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WO2002038179A1 - Preventifs ou remedes de maladies du reticulum endoblastique liees au stress - Google Patents

Preventifs ou remedes de maladies du reticulum endoblastique liees au stress Download PDF

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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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ask1
disease
recombinant vector
neurodegenerative disease
inhibitor
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PCT/JP2001/009792
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Japanese (ja)
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Hidenori Ichijyo
Atsushi Matsuzawa
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Kissei Pharmaceutical Co., Ltd.
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Priority to AU2002224022A priority Critical patent/AU2002224022A1/en
Priority to JP2002540761A priority patent/JPWO2002038179A1/ja
Publication of WO2002038179A1 publication Critical patent/WO2002038179A1/fr

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out

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

L'invention porte sur des préventifs ou remèdes de maladies du reticulum endoplasmique liées au stress (par exemple les maladies neurodégénératives dont la maladie due à la polyglutamine, la maladie d'Alzheimer, la maladie de Parkinson, la maladie due au prion, la sclérose amyotrophique latérale et la FTDP-17) contenant des substances chimiques inhibant l'apoptose du reticulum endoplasmique liées au stress et l'effet inhibiteur de l'ASK1 (dû par exemple aux composés négatifs dominant de l'ASK1, aux oligonucléotides antisens de l'ASK1, au glutathione, aux S transférases (Mul-1 etc.), au Nef, à la protéine 14-3-3, et à la thiorédoxyne). L'invention porte également sur leurs oligonucléotides, leurs vecteurs d'expression, ou les cellules hôtes transformées par lesdits vecteurs d'expression.
PCT/JP2001/009792 2000-11-10 2001-11-08 Preventifs ou remedes de maladies du reticulum endoblastique liees au stress WO2002038179A1 (fr)

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AU2002224022A AU2002224022A1 (en) 2000-11-10 2001-11-08 Preventives or remedies for endoplasmic reticulum stress-associated diseases
JP2002540761A JPWO2002038179A1 (ja) 2000-11-10 2001-11-08 小胞体ストレス関連疾患の予防又は治療剤

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JP2000-344516 2000-11-10
JP2000344516 2000-11-10

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WO2003063905A1 (fr) * 2002-01-31 2003-08-07 Center For Advanced Science And Technology Incubation, Ltd. Medicaments preventifs ou remedes pour maladies immunologiques
JP2004010574A (ja) * 2002-06-10 2004-01-15 Redox Bioscience Inc 神経再生因子
WO2004048565A1 (fr) * 2002-11-22 2004-06-10 Takeda Pharmaceutical Company Limited Proteine associee a l'apoptose et son utilisation
JP2004180680A (ja) * 2002-11-22 2004-07-02 Takeda Chem Ind Ltd アポトーシス関連蛋白質およびその用途
WO2005009470A1 (fr) * 2003-07-28 2005-02-03 Osaka Industrial Promotion Organization Traitement de l'insuffisance cardiaque contenant comme principe actif un inhibiteur d'ask1 et procede de criblage de celui-ci
WO2005103288A1 (fr) 2004-04-27 2005-11-03 Takeda Pharmaceutical Company Limited Procédé de criblage
WO2008016131A1 (fr) 2006-08-04 2008-02-07 Takeda Pharmaceutical Company Limited Composé hétérocyclique à cycles fusionnés
JP2011518566A (ja) * 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コンディショナルノックアウトマウス
JP2017071647A (ja) * 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 アポトーシスシグナル調節キナーゼ阻害剤
US10604490B2 (en) 2014-12-23 2020-03-31 Gilead Sciences, Inc. Processes for preparing ASK1 inhibitors

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003063905A1 (fr) * 2002-01-31 2003-08-07 Center For Advanced Science And Technology Incubation, Ltd. Medicaments preventifs ou remedes pour maladies immunologiques
JP2004010574A (ja) * 2002-06-10 2004-01-15 Redox Bioscience Inc 神経再生因子
US7390625B2 (en) 2002-11-22 2008-06-24 Takeda Pharmaceutical Company Limited Apoptosis-associated protein and use thereof
WO2004048565A1 (fr) * 2002-11-22 2004-06-10 Takeda Pharmaceutical Company Limited Proteine associee a l'apoptose et son utilisation
JP2004180680A (ja) * 2002-11-22 2004-07-02 Takeda Chem Ind Ltd アポトーシス関連蛋白質およびその用途
WO2005009470A1 (fr) * 2003-07-28 2005-02-03 Osaka Industrial Promotion Organization Traitement de l'insuffisance cardiaque contenant comme principe actif un inhibiteur d'ask1 et procede de criblage de celui-ci
WO2005103288A1 (fr) 2004-04-27 2005-11-03 Takeda Pharmaceutical Company Limited Procédé de criblage
WO2008016131A1 (fr) 2006-08-04 2008-02-07 Takeda Pharmaceutical Company Limited Composé hétérocyclique à cycles fusionnés
JP2011518566A (ja) * 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コンディショナルノックアウトマウス
JP2017071647A (ja) * 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 アポトーシスシグナル調節キナーゼ阻害剤
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

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