this specification, `` + / + '' means that two A SKI loci present on each of a pair of homologous chromosomes in a mouse or mouse-derived cells are both normal, that is, wild-type. `` -Z- '' indicates 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 the ASK1 protein is not expressed, That is, it indicates that ASK1 is a knocked out mouse or a mouse-derived cell.
本発明において、 小胞体ストレスに起因する疾患とは、 小胞体ストレスがそ の病態の発症または進展に関与している疾患をいい、 例えば、 ポリグルタミン 病、 アルツハイマー病、 プリオン病、 パーキンソン病、 筋萎縮性側索硬化症、 FTDP- 17等の神経変性疾患を挙げることができる。 In the present invention, the disease caused by endoplasmic reticulum stress refers to a disease in which endoplasmic reticulum stress is involved in the onset or progression of the pathological condition, such as polyglutamine disease, Alzheimer's disease, prion disease, Parkinson's disease, and muscle. Examples include neurodegenerative diseases such as amyotrophic lateral sclerosis and FTDP-17.
本発明において、 ASK1阻害物質とは、 それ自体 ASK 1阻害作用を有し ているものに限定されるものではなく、 生体内または培養系において ASK 1
阻害作用を有しているものを産生するものを含み、 例えば、 アミノ酸配列にお いて 1以上のアミノ酸を付加、 揷入、 置換および/または欠損 （例えば、 96 0番目のァラニン残基の欠損） させた誘導体を含む （例えば、 Wang X Set al., J. Biol. Chem., Vol.271, pp.31607-31611 (1996))、 ヒト A S K 1に対するドミ ナントネガティブ体 （例えば、 K709R、 K709M ;以下、 A S K 1ドミ ナントネガティブ体と称する）、ヒト ASK 1に対するアンチセンスオリゴヌク レ才チド (以下、 A S K 1ァンチセンスォリゴヌクレオチドと称する）、 ASK 1阻害作用を有する化学物質 （例えば、 ダルタチオン S—トランスフェラ一 ゼ （Mu l— l等)、 ネフ、 14一 3— 3蛋白質、 チォレドキシンなど） （例え ば、 Ssang-Goo Cho et al., J. Biol. Chem., Vol.276, No.16, pp.12749-12755 (2001)； Romas Geleziunas et al., Nature, Vol.410, pp.834-838 (2001)； FASEB Journal, Vol.15, No.4, pp.A235 (2001)； EMBO Journal, Vol.17, No.9, pp.2596-2606 (1998))やそのセンスオリゴヌクレオチド （それらの薬理学的に 許容される塩を含む）、並びに A S K 1ドミナントネガティブ体または A SKI アンチセンスオリゴヌクレオチドをコードする塩基配列を複製し、 標的組織ま たは宿主細胞内で当該ドミナントネガティブ体またはアンチセンスオリゴヌク レオチドを発現することができる組換えベクター （以下、 ASK1ドミナント ネガティブ体発現組換えベクターまたは ASK 1アンチセンスオリゴヌクレオ チド発現組換えベクターと称する）、 A S K 1阻害作用を有する化学物質または そのセンスオリゴヌクレオチドを標的組織または宿主細胞内で発現することが できる組換えベクター （以下、 ASK 1阻害作用を有する化学物質発現組換え ベクターまたは AS K 1阻害作用を有する化学物質のセンスオリゴヌクレオチ ド発現組換えベクターと称する）、それらの組換えベクターによって形質転換さ れた宿主細胞 （以下、 場合により ASK1ドミナントネガティブ体発現組換え ベクターにより形質転換された宿主細胞、 ASK1アンチセンスオリゴヌクレ ォチド発現組換えべクタ一により形質転換された宿主細胞、 A S K 1阻害作用 を有する化学物質発現組換えベクターにより形質転換された宿主細胞、 または
ASKl阻害作用を有する化学物質のセンスオリゴヌクレオチド発現組換えべ クタ一により形質転換された宿主細胞と称する） 等を例示することができる。 本発明の ASK 1阻害物質の使用方法としては、 ASK1ドミナントネガテ ィブ体発現組換えベクター、 A S K 1アンチセンスオリゴヌクレオチド発現組 換えベクター、 ASK 1阻害作用を有する化学物質発現組換えベクター、 また は A S K 1阻害作用を有する化学物質のセンスオリゴヌクレオチド発現組換え ベクタ一や、 それらの組換えベクターによって形質転換された宿主細胞を生体 内の標的組織に適当な方法により導入し、 所望の ASK 1ドミナントネガティ ブ体、 AS K1アンチセンスヌクレオチド、 ASK 1阻害作用を有する化学物 質またはそのセンスオリゴヌクレオチドを発現させることによる遺伝子的な予 防または治療剤としての使用や、 ASK1ドミナントネガティブ体、 ASK1 アンチセンスオリゴヌクレオチド、 ASK 1阻害作用を有する化学物質、 また は A S K 1阻害作用を有する化学物質のセンスオリゴヌクレオチドを有効成分 とする製剤を経口または非経口的に投与させることによる薬物的な予防または 治療剤としての使用を挙げることができる。 In the present invention, the ASK1 inhibitor is not limited to a substance having an ASK1 inhibitory action itself, and may be an ASK1 inhibitor in vivo or in a culture system. Includes those that produce inhibitors, including, for example, adding, inserting, substituting and / or deleting one or more amino acids in the amino acid sequence (eg, deletion of the 960th alanine residue) (For example, Wang X Set al., J. Biol. Chem., Vol.271, pp.31607-31611 (1996)), and a dominant negative form against human ASK1 (for example, K709R, K709M; Hereinafter, ASK1 dominant negative body), antisense oligonucleotide for human ASK1 (hereinafter, ASK1 antisense oligonucleotide), chemical substance having ASK1 inhibitory action (eg, daltathione S) —Transferase (Mul-l, etc.), Nef, 1413-3 protein, thioredoxin, etc. (for example, Ssang-Goo Cho et al., J. Biol. Chem., Vol.276, No. 16, pp.12749-12755 (2001); Romas Geleziunas et al., Nature, Vol. .410, pp.834-838 (2001); FASEB Journal, Vol.15, No.4, pp.A235 (2001); EMBO Journal, Vol.17, No.9, pp.2596-2606 (1998)) And its sense oligonucleotides (including their pharmacologically acceptable salts), and the nucleotide sequence encoding the ASK1 dominant negative or ASKI antisense oligonucleotide, and replicated in the target tissue or host cell. A recombinant vector capable of expressing the dominant negative or antisense oligonucleotide (hereinafter referred to as an ASK1 dominant negative expression recombinant vector or ASK1 antisense oligonucleotide expressing recombinant vector), ASK1 inhibition A recombinant vector capable of expressing a chemical substance having an action or its sense oligonucleotide in a target tissue or host cell (hereinafter referred to as a chemical substance having an ASK1 inhibitory action). Recombinant expression vectors or sense oligonucleotide-expressing recombinant vectors of chemicals having ASK1 inhibitory activity), and host cells transformed with those recombinant vectors (hereinafter ASK1 dominant-negative in some cases) A host cell transformed by an expression recombinant vector, a host cell transformed by an ASK1 antisense oligonucleotide-expressing recombinant vector, a host transformed by a chemical substance-expressing recombinant vector having ASK1 inhibitory action Cells, or (Referred to as a host cell transformed by a recombinant vector expressing a sense oligonucleotide having a chemical substance having ASKl inhibitory activity)). 