WO2009084232A1 - 糖鎖関連遺伝子、およびその利用 - Google Patents
糖鎖関連遺伝子、およびその利用 Download PDFInfo
- Publication number
- WO2009084232A1 WO2009084232A1 PCT/JP2008/004025 JP2008004025W WO2009084232A1 WO 2009084232 A1 WO2009084232 A1 WO 2009084232A1 JP 2008004025 W JP2008004025 W JP 2008004025W WO 2009084232 A1 WO2009084232 A1 WO 2009084232A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sirna
- tissue
- gene
- mouse
- acetylgalactosamine
- Prior art date
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/08—Drugs for disorders of the urinary system of the prostate
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/10—Drugs for disorders of the urinary system of the bladder
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
-
- 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
-
- 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
- A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
-
- 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
- A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
-
- 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
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/06—Antiarrhythmics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- 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
- 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
-
- 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.)
- C12N9/13—Transferases (2.) transferring sulfur containing groups (2.8)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
- G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/91194—Transferases (2.) transferring sulfur containing groups (2.8)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
- Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
- Y10T436/143333—Saccharide [e.g., DNA, etc.]
Definitions
- the present invention relates to an inhibitor of fibril formation at the level of biological tissue by inhibiting sugar chain-related genes.
- sugars constituting sugar chains
- various sugars have been known so far.
- glucose (Glc) galactose (Gal), mannose (Man), glucuronic acid (GlcUA), iduronic acid (IdoA), fucose (Fuc), glucosamine (GlcN), N-acetylglucosamine (GlcNAc), N-acetyl Galactosamine (GalNAc), xylose (Xyl), sialic acid (SA) and the like are known.
- sugars constituting sugar chains undergo various chemical modifications. For example, methylation, acetylation, formylation, myristoylation, amidation, ubiquitination, acylation, phosphorylation, epimerization, sulfation and the like are known. Furthermore, chemical modifications such as sialylation, asialization, fucosylation, glycosylation, galactosylation, lactosylation, and mannanization can be exemplified. There are multiple sites in the sugar that undergo these chemical modifications. For example, in GlcNAc, it is known that all of the 1st to 6th carbons are chemically modified. It has been reported that other sugars undergo chemical modification at various sites.
- the present invention is based on knowledge newly found through research on genes related to sugar chains.
- An object of the present invention is to provide a new use of a sugar chain-related gene.
- an object of the present invention is to provide a drug that suppresses fibril formation at the level of biological tissue by inhibiting the function of a sugar chain-related gene, and a screening method for the drug.
- sugar chains in vivo Although it has been suggested that the role of sugar chains in vivo is very important, there are many unclear parts about the functions of sugar chains in vivo. As described above, multiple types of sugars constituting sugar chains are known, and sugars are considered to bear important physiological actions in vivo by undergoing various chemical modifications at multiple sites. ing. As described above, since there are many types of sugars constituting sugar chains, types of chemical modifications targeting sugars, and many sites that are chemically modified, it is considered that sugar chains are composed of countless variations. Therefore, it is very difficult to specify the structure of the sugar chain that plays a role, and it is extremely difficult to find the relationship between the pathology caused by a certain disease and the specific action of the sugar chain. That is.
- tissue fibrosis can be suppressed at the living body level by inhibiting sulfation at the 4-position or 6-position of GalNAc, which is one of the sugars constituting the sugar chain.
- GalNAc a sulfation inhibitor at the 4th or 6th position, from a study using various disease model animals, for a disease caused by tissue fibrosis (tissue fibrogenic disease), It has been demonstrated to have a therapeutic effect.
- the present invention relates to a drug that suppresses fibril formation at the level of biological tissue by inhibiting the function of a sugar chain-related gene, and more specifically, a screening method for the drug.
- a tissue fibrosis inhibitor comprising a 4- or 6-position sulfation inhibitor of N-acetylgalactosamine as a component
- the drug according to [1] which has an inhibitory effect on fiber formation in living tissue
- the drug according to [1] or [2] wherein the inhibitor has an activity to inhibit the function of sulfate group 4 or 6 of N-acetylgalactosamine
- the agent according to [3], wherein the inhibitor is an siRNA that suppresses the expression of a sulfate group transferase gene at position 4 or 6 of N-acetylgalactosamine
- the inhibitor is a desulfating enzyme of the 4- or 6-position sulfate group
- the present invention relates to the following.
- a method for suppressing tissue fibrosis comprising a step of administering a 4- or 6-position sulfation inhibitor of N-acetylgalactosamine to an individual.
- the enhanced expression of type I collagen (lower left) and ⁇ -SMA (lower right) is significantly suppressed by the silencing effect of GalNAcNA4S-6ST siRNA (upper). It is a photograph showing tissue deposition of collagen in a mouse intestinal fibril formation model. Masson-stained image of the large intestine tissue (blue). X100. Tissue deposition of collagen is reduced on GalNAc 4S-6ST siRNA. It is a photograph which shows fibroblast immersion in a mouse
- C6ST-1 and fibril formation markers ⁇ -SMA and type I collagen in lung tissue The graph shows the relative ratio between the target gene and house keeping Gene (ribosome 18S).
- the silencing effect of C6ST-1 siRNA (left) significantly suppresses enhanced expression of type I collagen (middle) and ⁇ -SMA (right). It is a photograph which shows fibroblast immersion in a mouse emphysema model. Fibroblast-stained image of lung tissue (brown). X200.
- C6ST-1 siRNA suppresses infiltration of fibroblasts into the alveolar stroma. It is a photograph which shows the macrophage infiltration image in a mouse emphysema model.
- C6ST-1 siRNA suppresses macrophage infiltration in the alveolar stroma. It is a graph which shows the static lung compliance (Cst) in a mouse emphysema model. C6ST-1 siRNA significantly reduces Cst. It is a graph which shows the volume (microliter) of the right lung in a mouse emphysema model. C6ST-1 siRNA significantly reduces lung volume. It is a graph which shows the expression of the fibrogenesis related gene in a mouse emphysema model.
- ⁇ -SMA type I collagen and TGF- ⁇ , which are fibrosis markers in lung tissue.
- the graph shows the relative ratio between the target gene and house keeping Gene (ribosome 18S).
- silencing GalNACST siRNA By silencing GalNACST siRNA, the enhanced expression of each fibrosis marker is significantly suppressed. It is a graph which shows the static lung compliance (Cst) in a mouse emphysema model. GalNAcST siRNA significantly reduces Cst. It is a graph which shows the volume (microliter) of the right lung in a mouse emphysema model. GalNAcST siRNA significantly reduces lung volume. It is a graph which shows transition of obesity in a mouse type 2 diabetes model. C4ST-1 siRNA, C4ST-2 siRNA and C4ST-3 siRNA show a tendency to suppress obesity.
- C4ST-2 siRNA and C4ST-3 siRNA exhibit a significant anti-obesity effect. It is a graph which shows the insulin resistance in a mouse
- Cell nucleus (red), x400 It is a photograph which shows the fibroblast infiltration to a pancreatic islet in a mouse type 2 diabetes model.
- C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA suppress fibroblast (brown) infiltration into the islet.
- X200 It is a photograph which shows the macrophage infiltration to the pancreatic islet in a mouse type 2 diabetes model.
- C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA suppress macrophage (brown) infiltration into the islet.
- GalNAcST siRNA it shows that administration of GalNAcST siRNA favorably lowers blood glucose after insulin loading, that is, insulin resistance is improved. It is the graph and photograph which show the renal tissue stromal accumulation of the fibroblast in a mouse diabetic nephropathy model. C4ST-1 siRNA significantly suppresses the accumulation of fibroblasts (brown). X200. It is the graph and photograph which show the renal tissue stromal accumulation of the macrophage in a mouse diabetic nephropathy model. Accumulation of macrophages (brown) is significantly suppressed by C4ST-1 siRNA. X200. It is a photograph which shows the interstitial accumulation of kidney tissue of ⁇ SMA positive cells in a mouse diabetic nephropathy model.
- ⁇ SMA positive cells (brown) is significantly suppressed by C4ST-1 siRNA.
- X200 It is a graph which shows the anti-fibrogenic effect in a mouse diabetic nephropathy model.
- Administration of GalNAc4S-6ST (G # 1) siRNA significantly suppresses the expression enhancement of GalNAc4S-6ST (G # 1), ⁇ SMA, and TGF ⁇ in renal tissue.
- ARB Angiotensin receptor antagonist (valsartan). It is the photograph and graph which show the renal tissue stromal accumulation of the fibroblast in a mouse diabetic nephropathy model.
- GalNAc4S-6ST (G # 1) siRNA significantly suppresses the accumulation of fibroblasts (brown).
- GalNAc4S-6ST (G # 1) siRNA significantly suppresses angiotensinogen and ACE expression enhancement in renal tissue. It is a graph which shows the renal function protective effect in a mouse diabetic nephropathy model.
- GalNAc4S-6ST (G # 1) siRNA suppresses an increase in serum creatinine, that is, a decrease in renal function. It is a graph which shows the gene expression in a mouse diabetic nephropathy model.
- GalNAcST siRNA significantly suppresses the enhanced expression of GalNAc4ST-1, GalNAc4ST-2, and GalNAc4S-6ST in kidney tissue. It is a graph which shows the anti-fibrogenic effect in a mouse diabetic nephropathy model.
- GalNAcST siRNA significantly suppresses the enhanced expression of CTGF, ⁇ SMA, type I collagen, and ACE in renal tissue. It is a graph which shows the gene expression in mouse drug-related interstitial nephritis.
- GalNAc4S-6ST (G # 1) siRNA significantly suppresses the enhanced expression of GalNAc4S-6ST (G # 1) in kidney tissue. It is a photograph which shows collagen deposition in mouse drug-related interstitial nephritis.
- GalNAc4S-6ST (G # 1) siRNA clearly reduces type I collagen (brown) deposition in the renal interstitium. X200. It is a graph which shows the anti-fibrosis effect in a mouse
- C6ST siRNA significantly suppresses the enhanced expression of C6ST-2 (G # 10), TGF ⁇ , ⁇ SMA, type I collagen, and CTGF in renal tissue.
- 2 is a graph and a photograph showing stromal accumulation of fibroblasts in a mouse UUO fibrosis model.
- C6ST siRNA significantly suppresses the accumulation of fibroblasts (brown) from the paraglomerular to the stroma.
- X200 It is the graph and photograph which show the stromal accumulation of the macrophage in a mouse
- C6ST siRNA significantly suppresses the accumulation of macrophages (brown) from the paraglomerus to the stroma. X200.
- GalNac4S-6ST (G # 1) siRNA significantly increases GFAP-positive cells (brown) from INL to GCL. X200. It is a graph which shows the number of ganglion cells in a mouse diabetic retinopathy model. Counting the number of ganglion cells in GGL. GalNac4S-6ST (G # 1) siRNA can significantly suppress the decrease in the number of ganglion cells. It is a graph which shows the optic nerve regeneration effect in a mouse diabetic retinopathy model. GalNac4S-6ST (G # 1) siRNA significantly increases ocular tissue GS expression. It is a graph which shows gene expression in a mouse fatty liver disorder model.
- GalNAcST siRNA Increased expression of GalNAc4S-6ST in liver tissue and significant suppression by GalNAcST siRNA. It is a graph which shows the fibril formation related gene expression in a mouse fatty liver disorder model. Increased expression of type I collagen and ⁇ SAM in liver tissue and significant suppression by GalNAcST siRNA. It is a photograph which shows fibrogenic cell infiltration in a mouse fatty liver injury model. GalNAcST siRNA suppresses bridge-like accumulation in fibroblast (brown) liver tissue. X100. It is a graph which shows the clinical fibrosis score in a mouse fatty liver disorder model. GalNAcST siRNA significantly suppresses an increase in fibrosis score of liver tissue.
- C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA significantly suppresses the increase in fibrosis score of liver tissue. It is a graph which shows the clinical liver disorder in a mouse
- C6ST siRNA significantly suppresses an increase in fibrosis score of liver tissue. It is a graph which shows the anti-fibrogenic effect in a mouse liver fibrosis model. C6ST siRNA significantly suppresses the expression of certain ⁇ SMA, type I collagen, CTGF, and TGF ⁇ . It is a graph which shows the anti-fibrogenic effect in a mouse Parkinson's disease model.
- GalNAc4S-6ST siRNA significantly suppresses the expression of GalNAc4S-6ST, TGF ⁇ , type I collagen and ⁇ SMA in brain tissue. It is a photograph which shows fibroblast accumulation in a mouse Parkinson's disease model.
- GalNAc4S-6ST siRNA drastically reduces brain tissue accumulation of fibroblasts (brown).
- X200 It is a graph which shows the neuroprotective effect in a mouse Parkinson's disease model.
- GalNAc4S-6ST siRNA significantly enhances the expression of GDNF and Nurr1. It is a photograph which shows the dopamine nerve regeneration effect in a mouse Parkinson's disease model.
- GalNAc4S-6ST siRNA suppresses loss of TH-positive dopamine nerve (green).
- X200 It is a photograph which shows the dopamine nerve regeneration effect in a mouse Parkinson's disease model.
- GalNAc4ST siRNA suppresses loss of TH-positive dopamine nerve (green).
- a type IV collagen stained image (brown, arrow) in the type 2 diabetes model mouse retina is shown. It is the photograph shown about the fibroblast accumulation inhibitory effect of C4-sulfatase in the liver of a mouse type 2 diabetes model. Magnification 50x and 100x. It is the photograph shown about the macrophage infiltration suppression effect in the liver of a mouse type 2 diabetes model. Magnification 50x and 100x. It is a graph shown about the result of the biochemical test (AST, ALT, TG) in the serum of a mouse type 2 diabetes model. In the graph, the untreated group is expressed as unt, the control group is expressed as nor, and the C4-sulfatase is expressed as C4sul.
- the present invention relates to a tissue fibrosis inhibitor that inhibits sulfation at the 4- or 6-position of N-acetylgalactosamine, which is one of the sugars constituting the sugar chain. . That is, the present invention relates to tissue fibrosis which comprises a sulfation inhibitor at the 4-position or 6-position of N-acetylgalactosamine (sometimes referred to herein as “inhibitor of the present invention” or simply “inhibitor”). Provide an inhibitor.
- N-acetylgalactosamine (GalNAc)” of the present invention is an N-acetyl form of galactosamine which is a kind of hexosamine. N-acetylgalactosamine is known to undergo chemical modification at positions 1-6.
- the present invention is characterized by inhibiting sulfation at the position 4 or 6 of N-acetylgalactosamine.
- the site inhibited by the inhibitor of the present invention is a site represented by an arrow in the following chemical formula of GalNAc.
- the site on GalNAc where sulfation is inhibited in the inhibitor of the present invention is at the 4th or 6th position, but the case where both the 4th and 6th positions are inhibited is also included in the present invention.
- the GalNAc of the present invention is preferably a sugar contained in chondroitin sulfate proteoglycan (CSPG).
- inhibition of sulfation means that the sulfate group is inhibited from being transferred to the 4th or 6th position on GalNAc, or the sulfate group is removed from the already sulfated site. Or it means to be substituted with another chemical modification.
- tissue fibrosis inhibitor in the present specification, sometimes described as “the drug of the present invention” in the present invention preferably has an inhibitory effect on fibrosis in a living tissue (in vivo).
- the tissue in which fibrosis is suppressed by the agent of the present invention is not particularly limited.
- tissue for example, heart tissue, gastrointestinal tissue, lung tissue, pancreatic tissue, kidney tissue, eye tissue, liver tissue, cranial nerve tissue, or skin tissue. Etc.
- fibrogenesis can also be expressed as “Fibrogenesis”. It can also be expressed using expressions such as “fibrogenic lesion on tissue”, “fibrotic tissue change”, “tissue fibrogenesis”, and the like.
- the inhibitor of the present invention is not particularly limited as long as it has a function of inhibiting sulfation at the 4-position or 6-position of N-acetylgalactosamine.
- a substance having an activity of inhibiting the function of sulfate group 4 or 6 of N-acetylgalactosamine can be mentioned.
- a compound (nucleic acid) selected from the group consisting of the following (a) to (c) can be mentioned.
- Sulfyltransferase at position 4 or 6 of N-acetylgalactosamine Nucleic acid with ribozyme activity that specifically cleaves the transcript of the gene encoding the enzyme
- c The action of inhibiting the expression of the gene encoding the sulfotransferase at position 4 or 6 of N-acetylgalactosamine by the RNAi effect Nucleic acid (siRNA that suppresses the expression of the sulfotransferase gene)
- Examples of the “substance having a sulfation inhibiting action” include compounds selected from the group consisting of the following (a) to (c).
- a substance having an action of desulfating a 4- or 6-position sulfate group of N-acetylgalactosamine can be mentioned.
- the substance include an enzyme (desulfating enzyme) that desulfates the 4- or 6-position sulfate group of N-acetylgalactosamine.
- “Desulfation” of the 4- or 6-position sulfate group of N-acetylgalactosamine refers to the removal of the 4- or 6-position sulfate group of N-acetylgalactosamine.
- desulfating enzymes include Chondroitin-4-sulfatase (C4-sulfatase), Chondroitin-6-sulfatase, and the like.
- the sulfate group transferase in the present invention is not particularly limited as long as it is an enzyme having an activity of transferring a sulfate group to the 4-position or 6-position of GalNAc, and examples thereof include the following enzymes.
- GalNAc4ST-1 N-acetylgalactosamine 4-sulfotransferase-1 Also known as CHST8: Carbohydrate (N-acetylgalactosamine 4-O) sulfotransferase 8
- GalNAc4ST-2 CHST9: Carbohydrate (N-acetylgalactosamine 4-O) sulfotransferase 9
- C4ST-1 chondroitin-4-O-sulfotransferase-1
- CHST11 Carbohydrate (chondroitin 4) sulfotransferase 11 4)
- C4ST-2 Also known as CHST12
- C4ST-3 CHST13 6)
- C6ST-1 chondroit
- chondroitin-4-sulfatase and chondroitin-6-sulfatase are sequences referred to by a plurality of accession numbers on the genome database (for example, Genbank accession number NT_039500 (part of which is accession number CAAA01098429 (SEQ ID NO: 1)), and NT_078575, NT_039353, NW_001030904, NW_001030811, NW_001030796, NW_000349) can be obtained from the public gene database Genbank.
- GalNAc4ST-1 accession number NM_175140, nucleotide sequence SEQ ID NO: 2, amino acid sequence SEQ ID NO: 3
- GalNAc4ST-2 accesion number NM_199055, nucleotide sequence SEQ ID NO: 4, amino acid sequence SEQ ID NO: 5
- C4ST-1 accesion number NM_021439, nucleotide sequence SEQ ID NO: 6, amino acid sequence SEQ ID NO: 7)
- C4ST-2 accesion number NM_021528, nucleotide sequence SEQ ID NO: 8, amino acid sequence SEQ ID NO: 9)
- C4ST-3 accession number XM_355798, base sequence SEQ ID NO: 10, amino acid sequence SEQ ID NO: 11)
- D4ST accession number NM_028117, base sequence SEQ ID NO: 12, amino
- proteins other than the above have high homology (usually 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more) with the sequences described in the sequence listing, for example.
- the protein which has the function (For example, the function etc. which couple
- the protein is, for example, one or more amino acids added, deleted, substituted or inserted in the amino acid sequence described in any of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, and 17. It is a protein comprising an amino acid sequence, and the number of amino acids that normally change is within 30 amino acids, preferably within 10 amino acids, more preferably within 5 amino acids, and most preferably within 3 amino acids.
- the gene in the present invention includes, for example, an endogenous gene in another organism corresponding to the DNA comprising the base sequence described in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 16. (Such as homologues of the above human genes).
- the endogenous DNA of other organisms corresponding to the DNA consisting of the base sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16 are generally sequenced respectively. Numbers: 2, 4, 6, 8, 10, 12, 14, and 16 have high homology with the DNA. High homology means 50% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more (for example, 95% or more, further 96%, 97%, 98% or 99% or more). ) Homology. This homology is determined by the mBLAST algorithm (Altschul et al. (1990) Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci.
- a person skilled in the art will determine a method for measuring the activity of the desulfation action or sulfation inhibitory action at the 4th or 6th position of N-acetylgalactosamine from the above highly homologous proteins. It can be appropriately acquired by using.
- the above-mentioned protein of the present invention can be prepared not only as a natural protein but also as a recombinant protein using genetic recombination technology.
- the natural protein can be prepared, for example, by a method using affinity chromatography using an antibody against the protein against an extract of a cell (tissue) considered to express the protein.
- the recombinant protein can be prepared, for example, by culturing cells transformed with DNA encoding the protein.
- the above protein of the present invention is suitably used, for example, in the screening method described below.
- nucleic acid means RNA or DNA.
- Chemically synthesized nucleic acid analogs such as so-called PNA (peptide nucleic acid) are also included in the nucleic acids of the present invention.
- PNA is obtained by replacing the pentose / phosphate skeleton, which is the basic skeleton structure of nucleic acids, with a polyamide skeleton having glycine as a unit, and has a three-dimensional structure very similar to nucleic acids.
- antisense nucleic acids inhibit the expression of target genes by inhibiting various processes such as transcription, splicing or translation (Hirashima and Inoue, Nusei Chemistry Laboratory 2 Nucleic acid IV gene replication and expression, (Academic Society, Tokyo Chemical Doujin, 1993, 319-347).
- the antisense nucleic acid used in the present invention may inhibit the expression and / or function of a gene encoding any of the above-mentioned sulfotransferases by any of the above-described actions.
- an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of the gene encoding the above-mentioned sulfate transferase is designed, it is considered effective for inhibiting translation of the gene.
- a sequence complementary to the coding region or the 3 ′ untranslated region can also be used.
- the nucleic acid containing the antisense sequence of the sequence of the untranslated region is included in the antisense nucleic acid used in the present invention.
- the antisense nucleic acid used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side.
- the nucleic acid thus prepared can be transformed into a desired animal (cell) using a known method.
- the sequence of the antisense nucleic acid is preferably a sequence complementary to a gene encoding an endogenous sulfate transferase or a part thereof, which is possessed by the animal (cell) to be transformed. As long as it can be suppressed, it may not be completely complementary.
- the transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcript of the target gene.
- the length of the antisense nucleic acid is preferably at least 15 bases and less than 25 bases, but the antisense nucleic acid of the present invention is not necessarily of this length. For example, it may be 100 bases or more, or 500 bases or more.
- the antisense nucleic acid of the present invention is not particularly limited.
- C4ST-1 GenBank accession number NM_021439, SEQ ID NO: 6
- C4ST-2 GenBank accession number NM_021528, SEQ ID NO: 8
- C4ST-3 GenBank accession number XM_355798, SEQ ID NO: 10.
- Inhibition of the expression of the gene encoding the above-mentioned sulfotransferase can also be carried out using a ribozyme or a DNA encoding a ribozyme.
- a ribozyme refers to an RNA molecule having catalytic activity. Some ribozymes have a variety of activities, but research focusing on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes that cleave RNA in a site-specific manner.
- ribozymes have a size of 400 nucleotides or more, such as group I intron type and M1 RNA contained in RNase P, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type. (Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzyme, 1990, 35, 2191.)
- the self-cleaving domain of the hammerhead ribozyme cleaves 3 ′ of C15 in the sequence G13U14C15, but base pairing between U14 and A9 is important for its activity, and instead of C15, A15 or U15 But it has been shown that it can be cut (Koizumi, M. et al., FEBS Lett, 1988, 228, 228.).
- a ribozyme whose substrate binding site is complementary to the RNA sequence in the vicinity of the target site, it is possible to create a restriction RNA-cleaving ribozyme that recognizes the sequence UC, UU or UA in the target RNA (Koizumi, M.
- Hairpin ribozymes are also useful for the purposes of the present invention.
- This ribozyme is found, for example, in the minus strand of satellite RNA of tobacco ring spot virus (Buzayan, JM., Nature, 1986, 323, 349.). It has been shown that target-specific RNA-cleaving ribozymes can also be produced from hairpin ribozymes (Kikuchi, Y. and Sasaki, N., Nucl AcidsidRes, 1991, 19, 6751., Hiroshi Kikuchi, 1992, 30, 112.). Thus, the expression of the gene can be inhibited by specifically cleaving the transcription product of the gene encoding the above-mentioned sulfotransferase using a ribozyme.
- RNA-interference RNA-interference
- RNAi RNA interference
- RNAi small interfering RNA
- shRNAi small interfering RNA
- RNAi is a phenomenon discovered by Fire et al. In 1998 (Fire A, Nature (1998) 391,: 806-811) that strongly suppresses the expression of target genes with double-stranded RNA homology. That's it. It has recently been attracting attention because it is simpler than conventional gene transfer methods using vectors and the like, has high specificity for a target, and can be applied to gene therapy.
- siRNA short dsRNA
- RNAi is a short double-stranded RNA (hereinafter abbreviated as “dsRNA”) consisting of a sense RNA consisting of a sequence homologous to the mRNA of the target gene and an antisense RNA consisting of a complementary sequence to the cell.
- dsRNA short double-stranded RNA
- This is a phenomenon that induces destruction by specifically and selectively binding to the target gene mRNA, and efficiently inhibiting (suppressing) the expression of the target gene by cleaving the target gene.
- dsRNA when dsRNA is introduced into a cell, expression of a gene having a homologous sequence with that RNA is suppressed (knocked down). Since RNAi can suppress the expression of target genes in this way, it can be used as a simple gene knockout method to replace conventional complicated and low efficiency homologous recombination methods, or a method applicable to gene therapy. It is attracting attention as.
- the RNA used for RNAi is not necessarily completely identical to the above-mentioned gene encoding the sulfotransferase or a partial region of the gene, but preferably has complete homology.
- the terminal part may have an overhang of about 2 bases.
- the target is not particularly limited as long as it is a gene encoding the above-mentioned sulfate transferase, and any region can be a target candidate.
- the base sequence of the C4ST-1 gene (SEQ ID NO: 6), the base sequence of the C4ST-2 gene (SEQ ID NO: 8), the base sequence of the C4ST-3 gene (SEQ ID NO: 10), etc. be able to. More specifically, a partial region of the sequence can be used as a target candidate.
- a partial region of the base sequence of the C4ST-1 gene (SEQ ID NO: 20), a base of the C4ST-2 gene Partial region of sequence (SEQ ID NO: 21), partial region of base sequence of C4ST-3 gene (SEQ ID NO: 22), partial region of base sequence of C6ST-1 gene (SEQ ID NO: 23), C6ST- It can be created based on a partial region (SEQ ID NO: 24) of the base sequence of two genes. More specifically, siRNA targeting the DNA sequence specifically shown by this specification (SEQ ID NO: 25, 26, 35-50, 55-65, 82-88) can be exemplified.
- siRNA synthesized in vitro is linked to plasmid DNA and introduced into the cell
- a method in which two RNAs are annealed, or the like can be employed.
- the two RNA molecules may be molecules having a structure in which one end is closed, for example, siRNA (shRNA) having a hairpin structure.
- shRNA is an RNA molecule called a short hairpin RNA (short hairpin RNA) and having a stem-loop structure so that a part of a single strand forms a complementary strand with another region. That is, molecules capable of forming a double-stranded RNA structure within the molecule are also included in the siRNA of the present invention.
- an RNA capable of suppressing the expression of C4ST-1, C4ST-2, C4ST-3 and the like by the RNAi effect, and a DNA sequence specifically shown by the present specification
- siRNA targeting SEQ ID NO: 25, 26, 35-50, 55-65, 82-88
- a double-stranded RNA having a structure in which one or a few RNAs are added or deleted.
- siRNA of the present invention are also included in the siRNA of the present invention as long as they have a function of suppressing the expression of the gene encoding the above-mentioned sulfate group transferase.
- RNA used for RNAi does not have to be completely identical (homologous) to the gene encoding the protein or a partial region of the gene, but may have perfect identity (homology) preferable.
- the double-stranded RNA having the RNAi effect in the present invention includes a double-stranded RNA before being degraded by DICER as described above. That is, even if a long-chain RNA that does not have an RNAi effect at its original length is expected to be degraded into siRNA having an RNAi effect in cells, the double-stranded RNA of the present invention The length is not particularly limited.
- RNAi double-stranded RNA molecules having the RNAi effect.
- a region on mRNA that is expected to have an RNAi effect need not be selected. That is, the region on the mRNA of the above-mentioned gene of the present invention having the RNAi effect does not necessarily need to be accurately defined.
- double-stranded RNA that can be suppressed by the RNAi effect is appropriately determined by those skilled in the art based on the base sequence of the gene encoding the above-mentioned sulfate transferase that is the target of the double-stranded RNA.
- the double-stranded RNA of the present invention can be prepared based on the base sequence shown in SEQ ID NO: 25.
- RNA sequence set forth in SEQ ID NO: 25 selecting an arbitrary continuous RNA region of mRNA that is a transcription product of the sequence, and preparing a double-stranded RNA corresponding to this region, For those skilled in the art, it can be appropriately performed within the range of normal trials. In addition, selection of siRNA sequences having a stronger RNAi effect from mRNA sequences that are transcripts of the sequences can also be appropriately performed by those skilled in the art by known methods. If one strand is known, those skilled in the art can easily know the base sequence of the other strand (complementary strand). The siRNA can be appropriately prepared by those skilled in the art using a commercially available nucleic acid synthesizer. In addition, for synthesis of a desired RNA, a general synthesis contract service can be used.
- the siRNA in the present invention is not necessarily a set of double-stranded RNAs for the target sequence, and may be a mixture of a plurality of sets of double-stranded RNAs for the region containing the target sequence.
- siRNA as a nucleic acid mixture corresponding to the target sequence can be appropriately prepared by a person skilled in the art using a commercially available nucleic acid synthesizer and a DICER enzyme. Synthetic contract service can be used.
- the siRNA of the present invention includes so-called “cocktail siRNA”.
- RNA ribonucleotides
- the one or more ribonucleotides constituting the siRNA may be corresponding deoxyribonucleotides.
- This “corresponding” refers to the same base species (adenine, guanine, cytosine, thymine (uracil)) although the structures of the sugar moieties are different.
- deoxyribonucleotide corresponding to ribonucleotide having adenine refers to deoxyribonucleotide having adenine.
- the “plurality” is not particularly limited, but preferably refers to a small number of about 2 to 5.
- a DNA (vector) capable of expressing the RNA of the present invention is also included in a preferred embodiment of the compound capable of suppressing the expression of the gene encoding the above-described protein of the present invention.
- the DNA (vector) capable of expressing the double-stranded RNA of the present invention is a DNA encoding one strand of the double-stranded RNA and a DNA encoding the other strand of the double-stranded RNA, Each DNA has a structure linked to a promoter so that it can be expressed.
- the expression vector of the present invention can be prepared by appropriately inserting DNA encoding the RNA of the present invention into various known expression vectors.
- the expression inhibitory substance of the present invention binds to the expression regulatory region (for example, promoter region) of the gene encoding the above-mentioned sulfate group transferase to thereby express the gene encoding the above-mentioned sulfate group enzyme.
- Compounds that inhibit are included.
- the compound can be obtained, for example, by a screening method using a promoter DNA fragment of a gene encoding the above-mentioned sulfate transferase and using the binding activity with the DNA fragment as an index. Further, those skilled in the art can appropriately determine whether or not the desired compound inhibits the expression of the gene encoding the above-mentioned sulfotransferase by a known method such as a reporter assay method.
- a DNA (vector) capable of expressing the RNA of the present invention is also included in a preferable embodiment of the compound capable of inhibiting the expression of the gene encoding the above-mentioned sulfate group transferase of the present invention.
- the DNA (vector) capable of expressing the double-stranded RNA of the present invention is a DNA encoding one strand of the double-stranded RNA and a DNA encoding the other strand of the double-stranded RNA, respectively. It is a DNA having a structure linked to a promoter so that it can be expressed.
- the expression vector of the present invention can be prepared by appropriately inserting DNA encoding the RNA of the present invention into various known expression vectors.
- a preferred embodiment of the vector of the present invention includes a vector that expresses RNA (siRNA) capable of suppressing the expression of C4ST-1, C4ST-2, C4ST-3, etc. by the RNAi effect.
- siRNA RNA
- An antibody that binds to the above-mentioned sulfotransferase can be prepared by methods known to those skilled in the art. If it is a polyclonal antibody, it can obtain as follows, for example.
- a small animal such as a rabbit is immunized with a recombinant (recombinant) protein expressed in a microorganism as a fusion protein with the above-mentioned natural protein or a GST, or a partial peptide thereof to obtain serum.
- This is prepared by, for example, purification using ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, affinity column coupled with the above-mentioned sulfate group transferase or synthetic peptide, or the like.
- a monoclonal antibody for example, a small animal such as a mouse is immunized with the above-mentioned sulfate transferase or its partial peptide, and the spleen is removed from the mouse and ground to separate the cells.
- a myeloma cell is fused with a reagent such as polyethylene glycol, and a clone producing an antibody that binds to the above-mentioned sulfotransferase is selected from the resulting fused cell (hybridoma).
- a clone producing an antibody that binds to the above-mentioned sulfotransferase is selected from the resulting fused cell (hybridoma).
- the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is obtained by, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, the above-mentioned sulfate. It can be prepared by purification using an affinity column coupled with a protein of a transferase or a synthetic peptide.
- the antibody of the present invention is not particularly limited as long as it binds to the above-described sulfotransferase of the present invention.
- a human antibody, a humanized antibody obtained by genetic recombination, and an antibody thereof may be a fragment or a modified antibody.
- the protein of the present invention used as a sensitizing antigen for antibody acquisition is not limited with respect to the animal species from which it is derived, but is preferably a protein derived from a mammal such as a mouse or a human, and particularly preferably a protein derived from a human.
- Human-derived proteins can be appropriately obtained by those skilled in the art using the gene sequences or amino acid sequences disclosed in the present specification.
- the protein used as the sensitizing antigen may be a complete protein or a partial peptide of the protein.
- the partial peptide of protein include an amino group (N) terminal fragment and a carboxy (C) terminal fragment of protein.
- antibody means an antibody that reacts with the full length or fragment of a protein.
- human lymphocytes such as human lymphocytes infected with EB virus are sensitized with proteins, protein-expressing cells or lysates thereof in vitro. Lymphocytes can be fused with human-derived myeloma cells having permanent mitotic activity, such as U266, to obtain hybridomas that produce desired human antibodies having binding activity to proteins.
- the antibody against the above-mentioned sulfotransferase of the present invention is expected to have an effect of inhibiting the expression or function of the protein by binding to the protein.
- a human antibody or a humanized antibody is preferred in order to reduce immunogenicity.
- the present invention also includes a low molecular weight substance (low molecular weight compound) that binds to the above-mentioned sulfotransferase as a substance that can inhibit the function of the above-mentioned sulfotransferase.
- the low molecular weight substance may be a natural or artificial compound. Usually, it is a compound that can be produced or obtained by using methods known to those skilled in the art. The compound of the present invention can also be obtained by the screening method described later.
- a mutant (dominant negative protein) having a dominant negative property with respect to the above-mentioned sulfate group transferase can be exemplified.
- the protein mutant having a dominant negative property with respect to the 4- or 6-position sulfotransferase of N-acetylgalactosamine is a gene encoding the 4- or 6-position sulfate group of N-acetylgalactosamine Is a protein having a function of eliminating or reducing the activity of the endogenous wild-type protein.
- the sulfation inhibitor at the 4th or 6th position of N-acetylgalactosamine, which is a drug of the present invention, has a therapeutic or prophylactic effect on fibrogenic diseases. Therefore, a preferred embodiment of the drug of the present invention is for treatment or prevention of fibrogenic diseases.
- treatment or prevention does not necessarily need to have a complete therapeutic effect or preventive effect on an organ or tissue exhibiting tissue fibrosis, and may be a partial effect.
- tissue fibrosis inhibitor of the present invention has an action of suppressing fibrosis by inhibiting sulfation at the 4th or 6th position of N-acetylgalactosamine which is the cause of fibrosis. Therefore, as a preferred embodiment of the present invention, for example, a therapeutic or prophylactic agent for a tissue fibrosis disease comprising the tissue fibrosis inhibitor of the present invention as an active ingredient is provided.
- tissue fibrosis disease therapeutic agent” of the present invention can also be expressed as “tissue fibrosis disease ameliorating agent”, “anti-tissue fibrosis agent” and the like.
- the drug in the present invention can also be expressed as “pharmaceutical agent”, “pharmaceutical composition”, “therapeutic drug”, and the like.
- the “treatment” in the present invention includes a preventive effect that can suppress the occurrence of fibrosis in advance. Moreover, it is not necessarily limited to having a complete therapeutic effect on a fibrogenic organ (tissue), and may have a partial effect.
- the drug of the present invention can be administered orally or parenterally as a pharmaceutical composition by mixing with a physiologically acceptable carrier, excipient, diluent or the like.
- dosage forms such as granules, powders, tablets, capsules, solvents, emulsions or suspensions can be used.
- parenteral agents dosage forms such as injections, instillations, external drugs, inhalants (nebulizers) or suppositories can be selected.
- Examples of the injection include subcutaneous injection, intramuscular injection, intraperitoneal injection, intracranial injection, intranasal injection, and the like.
- medicine for external use include a nasal administration agent or an ointment.
- the preparation technology for making the above-mentioned dosage form so as to include the drug of the present invention as the main component is known.
- a tablet for oral administration can be produced by adding an excipient, a disintegrant, a binder, a lubricant, and the like to the drug of the present invention, mixing, and compressing and shaping.
- an excipient lactose, starch, mannitol or the like is generally used.
- disintegrant calcium carbonate, carboxymethyl cellulose calcium and the like are generally used.
- binder gum arabic, carboxymethylcellulose, or polyvinylpyrrolidone is used.
- lubricant talc, magnesium stearate and the like are known.
- the tablet containing the drug of the present invention can be coated with a known coating for masking or enteric preparation.
- the coating agent ethyl cellulose, polyoxyethylene glycol, or the like can be used.
- the injection can be obtained by dissolving the agent of the present invention as a main component together with an appropriate dispersant, dissolving in a dispersion medium, or dispersing.
- an appropriate dispersant dissolving in a dispersion medium, or dispersing.
- either a water-based solvent or an oil-based solvent can be used.
- an aqueous solvent distilled water, physiological saline, Ringer's solution, or the like is used as a dispersion medium.
- various vegetable oils and propylene glycol are used as a dispersion medium.
- a preservative such as paraben may be added as necessary.
- known isotonic agents such as sodium chloride and glucose can be added to the injection.
- a soothing agent such as benzalkonium chloride or procaine hydrochloride can be added.
- an external preparation can be obtained by making the agent of the present invention into a solid, liquid or semi-solid composition.
- a solid or liquid composition it can be set as an external preparation by setting it as the composition similar to what was described previously.
- a semi-solid composition can be prepared by adding a thickener to an appropriate solvent as required.
- the solvent water, ethyl alcohol, polyethylene glycol, or the like can be used.
- the thickener bentonite, polyvinyl alcohol, acrylic acid, methacrylic acid, or polyvinylpyrrolidone is generally used.