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 substance expression vector having ASK1 inhibitory activity, or Recombinant expression of a sense oligonucleotide expressing a chemical substance having ASK 1 inhibitory activity or a host cell transformed with such a recombinant vector into a target tissue in a living body by an appropriate method to obtain a desired ASK 1 dominant Negative form, ASK1 antisense nucleotide, use as a genetic preventive or therapeutic agent by expressing a chemical substance having ASK1 inhibitory activity or its sense oligonucleotide, ASK1 dominant negative form, ASK1 antisense Oligonucleotides, ASK1 inhibitory chemicals, or ASK (1) Use of a preparation containing a sense oligonucleotide, which is a chemical substance having an inhibitory action, as an active ingredient orally or parenterally as a medicinal prophylactic or therapeutic agent can be mentioned.
ベクタ一としては、 プラスミドベクタ一、 ウィルスベクタ一 （例えば、 レト ロウィルスベクター、 アデノウイルスベクタ一、 ヘルぺスウィルスベクタ一、 センダイウィルスベクター、 ワクシニアウィルスベクター）、 リボソームベクタ 一 (例えば、 カチォニックリボソームベクター） 等を挙げることができる。 組換えべクタ一においては、 ASK1ドミナントネガティブ体や ASK1ァ ンチセンスオリゴヌクレオチドをコ一ドする塩基配列の他に、 これを実際に標 的組織または宿主細胞に導入して所望の当該ドミナントネガティブ体やアンチ センスオリゴヌクレオチドを発現させるために、 その発現を制御する塩基配列 (例えば、 プロモーター配列、 ターミネ一ター配列、 ェンハンサー配列） ゃ微 生物、 昆虫細胞または動物培養細胞等を選択するための遺伝子マーカ一 （例え ば、 ネオマイシン耐性遺伝子、 カナマイシン耐性遺伝子） 等を含んでいてもよ い。
宿主細胞としては、 例えば、 大腸菌、 酵母、 昆虫細胞、 並びに CHO細胞、 COS細胞、 ミンク肺上皮細胞（例えば、 MvlLu)、 リンパ球、 繊維芽細胞、 血液系細胞および腫瘍細胞等の動物細胞を挙げることができる。 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, a vaccinia virus vector), and a ribosome vector (for example, Kachonic Ribosome vector). In the 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. And nucleotide sequences that control the expression to express antisense oligonucleotides (eg, promoter sequence, terminator sequence, enhancer sequence) 遺 伝 子 Gene markers for selecting microorganisms, insect cells, animal cultured cells, etc. (Eg, a neomycin resistance gene, a kanamycin resistance gene) and the like. Examples of host cells include Escherichia coli, yeast, insect cells, and animal cells such as CHO cells, COS cells, mink lung epithelial cells (eg, MvlLu), lymphocytes, fibroblasts, blood cells, and tumor cells. be able to.
組換えベクターの標的組織または宿主細胞への導入方法としては、 HV Jリ ポソ一ム法 （金田， 実験医学， Vol.12, No.2, p.78 (1994); 森下等， 実験医学， Vol.12, No.15, p.158 (1994))、 ASK 1阻害物質を注射等により直接投与する 方法、 リン酸カルシウム法、 DEAE—デキストラン法、 エレクトロボレ一シ ョン法、 遺伝子銃による方法 （T. M. Klein et al.， Bio/Technology 10, pp.286- 291 (1992))、リポフエクシヨン法によつて投与する方法（Nabel et al.， Science, Vol.244, p.1285 (1990)) ベクター （例えば、 レトロウイルスベクタ一、 アデ ノウィルスベクタ一、 ヘルぺスウィルスベクター、 ワクシニアウィルスベクタ 一等） を使う方法等を挙げることができる。 Methods for introducing a recombinant vector into a target tissue or host cell include the HV J liposome method (Kaneda, Experimental Medicine, Vol. 12, No. 2, p. 78 (1994); Morishita et al., Experimental Medicine , Vol. 12, No. 15, p. 158 (1994)), a method of directly administering an ASK1 inhibitor by injection, etc., a calcium phosphate method, a DEAE-dextran method, an electroporation method, and a method using a gene gun. (TM Klein et al., Bio / Technology 10, pp. 286-291 (1992)), a method of administration by the lipofection method (Nabel et al., Science, Vol. 244, p. 1285 (1990)) (For example, a retrovirus vector, an adenovirus vector, a herpes virus vector, a vaccinia virus vector, etc.).
ASK1阻害物質を有効成分とする製剤を実際に小胞体ストレスに起因する 疾患の予防または治療において用いる場合、 用法に応じ種々の製剤形態のもの が使用される。 製剤形態としては、 例えば、 錠剤、 カプセル剤、 顆粒剤、 散剤、 丸剤、 細粒剤、 トローチ剤、 注射剤、 直腸投与剤、 座剤等を挙げることができ、 経口または非経口的に投与される。 When a preparation containing an ASK1 inhibitor as an active ingredient is actually used in the prevention or treatment of a disease caused by endoplasmic reticulum stress, various preparation forms are used depending on the usage. Examples of the formulation include tablets, capsules, granules, powders, pills, fine granules, troches, injections, rectal administration, suppositories, etc., and are administered orally or parenterally Is done.