- a preservative such as benzalkonium chloride can be added to the composition.
- a suppository can be obtained by combining an oily base material such as cacao butter or an aqueous gel base material such as a cellulose derivative as a carrier.
- the agent of the present invention When the agent of the present invention is used as a gene therapy agent, a method of administering a vector incorporating a nucleic acid in addition to a method of directly administering the agent of the present invention by injection can be mentioned.
- the vector include an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a vaccinia virus vector, a retrovirus vector, a lentivirus vector, and the like, and can be efficiently administered by using these virus vectors.
- siRNAs have a very specific post-transcriptional repression effect in vitro, but in in vivo, they are rapidly degraded by nuclease activity in the serum, so their duration is limited and more optimal and effective delivery System development has been demanded.
- biocompatible material atelocollagen is mixed with nucleic acid and combined.
- the body it has been reported that it is a carrier suitable for siRNA and has a function of protecting nucleic acid from degrading enzymes in the living body. Not limited.
- the necessary amount (effective amount) of the drug of the present invention is administered to mammals including humans within the safe dose range.
- the dosage of the drug of the present invention can be appropriately determined finally based on the judgment of a doctor or veterinarian in consideration of the type of dosage form, administration method, patient age and weight, patient symptoms, and the like.
- the dose varies depending on age, sex, symptoms, administration route, administration frequency, and dosage form.
- the dose is about 10 6 to 10 13 per day.
- a commercially available gene introduction kit for example, Adeno Express: Clontech
- a commercially available gene introduction kit for example, Adeno Express: Clontech
- the disease to be treated or prevented by the drug of the present invention is not particularly limited as long as it is a disease caused by tissue fibrosis, but preferably, heart disease, digestive tract disease, liver disease, lung disease, kidney disease, A cranial nerve disease, an eye disease, a pancreas disease, etc. can be mentioned.
- Disease caused by fibrosis are not particularly limited, but include elastic fibrosis, scleroderma, chronic peritonitis, retroperitoneal cavity fibrosis in the outer skin and epithelial tissues including skin, etc.
- Supporting tissues such as connective tissue, polymyositis in muscle, dermatomyositis, nodular polyarteritis, soft tissue fibrosis, rheumatoid arthritis, palmar fibroma, tendonitis, tendonitis, Achilles tendonitis, foot mycosis, bone marrow, Blood tissues such as the heart, myelofibrosis in the vascular system, hypersplenism, vasculitis, bradyarrhythmias, arteriosclerosis, obstructive thromboangiitis, nodular fibrosis, angina pectoris, dilated congestion Cardiomyopathy, heart failure, restrictive cardiomyopathy, diffuse non-occlusive cardiomyopathy, obstructive cardiomyopathy, pulmonary heart, mitral stenosis, aortic stenosis, chronic pericarditis, endocardial fibrosis, intracardiac Chronic pancreatitis in the digestive system such as liver, including membrane
- fibrosis Cranial nerve disease, posterior lens fibrosis in the eyeball, proliferative retinopathy, sarcoidosis that causes lesions throughout the body, fibrosis due to systemic lupus erythematosus, systemic sclerosis, polymyositis, dermatomyositis, etc.
- the “disease caused by fibrosis” in the present invention is not limited to these, and includes diseases caused by fibrosis in various living tissues such as skin and organs.
- the present invention also provides a method for screening a tissue fibrosis inhibitor using the degree of sulfation at the 4-position or 6-position of N-acetylgalactosamine as an index (in the present specification, the case where “the method of the present invention” is described). Yes).
- a preferred embodiment of the method of the present invention is a method comprising a step of selecting a compound that inhibits sulfation at the 4-position or 6-position of N-acetylgalactosamine constituting the sugar chain.
- a preferred embodiment of the screening method of the present invention is a screening method for a tissue fibrosis inhibitor comprising the following (a) to (c).
- c A step of selecting a compound that reduces the degree of sulfation compared to the case where the test compound is not contacted.
- examples of the origin of the N-acetylgalactosamine, sulfate transferase, desulfase, etc. used include those derived from humans, mice, rats, and the like. Not.
- test compound used in the embodiments described below is not particularly limited.
- a single compound such as a natural compound, an organic compound, an inorganic compound, a protein, and a peptide, and expression of a compound library and a gene library Examples include products, cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts and the like.
- contact with the test compound is usually carried out by mixing N-acetylgalactosamine, sulfotransferase, or desulfating enzyme with the test compound, but is not limited to this method.
- the above “contact” can be performed by bringing a cell expressing these proteins or a part thereof into contact with a test compound.
- examples of the origin of the “cell” in the embodiments described below include cells derived from human, mouse, rat, etc., but are not particularly limited to cells derived from these, and express proteins used in each embodiment. It is also possible to use microbial cells such as Escherichia coli and yeast transformed as described above. For example, “cells that express a gene encoding the 4- or 6-position sulfotransferase of N-acetylgalactosamine” encode the endogenous 4 or 6-sulfate transferase of N-acetylgalactosamine.
- Cells expressing the gene to be expressed, or cells in which the gene encoding the 4- or 6-position sulfate transferase of exogenous N-acetylgalactosamine is introduced and the gene is expressed can be used .
- Cells expressing a gene encoding sulfate group 4 or 6 of exogenous N-acetylgalactosamine usually contain a gene encoding sulfate group 4 or 6 of N-acetylgalactosamine It can be produced by introducing the expressed expression vector into a host cell.
- the expression vector can be prepared by general genetic engineering techniques.
- the measurement of the degree of sulfation in the method of the present invention can be carried out by methods known to those skilled in the art. For example, it can be detected by measuring the amount of labeling using a labeled compound or an antibody that binds to the sulfated structure at position 4 or 6 of N-acetylgalactosamine or a part thereof. Moreover, it can also detect using a chromatography method, a mass spectrometry, etc.
- a person skilled in the art can appropriately evaluate the degree of sulfation at the 4-position or 6-position of N-acetylgalactosamine by, for example, the following known methods.
- Quantitative dye binding method using labeled dye (1-9-dimethylmethylene blue) (Nature. 1998 Feb 26; 391 (6670): 908-11)
- (32 P) 3 ' the method according to photoaffinity labeling with 5'-ABP (Mandon, EC, Milla, ME, Kempner, E., and Hirschberg, CB (1994) Proc. Natl. Acad. Sci.
- a method comprising a step of selecting a substance (compound) that reduces the transsulfation activity at the 4-position or 6-position of N-acetylgalactosamine.
- the above method of the present invention comprises, for example, the following steps. (A) a step of bringing a test compound into contact with the 4-position or 6-position sulfotransferase of N-acetylgalactosamine and (b) a step of measuring the transsulfation activity of the enzyme (c) not contacting the test compound Selecting a compound that reduces the activity compared to the case
- test compound is brought into contact with the 4- or 6-position sulfotransferase of N-acetylgalactosamine.
- the sulfotransferase activity of the enzyme is measured.
- the compound which reduces the said activity is selected compared with the case where a test compound is not contacted (control).
- the compound to be reduced becomes a fibrosis inhibitor or a drug for treating fibrotic diseases. Examples of methods that can be evaluated (measured) as to whether a test compound has the above-described sulfate group transfer activity include the following methods.
- test compounds are mixed in the process of culturing cells or cell lines that promote sulfation at position 4 or 6 of N-acetylgalactosamine for a certain period of time.
- Antibody clone; LY111, 2H6
- an antibody that detects 6-position sulfation clone: MC21C, MO225, CS-56, all manufactured by Seikagaku Corporation.
- fluorescently labeled antibodies the fluorescence values before and after the culture may be compared, or a detection method using 2-B-6 or 3-B-3 antibody may be performed before and after the culture.
- a cell line in which a sulfated transferase gene such as C4ST-1 or C6ST-1 is introduced into CHO cells or L cells by a well-known method and expressed constantly is prepared.
- a treatment candidate compound can be determined more clearly.
- Another preferred embodiment of the present invention is the suppression of tissue fibrosis comprising the following steps (a) to (c), wherein a compound that decreases the expression level of the N-acetylgalactosamine sulfate transferase gene of the present invention is selected.
- This is a screening method for agents.
- A a step of bringing a test compound into contact with a cell that expresses a gene encoding the 4-position or 6-position sulfotransferase of N-acetylgalactosamine
- b a step of measuring the expression level of the gene in the cell
- a test compound is brought into contact with a cell expressing a gene encoding the 4- or 6-position sulfotransferase of N-acetylgalactosamine.
- the expression level of the gene encoding the 4- or 6-position sulfotransferase of N-acetylgalactosamine is then measured.
- gene expression includes both transcription and translation.
- the gene expression level can be measured by methods known to those skilled in the art.
- mRNA is extracted from cells expressing any of the above proteins according to a standard method, and the transcription amount of the gene is determined by performing Northern hybridization, RT-PCR, DNA array method, etc. using this mRNA as a template. Measurements can be made. In addition, by collecting a protein fraction from a cell expressing a gene encoding any of the above proteins and detecting the expression of any of the above proteins by electrophoresis such as SDS-PAGE, the amount of translation of the gene can be reduced. Measurements can also be made. Furthermore, the amount of translation of a gene can be measured by detecting the expression of the protein by performing Western blotting using an antibody against any of the above proteins.
- the antibody used for detection of the protein is not particularly limited as long as it is a detectable antibody. For example, both a monoclonal antibody and a polyclonal antibody can be used.
- the expression level of the gene is then compared with that when the test compound is not contacted (control).
- a compound that reduces (suppresses) the expression level of the gene is then selected as compared with the case where the test compound is not contacted.
- a compound that decreases (suppresses) becomes a tissue fibrosis inhibitor or a candidate compound for treating a fibrotic disease.
- a compound that decreases the expression level (level) of the gene encoding the 4- or 6-position sulfotransferase of N-acetylgalactosamine of the present invention is used as a reporter gene.
- the above method of the present invention includes, for example, the following steps (a) to (c).
- A a cell or a cell extract containing DNA having a structure in which a transcriptional regulatory region of a gene encoding the 4- or 6-position sulfate transferase of N-acetylgalactosamine and a reporter gene are functionally linked;
- B a step of measuring the expression level (level) of the reporter gene (c) a compound that decreases the expression level (level) of the reporter gene compared to a case where the test compound is not contacted The process of selecting
- “functionally linked” means that expression of the reporter gene is induced by binding of a transcription factor to the transcriptional regulatory region of the gene encoding the sulfotransferase at position 4 or 6 of N-acetylgalactosamine. As mentioned above, it means that the transcriptional regulatory region of the gene encoding the 4- or 6-position sulfate transferase of N-acetylgalactosamine is linked to the reporter gene.
- the gene encoding sulfate group 4 or 6 of N-acetylgalactosamine If the expression of the fusion protein is induced by binding of a transcription factor to the transcriptional regulatory region, it is included in the meaning of “functionally bound”.
- the sulfate group at the 4- or 6-position of N-acetylgalactosamine present in the genome. It is possible to obtain a transcriptional regulatory region of a gene encoding a transferase by a known method.
- the reporter gene used in the present method is not particularly limited as long as its expression can be detected, and examples thereof include CAT gene, lacZ gene, luciferase gene, and GFP gene.
- a cell containing DNA having a structure in which a transcriptional regulatory region of a gene encoding sulfate group 4 or 6 of N-acetylgalactosamine and a reporter gene is functionally linked is described as such a structure.
- a cell into which a vector into which is inserted is introduced.
- Such vectors can be prepared by methods well known to those skilled in the art. Introduction of the vector into the cells can be performed by a general method such as calcium phosphate precipitation, electric pulse perforation, lipofection, microinjection and the like.
- a cell containing a DNA having a structure in which a transcriptional regulatory region of a gene encoding the 4- or 6-position sulfate transferase of N-acetylgalactosamine and a reporter gene is functionally linked includes the structure on a chromosome. Inserted cells are also included. The DNA structure can be inserted into the chromosome by a method generally used by those skilled in the art, for example, a gene introduction method using homologous recombination.
- Cell extract containing a DNA having a structure in which a transcriptional regulatory region of a gene encoding sulfate group 4 or 6 of N-acetylgalactosamine and a reporter gene is functionally linked is, for example, commercially available
- the cell extract contained in the in vitro transcription and translation kit has a structure in which the transcriptional regulatory region of the gene encoding the sulfotransferase at position 4 or 6 of N-acetylgalactosamine and the reporter gene are functionally linked. Examples include those to which DNA has been added.
- contact means “a cell containing a DNA having a structure in which a transcriptional regulatory region of a gene encoding a sulfotransferase at position 4 or 6 of N-acetylgalactosamine and a reporter gene are functionally linked”. It can be carried out by adding a test compound to the culture solution or adding the test compound to the above-mentioned commercially available cell extract containing the DNA.
- the test compound is a protein, for example, it can be performed by introducing a DNA vector expressing the protein into the cell.
- the expression level of the reporter gene is then measured.
- the expression level of the reporter gene can be measured by methods known to those skilled in the art depending on the type of the reporter gene. For example, when the reporter gene is a CAT gene, the expression level of the reporter gene can be measured by detecting acetylation of chloramphenicol by the gene product.
- the reporter gene is a lacZ gene, by detecting the color development of the dye compound catalyzed by the gene expression product, and when the reporter gene is a luciferase gene, the fluorescent compound catalyzed by the gene expression product By detecting fluorescence, and in the case of a GFP gene, the expression level of the reporter gene can be measured by detecting fluorescence due to the GFP protein.
- the expression level (level) of the measured reporter gene is then compared with that measured in the absence of the test compound (control).
- the reporter gene is operably linked to a gene encoding sulfate group 4 or 6 of N-acetylgalactosamine
- the expression level of the reporter gene is reduced (suppressed) compared to the control.
- a compound that decreases (suppresses) becomes a drug for suppressing tissue fibrosis or a candidate compound for treating fibrotic diseases.
- the tissue fibrosis inhibitor found in the screening method of the present invention is preferably used for treating or preventing fibrotic diseases.
- the present invention also provides a method for producing a pharmaceutical composition for treating or preventing a fibrogenic disease.
- the production method of the present invention includes, for example, the following steps (a) and (b). (A) a step of selecting a tissue fibrosis inhibitor from the test sample by the screening method for the tissue fibrosis inhibitor, and (b) a step of mixing the drug and a pharmaceutically acceptable carrier.
- a tissue fiber formation inhibitor is selected from the test sample by the screening method for the tissue fiber formation inhibitor.
- the method then mixes the selected drug with a pharmaceutically acceptable carrier.
- a pharmaceutically acceptable carrier for example, the above-mentioned carriers can be used.
- the present invention also provides a kit containing various drugs / reagents and the like used for carrying out the screening method of the present invention.
- the kit of the present invention can be appropriately selected from, for example, the above-mentioned various reagents of the present invention according to the screening method to be performed.
- the kit of the present invention can comprise a sulfotransferase at the 4th or 6th position of the N-acetylgalactosamine of the present invention as a component.
- the kit of the present invention can further contain various reagents, containers and the like used in the method of the present invention.
- an antibody, a probe, various reaction reagents, cells, a culture solution, a control sample, a buffer solution, instructions describing how to use, and the like can be appropriately included.
- the present invention also provides a method for treating or preventing a fibrogenic disease, which comprises the step of administering the agent of the present invention to an individual (for example, a patient).
- the subject of the prevention or treatment method of the present invention is not particularly limited as long as it is an organism that can develop a fibrogenic disease, but is preferably a human.
- Administration to an individual can be generally performed by methods known to those skilled in the art, such as intraarterial injection, intravenous injection, and subcutaneous injection.
- the dosage varies depending on the weight and age of the patient, the administration method, etc., but a person skilled in the art (such as a doctor, veterinarian, pharmacist, etc.) can appropriately select an appropriate dosage.
- the present invention further relates to the use of the agent of the present invention in the production of a tissue fibrosis inhibitor. It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.
- siRNA may be expressed by describing the DNA region of a target gene.
- a person skilled in the art can easily recognize the structure of siRNA composed of double-stranded RNA corresponding to the DNA sequence based on the information of the DNA sequence described as the target sequence.
- Example 1 Examination of siRNA knockdown effect of target sugar chain gene in mouse cardiomyopathy model and examination of anti-fibrogenic effect at gene level
- mouse cardiomyopathy model An intraperitoneal injection model of doxylubicin hydrochloride (DOX: manufactured by Kyowa Hakko) was used.
- DOX doxylubicin hydrochloride
- this mouse model is classic, it has excellent reproducibility and is easy to use, so it is widely used as a cardiomyopathy model for elucidation of disease states and new treatment experiments (Longhu Li, Circulation.
- myocardial interstitial fibrosis is present, so in addition to dilated cardiomyopathy, restrictive cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic primary right ventricular cardiomyopathy (ARVC), acute myocardial infarction / stable angina Pathologic findings that are common to left ventricular remodeling secondary to infectious diseases, unstable angina pectoris, myocarditis, valvular heart disease, arrhythmia, and hypertension, and responsible for myocardial dysfunction in chronic heart failure based on the aforementioned diseases (Jugdutt BI, Circulation. 108: 1395-1403, 2003).
- DOX 15 mg / kg; manufactured by Kyowa Hakko
- C57BL6 / J mice male, 8 weeks old, manufactured by CLEA Japan
- the animals were raised for 1 week, and heart tissue was collected.
- the same mice that were purchased and raised at the same time without using DOX were used.
- GalNac 4S-6ST siRNA drug is obtained by using 1 ⁇ g of GalNac 4S-6ST siRNA (Hokkaido System Science Co., Ltd.) and 1% Athenocollagen (Koken Co., Ltd.) 200 ⁇ L per mouse 24 hours prior to DOX administration. The mixture was administered intraperitoneally.
- the base sequence of the GalNac 4S-6ST siRNA drug used in this example is shown below. The arrangement is not necessarily limited to this example.
- RNA iso (Takara Bio) to 50 mg of an organ (heart) extracted from a cardiomyopathy model mouse, pulverize with an electric homogenizer (DIGITAL HOMOGENIZER, AS ONE), and then chloroform 200 ⁇ L (Sigma-Aldrich-Japan Co., Ltd.) Gently added and mixed gently, then ice-cooled for about 5 minutes, and centrifuged at 12,000 rpm, 4 ° C., 15 minutes using a centrifuge (Centrifuge® 5417R, eppendorf).
- RNA concentration in the sample extracted by Wave XS, manufactured by BIO-TEK was calculated.
- RNA sample was adjusted to a concentration of 500 ng / 20 ⁇ L, heated at 68 ° C. for 3 minutes with BLOCK INCUBATOR (manufactured by ASTEC), and ice-cooled for 10 minutes.
- RT Pre Mix solution composition: 25 mM MgCl 2 18.64 ⁇ L (Invitrogen), 5 ⁇ Buffer 20 ⁇ L (Invitrogen), 0.1 M DTT 6.6 ⁇ L (Invitrogen), 10 mM dNTP mix 10 ⁇ L (Invitrogen), RNase Inhibitor 2 ⁇ L (Invitrogen), MMLV Reverse transcriptase 1.2 ⁇ L (Invitrogen), Random primer 2 ⁇ L (Invitrogen), sterile distilled water 19.56 ⁇ L (Otsuka distilled water: Otsuka (Manufactured by Pharmaceutical Co., Ltd.) was added 80 ⁇ L and reacted with BLOCK INCUBATOR (manufactured by ASTEC) at 42 ° C.
- BLOCK INCUBATOR manufactured by ASTEC
- the synthesized cDNA was used for quantitative PCR reaction with the following composition: For quantitative PCR, SYBR premix kit (manufactured by Takara Bio Inc.) and Real-time PCR thermal cycler DICE (manufactured by Takara Bio Inc.) was used for PCR reaction conditions: 95 ° C for 10 seconds, 95 ° C for 5 seconds and 60 ° C for 30 seconds for 40 cycles. Finally, a melting curve analysis was performed, and the base sequence of the primer used for quantitative PCR is shown below.