ASK1阻害物質を含有するこれらの各種製剤は、 上記 A S K 1阻害物質を 有効成分として、 通常用いられている賦形剤、 崩壊剤、 結合剤、 滑沢剤、 希釈 剤、 緩衝剤、 等張化剤、 防腐剤、 湿潤剤、 乳化剤、 分散剤、 安定化剤、 溶解補 助剤等の医薬品添加物と適宜混合または希釈若しくは溶解し、 常法に従い調剤 することにより製造することができる。 These preparations containing an ASK1 inhibitor can be used as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonic agents, etc., using the ASK1 inhibitor as an active ingredient. 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.
ASK1阻害物質の小胞体ストレスに起因する疾患の薬物的な予防または治 療における投与量は、 用法、 患者の年齢、 性別、 症状の程度や疾患の種類等を 考慮して適宜決定されるが、 通常成人 1日当たり約 0. l〜500mg、 好ま しくは約 0. 5〜10 Omg程度とするのがよく、 1日一回または数回に分け て投与することができる。 また、 AS K1阻害物質の小胞体ストレスに起因す
る疾患の遺伝的な予防または治療における投与量もこれに準じて適宜決定する ことができる。 The dosage of the ASK1 inhibitor in the pharmacological prevention or treatment of a disease caused by endoplasmic reticulum stress is appropriately determined in consideration of the usage, patient age, gender, degree of symptoms, type of disease, etc. Usually, the dose is about 0.1 to 500 mg, preferably about 0.5 to 10 Omg per day for an adult, and it can be administered once or several times a day. In addition, AS K1 inhibitors cause ER stress. The dose for genetic prevention or treatment of a disease can be determined accordingly.
本発明によれば、 ASK1ドミナントネガティブ体、 ASK1アンチセン スオリゴヌクレオチド、 A S K 1阻害作用を有する化学物質またはそのセンス オリゴヌクレオチドをコードする塩基配列またはこれを含むベクター、 または そのベクターにより形質転換させた宿主細胞を用いて標的組織に導入すること により、 神経変性疾患等の小胞.体ストレスに起因する疾患の発症または進展を 抑制することができる。 また、 AS K1ドミナントネガティブ体、 ASK1ァ ンチセンスオリゴヌクレオチド、 ASK 1阻害作用を有する化学物質又はその センスオリゴヌクレオチドを含む製剤を経口または非経口的に投与することに より、 神経変性疾患等の小胞体ストレスに起因する疾患の発症または進展を抑 制することができる。 本発明の内容を以下の試験例でさらに詳細に説明するが、 本発明はその内容 に限定されるものではない。 試験例 1 According to the present invention, an ASK1 dominant negative, an ASK1 antisense oligonucleotide, a chemical substance having an ASK1 inhibitory action or a nucleotide sequence encoding a sense oligonucleotide thereof, or a vector containing the same, or a host transformed with the vector By introducing cells into target tissues using cells, it is possible to suppress the development or progression of vesicles such as neurodegenerative diseases and diseases caused by body stress. In addition, oral administration or parenteral administration of an ASK1 dominant negative body, an ASK1 antisense oligonucleotide, a chemical substance having ASK1 inhibitory activity, or a preparation containing the sense oligonucleotide thereof may reduce the risk of neurodegenerative diseases. It can suppress the onset or progress of diseases caused by endoplasmic reticulum stress. 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
ASK 1ノックァゥトマウスの作製 Preparation of ASK 1 knockout mouse
129ZSv Jマウスの染色体 （ジエノミック） DNAライブラリー （S t r a t e g e n e社） から、 ASK 1の第 1ェクソンを含む幾つかの染色体 D NA断片をクローン化した。 そのうちの一つのクローンを用いて、 第 1ェクソ ンより上流 10 kb及び下流 2 kbを相同組換え領域とし、 第 1ェクソン及び 隣接するイントロン領域を、 GFP (g r e en f l uo r e s c enc e r o t e i n) とポジティブセレクション用のネオマイシン耐性遺伝子とに 置換したターゲッティングベクターを構築した。 ターゲッティングベクターに は、 ネガティブセレクション用の DT— A (ジフテリアトキシンひ鎖） を含む pB l ue s c r i p t SK (S t r a t e gene社） を用いた。 ターゲ
ッティングベクターは、 エレクト口ポーレーシヨン法によって J 1 ES細胞 (emb ryon i c s t em c e 1 1 ) に導入した。 ネオマイシン ffiH生 の ES細胞コロニーを選択した後、 更にサザンブロット法により相同組換え細 胞の確認を行った。 ヘテロ接合変異体 ES細胞は、 マイクロインジェクション 法により C 57BL/6 Jの胚盤胞に導入した。 生まれてきたキメラマウスと C57BL/6 Jマウスとの戻し交配によって、 F 1 (第 1世代） ヘテロ接合 体マウスが得られた。 遺伝子変異が生殖細胞に導入されているか否かをサザン ブロット法により確認した後、 それら F 1ヘテロ接合体マウス同士の掛け合わ せから、 ASK 1のホモ接合体である ASK 1ノックァゥトマウスを樹立した。 試験例 2 Several chromosomal DNA fragments, including the first exon of ASK1, were cloned from the chromosome (dienomic) DNA library of 129ZSv J 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 positive with GFP (green fluo rescencerotein). A targeting vector was constructed by substituting the neomycin resistance gene for selection. As a targeting vector, pBluescript SK (Strate gene) containing DT-A (diphtheria toxin heavy chain) for negative selection was used. Target The setting vector was introduced into J1 ES cells (embryon icst emce11) by an electoral poration method. After selecting a neomycin ffiH live ES cell colony, 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 C57BL / 6J 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 2
小胞体ストレスによるアポトーシス誘導 Apoptosis induction by endoplasmic reticulum stress