- the GalNac 4S-6ST siRNA treatment group was more significant than the untreated group (P ⁇ 0.001, vs untreated group).
- Suppressed GalNac4S-6ST expression As shown in FIG. 2, as a result of examining the gene expression of GalNac4S-6ST, TypeI collagen, and ⁇ -SMA, the GalNac 4S-6ST siRNA treatment group was more significant than the untreated group (P ⁇ 0.001, vs untreated group).
- Suppressed GalNac4S-6ST expression As a result of examining the gene expression of ⁇ -SMA and Type I collagen as fibrosis indicators that are important for cardiomyopathy pathology, the GalNac 4S-6ST siRNA treatment group showed a significant decrease in expression compared to the untreated group (P ⁇ 0.001 vs untreated group).
- the agent of the present invention is useful, for example, as a myocardial fiber formation inhibitor.
- Example 2 Inhibitory effect of GalNAc4S-6ST siRNA on cardiac hypertrophy in mouse cardiomyopathy model
- heart weight mg
- body weight g
- Cardiac hypertrophy is also an indicator of tissue fibrotic changes.
- the ratio value in the untreated group was 6.376 ⁇ 0.484, and the value in the siRNA treated group was 5.442 ⁇ 0.203.
- there was a significant decrease in the siRNA treated group compared to the untreated group p ⁇ 0.05: t test.
- the drug of the present invention is useful, for example, as a cardiac hypertrophy inhibitor (cardiac hypertrophy therapeutic agent).
- Example 3 Examination of the inhibitory effect of GalNAc 4S-6ST siRNA on type I collagen deposition in a mouse cardiomyopathy model
- GalNac 4S-6ST siRNA type I collagen deposition A comparative study was conducted on the inhibitory effect of fibrosis index.
- a heart tissue sample collected from the same mouse as in Example 1 was embedded in a freezing embedding agent OCT compound (manufactured by Miles), sliced with a cryostat (manufactured by Carl Zeiss), and the obtained section was obtained.
- the agent of the present invention is useful, for example, as a type I collagen deposition inhibitor in myocardial tissue.
- Example 4 Examination of the inhibitory effect of GalNAc 4S-6ST siRNA on type III collagen deposition in a mouse cardiomyopathy model
- GalNac 4S-6ST siRNA type III collagen deposition A comparative study was conducted on the inhibitory effect of the fibrogenesis activity index.
- a tissue section obtained in the same manner as in Example 3 was fixed with acetone (manufactured by Sigma-Aldrich Japan) for 10 minutes, washed with a phosphate buffer, and further anti-type III collagen rabbit antiserum (rabbit as a primary antibody).
- Polyclonal antibody (1: 2000 dilution; manufactured by LSL) was added and allowed to react at room temperature for 1 hour.
- GalNac 4S-6ST siRNA suppresses type III collagen deposition in myocardial tissue, that is, effective suppression of active collagen deposition. It became clear to have.
- the agent of the present invention is useful, for example, as a type III collagen deposition inhibitor in myocardial tissue.
- Example 5 Examination of fibroblast infiltration suppression effect of GalNAc 4S-6ST siRNA in mouse cardiomyopathy model
- acetone manufactured by Sigma-Aldrich Japan
- ER- anti-mouse fibroblast antibody
- TR7 rat monoclonal antibody, 1: 400 dilution; manufactured by BMA Biomedicals) was added and allowed to react at room temperature for 1 hour. Subsequently, a peroxidase-labeled goat-derived anti-rat immunoglobulin antibody (1: 200 dilution; manufactured by Biosource) as a secondary antibody was added and reacted at room temperature for 30 minutes. DAB substrate (Nichirei) was added to the sample after the reaction. This specimen was observed using an optical microscope (Leica). The tissue findings are shown in FIG. The photo is focused on the ventricular septum. In the untreated group, more fibroblasts were infiltrated than in the control group.
- the degree of fibroblast infiltration in the siRNA treated group was less than that in the untreated group.
- This result indicates that GalNac 4S-6ST siRNA has a pharmacological effect of suppressing fibroblast infiltration in myocardial tissue, and this effect is one end of the anti-fibrogenic effect.
- the agent of the present invention is useful, for example, as a fibroblast infiltration inhibitor in myocardial tissue.
- Example 6 Clinical fibrosis inhibitory effect of GalNAc4S-6ST in mouse intestinal fibrosis model C57BL / 6J mice ( ⁇ , 6 weeks old, manufactured by Clea Japan) 3% dextran sulfate sodium (DSS; manufactured by Wako) A colitis model was created by allowing the high-chlorine water contained to freely drink for 8 days.
- This DSS-induced colitis model is highly reproducible and is widely used as a standard experimental model for inflammatory bowel diseases such as mouse ulcerative colitis and Crohn's disease, and is a histological feature of intestinal stenosis It is also a model exhibiting full-thickness inflammation and fibrotic changes and muscle layer thickening (Sasaki N, J Inflamm. 2005 2:13, review: Pucilowska JB et al. Am J Physiol Gastroenterol Liver Physiol. 279: G653-G659 , 2000).
- intestinal stenosis in addition to inflammatory bowel disease, pathological conditions presenting intestinal stenosis on the tissue, ie intestinal behcet (simple ulcer), irritable bowel syndrome, ischemic enteritis, drug enteritis, radiation enteritis, esophageal achalasia, scleroderma Esophageal stenosis associated with infectious disease, intestinal stenosis associated with systemic lupus erythematosus (SLE), Hills-Spring disease, stenosis after intestinal excision (postoperative stenosis), cancer of the digestive tract (tongue cancer, nasopharyngeal cancer)
- This is a histological finding common to a wide range of diseases such as intestinal stenosis and intestinal obstruction after endoscopic mucosal resection for laryngeal cancer, esophageal cancer, stomach cancer, small intestine cancer, colon cancer, and rectal cancer).
- mice were mixed with the same GalNAc4S-6ST siRNA (1 ⁇ g / mouse) as in Example 1 in atelocollagen (manufactured by Koken Co., Ltd.) diluted 10-fold in PBS and 200 ⁇ l intraperitoneally. Injected.
- the group of mice treated with this treatment was named the GalNAc4S-6STRNAsiRNA group, and the group treated with atelocollagen alone without mixing GalNAc4S-6ST siRNA was named the control group.
- Activity index (DAI) scores were recorded (Kihara M, Gut.ut2003 52: 713-9). The evaluation criteria for DAI are as follows.
- FIG. 6 shows the results of recording the DAI of each mouse, with the first day of DSS water supply (day 0) being 1.
- the GalNAc4S-6ST siRNA-administered group showed significantly lower values than the control group (p ⁇ 0.001, t test). From these results, it was shown that suppression of GalNAc4S-6ST gene expression has an effect of suppressing inflammation activity from a relatively early stage.
- the colon length was measured after the mice were sacrificed on the fifth day, the shortening of the colon was significantly suppressed (p ⁇ 0.005, t test) in the group administered with GalNAc4S-6ST siRNA (FIG. 6).
- the length of the large intestine is a critical indicator that reflects intestinal fibril formation or stenosis. Therefore, it was clarified that in the group administered with GalNAc4S-6ST siRNA, intestinal fibrotic changes were strongly suppressed clinically.
- the agent of the present invention is useful, for example, as an intestinal fibrotic change inhibitor.
- Example 7 Intestinal Fibrosis Inhibition Effect by GalNAc4S-6ST siRNA in Mouse Intestinal Fibrosis Model
- expression of local fibrosis-related genes by administration of GalNAc4S-6ST siRNA was examined by real-time quantitative PCR.
- An intestinal fibrosis model was prepared in the same manner as in Example 6, and the mice were sacrificed on the seventh day. Part of the collected large intestine was frozen in a 1.5 ml tube, especially with liquid nitrogen.
- CDNA synthesis was performed in the same manner as in Example 1, and quantitative PCR was performed. The same applies to primer sequences and PCR cycles. The results are shown in FIG.
- GalNAc4S-6ST gene was enhanced, but it was confirmed that the knockdown was significant (p ⁇ 0.001, t test) by GalNAc4S-6ST siRNA treatment.
- the agent of the present invention is useful, for example, as a fibrotic change inhibitor in the large intestine.
- Example 8 Inhibitory effect of GalNAc4S-6ST siRNA on tissue fibrosis in mouse intestinal fibrosis model
- An intestinal fibrosis model was prepared in the same manner as in Example 6, and the mice were sacrificed on the seventh day.
- a frozen block and a tissue section were prepared from the collected large intestine in the same manner as in Example 3.
- a Masson-stained image of a colon tissue section is shown in FIG. Masson staining is an indicator for visualizing collagen fibers and evaluating changes in tissue fibrosis.
- the agent of the present invention is useful, for example, as a full-layer (mucosal lamina limbal, submucosa, muscle layer) collagen fiber deposition inhibitor.
- Example 9 Histological fibroblast infiltration inhibitory effect of GalNAc4S-6ST siRNA in mouse intestinal fibrosis model
- An intestinal fibrosis model was prepared in the same manner as in Example 6, and the mice were sacrificed on the seventh day.
- a frozen block and a tissue section were prepared from the collected large intestine in the same manner as in Example 3.
- the obtained section was fixed with acetone (manufactured by Wako) for 10 minutes, washed with a phosphate buffer, anti-ER-TR7 antibody (rat monoclonal antibody, 1 ⁇ g / ml, manufactured by BMA) was added as a primary antibody, The reaction was allowed to proceed for 1 hour at room temperature.
- anti-ER-TR7 antibody rat monoclonal antibody, 1 ⁇ g / ml, manufactured by BMA
- the agent of the present invention is useful, for example, as a fibroblast infiltration inhibitor or a fibroblast colonization inhibitor.
- Example 10 Inhibitory effect of GalNAc4S-6ST siRNA on histological macrophage infiltration in mouse intestinal fibrosis model
- An intestinal fibrosis model was prepared in the same manner as in Example 6, and the mice were sacrificed on the seventh day.
- a frozen block and a tissue section were prepared from the collected large intestine in the same manner as in Example 3.
- the obtained section was fixed with acetone (manufactured by Wako) for 10 minutes, washed with a phosphate buffer, and anti-F4 / 80 antibody (clone A3-1, rat monoclonal antibody, 2 ⁇ g / ml: CALTAG LABORATORIES, Inc.) as the primary antibody. And made to react at room temperature for 1 hour.
- the agent of the present invention is useful, for example, as an infiltration suppressor for macrophages or fibroblasts.
- Example 11 Histological macrophage infiltration suppression effect of GalNAcST siRNA in mouse intestinal fibril formation model
- GalNAc4S-6ST is an enzyme that transfers a sulfate group to the 6-position of N-acetylgalactosamine sulfated at the 4-position.
- the role of the family of GalNAc-4ST1 and GalNAc-4ST2 which are 4-position sulfotransferases was examined.
- An intestinal fibril formation model was prepared in the same manner as in Example 6. On day 7, the mice were sacrificed.
- GalNAc 4S-6ST siRNA not only GalNAc 4S-6ST siRNA but also GalNAc 4ST-1 and GalNAc 4ST-2 (Gene World) were mixed and 1 ⁇ g per mouse was added to 1% atenocollagen (Koken) as a medium. A mixture of 200 ⁇ L was intraperitoneally administered.
- This siRNA administration group is expressed as a GalNAc ST siRNA administration group.
- the control group is the same as in Example 6.
- the nucleotide sequences of GalNAc 4S-6ST siRNA, GalNAc 4ST-1 and GalNAc 4ST-2 used in this example are shown below. The arrangement is not necessarily limited to this example.
- [GalNAc4ST-1 siRNA cocktail sequence] (GenBank accession number NM_175140) (Gene world) 5'-ACCCCCAACTCGGAACGATGCGGCT-3 '(SEQ ID NO: 35) 5'-TGCATGTTCTCGTCCATCCTGCTG-3 '(SEQ ID NO: 36) 5'-CGCCACCGTGTACTGTACTGTGAAGT-3 '(SEQ ID NO: 37) 5'-AGGCT GCTCCAACTG GAAGAGGGTG-3 '(SEQ ID NO: 38) [GalNAc4ST-2 siRNA cocktail sequence] (GenBank accession number NM_199055) (Gene world) 5'-ATATAGTATCTAGGATATATGTAG-3 '(SEQ ID NO: 39) 5'-GAAGTACCAAAAGCTGGCTGCTCTA-3 '(SEQ ID NO: 40) 5'-TTCTATCACTTGGACTATTTGATGTT-3 '(SEQ ID NO: 41) 5'-TACACAACT
- the agent of the present invention is useful, for example, as an intestinal fibril formation inhibitor.
- Example 12 Effect of C6ST-1 siRNA on alveolar stroma in a mouse emphysema model
- a porcine pancreatic elastase (PPE) intratracheal administration model which is a basic mouse model of emphysema, was used.
- PPE pancreatic elastase
- hematoxylin and eosin staining was performed using a lung tissue sample of an emphysema model mouse, and a comparative study was conducted on the effect of C6ST-1 siRNA on emphysematous lesion suppression.
- P57 (4 units; Calbiochem-Novabiochem) is intratracheally administered to C57BL6 / J mice (female, 5-6 weeks old, manufactured by CLEA Japan). Lung tissue was collected for 3 weeks after administration.
- the same mice with no PPE administered were used.
- C6ST-1 siRNA was administered in the same manner as in Example 1.
- a 6 ⁇ m-thick section was prepared using a cryostat (manufactured by Microm).
- the obtained sections were fixed with 1% glutaraldehyde (manufactured by Nacalai Tesque) for 10 minutes, and further fixed with formol calcium solution for 10 minutes.
- stain with Lilymeyer's hematoxylin solution (Sigma Aldrich Japan) at room temperature for 5 minutes and use as a decolorizing solution (70% ethanol containing 0.5% HCL, all made using Nacalai Tesque reagent) And lightly washed. Thereafter, it was washed with water for 10 minutes.
- Example 13 Inhibitory effect of C6ST-1 siRNA on pulmonary stromal fibrosis in mouse emphysema model
- the expression of alveolar stromal fibrosis-related genes by C6ST-1 siRNA administration was examined by real-time quantitative PCR.
- a COPD model was created in the same manner as in Example 12. Part of the collected lung tissue was frozen in 1.5 ml tubes, especially with liquid nitrogen.
- CDNA synthesis was performed in the same manner as in Example 1, and quantitative PCR was performed. Primer type I and ⁇ -SMA primer sequences and PCR cycles are similar. The primer sequence of C6ST-1 is shown below.
- Example 14 Histological fibroblast infiltration suppression effect of C6ST-1 siRNA in mouse emphysema model
- An intestinal fibrosis model was prepared in the same manner as in Example 12.
- Frozen blocks and tissue sections were prepared from the collected lung tissues in the same manner as in Example 3. The obtained section was fixed with acetone (manufactured by Wako) for 10 minutes, washed with a phosphate buffer, anti-ER-TR7 antibody (rat monoclonal antibody, 1 ⁇ g / ml, manufactured by BMA) was added as a primary antibody, The reaction was allowed to proceed for 1 hour at room temperature.
- anti-ER-TR7 antibody rat monoclonal antibody, 1 ⁇ g / ml, manufactured by BMA
- the agent of the present invention is useful, for example, as an agent for suppressing fibroblast infiltration or colonization of alveolar tissue stroma.
- Example 15 Histological macrophage infiltration suppression effect of C6ST-1 siRNA in mouse emphysema model
- a COPD model was prepared in the same manner as in Example 12.
- Frozen blocks and tissue sections were prepared from the collected lung tissues in the same manner as in Example 3. The obtained section was fixed with acetone (manufactured by Wako) for 10 minutes, washed with a phosphate buffer, and anti-F4 / 80 antibody (clone A3-1, rat monoclonal antibody, 2 ⁇ g / ml: CALTAG LABORATORIES, Inc.) as the primary antibody. And made to react at room temperature for 1 hour.
- suppression of C6ST-1 gene expression suppresses infiltration of responsible cell groups that sustain and enhance fibrotic changes such as macrophages and fibroblasts, and comprehensively suppresses fibrous changes in tissues.
- the agent of the present invention is useful, for example, as an inhibitor of macrophage infiltration into alveolar stroma.
- Example 16 Respiratory function preservation effect of C6ST-1 siRNA in mouse pulmonary emphysema model
- static lung compliance status compliance: Cst
- Cst statistic compliance
- the effect on respiratory function was evaluated.
- Cst represents the ease of lung tissue growth, but Cst rises in emphysema, a disease involving tissue destruction in the alveolar region.
- An emphysema model mouse was prepared by the same procedure as that shown in Example 12, and treated with C6ST-1 siRNA.
- Cst was measured using PV roop mode of FlexiVent (manufactured by SCIREQ) respiratory function analyzer.
- the connection between the mouse and FlexiVent was made by making a midline incision after stopping spontaneous breathing of the mouse, inserting a special cannula into the trachea, and ligating the bronchi.
- the results shown in this example show that not only the alveolar destruction associated with the interstitial fibrosis change of lung tissue is suppressed, but also the actual clinical symptoms (respiratory state) are suppressed by suppressing the gene expression of C6ST-1. ) was suggested to have an effect of improving.
- the agent of the present invention is useful, for example, as an alveolar destruction inhibitor.
- Example 17 Tissue preservation effect of C6ST-1 siRNA in mouse pulmonary emphysema model
- lung volume increases as the emphysematous lesion progresses.
- the purpose of this example was to verify that the therapeutic effect of C6ST-1 siRNA extends not only to the destruction at the cellular level but also to the effect of maintaining and preserving organ morphology.
- the lung tissue used in this example is the right lung of the lung tissue used in Example 12. The lung tissue removed from the mouse was gently washed with a phosphate buffer, and then applied to a phosphate buffer filled in a glass container.
- the weight of the glass container filled with the phosphate buffer was measured in advance, and the weight increased after the lung tissue was added to the container was converted to the liquid volume to obtain the lung volume.
- the results of this example are shown in FIG.
- Example 18 Inhibition effect of pulmonary interstitial fibrosis by GalNAcST siRNA in mouse emphysema model It is shown in another example that sulfation at positions 4 and 6 is important.
- fibrosis-related gene expression in the alveolar stroma by administration of GalNAcST siRNA was examined by real-time quantitative PCR in the same manner as in Example 11.
- the sequence of siRNA is the same as in Example 11.
- a pulmonary emphysema model was prepared in the same manner as in Example 12. Part of the collected lung tissue was frozen in 1.5 ml tubes, especially with liquid nitrogen. CDNA synthesis was performed in the same manner as in Example 1, and quantitative PCR was performed.
- TGF- ⁇ The sequence of TGF- ⁇ is shown below.
- the results are shown in FIG.
- the expression enhancement of type I collagen, ⁇ -SMA, and TGF- ⁇ which are fibrosis indicators, was significantly suppressed (both p ⁇ 0.01, t test) by GalNAcST siRNA.
- the agent of the present invention is useful, for example, as a pulmonary interstitial fibrotic change inhibitor.
- Example 19 Effect of preserving respiratory function of GalNAcST siRNA in mouse emphysema model
- static lung compliance status compliance: Cst
- Cst statistic compliance
- An emphysema model mouse was prepared by the same procedure as that shown in Example 12, and treated with GAlNAcST siRNA. After stopping spontaneous breathing of these mice with an anesthetic, Cst was measured using PV roop mode of FlexiVent (manufactured by SCIREQ) respiratory function analyzer.