胎生 12. 5日目の ASK 1ノックアウトマウス由来の胎児線維芽細胞 （m 0 u s e emb ryon i c f i b r ob l a s t s ； MEF s) を用い て、 AS K 1が小胞体ストレス誘導性のアポト一シスに関与しているか否かに ついて検討を行った。 24ゥエルプレートに 1 X 105c e 1 1 s /we 1 1の 濃度で M E F sをー晚培養し、 刺激前 2時間に無血清培地に変換後、 2 ιι の タプシガルギン、 2. 5 a gZmLのッニカマイシン又は 1 OmMのジチォス レイトールを培地に添加することによって小胞体ストレスを誘導した。 タプシ ガルギンは、 小胞体へのカルシウム取り込みを阻害し、 小胞体内のカルシウム ホメォスタシスを乱すことによって小胞体ストレスを誘導する薬剤である。 ま た、 ッニカマイシンは、 蛋白質糖鎖付加を阻害することにより、 ジチオスレィ トールは、 ジスルフイド結合を阻害することにより、 それぞれ小胞体ストレス を誘導する薬剤である。 アポト一シスによる細胞死の検出は、 TUNEL (夕 一ミナル ·デォキシヌクレオチジル · トランスフェラーゼーメディエーティッ ド dUTPニック ·エンド ·ラベリング） 法および MTT (3 - 〔4, 5 - ジメチルチアゾール— 2—ィル〕 一 2, 5ージフエ二ルー 2 J-J—テトラゾリウ
ム ブロマイド） 法によって行った。 ASK1 is involved in endoplasmic reticulum stress-induced apoptosis using fetal fibroblasts (m0 use embryon icfibr ob lasts; MEFs) derived from ASK1 knockout mice on day 5. It was examined whether or not it was. Incubate MEFs at a concentration of 1 x 10 5 ce 11 s / we 11 in a 24-well plate, convert to serum-free medium 2 hours before stimulation, and 2 ιι thapsigargin, 2.5 agZmL Endoplasmic reticulum stress was induced by adding tunicamycin or 1 OmM dithiothreitol to the medium. Thapsigargin is a drug that inhibits calcium uptake into the endoplasmic reticulum and induces endoplasmic reticulum stress by disrupting calcium homeostasis in the endoplasmic reticulum. Also, tunicamycin is an agent that induces endoplasmic reticulum stress by inhibiting protein glycosylation and dithiothreitol by inhibiting disulfide bonds. Detection of cell death by apoptosis was performed using the TUNEL (Evening Minal Deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling) method and the MTT (3- [4,5-dimethylthiazole-2 1) 2,5 di-Fe2Lu 2 JJ—Tetrazoliu (Bromide) method.
1) TUNEL法 1) TUNEL method
アポトーシスによる細胞死の大きな特徴の一つは、 DN Aの断片化であり、 TUNEL法は、 その断片化された DN Aを特異的に染色できる方法である。 夕プシガルギン添加 2、 6時間後、 細胞を 4%パラホルムアルデヒドによって 30分間、 室温で固定し、 0. 1 %トライトン X_ 100で 4° (：、 2分間処理 し細胞膜透過性を亢進させた。アポトーシス細胞の染色のために、 F I TC (f l uo r e s c e i n i s o t h i o cyana t e) — c on j uga t e d TUNEL r e a c t i on mi t u r e (ロシュ社） を加え、 室温で 1時間静置した。 全体の細胞数を数えるため、 生細胞と死細胞の両者を 染色できる H o e c h s t 33258 (I n g/mL) と p r o p i d i u m i od i de (10 g/mL) を用いた。 全ての溶液の希釈および洗浄 は、 PBS (pho s ph a t e— bu f f e r e d s a l i ne) によつ て行った。 染色後の細胞は、 Mowi o 1 (和光純薬工業 （株)） で包埋し、 蛍 光顕微鏡観察により死細胞を同定した。 アポトーシス細胞の割合 （％) は、 全 体の細胞数として少なくとも 3つ以上の視野でそれぞれ 100個以上の細胞を 数え、 そのうちの TUNEL染色陽性の細胞数の割合として算出した。 One of the major characteristics of cell death due to apoptosis is fragmentation of DNA, and the TUNEL method is a method that can specifically stain the fragmented DNA. Two to six hours after addition of psigargin, the cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature, and treated with 0.1% Triton X_100 at 4 ° (:, 2 minutes to enhance cell membrane permeability. To stain the cells, add FITC (fl uo resceinisothio cyanate) — conjugated TUNEL reacti on miture (Roche) and allow to stand at room temperature for 1 hour to count the total number of cells. Hoechst 33258 (Ing / mL) and propidiumiodide (10 g / mL), which can stain both live and dead cells, were used. The stained cells were embedded with Mowio 1 (Wako Pure Chemical Industries, Ltd.), and dead cells were identified by fluorescence microscopy. %) Means at least 3 or more cells as a whole More than 100 cells were counted in each field of view, and the ratio was calculated as the ratio of the number of cells positive for TUNEL staining.
2) MTT法 2) MTT method
MTT法は、 me t hy l t h'i a z o l t e t r a z o l i umのフォ ルマザンへの還元による吸光度の変化を指標に、 生細胞の割合を検出するアツ セィ法である。 夕プシガルギンを加えて 8、 16、 24時間後に、 また、 その 他の小胞体ストレス誘発剤については 8時間後に、 それぞれ MTTアツセィを 行った。 MTTアツセィ溶液原液 （Do j i ndo社） を細胞培養液中に 10 倍希釈で添加し、 37°C、 1時間静置し、 その培養液中の吸光度を 45 Onm にて測定した。 無処理の細胞培養液中の吸光度値を 100 %として、 生細胞率 ( ) を計算した。 The MTT method is an Atsey method for detecting the ratio of living cells using the change in absorbance due to reduction of methylth'iazolteletrazolium to formazan as an index. At 8, 16, and 24 hours after addition of evening psigargin, and after 8 hours for other ER stress inducers, MTT assy was performed. MTT Atsushi solution stock solution (Dojindo) was added to the cell culture at a 10-fold dilution, allowed to stand at 37 ° C for 1 hour, and the absorbance in the culture was measured at 45 Onm. The percentage of viable cells () was calculated assuming the absorbance value in the untreated cell culture solution as 100%.
第 1図は、 TUNEL法により、アポトーシス細胞を視覚化したものである。
緑色蛍光染色されているものが、 アポトーシス細胞である。 細胞の全体像は、 青色の Ho e c h s t 33258及び赤色の p r 0 ρ i d i um i od i d eによって染色されている。 第 1図は刺激 6時間後の代表的な視野であり、 更に 3つ以上の視野で観察した結果をグラフにまとめたものが第 2図である。 野生型の ME F s (+/+) と比較して、 ASK 1ノックアウトマウス由来の胎 児線維芽細胞 ME F s (-/-) は、 夕プシガルギンによる小胞体ストレス誘導 性の細胞死に対して、 顕著に耐性を示した。 同様の結果は、 MTT法によって も確認された（第 3図）。 また、 夕プシガルギンとは別なメカニズムで小胞体ス トレスを誘発する代表的な化合物であるッニカマイシンゃジチォスレイトール によるアポトーシスに対しても同様に、 ASK1を欠損した ME F s細胞は耐 性を示した （第 4図)。 FIG. 1 is a visualization of apoptotic cells by the TUNEL method. Apoptotic cells are stained with green fluorescence. The whole picture of the cells is stained with blue Hoechst 33258 and red pr0pidimuiodide. FIG. 1 is a representative visual field 6 hours after stimulation, and FIG. 2 is a graph summarizing the results of observations in three or more visual fields. Compared to wild-type ME F s (+ / +), fetal fibroblast ME F s (-/-) from ASK 1 knockout mice were more resistant to endoplasmic reticulum stress-induced cell death by evening pshigargin. , Markedly resistant. Similar results were confirmed by the MTT method (Fig. 3). Similarly, ASK1-deficient MEFS cells are also resistant to apoptosis by tunicamycin dithiothreitol, a representative compound that induces endoplasmic reticulum stress by a mechanism different from evening psigargin. (Figure 4).
以上の結果は、 小胞体ストレス誘導性のアポトーシスは A S K 1によって実 行されることを示している。 ASK1の欠損によって、 小胞体ストレス誘導性 のアポトーシスが消失する事実は、 ASK 1阻害物質のアポトーシス関連性疾 患への有用性を顕著に証明するものである。 試験例 3 These results indicate that ER stress-induced apoptosis is performed by ASK1. The fact that ASK1 deficiency abolishes endoplasmic reticulum stress-induced apoptosis demonstrates the usefulness of ASK1 inhibitors in apoptosis-related diseases. Test example 3
神経変性蛋白質 Amy 1 o i d— |Sによる初代培養神経細胞へのアポトーシス 胎生 14. 5日目の ASK 1ノックァゥトマウス大脳由来の初代培養神経細 胞を用いて、 AS K1が神経変性蛋白質 Amy 1 o i d— )3によるアポトーシ スに関与しているか否かについて検討を行った。 マウス大脳半球を単離し培地 中で組織をピベッティングによりほぐした後、ポリ- L-リジンおよびフイブロネ クチンをコートした 24ゥエルプレート上におよそ 1 X 105 c e 1 1 s /w e 1 1の濃度で播種した。 培養 1 日目および 3日目に 10118 1111^の ？0 F (basic-Fibroblast Grouwth Factor) を培地に添加し、 4日間培養したもの を初代培養神経細胞として用いた。 Amy 1 o i d— /3はアルツハイマー病患
者の脳組織に多量に凝集する変性蛋白質であり、 強い神経毒性を有し、 ァルツ ハイマー病における神経変性の原因蛋白質である。 Amy 1 o i d - /3 ( 2 5 - 3 5 ) (Amy 1 o i d— /3蛋白質のアミノ酸配列で 2 5番目から 3 5番目 までのペプチドで、 最も神経毒性の強い部分と言われている） を 2 5 Mの濃 度で培地に加え、 3日後の生存率を、 試験例 2記載の MT T法と同様の方法に より、 無処理の初代培養神経細胞に対する生細胞率 （％) として計算した。 図 5は、 Amy 1 o i d— ;3による神経毒性に対する生存率を、 野生型マウ ス由来の神経細胞（+/+) と A S K 1ノックアウトマウス由来の神経細胞（一 /-) とで比較したものである。野生型の神経細胞のほとんどが Amy 1 o i d _ j3誘導性細胞死を起こすのに対して、 A S K 1ノックアウトマウス由来の神 経細胞は、 Amy 1 o i d— によって誘導される神経細胞死に対して、 顕著 に »†生を示した。 Amy 1 oid—Apoptosis of primary cultured neurons by the neurodegenerative protein Embryos 14. Using primary cultured neurons derived from the cerebrum of the ASK 1 knockout mouse on day 5, ASK1 has been converted to the neurodegenerative protein Amy 1 oid —) We examined whether we are involved in apoptosis by (3). After isolating the mouse cerebral hemisphere and dislodging the tissue by pipetting in the medium, a concentration of approximately 1 x 10 5 ce 11 s / we 11 on a 24-L plate coated with poly-L-lysine and fibronectin And sowed. On day 1 and day 3 of culture 10118 1111 ^? 0 F (basic-Fibroblast Grouwth Factor) was added to the medium, and the cells cultured for 4 days were used as primary cultured neurons. Amy 1 oid— / 3 is Alzheimer's disease It is a denatured protein that aggregates in large amounts in the brain tissue of the elderly, has strong neurotoxicity, and is a causative protein of neurodegeneration in Alzheimer's disease. Amy 1 oid-/ 3 (25-35) (The amino acid sequence of the Amy 1 oid- / 3 protein from the 25th to the 35th peptide, which is said to be the most neurotoxic part) The viability after 3 days was added to the medium at a concentration of 25 M and the viability was calculated as the viable cell ratio (%) to the untreated primary cultured neurons by the same method as the MTT method described in Test Example 2. . Figure 5 shows the survival rate against neurotoxicity caused by Amy 1 oid—; 3 in neurons derived from wild-type mouse (+ / +) and neurons derived from ASK 1 knockout mouse (1-/-). It is. While most wild-type neurons undergo Amy 1 oid_j3-induced cell death, neurons derived from ASK 1 knockout mice are significantly more prone to Amy 1 oid-induced neuronal death. In »showed a life.