- the agent of the present invention is useful, for example, as an alveolar destruction inhibitor or a respiratory condition improving agent.
- Example 20 Tissue preservation effect of GalNAcST siRNA in mouse emphysema model
- the therapeutic effect of GalNAcST siRNA extends not only to the destruction at the cellular level but also to the effect of maintaining and preserving organ morphology. It was done for the purpose of proving that.
- the lung tissue used in this example is the right lung of the lung tissue used in Example 12.
- the lung tissue removed from the mouse was gently washed with a phosphate buffer, and then applied to a phosphate buffer filled in a glass container.
- the weight of the glass container filled with the phosphate buffer was measured in advance, and the weight increased after the lung tissue was added to the container was converted to the liquid volume to obtain the lung volume.
- the results of this example are shown in FIG.
- the agent of the present invention is useful, for example, as a lung shape maintenance agent or a lung shape preservative.
- a type 2 diabetes model was prepared by administration of Streptozotocin in day 2 postnatal day C57BL / 6JcL mice (female, manufactured by CLEA Japan), and C4ST-1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1) siRNA , C4ST-2 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2) siRNA, C4ST-3 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 3) siRNA treatment, body weight fluctuation, blood sugar level fluctuation, gene expression and pancreatic tissue The fibrotic changes in were studied.
- C4ST-1 siRNA cocktail sequence C4ST1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1) (GenBank accession number NM_021439) 5'-ACAAAGCCATGAAGCCGGCGCTGCTGGAAGTGATGAGGATGAACAGAATT-3 '(SEQ ID NO: 55) 5'-CAACCTGAAGACCCTTAACCAGTACA-3 '(SEQ ID NO: 56) 5'-GCATCCCAGAGATCAACCACCGCTTG-3 '(SEQ ID NO: 57) * [C4ST-2 siRNA cocktail sequence] C4ST2 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2) (GenBank accession number NM_021528) 5'-GCCAGGAGTGGGCCCAGCCCAGGGC -3 '(SEQ ID NO: 57)
- Example 21 Examination of anti-obesity effect in C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA treatment with Streptozocin-induced C57BL / 6JcL type 2 diabetes model mice Day 14 of pregnancy C57BL / 6JcL mice (manufactured by CLEA Japan) ) 2 days after birth C57BL / 6JcL female mice, each injected subcutaneously into Streptozocin 10 mg / mL (SIGMA) 20 ⁇ L / head, and CE-2 (made by Claire Japan) until 4 weeks of age ) Feed and sterilized water, and was bred for 4 weeks after being fed with a High Fat Diet diet (manufactured by Claire Japan) and sterilized water for 2 weeks.
- SIGMA Streptozocin 10 mg / mL
- CE-2 made by Claire Japan
- siRNA C4ST-1 Chodroitin DN-acetylgalactosamine-4-O-sulfotransferase 1
- C4ST-2 Chodroitin DN-acetylgalactosamine-4-O-sulfotransferase 2
- C4ST-3 Chodroitin DN-acetylgalactosamine-4-
- O-sulfotransferase 3 1 ⁇ g (GeneWorld) mixed with 1% Atelocollagen (manufactured by Koken) siRNA medium once / week 200 ⁇ L intraperitoneally administered (once / week) twice Treatment was performed (2 weeks).
- the C4ST-1 siRNA-treated group, C4ST-2 siRNA-treated group, and C4ST-3 siRNA-treated group showed a tendency to suppress weight gain after the 10th day of treatment.
- weight gain was significantly suppressed on the 18th day of treatment (both p ⁇ 0.05). This result indicates that obesity associated with type 2 diabetes can be suppressed by suppressing the expression of C4ST-1, C4ST-2, and C4ST-3.
- the agent of the present invention is useful, for example, as a weight gain inhibitor or an obesity inhibitor associated with type 2 diabetes.
- Example 22 Streptozocin-induced C57BL / 6JcL Type 2 diabetes model mouse study of insulin resistance in C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA treatment Insulin-tolerance test the day before dissection Human crystalline insulin (0.75 U / kg) was administered intraperitoneally, and blood glucose levels after 0, 15, and 60 minutes were measured using a Glutest Ace blood glucose meter (manufactured by Bombyx Pharmaceutical). Evaluation was performed. The fluctuation of blood glucose level after 0 minutes, 15 minutes, and 60 minutes after siRNA treatment is shown in FIG.
- FIG. 22 shows a significant decrease in blood glucose level after 0 minutes and 15 minutes after treatment in the C4ST-1 siRNA treatment group, C4ST-2 siRNA treatment group, and C4ST-3 siRNA treatment group as compared with the Control group.
- a significant decrease in blood glucose level was observed in each of the C4ST-1 siRNA treatment group, the C4ST-2 siRNA treatment group and the C4ST-3 siRNA treatment group.
- This result shows that suppression of C4ST-1, C4ST-2, and C4ST-3 expression can effectively improve insulin resistance, which is an essential dysfunction of type 2 diabetes.
- the present invention is useful, for example, as an insulin resistance improving agent for type 2 diabetes.
- Example 23 Examination of pancreatic tissue gene expression in Streptozocin-induced C57BL / 6JcL type 2 diabetes model mice treated with C4ST-1 siRNA, C4ST-2 siRNA, C4ST-3 siRNA Streptozocin- induced C57BL / 6JcL mice, isolated from females
- cDNA was prepared in the same manner as in Example 1, and PCR was performed with the following composition.
- PCR Buffer 2 [mu] L [Composition: 166 mM (NH 4) (manufactured by Sigma Aldrich Japan Co.) 2 SO 4, (manufactured by Invitrogen Corp.) 670 mM Tris pH8.8, 67 mM MgCl 2 ⁇ 6H 2 O ( manufactured by Sigma Aldrich Japan), 100 mM 2-mercaptoethanol] (WAKO)], 25 mM dNTP mix 0.8 ⁇ L (Invitrogen), DMSO 0.6 ⁇ L (Sigma Aldrich Japan), Primer Forward 0.2 ⁇ L (GeneWorld), Primer Reverse 0.2 ⁇ L (GeneWorld), Otsuka distilled water 15.7 ⁇ L (Otsuka Pharmaceutical), Taq polymerase 0.1 ⁇ L (Perkin Elmer), and cDNA 1 ⁇ L obtained above were mixed and 94 ° C.
- GAPDH which is a positive control by RT-PCR
- C4ST1 Chodroitin DN-acetylgalactosamine-4-O-sulfotransferase-1
- C4ST2 Chodroitin DN-acetylgalactosamine-4-O-sulfotransferase IV-2
- C4ST3 Choondroitin DN- C4ST1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1) siRNA
- C4ST2 Choondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2
- siRNA treatment group showed decreased expression, Atellocollagen medium C4ST1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 3) siRNA treatment group showed decreased expression, Atellocollagen medium C4ST1 (Chondroitin DN-acetylgalacto
- Example 24 Examination of pancreatic amyloid precursor protein accumulation in Streptozocin-induced C57BL / 6JcL type 2 diabetes model mice treated with C4ST-1 siRNA, C4ST-2 siRNA, C4ST-3 siRNA
- mice of type 2 diabetes model Using pancreas tissue samples, C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA were compared for amyloid precursor protein (APP) deposition inhibitory effects.
- APP amyloid precursor protein
- FIG. 24 shows tissue images of a normal mouse group, a control group, and a C4ST-2 siRNA treatment group. APP deposition was enhanced in the islet of type 2 diabetes model mice, but it was confirmed that it was clearly suppressed in the C4ST-2 siRNA-treated group compared to the Control group.
- the agent of the present invention is useful, for example, as an amyloid fibril deposition inhibitor.
- Example 25 Streptozocin-induced C57BL / 6JcL Type 2 diabetes model mouse treated with C4ST-1 siRNA, C4ST-2 siRNA, C4ST-3 siRNA histological fibroblast invasion inhibitory effect
- Pancreatic tissue was collected in Example 3 A frozen block and a tissue section were prepared in the same manner. The obtained section was fixed with acetone (manufactured by Wako) for 10 minutes, washed with a phosphate buffer, anti-ER-TR7 antibody (rat monoclonal antibody, 1 ⁇ g / ml: manufactured by BMA) was added as a primary antibody, The reaction was allowed to proceed for 1 hour at room temperature.
- acetone manufactured by Wako
- anti-ER-TR7 antibody rat monoclonal antibody, 1 ⁇ g / ml: manufactured by BMA
- the agent of the present invention is useful as, for example, an inhibitor of fibroblast infiltration into an islet.
- Example 26 Streptozocin-induced C57BL / 6JcL type 2 diabetes model mouse treated with C4ST-1 siRNA, C4ST-2 siRNA, C4ST-3 siRNA histological macrophage infiltration inhibitory effect
- the collected pancreatic tissue was the same as in Example 3.
- a frozen block and a tissue section were prepared by the method. The obtained sections were fixed with acetone (manufactured by Wako) for 10 minutes and then washed with a phosphate buffer.
- anti-F4 / 80 antibody clone A3-1, rat monoclonal antibody, 2 ⁇ g / ml: CALTAG LABORATORIES
- C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA treatment groups infiltration of macrophages into the alveolar stroma was clearly suppressed as compared to the control group (FIG. 26). Therefore, it was found that suppression of C6ST-1 gene expression suppresses infiltration of responsible cell groups that sustain and enhance fibrotic changes such as macrophages and fibroblasts, and comprehensively suppresses fibrous changes in tissues. .
- the agent of the present invention is useful, for example, as an inhibitor of macrophage infiltration into alveolar stroma.
- Example 27 Examination of insulin resistance in Streptozocin-induced C57BL / 6JcL type 2 diabetes model mouse treatment with GalNAcST siRNA Another example is shown to show that sulfation at positions 4 and 6 is important.
- improvement of insulin resistance by administration of GalNAcST siRNA was examined in the same manner as in Example 11 and Example 18.
- a type 2 diabetes model was prepared in the same manner as in Example 21, and insulin resistance was examined in the same manner as in Example 22. The results are shown in FIG. The administration of GalNAcST siRNA showed a good hypoglycemic effect after insulin loading.
- the agent of the present invention is useful, for example, as a hypoglycemic agent or an agent for improving pancreatic tissue insulin resistance.
- kidney tissue Fibrous changes in kidney tissue are considered the endpoint of any kidney disease.
- Interstitial kidney disease tubulointerstitial disease
- 2) vascular renal disease can be mentioned.
- fibrosis associated with arteriosclerosis and metabolic syndrome is also included.
- primary glomerular diseases also cause interstitial fibrosis as a result of proteinuria, etc., leading to chronic renal failure.
- Primary glomerular disease and 4) Renal fibrosis in secondary glomerular disease. There is a change.
- IgA nephropathy minimal change nephrotic syndrome, membranous nephropathy, membranous proliferative nephritis, focal segmental glomerulosclerosis (FGFS), 4) diabetic nephropathy, SLE Lupus nephritis associated with (systemic lupus erythematosus), nephropathy associated with rheumatoid arthritis, amyloid nephropathy, nephropathy associated with hepatitis B and hepatitis C, and the like. Finally, 5) interstitial kidney disease associated with urinary tract obstruction, including urinary calculi, tumors, neurogenic bladder and the like.
- CKD chronic kidney disease
- ESRD end-stage renal disease
- CKD is reported to have 500 million patients worldwide, and the number of patients is expected to increase further with changes in lifestyle, and even cardiovascular disorders before artificial dialysis
- Example 28 Examination of anti-fibrogenic effect of C4ST-1 siRNA in mouse diabetic nephropathy model A type 2 diabetes model was prepared in the same manner as in Example 28. Day 14 of Pregnancy C57BL / 6JcL mice (CLEA Japan) were bred and born, and streptozotocin (STZ: Sigma) was administered to 2-day-old C57BL / 6JcL mice after birth to create an STZ-induced diabetes model. Mice were injected subcutaneously with 20 ⁇ l of STZ (10 mg / ml) for a total of 3 times over 2 days for a total of 60 ⁇ l.
- STZ streptozotocin
- C4ST-1 Chodroitin DN-acetylgalactosamine-4-O-sulfotransferase 1 siRNA 1 ⁇ g (GeneWorld) mixed with 0.1% Atelocollagen (Koken) siRNA medium once /
- the treatment was performed twice (2 weeks) by intraperitoneal administration (1 shot / week) of 200 ⁇ L per week. The animal was dissected on the 14th day of the experiment, the kidney was removed, and a sample for immunostaining was obtained.
- kidney tissue sample section was fixed with acetone (manufactured by Wako) for 10 minutes, washed with phosphate buffer, and anti-ER-TR7 antibody (rat monoclonal antibody, 1 ⁇ g / ml, manufactured by BMA) was used as the primary antibody.
- anti-ER-TR7 antibody rat monoclonal antibody, 1 ⁇ g / ml, manufactured by BMA
- the mixture was added and allowed to react at room temperature for 1 hour.
- a secondary antibody reaction was performed using peroxidase-labeled anti-rat IgG (1: 200 dilution), and then DAB substrate (manufactured by Nichirei Bioscience) was added to cause color development.
- Example 29 Examination of macrophage infiltration effect of C4ST-1 siRNA in mouse diabetic nephropathy model
- the obtained kidney tissue sample section was fixed with acetone (manufactured by Wako) for 10 minutes, washed with phosphate buffer, and primary Anti-ER-TR7 antibody (rat monoclonal antibody, 1 ⁇ g / ml, manufactured by BMA) was added as an antibody and reacted at room temperature for 1 hour.
- primary Anti-ER-TR7 antibody rat monoclonal antibody, 1 ⁇ g / ml, manufactured by BMA
- a secondary antibody reaction was performed using peroxidase-labeled anti-rat IgG (1: 200 dilution), and then DAB substrate (manufactured by Nichirei Bioscience) was added to cause color development.
- Example 30 Examination of fibroblast activation in tissue of C4ST-1 siRNA in mouse diabetic nephropathy model A kidney tissue sample section was fixed with acetone (Wako) for 10 minutes and then washed with phosphate buffer.
- Anti-human smooth muscle fiber actin antibody ( ⁇ SMA: mouse monoclonal antibody, 1: 100, manufactured by DACO) was added as a primary antibody and allowed to react at room temperature for 1 hour. Subsequently, a secondary antibody reaction was performed using a histofine mouse stain kit (manufactured by Nichirei Bioscience), and then a DAB substrate (manufactured by Nichirei Bioscience) was added to cause color development.
- Example 31 Examination of fibrotic changes in tissues of GalNAc4S-6ST siRNA in mouse diabetic nephropathy model
- a diabetic nephropathy model was prepared in the same manner as in Example 28, and the effect of GalNAc4S-6ST siRNA was examined. . In this example, the progress was observed over a longer period.
- the sequence of GalNAc4S-6ST siRNA was the same as in Example 1, and was administered intraperitoneally once each at 8 and 9 weeks of age. Further, as a comparison object, Valsartan, an angiotensin II receptor inhibitor (ARB), was orally administered in a volume of 30 mg / kg on the same schedule. Kidney tissues were collected at 10 weeks of age for immunohistological examination and gene expression examination.
- ARB angiotensin II receptor inhibitor
- GalNAc4S-6ST siRNA In the diabetic nephropathy model, expression of GalNAc4S-6ST in kidney tissue was enhanced, but GalNAc4S-6ST gene expression in kidney tissue was significantly suppressed by administration of GalNAc4S-6ST siRNA. Furthermore, the expression enhancement of ⁇ SMA and TGF ⁇ , which are fibrosis markers, was significantly suppressed. Comparing ARB and therapeutic effect, both groups showed inhibitory effect on TGF ⁇ , and there was no significant difference, but on ⁇ SMA, the GalNAc4S-6ST siRNA administration group showed a significant inhibitory effect compared to the ARB administration group. It was. This result shows that by suppressing the gene expression of GalNAc4S-6ST, a marker indicating a fibrous change in kidney tissue could be suppressed. Furthermore, it was also shown that the effect of GalNAc4S-6ST siRNA is remarkably superior with respect to activation of fibroblasts (enhancement of ⁇ SMA).
- Example 32 Examination of GalNAc4S-6ST siRNA in fibroblast infiltration in mouse diabetic nephropathy model The degree of infiltration of fibroblasts into renal tissue was examined by immunohistological examination in the same manner as in Example 28. Quantitative evaluation was performed. The results are shown in FIG. GalNAc4S-6ST siRNA administration significantly suppressed fibroblast infiltration into the renal interstitium. In the quantitative search of the entire stroma, there was no significant difference from ARB. showed that.
- the agent of the present invention is useful, for example, as an inhibitor of fibroblast infiltration into the renal interstitium.
- Example 33 Examination of GalNAc4S-6ST siRNA in macrophages in a mouse diabetic nephropathy model The degree of infiltration of fibroblasts into renal tissue was determined quantitatively by adding immunohistological examination in the same manner as in Example 29. Went. The results are shown in FIG. GalNAc4S-6ST siRNA administration significantly suppressed macrophage infiltration into the renal interstitium. A quantitative search across the stroma showed no significant difference from ARB, but in studies limited to macrophages infiltrating the paraglomerular region, the GalNAc4S-6ST siRNA administration group showed a clear inhibitory effect as shown in the figure. It was.
- the drug of the present invention is useful, for example, as an inhibitor of macrophage infiltration into the renal interstitium.
- Example 34 Examination of glNAc4S-6ST siRNA in glomerular basement membrane thickening in a mouse diabetic nephropathy model
- type IV collagen was immunostained.
- Anti-mouse type IV collagen rabbit antiserum manufactured by LSL
- DAB substrate manufactured by Nichirei Bioscience was added to cause color development.
- the agent of the present invention is useful, for example, as a glomerular basement membrane thickening inhibitor.
- Example 35 Examination of GalNAc4S-6ST siRNA in angiotensin pathway in mouse diabetic nephropathy model Angiotensin II has been reported to be involved in fibrosis of diabetic nephropathy.
- the angiotensin pathway was examined by quantitative PCR of kidney tissue.
- the expression of angiotensinogen and angiotensin converting enzyme (ACE) was enhanced (FIG. 35), and this was considered to be a factor to enhance angiotensin II in the local kidney.
- ACE angiotensinogen and ACE inhibitory effects were observed (FIG. 35).
- the agent of the present invention is useful, for example, as an angiotensinogen expression inhibitor or an angiotensin converting enzyme inhibitor.
- Example 36 In the process leading from a mouse diabetic nephropathy model in GalNAc4S-6ST siRNA considered diabetic nephropathy in serum creatinine concentration in ESRD, since the serum creatinine concentration with the decrease in renal function increases, the most clinically It is a commonly used renal function marker. However, when serum creatinine level rises, it has already been revealed that 50% of functional nephron has been reduced, and how to protect kidney function before creatinine level is clinically significant. It is a problem. In this model, an increase in serum creatinine concentration was finally observed at 18 weeks of age when histological fibrosis had already progressed (FIG. 36).
- the drug of the present invention is useful, for example, as a renal function lowering inhibitor or a renal protective agent.
- Example 37 Examination of anti-fibrotic effect of GalNAcST siRNA in mouse diabetic nephropathy model It is shown in another example that 4th and 6th sulfation is important. In the same manner as in Example 11, the change in renal interstitial fibrosis caused by administration of GalNAcST siRNA was examined. The administration schedule of GalNAcST siRNA is the same as in Example 28. Quantitative PCR using kidney tissue was performed in the same manner as in Example 31. In this example, ⁇ -actin is used as an internal control, and its sequence is shown below.