神経変性蛋白質である Amy 1 o i d— ]3は小胞体ストレスを誘導すること が分かっていることから、 第 5図の結果は、 実際に小胞体ストレス誘導性の神 経細胞アポトーシスは A S K 1によって実行されることを示している。 また、 A S K 1の欠損によって、 Amy 1 o i d— /3誘導性誘導性のアポト一シスが 消失する事実は、 A S K 1阻害物質の神経変性疾患への効果を顕著に証明する ものである。 〔産業上の利用可能性〕 Amy1 oid—] 3, a neurodegenerative protein, has been shown to induce ER stress, and the results in Figure 5 show that ASK1 is actually responsible for ER stress-induced neuronal apoptosis. It is shown that it is done. In addition, the fact that ASK1 deficiency abolishes Amy1oid-3 / 3-induced apoptosis demonstrates the effect of ASK1 inhibitors on neurodegenerative diseases. [Industrial applicability]
本発明は、 A S K 1を阻害することによる小胞体ストレス誘導性のアポト一 シスの抑制に関するものである。本発明により小胞体ス卜レスに起因する疾患、 特には、 ポリグルタミン病、 アルツハイマー病、 パーキンソン病、 プリオン病、 筋萎縮性側索硬化症、 F T D P— 1 7などの神経変性疾患の予防または治療剤 或いは予防または治療方法を提供することができる。
The present invention relates to suppression of ER stress-induced apoptosis by inhibiting ASK1. Prevention or treatment of diseases caused by endoplasmic reticulum stress, in particular, neurodegenerative diseases such as polyglutamine disease, Alzheimer's disease, Parkinson's disease, prion disease, amyotrophic lateral sclerosis, and FTDP-17 according to the present invention. An agent or a prophylactic or therapeutic method can be provided.
Claims
1. ASKl阻害物質を含む、 小胞体ストレスに起因する疾患の予防また は治療剤。 1. A preventive or therapeutic agent for diseases caused by endoplasmic reticulum stress, including ASKl inhibitors.
2. ASK1阻害物質が ASK1ドミナントネガティブ体、 ASK1ドミ ナントネガティブ体発現組換えべクタ一または ASK 1ドミナントネガティブ 体発現組換えベクターにより形質転換された宿主細胞である請求項 1記載の予 防または治療剤。 2. The prevention or treatment according to claim 1, wherein the ASK1 inhibitor is an ASK1 dominant negative body, a recombinant vector expressing the ASK1 dominant negative body, or a host cell transformed with the recombinant vector expressing the ASK1 dominant negative body. Agent.
3. ASK 1ドミナントネガティブ体が K709 Rまたは K709Mであ る請求項 2記載の予防または治療剤。 3. The preventive or therapeutic agent according to claim 2, wherein the ASK1 dominant negative is K709R or K709M.
4. ASK 1阻害物質が ASK 1アンチセンスオリゴヌクレオチド、 AS K 1アンチセンスオリゴヌクレオチド発現組換えベクターまたは ASK 1アン チセンスオリゴヌクレオチド発現組換えベクターにより形質転換された宿主細 胞である請求項 1記載の予防または治療剤。 4. The ASK1 inhibitor is an ASK1 antisense oligonucleotide, an ASK1 antisense oligonucleotide-expressing recombinant vector or a host cell transformed with an ASK1 antisense oligonucleotide-expressing recombinant vector. The prophylactic or therapeutic agent according to the above.
5. AS K1阻害物質がダルタチオン S—トランスフェラ一ゼ、 ネフ、 14-3-3蛋白質およびチォレドキシンから選択される A S K 1阻害作用を 有する化学物質、 その A S K 1阻害作用を有する化学物質のセンスオリゴヌク レオチド、 その ASK 1阻害作用を有する化学物質発現組換えべクタ一、 その ASK1阻害作用を有する化学物質のセンスオリゴヌクレオチド発現組換えべ クタ一、 またはそれらの組換えベクターにより形質転換された宿主細胞である 請求項 1記載の予防または治療剤。 5. ASK1 inhibitory chemical substance whose AS K1 inhibitor is selected from daltathione S-transferase, Nef, 14-3-3 protein and thioredoxin, and sense oligo of the ASK1 inhibitory chemical substance Nucleotide, a recombinant vector expressing a chemical substance having ASK1 inhibitory activity, a recombinant vector expressing a sense oligonucleotide having a chemical substance having ASK1 inhibitory action, or a host transformed with such a recombinant vector 2. The preventive or therapeutic agent according to claim 1, which is a cell.
6. 小胞体ストレスに起因する疾患が神経変性疾患である請求項 1、 2、 3、 4または 5記載の予防または治療剤。 6. The preventive or therapeutic agent according to claim 1, 2, 3, 4, or 5, wherein the disease caused by endoplasmic reticulum stress is a neurodegenerative disease.
7. 神経変性疾患がポリグルタミン病である請求項 6記載の予防または治 療剤。 7. The preventive or therapeutic agent according to claim 6, wherein the neurodegenerative disease is polyglutamine disease.
8. 神経変性疾患がァルツハイマー病である請求項 6記載の予防または治
8. The prevention or treatment according to claim 6, wherein the neurodegenerative disease is Alzheimer's disease.
9. 神経変性疾患がパ一キンソン病である請求項 6記載の予防または治療 剤。 9. The preventive or therapeutic agent according to claim 6, wherein the neurodegenerative disease is Parkinson's disease.
10. 神経変性疾患がプリオン病である請求項 6記載の予防または治療剤。 10. The preventive or therapeutic agent according to claim 6, wherein the neurodegenerative disease is prion disease.
11. 神経変性疾患が筋萎縮性側索硬化症である請求項 6記載の予防または 治療剤。 11. The preventive or therapeutic agent according to claim 6, wherein the neurodegenerative disease is amyotrophic lateral sclerosis.
12. 神経変性疾患が F TD P— 17である請求項 6記載の予防または治療 剤。 12. The preventive or therapeutic agent according to claim 6, wherein the neurodegenerative disease is FTDP-17.
13. ASK1阻害物質を有効量使用することを含む、 小胞体ストレスに起 因する疾患の予防または治療方法。 13. A method for preventing or treating a disease caused by endoplasmic reticulum stress, comprising using an effective amount of an ASK1 inhibitor.
14. ASK 1阻害物質が A SKIドミナントネガティブ体、 ASK 1ドミ ナントネガティブ体発現組換えベクターまたは AS K 1ドミナントネガティブ 体発現組換えベクターにより形質転換された宿主細胞である請求項 13記載の 予防または治療方法。 14. The method according to claim 13, wherein the ASK1 inhibitor is a host cell transformed with an ASK1 dominant negative body, an ASK1 dominant negative body expression recombinant vector or an ASK1 dominant negative body expression recombinant vector. Method of treatment.