- Example 38 Examination of anti-fibrogenesis and nephroprotective action of GalNAcST siRNA in mouse diabetic nephropathy model
- ⁇ SMA, TGF ⁇ which are markers of fibrosis in renal tissue
- CTGF CTGF
- ACE fibrosis of the renal tissue
- Example 39 Examination of gene expression of GalNAc4S-6ST siRNA in mouse drug-induced interstitial nephritis model
- adriamycin (15 mg / kg; manufactured by Kyowa Hakko) is intraperitoneally administered to C57BL6 / J mice (male, 8 weeks old, manufactured by CLEA Japan). After the administration, the animals were raised for 1 week, and kidney tissues were collected. The control group used was a similar mouse that was purchased and raised at the same time without adriamycin.
- GalNac 4S-6ST siRNA was prepared in the same manner as in Example 1, with 1 ⁇ g of GalNac 4S-6ST siRNA (Hokkaido System Science Co., Ltd.) and 1% Atheno as a vehicle per mouse 24 hours before administration of adriamycin. A mixture of 200 ⁇ L of collagen (manufactured by Koken) was administered intraperitoneally. In the typical model of drug-induced interstitial nephritis in this example, expression of GalNac 4S-6ST was enhanced in kidney tissue (FIG. 39), and its expression was significantly suppressed by administration of GalNac 4S-6ST siRNA. .
- Example 40 Examination of gene expression of GalNAc4S-6ST siRNA in mouse drug-induced interstitial nephritis model
- immunostaining of type I collagen was performed in the same manner as in Example 34.
- Anti-rat type I collagen rabbit antiserum manufactured by LSL
- DAB substrate manufactured by Nichirei Bioscience was added to cause color development.
- the agent of the present invention is useful, for example, as a renal tissue fibrosis change inhibitor.
- Example 41 Examination of anti-fibrotic effect of C6ST siRNA in renal fibrosis model mice by unilateral ureteral ligation (UUO)
- UUO unilateral ureteral ligation
- renal fibrosis model mice As a typical example of renal fibrosis model mice, renal fibrosis model mice by unilateral ureteral ligation (UUO) were used, and the inhibitory effect of C6ST expression by C6ST siRNA administration was confirmed by PCR. U57O was applied to C57BL / 6JcL mice ( ⁇ , 8 weeks old, manufactured by CLEA Japan, Inc.) to create a renal fibrosis model.
- UUO unilateral ureteral ligation
- mice were treated with C6ST-1 siRNA, C6ST-2 siRNA mixture (1 ⁇ g / mouse, Geneworld), or PBS with 0.1% Atelocollagen (Koken) as siRNA medium. After mixing, 200 ⁇ l was injected intraperitoneally. The group of mice treated with this treatment was named C6ST siRNA group and control group.
- C6ST siRNA group and control group On the 8th day of the experiment, the UUO-treated kidney was removed, and a sample for immunostaining and a sample for gene expression analysis were obtained. Quantitative PCR was performed in the same manner as in Example 1.
- the C6ST-1 siRNA and C6ST-2 Primer (Forward, Reverse) (GeneWorld) used here are shown below.
- C6ST-2 was expressed as G # 10). Decreased expression was observed in the C6ST siRNA treatment group, and knockdown of the C6ST-2 gene was confirmed by administration of Atellocollagen vehicle C6ST siRNA (FIG. 41). In addition, the enhanced expression of fibrogenic markers such as TGF ⁇ , ⁇ SMA, type I collagen, and CTGF was also significantly suppressed by C6ST siRNA administration.
- Example 42 Examination of C6ST siRNA fibroblast infiltration in renal fibrosis model mice by unilateral ureteral ligation (UUO)
- UUO unilateral ureteral ligation
- the excised tissue was embedded with a freezing embedding agent OCT compound (manufactured by Sakura).
- a frozen block was prepared with liquid nitrogen, and a 6 ⁇ m-thick section was prepared using a cryostat (manufactured by Microedge).
- the obtained sections were immunostained using an anti-ER-TR7 antibody in the same manner as in Example 28.
- the sample was observed under an optical microscope (manufactured by Leica), and the antibody binding visualized with a brown signal was observed for quantification.
- the agent of the present invention is useful, for example, as a fibroblast accumulation inhibitor in the renal stroma.
- Example 43 Examination of C6ST siRNA macrophage infiltration in renal fibrosis model mice by unilateral ureteral ligation (UUO) In the same manner as in Example 42, immunostaining and quantification were performed with anti-F4 / 80 antibody. As a result, in the UUO-treated kidney, macrophage accumulation in the renal stroma was significantly suppressed in the C6ST siRNA-treated group compared to the control group (FIG. 43).
- the drug of the present invention is useful, for example, as a macrophage accumulation inhibitor in the renal stroma.
- Example 44 Examination of collagen accumulation of C6ST siRNA in renal fibrosis model mice by unilateral ureteral ligation (UUO) As in Example 42, immunostaining was performed with an anti-type IV collagen antibody. Quantification was performed. As a result, in the UUO-treated kidney, the fibrous thickening of the glomerular basement membrane was significantly suppressed in the C6ST siRNA-treated group compared to the control group (FIG. 44). From these results, it was confirmed that by suppressing the gene expression of C6ST-2, infiltration of inflammatory cells and fibrosis could be suppressed.
- the agent of the present invention is useful, for example, as an inflammatory cell infiltration inhibitor.
- Example 45 Examination of fibroblast activation of C6ST siRNA in renal fibrosis model mice by unilateral ureteral ligation (UUO) Immunostaining was performed with anti- ⁇ SMA antibody in the same manner as in Example 30.
- UUO unilateral ureteral ligation
- the C6ST siRNA-treated group showed a clear decrease in ⁇ SMA-positive cells in the stroma, particularly in the paraglomerular region, compared to the control group (FIG. 45).
- the agent of the present invention is useful, for example, as a fibroblast activation inhibitor in the renal stroma.
- Example 46 Immunostaining was performed using an anti-human ACE rabbit antibody (manufactured by Santa Cruz) in the same manner as in the examination of ACE expression of C6ST siRNA in renal fibrosis model mice by unilateral ureteral ligation (UUO) .
- UUO unilateral ureteral ligation
- the C6ST siRNA-treated group showed a clear decrease in ACE-positive cells in the stroma, particularly in the paraglomerular region, compared to the control group (FIG. 46).
- This result shows that by suppressing the gene expression of C6ST-2, activation of fibroblasts accumulated in the renal interstitium is suppressed, and accordingly, the expression of ACE is also suppressed.
- ACE suppression has a blood pressure lowering effect
- this result also has a blood pressure lowering or arteriosclerosis suppressing effect.
- the agent of the present invention is useful, for example, as an arteriosclerosis inhibitor.
- Eye tissue In the eye tissue, as in other organs, fibrotic changes occur due to invasion due to various causes, resulting in a reduction or loss of visual acuity.
- Major diseases include diabetic retinopathy, retinal vein occlusion, retinopathy of prematurity, age-related macular degeneration, retinitis pigmentosa, etc., but fibrosis associated with corneal inflammation, fibrosis associated with glaucoma Also included are fibrosis associated with cataracts (Review: Fiedlander M. J Clin Invest 117: 576-586, 2007, Harada T et al. Genes and Dev. 21: 367-378, 2007). Histopathologically, the damage and loss of photoreceptor cells associated with fibrosis is a major factor in reducing visual acuity. Therefore, suppressing fibrosis in ocular tissue is a new treatment to prevent visual loss in all eye diseases. Expected as a strategy. In this example, examination was made focusing on retinal fibrosis and associated loss of photoreceptor cells in a model of diabetic retinopathy.
- Example 47 Examination of GalNAc4S-6ST (G # 1) siRNA collagen accumulation in a mouse diabetic retinopathy model : Day 14 of pregnancy C57BL6J / JcL mice (CLEA Japan) were bred and given birth, and two days after birth C57BL6J / JcL mice female (CLEA Japan), each with Streptozocin 10 mg / mL (SIGMA) 20 ⁇ L / head is subcutaneously injected and fed with CE-2 (CLEA Japan) feed and sterilized water until 4 weeks of age. High Fat Diet diet (CLEA Japan) and sterilized water are supplied after 4 weeks of age. Raised for 2 weeks.
- GalNac4S-6ST G # 1 siRNA (Hokkaido System Science) per mouse and 1% atenocollagen (high) A mixture of 200 ⁇ L) was administered intraperitoneally.
- G # 1 siRNA Hokkaido System Science
- the eyes of both groups of mice were removed and subjected to immunohistological studies. Frozen blocks and sections were prepared with the removed eyes.
- the section was fixed with acetone (Sigma Aldrich Japan) for 10 minutes, washed with a phosphate buffer, and then added with a rabbit-derived anti-type IV collagen antiserum (1: 2000 dilution; LSL) as a primary antibody. The reaction was allowed to proceed for 1 hour at room temperature. Subsequently, peroxidase-labeled anti-rabbit IgG (1:25 dilution; manufactured by Cappel), which is a secondary antibody, was added and reacted at room temperature for 30 minutes. went. This specimen was observed using an optical microscope (manufactured by Leica). The obtained retinal tissue image is shown in FIG.
- GalNac4S-6ST compared to the increased deposition of type IV collagen from the ganglion cell layer (GCL), which is important for visual acuity, to the inner nuclear layer (INL).
- GCL ganglion cell layer
- INL inner nuclear layer
- Example 48 Examination of GalNac4S-6ST (G # 1) siRNA in chondroitin sulfate sodium proteoglycan accumulation in a mouse diabetic retinopathy model : The immunohistological examination of the retina was performed in the same manner as in Example 47.
- An anti-chondroitin sulfate proteoglycan (CSPG) antibody (clone CS56, mouse monoclonal antibody, 1: 100; manufactured by Seikagaku Corporation) was added as a primary antibody and allowed to react at room temperature for 1 hour. Subsequently, a secondary antibody reaction was performed using a histofine mouse stain kit (manufactured by Nichirei; used for mouse monoclonal antibodies). As shown in FIG.
- Example 49 Examination on glial cell accumulation of GalNac4S-6ST (G # 1) siRNA in a mouse diabetic retinopathy model : Regeneration of the optic nerve has been reported as part of the defense mechanism during retinal damage, but it has been reported that optic nerve progenitor cells involved in such post-injury regeneration are glial cells (Fischer AJ et al. Nature neuroscience 4: 247, 2001, Ooto S et al. PNAS 101: 13645, 2004).
- anti-GFAP goat antibody manufactured by Santa Cruz
- immunostaining was performed in the same manner as in Example 47. The results are shown in FIG.
- the agent of the present invention is useful, for example, as an optic nerve regeneration agent.
- Example 50 Examination of GalNac4S-6ST (G # 1) siRNA in ganglion cells in a mouse diabetic retinopathy model : Using the sample prepared in Example 47, the number of ganglion cells was quantified. As a result, ganglion cell decrease in GCL was also observed in diabetic retinopathy model, but it became clear that such loss was significantly recovered by administration of GalNac4S-6ST (G # 1) siRNA ( FIG. 50).
- Example 51 Examination of gene expression of GalNac4S-6ST (G # 1) siRNA in a mouse diabetic retinopathy model : Eye tissue RNA was extracted in the same manner as in Example 1, and quantitative PCR was performed. We examined the ability of optic nerve regeneration by analyzing changes in the expression of glutamate synthetase (GS), a marker of Mueller cells. PCR primer sequences are listed below. [Quantitative PCR Primer sequence] * mouse GS (Takara Bio) forward: 5'-CTGTGAGCCCAAGTGTGTGGA -3 '(SEQ ID NO: 89) reverse: 5'-GTCTCGAAACATGGCAACAGGA -3 '(SEQ ID NO: 90) The results are shown in FIG.
- GalNac4S-6ST (G # 1) siRNA administration significantly suppressed the expression enhancement of GalNAc4S-6ST in ocular tissues. Accordingly, in the GalNac4S-6ST (G # 1) siRNA administration group, the expression of GS in the ocular tissue was significantly increased. This result indicates that administration of GalNac4S-6ST (G # 1) siRNA leads to the regeneration of Mueller cells, that is, the recovery of the optic nerve. According to the present example, it was found that by suppressing the gene expression of GalNAc4S-6ST, the fibrous change of the retinal tissue can be suppressed, resulting in avoidance of photoreceptor loss due to regeneration of ganglion cells.
- the agent of the present invention is useful, for example, as a Mueller cell regenerative agent or a ganglion cell regenerative agent.
- liver fibrotic changes in the liver are the progression or terminal picture of any liver disease. Not only liver fibrosis derived from viral hepatitis (hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, hepatitis G virus), but also alcoholic liver injury, non-alcohol Fatty liver disorder (NAFLD and NASH), metabolic liver disorder, drug-induced liver disorder, idiopathic portal hypertension, Budd-Chiari syndrome, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing bile duct Wide range of diseases, including biliary tract obstruction due to inflammation, biliary tract diseases (including biliary atresia and biliary dilatation), pancreatic diseases such as tumors, graft-versus-host reaction, chronic rejection (Bataller R et al. J Clin Invest 115: 209, 2005. I
- Example 52 Examination of effect on gene expression of GalNAcST siRNA in mouse fatty liver injury model Day 14 of pregnancy C57BL / 6JcL mice (manufactured by CLEA Japan, Inc.) are bred and given birth, and 2 days after birth to C57BL / 6JcL mice Streptozotocin (STZ: manufactured by Sigma) was administered. Mice were injected subcutaneously with 20 ⁇ l of STZ (10 mg / ml) for a total of 3 times over 2 days for a total of 60 ⁇ l. This was reared on a normal diet with parent mice until 4 weeks of age, weaned at 4 weeks of age and then fed with a high fat diet (manufactured by Claire Japan) for 2 weeks.
- STZ manufactured by Sigma
- GalNAcST siRNA described in Example 11 was treated twice (2 weeks) by intraperitoneal administration (1 shot / 1 week) once / week for 200 ⁇ L.
- the animal was dissected on the 14th day of the experiment, and the liver was removed to obtain gene expression analysis and immunostaining samples.
- quantitative PCR of liver tissue was performed in the same manner as in Example 1. The results are shown in FIG.
- the expression of GalNAc4S-6ST was enhanced in the liver tissue of this model, but was significantly suppressed by administration of GalNAcST siRNA.
- Example 53 Examination of anti-fibrogenic effect of GalNAcST siRNA in mouse fatty liver injury model In the same manner as in Example 52, the expression of fibrogenic marker in liver tissue was examined. In this model, the expression of type I collagen and ⁇ SMA were significantly increased (FIG. 53). Thus, it was shown that fibrotic changes in liver tissue are enhanced. In contrast, the administration of GalNAcST siRNA significantly suppressed the enhancement of these fibrogenesis markers.
- Example 54 Examination of fibroblast infiltration of GalNAcST siRNA in mouse fatty liver injury model Next, immunostaining of liver tissue was performed in the same manner as in Example 3. Rat anti-mouse fibroblast antibody (clone ER-TR7; 1: 500 dilution; manufactured by BMA) was added as a primary antibody and allowed to react at room temperature for 1 hour. Subsequently, peroxidase-labeled anti-rat IgG (1: 200 dilution; manufactured by Biosource) was added as a secondary antibody and further reacted at room temperature for 30 minutes, and then DAB substrate (manufactured by Nichirei) was added.
- Rat anti-mouse fibroblast antibody (clone ER-TR7; 1: 500 dilution; manufactured by BMA) was added as a primary antibody and allowed to react at room temperature for 1 hour. Subsequently, peroxidase-labeled anti-rat IgG (1: 200 dilution; manufactured by Biosource) was
- the sample was observed under an optical microscope (manufactured by Leica), and antibody binding visualized with a brown signal was confirmed.
- An example of the obtained immunostained image of liver tissue is shown in FIG.
- fibroblast accumulation was clear, and a histological image forming bridging was observed. Compared to this, fibroblast accumulation was hardly observed in the GalNAcST siRNA administration group.
- Example 55 Examination of fibrosis score of GalNAcST siRNA in mouse fatty liver injury model Based on the immunohistological staining performed in Example 54, the degree of liver fibrosis in each sample has been reported (Dai K, et al. World J Gactroenterol. 31: 4822-4826, 2005, Hillebrandt S, et al. Nature Genetics 37: 835-843, 2005).
- the evaluation criteria for the degree of fibril formation are as follows.
- Example 56 Examination of macrophage infiltration of GalNAcST siRNA in mouse fatty liver injury model Next, immunostaining of liver tissue was performed in the same manner as in Example 54. Rat anti-mouse F4 / 80 antibody (clone A3-1; 2 ⁇ g / ml; manufactured by CALTAG LABORATORIES) was added as a primary antibody and allowed to react at room temperature for 1 hour. Subsequently, peroxidase-labeled anti-rat IgG (1: 200 dilution; manufactured by Biosource) was added as a secondary antibody and further reacted at room temperature for 30 minutes, and then DAB substrate (manufactured by Nichirei) was added.
- Rat anti-mouse F4 / 80 antibody clone A3-1; 2 ⁇ g / ml; manufactured by CALTAG LABORATORIES
- peroxidase-labeled anti-rat IgG (1: 200 dilution; manufactured by Biosource) was added as a secondary antibody
- the sample was observed under an optical microscope (manufactured by Leica), and antibody binding visualized with a brown signal was confirmed.
- An example of the obtained immunostained image of liver tissue is shown in FIG.
- the control group the accumulation of macrophages was clear, and a histological image forming an inflammatory collecting layer was observed. In comparison with this, no excessive accumulation of macrophages was observed in the GalNAcST siRNA administration group.
- Example 57 Examination of liver lipid metabolism of GalNAcST siRNA in mouse fatty liver injury model Expression of liver lipid metabolism-related genes was examined in the same manner as in Example 52. Expression of ChREBP (carbohydrate response element-binding protein) and ACC2 (Acetyl-CoA carxylase-2) was enhanced, but was significantly suppressed in the GalNAcST siRNA administration group (FIG. 57). This result shows that by suppressing the gene expression of GANAc4ST-1, GalNAc4ST-2 and GalNAc4S-6ST, fibrotic changes in liver tissue can be suppressed, and as a result, glycolipid metabolism can be improved.
- the agent of the present invention is useful, for example, as a glycolipid metabolism improving agent.
- Example 58 Examination of fibroblast accumulation of C4ST-1 siRNA, C4ST-2 siRNA, C4ST-3 siRNA in mouse fatty liver injury model
- a fatty liver injury model was prepared in the same manner as in Example 52, and In this administration protocol, C4ST-1 siRNA, C4ST-2 siRNA and C4ST-3 siRNA described in Example 21 were administered, and liver tissue was collected. Next, liver tissue was immunostained in the same manner as in Example 3.
- Rat anti-mouse fibroblast antibody (clone ER-TR7; 1: 500 dilution; manufactured by BMA) was added as a primary antibody and allowed to react at room temperature for 1 hour.
- peroxidase-labeled anti-rat IgG (1: 200 dilution; manufactured by Biosource) was added as a secondary antibody and further reacted at room temperature for 30 minutes, and then DAB substrate (manufactured by Nichirei) was added.
- the sample was observed under an optical microscope (manufactured by Leica), and antibody binding visualized with a brown signal was confirmed.
- An example of the obtained immunostained image of the liver tissue is shown in FIG. In the control group, fibroblast accumulation was clear, and a histological image forming bridging was observed. In comparison, no accumulation of fibroblasts was observed in all groups in the C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA administration groups.
- Example 59 Examination of fiber formation scores of C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA in a mouse fatty liver injury model Based on the immunohistological staining performed in Example 58, liver fibers of each sample The degree of formation was evaluated by fibrosis frequency. The results are shown in FIG. Each bar shows the mean value ⁇ standard deviation of the fibrosis frequency in each group. Compared with the control group, fibrosis was statistically significantly reduced in all groups administered with C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA (p ⁇ 0.001, t test). From the above, it was shown that administration of C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA exerts a clinically excellent liver fibrogenesis inhibitory effect.
- Example 60 Examination of C4ST-1 siRNA, C4ST-2 siRNA and C4ST-3 siRNA in hepatic cell injury in a mouse fatty liver injury model Blood was collected at the time of sacrifice of mice according to the protocol of Example 58, and serum ALT (alanine transferase) ) The value was commissioned to SRL and measured. The results are shown in FIG. Serum ALT level is the most commonly used clinical indicator of hepatocyte destruction. The control group showed an increase in serum ALT level, whereas the C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA administration groups showed a 50% or lower decrease in average (FIG. 60).
- the agent of the present invention is useful, for example, as a hepatocyte disorder reducing agent.
- Example 61 Examination of anti-fibrogenic effect of C6ST siRNA in mouse liver fibrosis model
- an experiment was conducted using a mouse carbon tetrachloride-induced cirrhosis model most commonly used experimentally.
- the mouse model was prepared. Carbon tetrachloride (25 ⁇ L / 100 g body weight; Sigma-Aldrich) was applied twice a week to C57BL6 / J mice (female, 5-6 weeks old, manufactured by CLEA Japan). Injected intraperitoneally for 4 weeks each (8 times) to induce liver fibrosis. Further, carbon tetrachloride was administered twice a week for 2 weeks (12 times in total) to induce cirrhosis.
- livers were collected (cirrhosis liver).
- livers were collected from normal C57BL6 / J mice (female, manufactured by CLEA Japan, Inc.) that had not been administered carbon tetrachloride (normal liver).
- the same C6ST siRNA as in Example 41 and its control were administered intraperitoneally for a total of 4 times in the same manner as the additional administration of carbon tetrachloride (4 times in total from the 9th to the 12th time). After completion of the additional administration, mice in both groups were sacrificed, liver tissue sections were prepared, and immunohistological examination was performed.
- Rat anti-mouse fibroblast antibody (clone ER-TR7; 1: 500 dilution; manufactured by BMA) was added as a primary antibody and allowed to react at room temperature for 1 hour. Subsequently, peroxidase-labeled anti-rat IgG (1: 200 dilution; manufactured by Biosource) was added as a secondary antibody and further reacted at room temperature for 30 minutes, and then DAB substrate (manufactured by Nichirei) was added. The sample was observed under an optical microscope (manufactured by Leica), and antibody binding visualized with a brown signal was confirmed. An example of the obtained immunostained image of liver tissue is shown in FIG. In the control group, fibroblast accumulation was clear, and a histological image forming bridging was observed. In comparison, no accumulation of fibroblasts was observed in all groups in the C6ST siRNA administration group.
- Example 62 Examination of C6ST siRNA Fibrosis Score in Mouse Liver Fibrosis Model Based on the immunohistological staining performed in Example 61, the degree of liver fibrosis in each sample was evaluated by the fibrosis frequency. The results are shown in FIG. Each bar shows the mean value ⁇ standard deviation of the fibrosis frequency in each group. Compared with the control group, the C6ST siRNA-administered group (p ⁇ 0.05, t-test) all showed a statistically significant reduction in fibril formation. From the above, it was shown that administration of C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA exerts a clinically excellent liver fibrogenesis inhibitory effect.
- Example 63 Examination of anti-fibrogenic effect of C6ST siRNA in mouse liver fibrosis model RNA was extracted from liver tissue in the same manner as in Example 1, and quantitative PCR was performed. The results are shown in FIG. In the control group, expression of ⁇ SMA, type I collagen, CTGF, and TGF ⁇ , which are fibrosis markers, was enhanced, but was significantly suppressed in the C6ST siRNA-administered group. This result shows that suppression of C6ST-1 and C6ST-2 gene expression can suppress fibrotic changes in liver tissue. (Cranial nerve tissue) In this example, a basic MPTP-induced Parkinson's disease model was used as a mouse model of Parkinson's disease.
- This model is classic but has excellent reproducibility and is widely used as a Parkinson's disease model because of its simplicity. Histologically, it is characterized by the infiltration of inflammatory cells into the brain parenchyma and the reduction of dopaminergic neurons. Neurofibrillary tangles are classically considered as the cause of neuronal loss in such lesions.
- pathological conditions in which nerves are impaired ie Parkinson's disease, progressive supranuclear paralysis, basal ganglia degeneration, Alzheimer's disease, polyglutamine disease, amyotrophic lateral sclerosis (ALS), spinal progressive Not only typical neurodegenerative diseases such as muscle atrophy, bulbar spinal muscular atrophy, Huntington's disease, multiple sclerosis, but also multisystem atrophy (striatal nigra degeneration, olive bridge cerebellar atrophy, shy) ⁇ Drager syndrome), adrenoleukodystrophy, Guillain-Barre syndrome, myasthenia gravis, Fischer syndrome, chronic inflammatory degenerative polyneuritis, Lewis-Sumner syndrome, Crow-Fukase syndrome, normal pressure hydrocephalus, syringomyelia , Prion disease (Kreuzfeld-Jakob disease, Gerstmann-Streisler-Scheinker disease, fatal familial insomnia), subacute sclerosing panencephalitis (SSPE), progression It includes
- brain trauma sequelae cerebrovascular disorder (cerebral infarction, cerebral hemorrhage) sequelae, viral encephalitis sequelae, bacterial meningitis sequelae, spinal cord injury sequelae, spinal nerve, peripheral nerve, auditory nerve, optic nerve, etc. Changes are also included.
- neurofibrillary tangles are important as a cause of psychiatric symptoms since the above-mentioned sequelae have long been thought to be the basis of mental illness such as depression.
- Example 64 Examination of anti-fibrogenic effect in GalNAc4S-6ST siRNA treatment by MPTP-induced C57BL / 6JcL Parkinson's disease model mice
- MPTP 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine
- Example 1 was administered into the tail vein, and after 1 hour, dissection was performed, the brain was removed, and immunostaining samples and gene expression analysis samples were prepared. Obtained. Gene expression was examined quantitatively in the same manner as in Example 1. The results are shown in FIG. In this Parkinson's disease model, expression of GalNAc4S-6ST in brain tissue and fibrogenic markers TGF ⁇ , type I collagen, and ⁇ SMA were enhanced, but these expressions were significantly suppressed by administration of GalNAc4S-6ST siRNA. Therefore, it was revealed that by suppressing the expression of GalNAc4S-6ST, fibrotic changes in brain tissue can be suppressed.
- the agent of the present invention is useful, for example, as a fibrotic change inhibitor in brain tissue.
- Example 65 Examination of fibroblast accumulation in GalNAc4S-6ST siRNA treatment by MPTP-induced C57BL / 6JcL Parkinson's disease model mice Using the sample of brain tissue in the same manner as in Example 3, fibrogenesis in nerve cells in the brain On the other hand, the tissue findings obtained by in vivo administration of GalNAc4S-6ST were compared. The obtained sections were fixed with 4% PFA phosphate buffer (manufactured by Nacalai Tesque) for 10 minutes, washed with deionized water, and fibroblast antibody (ER-TR7; 1: 100 dilution; BMA) as the primary antibody. And reacted at 4 ° C. overnight.
- PFA phosphate buffer manufactured by Nacalai Tesque
- Alexa488-labeled anti-rat IgG goat antibody (1: 200 dilution; Invitrogen) as a secondary antibody was added and allowed to react at room temperature for 30 minutes.
- a tissue image obtained by the above method is shown in FIG.
- the strong positive signal in the untreated group was a result showing fibroblast infiltration in the brain in the vicinity of the posterior granular cortex of the corpus callosum than in the control group.
- the GalNAc4S-6ST siRNA treatment group the positive findings of fibroblasts were drastically reduced. From the above results, it was revealed that the positive signal of ER-TR7 in brain tissue induced by Parkinson's disease mouse model was significantly suppressed by in vivo administration of GalNAc4S-6ST siRNA.
- Example 66 Examination of neuroprotective effect in treatment with GalNAc4S-6ST siRNA by MPTP-induced C57BL / 6JcL Parkinson's disease model mouse Next, in order to examine whether the above-mentioned fibrosis is accompanied by a decrease in nerve cells, Example 64 In the same manner as above, the expression of genes related to nerve regeneration in brain tissue was quantified. The expression of GDNF, a factor that controls survival and differentiation of dopamine neurons and promotes regeneration, and Nurr1, which is a factor that forms dopamine neurons, were enhanced by administration of GalNAc4S-6ST siRNA (FIG. 66). This result shows that by suppressing the expression of GalNAc4S-6ST, regeneration of dopamine neurons in brain tissue can be promoted.
- the agent of the present invention is useful, for example, as a dopamine nerve regeneration promoter in brain tissue.
- Example 67 Examination of neuroprotective effect in treatment with GalNAc4S-6ST siRNA by MPTP-induced C57BL / 6JcL Parkinson's disease model mouse Tissue sample section obtained in order to finally clarify the results shown in the above Examples
- the dopamine neurons were stained with an anti-tyrosine hydroxylase antibody using as a marker of dopamine neurons, and the tissue findings were compared.
- This tyrosine hydroxylase (TH) is an enzyme that converts a dopamine precursor into dopamine.
- FIG. 67 shows tissue images of the control group, the untreated group, the GalNAc4S-6ST siRNA, and the treated group (the original figure is color).
- the agent of the present invention is useful, for example, as a dopamine neuron function restoring agent.
- Example 68 Examination of neuroprotective effect in treatment with GalNAcST siRNA using MPTP-induced C57BL / 6JcL Parkinson's disease model mice As in Example 64, C57BL / 6JcL mice (manufactured by CLEA Japan) were bred and given birth.
- Example 67 On the 8th day of the experiment, 100 ⁇ l of BrdU 5 mg / mL (ZyMED Laboratory. Inc.) was administered into the tail vein, and after 1 hour, dissection was performed, the brain was removed, and immunostaining and gene expression analysis samples Obtained. As in Example 67, the obtained sections were immunostained with anti-TH antibody. The results are shown in FIG. In the group treated with GalNAcST siRNA, it was confirmed that the decrease of TH positive dopamine neurons was suppressed. That is, it was concluded that suppression of expression of GalNAc4ST-1, GalNAc4ST-2, and GalNAc4S-6ST suppressed neurofibrillary changes and expected functional recovery of dopamine neurons as in Example 67.
- the agent of the present invention is useful, for example, as a neurofibrillary change inhibitor.
- Example 69 Effect of C4-sulfatase in Streptozotocin-induced type 2 diabetic retinopathy model mice: Decreased pregnancy 14 days of sulfated CSPG C57BL / 6JcL mice (manufactured by CLEA Japan, Inc.) are bred and born, after birth 2-day-old C57BL / 6JcL mouse female (manufactured by Claire Japan), Streptozocin 10 mg / mL (manufactured by SIGMA) 20 ⁇ L / head subcutaneously injected into each, and CE-2 (manufactured by Claire Japan) feed until 4 weeks old, Sterile water was given and bred.
- Sections were fixed with acetone (Sigma Aldrich Japan) for 10 minutes, washed with phosphate buffer, and anti-chondroitin sulfate proteoglycan (CSPG) antibody (clone CS56, mouse monoclonal antibody, 10 ⁇ g / mL; biochemistry as primary antibody) Kogyo Co., Ltd.) was added and reacted at room temperature for 1 hour. Subsequently, a secondary antibody reaction was performed using a histofine mouse stain kit (manufactured by Nichirei; used for mouse monoclonal antibody), and then DAB substrate (manufactured by Nichirei) was added to perform an enzyme dye reaction. This specimen was observed using an optical microscope (manufactured by Leica).
- CSPG chondroitin sulfate proteoglycan
- the obtained tissue image is shown in FIG. 69 (the original drawing is in color).
- a new CS56 positive finding is observed on the vitreous side of the retina.
- the CS56 intensity in the enzyme treatment group is decreased.
- CS56 is an antibody that recognizes sulfate groups, and this attenuation is suggested to reflect the attenuation of the 4-position sulfate group. From the above, it was revealed that CSPG deposition in retinal tissue induced in this model mouse was modified and suppressed by in vivo administration of C4-sulfatase.
- C4-sulfatase is an enzyme that desulfates the 4-position sulfate group of GalNAc.
- tissue fibrosis can be suppressed at the biological level by inhibiting sulfation at the 4-position of GalNAc. That is, the sulfate group desulfating enzyme at position 4 of GalNAc is useful as a tissue fibrosis inhibitor.
- Example 70 Effect of C4-sulfatase in Streptozotocin-induced type 2 diabetic retinopathy model mouse: Inhibition of vascular endothelial cell proliferation
- a section obtained by the same method as in Example 69 was made of acetone (manufactured by Sigma-Aldrich Japan). After fixing for 10 minutes, washed with a phosphate buffer, rat-derived anti-vascular endothelial cell antibody (CD31; 1: 200 dilution; manufactured by Pharmingen) was added as a primary antibody, and allowed to react at room temperature for 1 hour.
- CD31 rat-derived anti-vascular endothelial cell antibody
- Example 71 Effect of C4-sulfatase in Streptozotocin-induced type 2 diabetic retinopathy model mice: Inhibition of collagen-induced changes: A section obtained in the same manner as in Example 69 was fixed with acetone (manufactured by Sigma-Aldrich Japan) for 10 minutes, washed with a phosphate buffer, and a rabbit-derived anti-type IV collagen antibody (1: 250 dilution) as a primary antibody. And Sigma) were added and reacted at room temperature for 1 hour. Next, peroxidase-labeled anti-rabbit IgG (1: 200 dilution; manufactured by Jackson ImmunoResearch), which is a secondary antibody, was added and reacted at room temperature for 30 minutes.
- DAB substrate (Nichirei) was added to the sample after the reaction. This specimen was observed using an optical microscope (Leica). The obtained tissue image is shown in FIG. 71 (the original drawing is color).
- the untreated group positive findings of type IV collagen were observed on the retinal vitreous side, and findings parallel to the intraretinal boundary membrane were observed. This suggests an increase in collagen, that is, a change in fibrosis, along with abnormal venous morphology.
- the enzyme treatment group the growth of type IV collagen at the same site was remarkably suppressed. From the above results, it was revealed that retinal collagen growth was observed in the type 2 diabetes model, but it was suppressed by C4-sulfatase administration. That is, the 4-sulfate desulfating enzyme of GalNAc is useful as a therapeutic agent for type 2 diabetic retinopathy.
- Example 72 Localization of Fibroblasts in Liver of Type 2 Diabetes Model Mouse C4-sulfatase was obtained using the liver collected from the above-mentioned (Examples 69 to 71) type 2 diabetes model mouse.
- the preparation of the frozen block and the immunostaining technique were all carried out by the methods described above. As shown in FIG. 72, infiltration of a large number of fibroblasts was observed in the untreated group. In contrast, the degree of fibroblast infiltration in the C4-sulfatase treated group was less than in the untreated group.
- C4-sulfatase has a pharmacological action that suppresses fibroblast infiltration, and this effect is one end of the anti-fibrotic action. That is, the sulfate group desulfating enzyme at position 4 of GalNAc is useful as a fibroblast infiltration inhibitor.
- Example 73 Localization of Macrophages in Liver of Type 2 Diabetes Model Mice
- anti-antigens in the liver of C4-sulfatase using the liver collected from the above-described (Examples 69 to 71) type 2 diabetes model mouse were used.
- the inflammatory effect index of macrophage tissue infiltration
- the preparation of the frozen block and the immunostaining technique were all carried out by the methods described above.
- FIG. 73 in the untreated group, infiltration of a large number of macrophages with spot formation was observed. In contrast, the degree of macrophage infiltration in the C4-sulfatase treated group was less than in the untreated group.
- C4-sulfatase has a pharmacological action that suppresses macrophage infiltration, and this effect is a part of the anti-inflammatory action. That is, the sulfate desulfating enzyme at the 4-position of GalNAc is useful as a macrophage infiltration inhibitor or anti-inflammatory agent.
- Example 74 Serum Biochemical Test Findings of Type 2 Diabetes Model Mouse Example 74 was analyzed for the purpose of supplementing the results of Example 72 and Example 73.
- serum collected from the aforementioned type 2 diabetes model mice (Examples 69 to 71) was used to measure liver function indicators aspartate aminotransferase (AST), alanine aminotransferase (ALT). ), Triacylglycerol (TG), which is an index of lipid metabolism, was measured.
- FIG. 74 shows the result.
- Example 75 Comparison of expression of CSPG in brain tissue of MPDP-induced Parkinson model mouse
- MPDP which is a metabolite of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) was used.
- a model was created and this experiment was performed.
- RNA concentration in the sample extracted by Wave XS was calculated.
- the following procedure was performed to perform RT reaction (cDNA synthesis).
- the calculated RNA sample was adjusted to a concentration of 500 ng / 20 ⁇ l, heated at 68 ° C. for 3 minutes with BLOCK INCUBATOR (manufactured by ASTEC), and ice-cooled for 10 minutes.
- RT Pre Mix solution (composition: 25 mM MgCl 2 18.64 ⁇ l (Invitrogen)), 5 ⁇ Buffer 20 ⁇ l (Invitrogen), 0.1 M DTT 6.6 ⁇ l (Invitrogen), 10 mM dNTP mix 10 ⁇ l (Invitrogen), RNase Inhibitor 2 ⁇ l (Invitrogen), MMLV Reverse transcriptase 1.2 ⁇ l (Invitrogen), Random primer 2 ⁇ l (Invitrogen), sterile distilled water 19.56 ⁇ l (Otsuka distilled water: Otsuka 80 ⁇ l) was added and reacted with BLOCK INCUBATOR (ASTEC) at 42 ° C for 1 hour, and after 1 hour, heated with BLOCK INCUBATOR (ASTEC) for 99 minutes at 99 ° C, 100 ⁇ l of cDNA obtained by ice cooling was prepared, and a PCR reaction was performed using the cDNA obtained by synthesis with the following composition
- PCR Buffer 2 [mu] l Composition: (manufactured by Sigma Aldrich Japan Co.) 166 mM (NH 4) 2 SO 4, ( manufactured by Invitrogen Corporation) 670mM Tris -HCl pH8.8, 67 mM MgCl 2 ⁇ 6H 2 O (Sigma Aldrich Japan Co.
- FIG. 75 shows the results of examination of CSPG expression in the MPDP-inducible Parkinson model by immunostaining using CS-56 antibody (anti-CSPG antibody: manufactured by Seikagaku Corporation, 1: 100 dilution).
- anti-CSPG antibody manufactured by Seikagaku Corporation, 1: 100 dilution.
- the CSPG positive signal is strongly expressed in the untreated group.
- the expression of positive signals was decreased in the C4-sulfatase treatment group and gene therapy group.
- Example 76 Localization of dopamine neurons in brain tissue of MPDP-induced Parkinson model mice
- dopamine in MPDP-induced Parkinson model Fluorescence using the anti-tyrosine hydroxylase (TH 1:20 dilution) antibody and the secondary antibody using Alexa-488-labeled anti-rabbit IgG antibody (1: 200 dilution by Invitrogen) Analysis was performed using immunostaining.
- TH 1:20 dilution anti-tyrosine hydroxylase
- Alexa-488-labeled anti-rabbit IgG antibody (1: 200 dilution by Invitrogen
- Example 77 Expression analysis of inflammation-related genes in brain tissue of MPDP-induced Parkinson model mice
- tissue sections used in Examples 75 and 76 TNF- ⁇ expression was analyzed by quantitative PCR using total RNA extracted from the same sample by the method described above.
- SYBR premix kit manufactured by Takara Bio Inc.
- Real-time PCR thermal cycler DICE manufactured by Takara Bio Inc.