15. ASK 1ドミナントネガティブ体が K 709 Rまたは K709Mであ る請求項 14記載の予防または治療方法。 15. The method according to claim 14, wherein the ASK1 dominant negative body is K709R or K709M.
16. AS K1阻害物質が ASK 1アンチセンスオリゴヌクレオチド、 AS K 1ァンチセンスオリゴヌクレオチド発現組換えべク夕一または A S K 1アン チセンスオリゴヌクレオチド発現組換えベクターにより形質転換された宿主細 胞である請求項 13記載の予防または治療方法。 16. The ASK1 inhibitor is a host cell transformed with an ASK1 antisense oligonucleotide, a recombinant vector expressing the ASK1 antisense oligonucleotide or a recombinant vector expressing the ASK1 antisense oligonucleotide. The method for preventing or treating according to claim 13.
17. AS K1阻害物質がダル夕チオン S—トランスフェラーゼ、 ネフ、 14-3一 3蛋白質およびチォレドキシンから選択される ASK 1阻害作用を 有する化学物質、 その AS K 1阻害作用を有する化学物質のセンスオリゴヌク レオチド、 その ASK 1阻害作用を有する化学物質発現組換えベクター、 その ASK1阻害作用を有する化学物質のセンスオリゴヌクレオチド発現組換えべ クタ一、 またはそれらの組換えベクターにより形質転換された宿主細胞である 請求項 13記載の予防または治療方法。 17. ASK1 inhibitory chemical substance whose ASK1 inhibitor is selected from daryuthion S-transferase, Nef, 14-33 protein and thioredoxin, and sense oligo of the ASK1 inhibitory chemical substance 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 host cell transformed with such a recombinant vector. The method for preventing or treating according to claim 13.
18. 小胞体ストレスに起因する疾患が神経変性疾患である請求項 13、 1
4、 15、 16または 17記載の予防または治療方法。 18. Claims 13 and 1 wherein the disease caused by ER stress is a neurodegenerative disease. The prevention or treatment method according to 4, 15, 16 or 17.
19. 神経変性疾患がポリグルタミン病である請求項 18記載の予防または 治療方法。 19. The method according to claim 18, wherein the neurodegenerative disease is polyglutamine disease.
20. 神経変性疾患がアルツハイマー病である請求項 18記載の予防または 治療方法。 20. The method according to claim 18, wherein the neurodegenerative disease is Alzheimer's disease.
21. 神経変性疾患がパーキンソン病である請求項 18記載の予防または治 療方法。 21. The method according to claim 18, wherein the neurodegenerative disease is Parkinson's disease.
22. 神経変性疾患がプリオン病である請求項 18記載の予防または治療方 法。 22. The method according to claim 18, wherein the neurodegenerative disease is prion disease.
23. 神経変性疾患が筋萎縮性側索硬化症である請求項 18記載の予防また は治療方法。 23. The method according to claim 18, wherein the neurodegenerative disease is amyotrophic lateral sclerosis.
24. 神経変性疾患が FTDP— 17である請求項 18記載の予防または治 療方法。 24. The method according to claim 18, wherein the neurodegenerative disease is FTDP-17.
25. 小胞体ストレスに起因する疾患の予防または治療用の製剤を製造する ための A S K 1阻害物質の使用。 25. Use of an ASK1 inhibitor for the manufacture of a formulation for the prevention or treatment of a disease caused by ER stress.
26. ASK1阻害物質が A SKI ミナントネガティブ体、 A S K 1ドミ ナントネガティブ体発現組換えべク夕一または A S K 1ドミナントネガティブ 体発現組換えべクタ一により形質転換された宿主細胞である請求項 25記載の 使用。 26. The ASK1 inhibitor may be a host cell transformed by an ASK1 minant-negative body, an ASK1 dominant-negative body-expressing recombinant vector or an ASK1 dominant-negative body-expressing recombinant vector. Use as described.
27. ASK1ドミナントネガティブ体が K709 Rまたは K709Mであ る請求項 26記載の使用。 27. Use according to claim 26, wherein the ASK1 dominant negative body is K709 R or K709M.
28. AS K1阻害物質が ASK 1アンチセンスオリゴヌクレオチド、 AS K 1アンチセンスオリゴヌクレオチド発現組換えベクターまたは A S K 1アン チセンスオリゴヌクレオチド発現組換えベクターにより形質転換された宿主細 胞である請求項 25記載の使用。 28. The ASK1 inhibitor is an ASK1 antisense oligonucleotide, a recombinant ASK1 antisense oligonucleotide expression vector or a host cell transformed with the ASK1 antisense oligonucleotide expression recombinant vector. Use of the description.
29. ASK 1阻害物質がダルタチオン S—トランスフェラーゼ、 ネフ、 14-3 _ 3蛋白質およびチォレドキシンから選択される ASK 1阻害作用を
有する化学物質、 その ASK 1阻害作用を有する化学物質のセンスオリゴヌク レオチド、 その ASK 1阻害作用を有する化学物質発現組換えベクター、 その ASK1阻害作用を有する化学物質のセンスオリゴヌクレオチド発現組換えべ クタ一、 またはそれらの組換えベクターにより形質転換された宿主細胞である 請求項 25記載の使用。 29. ASK 1 inhibitor inhibits ASK 1 inhibitory activity selected from daltathione S-transferase, nef, 14-3_3 protein and thioredoxin Chemicals having the ASK1 inhibitory action, sense oligonucleotides of the chemicals having the ASK1 inhibitory action, recombinant vectors for expressing the chemicals having the ASK1 inhibitory action, sense oligonucleotide expression recombinant vectors for the chemicals having the ASK1 inhibitory action The use according to claim 25, which is a host cell transformed by one or a recombinant vector thereof.
30. 小胞体ストレスに起因する疾患が神経変性疾患である請求項 25、 2 6、 27、 28または 29記載の使用。 30. The use according to claim 25, 26, 27, 28 or 29, wherein the disease caused by ER stress is a neurodegenerative disease.
31. 神経変性疾患がポリグルタミン病である請求項 30記載の使用。 31. The use according to claim 30, wherein the neurodegenerative disease is polyglutamine disease.
32. 神経変性疾患がアルツハイマー病である請求項 30記載の使用。 32. The use according to claim 30, wherein the neurodegenerative disease is Alzheimer's disease.
33. 神経変性疾患がパーキンソン病である請求項 30記載の使用。 33. The use according to claim 30, wherein the neurodegenerative disease is Parkinson's disease.
34. 神経変性疾患がプリォン病である請求項 30記載の使用。 34. The use according to claim 30, wherein the neurodegenerative disease is Prion's disease.
35. 神経変性疾患が筋萎縮性側索硬化症である請求項 30記載の使用。 35. The use according to claim 30, wherein the neurodegenerative disease is amyotrophic lateral sclerosis.
36. 神経変性疾患が F TD P— 17である請求項 30記載の使用。
36. The use according to claim 30, wherein the neurodegenerative disease is FTDP-17.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2002224022A AU2002224022A1 (en) | 2000-11-10 | 2001-11-08 | Preventives or remedies for endoplasmic reticulum stress-associated diseases |
| JP2002540761A JPWO2002038179A1 (en) | 2000-11-10 | 2001-11-08 | Preventive or therapeutic agent for ER stress-related disease |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2000-344516 | 2000-11-10 | ||
| JP2000344516 | 2000-11-10 |
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| WO2002038179A1 true WO2002038179A1 (en) | 2002-05-16 |
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| PCT/JP2001/009792 WO2002038179A1 (en) | 2000-11-10 | 2001-11-08 | Preventives or remedies for endoplasmic reticulum stress-associated diseases |
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| Country | Link |
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| JP (1) | JPWO2002038179A1 (en) |
| AU (1) | AU2002224022A1 (en) |
| WO (1) | WO2002038179A1 (en) |
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| WO2003063905A1 (en) * | 2002-01-31 | 2003-08-07 | Center For Advanced Science And Technology Incubation, Ltd. | Preventives or remedies for immunological diseases |
| JP2004010574A (en) * | 2002-06-10 | 2004-01-15 | Redox Bioscience Inc | Nerve regeneration factor |
| WO2004048565A1 (en) * | 2002-11-22 | 2004-06-10 | Takeda Pharmaceutical Company Limited | Apoptosis-associated protein and use thereof |
| JP2004180680A (en) * | 2002-11-22 | 2004-07-02 | Takeda Chem Ind Ltd | Apoptosis-associated protein and use thereof |
| WO2005009470A1 (en) * | 2003-07-28 | 2005-02-03 | Osaka Industrial Promotion Organization | Remedy for cardiac failure comprising ask1 inhibitor as the active ingredient and method of screening the same |
| WO2005103288A1 (en) | 2004-04-27 | 2005-11-03 | Takeda Pharmaceutical Company Limited | Screening method |
| WO2008016131A1 (en) | 2006-08-04 | 2008-02-07 | Takeda Pharmaceutical Company Limited | Fused heterocyclic compound |
| JP2011518566A (en) * | 2008-04-25 | 2011-06-30 | ニューヨーク ブラッド センター， インコーポレイテッド | ABI1 / HSSH3BP1 conditional knockout mouse |
| JP2017071647A (en) * | 2009-07-13 | 2017-04-13 | ギリアード サイエンシーズ， インコーポレイテッド | Apoptosis signal-regulating kinase inhibitors |
| US10604490B2 (en) | 2014-12-23 | 2020-03-31 | Gilead Sciences, Inc. | Processes for preparing ASK1 inhibitors |
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| EP0896055A2 (en) * | 1996-04-19 | 1999-02-10 | The Cancer Institute Of Japanese Foundation For Cancer Research | Apoptosis inducing protein and gene encoding the same |
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003063905A1 (en) * | 2002-01-31 | 2003-08-07 | Center For Advanced Science And Technology Incubation, Ltd. | Preventives or remedies for immunological diseases |
| JP2004010574A (en) * | 2002-06-10 | 2004-01-15 | Redox Bioscience Inc | Nerve regeneration factor |
| JP4488720B2 (en) * | 2002-11-22 | 2010-06-23 | 武田薬品工業株式会社 | Apoptosis-related proteins and uses thereof |
| WO2004048565A1 (en) * | 2002-11-22 | 2004-06-10 | Takeda Pharmaceutical Company Limited | Apoptosis-associated protein and use thereof |
| JP2004180680A (en) * | 2002-11-22 | 2004-07-02 | Takeda Chem Ind Ltd | Apoptosis-associated protein and use thereof |
| US7390625B2 (en) | 2002-11-22 | 2008-06-24 | Takeda Pharmaceutical Company Limited | Apoptosis-associated protein and use thereof |
| WO2005009470A1 (en) * | 2003-07-28 | 2005-02-03 | Osaka Industrial Promotion Organization | Remedy for cardiac failure comprising ask1 inhibitor as the active ingredient and method of screening the same |
| WO2005103288A1 (en) | 2004-04-27 | 2005-11-03 | Takeda Pharmaceutical Company Limited | Screening method |
| WO2008016131A1 (en) | 2006-08-04 | 2008-02-07 | Takeda Pharmaceutical Company Limited | Fused heterocyclic compound |
| JP2011518566A (en) * | 2008-04-25 | 2011-06-30 | ニューヨーク ブラッド センター， インコーポレイテッド | ABI1 / HSSH3BP1 conditional knockout mouse |
| JP2017071647A (en) * | 2009-07-13 | 2017-04-13 | ギリアード サイエンシーズ， インコーポレイテッド | Apoptosis signal-regulating kinase inhibitors |
| USRE48711E1 (en) | 2009-07-13 | 2021-08-31 | Gilead Sciences, Inc. | Apoptosis signal-regulating kinase inhibitors |
| US10604490B2 (en) | 2014-12-23 | 2020-03-31 | Gilead Sciences, Inc. | Processes for preparing ASK1 inhibitors |
| US11261161B2 (en) | 2014-12-23 | 2022-03-01 | Gilead Sciences, Inc. | Processes for preparing ASK1 inhibitors |
Also Published As
| Publication number | Publication date |
|---|---|
| AU2002224022A1 (en) | 2002-05-21 |
| JPWO2002038179A1 (en) | 2004-03-11 |
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