- the PCR reaction conditions were 95 ° C for 10 seconds, 95 ° C for 5 seconds, and 60 ° C for 30 seconds for 40 cycles, and finally a melting curve analysis was performed.
- TNF- ⁇ Tumor Necrosis Factor
- Example 78 Analysis of Expression of Inflammation Related Genes in Brain Tissue of MPDP-Induced Parkinson Model Mice
- Nurr1 which is a gene involved in the expression of dopaminergic neurons is used as a marker in order to supplement the results of Example 73.
- the effects of C4-sulfatase and GalNAc4S-6ST will be described using the quantitative PCR method described above. The base sequences of the primers used are shown below.
- Nuclea receptor subfamily 4 GroupA member2 (Nurr1) : (Takara Bio) forward: 5'-CTGCCCTGGCTATGGTCACA-3 '(SEQ ID NO: 97) reverse: 5'-AGACAGGTAGTTGGGTCGGTTCA-3 '(SEQ ID NO: 98)
- the Nurr1 gene expression was significantly increased (P ⁇ 0.001) in the C4-sulfatase treatment group and the gene therapy group as compared to the untreated group. This result supports the result of Example 76, and the results showed that C4-sulfatase administration, siRNA administration dopaminergic neuroprotective action, or regeneration / repair promotion action.
- inhibitors at the 4th or 6th position of GalNAc are useful as dopaminergic neuroprotective agents or regeneration / repair promoters.
- the present invention provides a drug that suppresses fibril formation at the level of biological tissue by inhibiting the function of a sugar chain-related gene.
- the drug is useful for treating or preventing a tissue fibrosis disease.
- Chronic tissue fibrosis fibrogenic tissue change
- Fibrogenic diseases are considered to be the terminal image of chronic inflammation, and are a general term for diseases that occur in organs throughout the body and cause dysfunction of the organs or even death of individuals.
- the present invention provides a technology for suppressing fibrotic diseases (fibrotic lesions of tissues and dysfunction associated therewith) by a completely new method that targets sugar chain genes that have recently been identified and whose functions are unknown. provide.
- a fibrogenic disease that is clinically intractable dysfunction is not only a decisive factor of death, but also a disease that significantly inhibits daily quality of life (QOL).
- QOL daily quality of life
- Establishment of tissue fibrosis inhibitors is therefore extremely important, but there are no drugs currently on the market.
- Experimentally, with a focus on TGF- ⁇ inhibitors, angiotensin inhibitors, inflammatory cytokine inhibitors, TLR inhibitors, and MMP inhibitors have been actively researched. (Review: Wynn TA, J. Clin. Invest. 117: 524-529, 2007).
- the present inventor administered a target sugar chain gene inhibitor to a subject in a fibrogenic disease model animal of various organs to reduce fibrogenic lesions and restore organ function. Confirmation of the fibrogenic disease was definitively performed by pathological images (including immunostaining and masson staining of fibroblasts and collagen).
- the therapeutic effect was determined not only by the pathological image, but also by clinical symptoms and collagen expression level in each organ. As a technical method, a therapeutic effect was proved by a direct gene knockdown method with a nucleic acid drug (siRNA) for a sugar chain gene.
- siRNA nucleic acid drug
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Neurology (AREA)
- Analytical Chemistry (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Virology (AREA)
- Neurosurgery (AREA)
- Diabetes (AREA)
- Cardiology (AREA)
- Epidemiology (AREA)
- Physical Education & Sports Medicine (AREA)
- Heart & Thoracic Surgery (AREA)
- Plant Pathology (AREA)
Abstract
Description
糖において、これらの化学修飾を受ける部位は複数存在する。例えば、GlcNAcにおいては、1位~6位の炭素の全てが化学修飾を受けることが知られている。その他の糖においても、種々の部位において化学修飾を受けることが報告されている。
上述のように、糖鎖を構成する糖には複数の種類が知られており、糖は複数の部位において種々の化学修飾を受けることによって、生体内において重要な生理作用を担うものと考えられている。
上述のように、糖鎖を構成する糖の種類、糖を標的とする化学修飾の種類およびその化学修飾される部位は多数存在することから、糖鎖は無数のバリエーションからなるものと考えられる。従って、ある役割を担う糖鎖の構造を特定することは非常に困難であり、また、ある疾患に起因する病態と、糖鎖の具体的な作用との関連性を見出すことは極めて困難を伴うことである。
〔1〕 N-アセチルガラクトサミンの4位または6位の硫酸化インヒビターを成分とする、組織線維形成抑制剤、
〔2〕 生体組織の線維形成に対して抑制効果を有することを特徴とする、〔1〕に記載の薬剤、
〔3〕 前記インヒビターがN-アセチルガラクトサミンの4位または6位の硫酸基転移酵素の機能を阻害する活性を有する、〔1〕または〔2〕に記載の薬剤、
〔4〕 前記インヒビターが、N-アセチルガラクトサミンの4位または6位の硫酸基転移酵素遺伝子の発現を抑えるsiRNAである、〔3〕に記載の薬剤、
〔5〕 前記インヒビターがN-アセチルガラクトサミンの4位または6位の硫酸基の脱硫酸化酵素である、〔1〕または〔2〕に記載の薬剤、
〔6〕 線維形成性疾患の治療用または予防用の、〔1〕~〔5〕のいずれかに記載の薬剤、
〔7〕 糖鎖を構成するN-アセチルガラクトサミンの4位または6位の硫酸化を阻害する化合物を選択する工程を含む、組織線維形成抑制剤のスクリーニング方法、
〔8〕 以下の工程(a)~(c)を含む、組織線維形成抑制剤のスクリーニング方法、
(a)N-アセチルガラクトサミンまたはN-アセチルガラクトサミンを含有する糖鎖と、被検化合物を接触させる工程
(b)N-アセチルガラクトサミンの4位または6位について硫酸化の程度を測定する工程
(c)被検化合物を接触させない場合と比較して、硫酸化の程度を低下させる化合物を選択する工程
〔9〕 以下の工程(a)~(c)を含む、組織線維形成抑制剤のスクリーニング方法、
(a)N-アセチルガラクトサミンの4位または6位の硫酸基転移酵素と、被検化合物を接触させる工程
(b)前記酵素の硫酸基転移活性を測定する工程
(c)被検化合物を接触させない場合と比較して、前記活性を低下させる化合物を選択する工程
〔10〕 以下の工程(a)~(c)を含む、組織線維形成抑制剤のスクリーニング方法、
(a)N-アセチルガラクトサミンの4位または6位の硫酸基転移酵素をコードする遺伝子を発現する細胞に、被検化合物を接触させる工程
(b)前記細胞における遺伝子の発現量を測定する工程
(c)被検化合物を接触させない場合と比較して、前記遺伝子の発現量を低下させる化合物を選択する工程
〔11〕 以下の工程(a)~(c)を含む、組織線維形成抑制剤のスクリーニング方法、
(a)N-アセチルガラクトサミンの4位または6位の硫酸基転移酵素をコードする遺伝子の転写調節領域とレポーター遺伝子とが機能的に結合した構造を有するDNAを含む細胞または細胞抽出液と、被検化合物を接触させる工程
(b)前記レポーター遺伝子の発現量を測定する工程
(c)被検化合物を接触させない場合と比較して、前記レポーター遺伝子の発現量を低下させる化合物を選択する工程
〔12〕 以下の工程(a)および(b)を含む、線維形成性疾患の治療または予防のための医薬組成物の製造方法、
(a)被検試料から、〔7〕~〔11〕のいずれかに記載の方法により、組織線維形成抑制剤を選択する工程
(b)前記薬剤と医薬的に許容される担体とを混合する工程
を、提供するものである。
さらに本発明は以下に関する。
〔13〕N-アセチルガラクトサミンの4位または6位の硫酸化インヒビターを個体へ投与する工程を含む、組織線維形成を抑制する方法。
〔14〕N-アセチルガラクトサミンの4位または6位の硫酸化インヒビターの、組織線維形成抑制剤の製造における使用。
〔15〕組織線維形成抑制における使用のためのN-アセチルガラクトサミンの4位または6位の硫酸化インヒビター。
以下、本発明を詳細に説明する。
本発明は、糖鎖を構成する糖の中の一つであるN-アセチルガラクトサミンにおいて、その4位または6位の部位の硫酸化を阻害することを機序とする、組織線維形成抑制剤に関する。
即ち本発明は、N-アセチルガラクトサミンの4位または6位の硫酸化インヒビター(本明細書において、「本発明のインヒビター」あるいは単に「インヒビター」と記載する場合あり)を成分とする、組織線維形成抑制剤を提供する。
また、N-アセチルガラクトサミンは、1~6位の部位が化学修飾を受けることが知られている。
本発明は、N-アセチルガラクトサミンの4位または6位の部位の硫酸化を阻害することを特徴とする。
本発明のインヒビターによって阻害される部位は、具体的には下記のGalNAcの化学式において矢印で表される部位である。
本発明におけるインヒビターの好ましい態様としては、例えば、N-アセチルガラクトサミンの4位または6位の硫酸基転移酵素の機能を阻害する活性を有する物質を挙げることができる。
(a)N-アセチルガラクトサミンの4位または6位の硫酸基転移酵素をコードする遺伝子の転写産物またはその一部に対するアンチセンス核酸
(b)N-アセチルガラクトサミンの4位または6位の硫酸基転移酵素をコードする遺伝子の転写産物を特異的に開裂するリボザイム活性を有する核酸
(c)N-アセチルガラクトサミンの4位または6位の硫酸基転移酵素をコードする遺伝子の発現をRNAi効果により阻害する作用を有する核酸(硫酸基転移酵素遺伝子の発現を抑えるsiRNA)
(a)N-アセチルガラクトサミンの4位または6位の硫酸基転移酵素と結合する抗体
(b)N-アセチルガラクトサミンの4位または6位の硫酸基転移酵素変異体
(c)N-アセチルガラクトサミンの4位または6位の硫酸基転移酵素と結合する低分子化合物
N-アセチルガラクトサミンの4位または6位の硫酸基の「脱硫酸化」とは、N-アセチルガラクトサミンの4位または6位の硫酸基が除去されることを指す。
脱硫酸化酵素としては、例えば、Chondroitin-4-sulfatase(C4-サルファターゼ)、Chondroitin-6-sulfatase等が挙げられる。
1)GalNAc4ST-1: N-acetylgalactosamine 4-sulfotransferase-1
別名CHST8:Carbohydrate (N-acetylgalactosamine 4-O) sulfotransferase 8
2)GalNAc4ST-2
別名CHST9:Carbohydrate (N-acetylgalactosamine 4-O) sulfotransferase 9
3)C4ST-1: chondroitin-4-O-sulfotransferase-1
別名CHST11:Carbohydrate (chondroitin 4) sulfotransferase 11
4)C4ST-2
別名CHST12
5)C4ST-3
別名CHST13
6)C6ST-1: chondroitin-6-O-sulfotransferase-1
別名CHST3: Carbohydrate (chondroitin 6) sulfotransferase 3
7)GalNAc4S-6ST: N-acetylgalactosamine 4-sulfate 6-0 sulfotransferase
8)D4ST-1:dermatan 4 sulfotransferase 1
9)C6ST-2: chondroitin-6-O-sulfotransferase-2
別名CHST7: Carbohydrate (chondroitin 6) sulfotransferase 7
GalNAc4ST-1(アクセッション番号NM_175140、塩基配列の配列番号:2、アミノ酸配列の配列番号:3)
GalNAc4ST-2(アクセッション番号NM_199055、塩基配列の配列番号:4、アミノ酸配列の配列番号:5)
C4ST-1(アクセッション番号NM_021439、塩基配列の配列番号:6、アミノ酸配列の配列番号:7)
C4ST-2(アクセッション番号NM_021528、塩基配列の配列番号:8、アミノ酸配列の配列番号:9)
C4ST-3(アクセッション番号XM_355798、塩基配列の配列番号:10、アミノ酸配列の配列番号:11)
D4ST(アクセッション番号NM_028117、塩基配列の配列番号:12、アミノ酸配列の配列番号:13)
C6ST-1(アクセッション番号NM_016803、塩基配列の配列番号:14、アミノ酸配列の配列番号:15)
C6ST-2(アクセッション番号AB046929、塩基配列の配列番号:16、アミノ酸配列の配列番号:17)
GalNAc4S-6ST(アクセッション番号NM_015892、塩基配列の配列番号:18、アミノ酸配列の配列番号:19)
RNAiは1998年にFireらによって発見された現象(Fire A, Nature (1998) 391, :806-811)で、二本鎖RNA(double strand RNA)が相同な標的遺伝子の発現を強力に抑制するというものである。従来のベクター等を用いる遺伝子導入法に比べ簡便であり、標的に対する特異性が高く、遺伝子治療への応用が可能であるということで最近注目されている。また哺乳類細胞においては、短鎖dsRNA(siRNA)を用いることにより、RNAiを誘導する事が可能で、RNAiは、ノックアウトマウスと比較して、効果が安定、実験が容易、費用が安価であるなど、多くの利点を有している。
ここで「治療または予防」とは、組織線維形成を呈する臓器、組織に対して、必ずしも完全な治療効果または予防効果を有する必要はなく、部分的な効果を有する場合であってよい。
また本発明の「組織線維形成性疾患治療剤」は、「組織線維形成性疾患改善剤」、「抗組織線維形成剤」等と表現することも可能である。また、本発明における薬剤は、「医薬品」、「医薬組成物」、「治療用医薬」等と表現することもできる。
なお、本発明における「治療」には、線維形成の発生を予め抑制し得る予防的な効果も含まれる。また、線維形成性の臓器(組織)に対して、必ずしも、完全な治療効果を有する場合に限定されず、部分的な効果を有する場合であってもよい。
本発明の薬剤を含む錠剤は、マスキングや、腸溶性製剤とするために、公知のコーティングを施すことができる。コーティング剤には、エチルセルロースやポリオキシエチレングリコール等を用いることができる。
また、siRNAまたはshRNAを目的の組織または器官に導入するために、市販の遺伝子導入キット(例えばアデノエクスプレス:クローンテック社)を用いることもできる。
本発明の方法の好ましい態様としては、糖鎖を構成するN-アセチルガラクトサミンの4位または6位の硫酸化を阻害する化合物を選択する工程を含む方法である。
本発明のスクリーニング方法によって、組織線維形成抑制剤もしくは線維形成性疾患治療もしくは予防剤のための候補化合物を効率的に取得することができる。
(a)N-アセチルガラクトサミンまたはN-アセチルガラクトサミンを含有する糖鎖と、被検化合物を接触させる工程
(b)N-アセチルガラクトサミンの4位または6位について硫酸化の程度を測定する工程
(c)被検化合物を接触させない場合と比較して、硫酸化の程度を低下させる化合物を選択する工程
(1) 標識色素(1-9-ジメチルメチレンブルー)を用いた定量的色素結合による方法(Nature. 1998 Feb 26;391(6670):908-11)
(2) (32P) 3',5'-ABPによる光親和性標識による方法 (Mandon, E.C., Milla, M.E., Kempner, E., and Hirschberg, C.B. (1994) Proc. Natl. Acad. Sci. USA, 91, 10707-10711)
(3) 3'-(32P)-βmethyleneb PAPSによる光親和性標識による方法 (Ozeran, J.D., Wesley, J., and Schwarz, N.B. (1996) Biochemistry, 35, 3695-3703)
(4) 陰イオン交換樹脂(硫酸化糖蛋白質の分離法)による方法 (Vol.16, No.2(19860430) pp. 69-72、北里大学 ISSN:03855449)
(5) Alcian blue によるsGAG比色染色 (Anal Biochem. 1998 Feb 15; 256(2): 229-37)
本発明の上記方法は、例えば以下の工程からなる。
(a)N-アセチルガラクトサミンの4位または6位の硫酸基転移酵素と、被検化合物を接触させる工程
(b)前記酵素の硫酸基転移活性を測定する工程
(c)被検化合物を接触させない場合と比較して、前記活性を低下させる化合物を選択する工程
被検化合物が上記硫酸基転移活性を有しているか否かについて評価(測定)可能な方法としては、例えば、以下の方法が挙げられる。
(a)N-アセチルガラクトサミンの4位または6位の硫酸基転移酵素をコードする遺伝子を発現する細胞に、被検化合物を接触させる工程
(b)前記細胞における遺伝子の発現量を測定する工程
(c)被検化合物を接触させない場合と比較して、前記遺伝子の発現量を低下させる化合物を選択する工程
(a)N-アセチルガラクトサミンの4位または6位の硫酸基転移酵素をコードする遺伝子の転写調節領域とレポーター遺伝子とが機能的に結合した構造を有するDNAを含む細胞または細胞抽出液と、被検化合物を接触させる工程
(b)前記レポーター遺伝子の発現量(レベル)を測定する工程
(c)被検化合物を接触させない場合と比較して、前記レポーター遺伝子の発現量(レベル)を低下させる化合物を選択する工程
(a)被検試料から、上記組織線維形成抑制剤のスクリーニング方法により、組織線維形成抑制剤を選択する工程
(b)前記薬剤と医薬的に許容される担体とを混合する工程
本方法においては次いで、上記選択された薬剤と、医薬的に許容される担体とを混合する。医薬的に許容される担体としては、例えば上述の担体を用いることができる。
本発明のキットは、例えば本発明の上述の各種試薬の中から、実施するスクリーニング方法にあわせて適宜選択することができる。例えば本発明のキットは、本発明のN-アセチルガラクトサミンの4位または6位の硫酸基転移酵素を構成要素とすることができる。本発明のキットには、さらに、本発明の方法において使用される各種試薬、容器等を含めることができる。例えば、抗体、プローブ、各種反応試薬、細胞、培養液、対照サンプル、緩衝液、使用方法を記載した指示書等を適宜含めることができる。
本発明の予防もしくは治療方法の対象となる個体は、線維形成性疾患を発症し得る生物であれば特に制限されないが、好ましくはヒトである。
個体への投与は、一般的には、例えば、動脈内注射、静脈内注射、皮下注射など当業者に公知の方法により行うことができる。投与量は、患者の体重や年齢、投与方法などにより変動するが、当業者(医師、獣医師、薬剤師等)であれば適当な投与量を適宜選択することが可能である。
なお本明細書において引用された全ての先行技術文献は、参照として本明細書に組み入れられる。
なお、本明細書においてsiRNAの構造(配列)を、ターゲットとなる遺伝子のDNA領域を記載することによって表現する場合がある。当業者であれば、ターゲット配列として記載されたDNA配列の情報に基づいて、当該DNA配列に対応する二本鎖RNAからなるsiRNAの構造を認識することは容易である。
実施例1:マウス心筋症モデルにおける標的糖鎖遺伝子のsiRNAノックダウン効果の検討と、遺伝子レベルにおける抗線維形成効果の検討
この実施例及び以下の実施例では、マウス心筋症モデルとして標準化されている、塩酸ドキシルビシン(DOX:協和発酵社製)腹腔内投与モデルを使用した。このマウスモデルは古典的であるが再現性にすぐれ、簡便であることより心筋症モデルとして病態解明や、新規治療実験などに幅広く使用されている(Longhu Li, Circulation. 2006;113:535-543、Xiaoming Yi, Am J Physiol Heart Circ Physiol 290: H1098-H1102, 2006、Kang YJ, J Biol Chem. 2000 May 5; 275(18):13690-8、Nozaki N, Circulation. 2004;110:2869-2874、Fisher PW, Circulation. 2005;111:1601-1610)。
[human GalNac4-6STsiRNA](Gene Bank accession number NM_015892)
(北海道システムサイエンス社製)
5’-ggagcagagcaagaugaauacaauc-ag -3’(配列番号:25)
3’-ua-ccucgucucguucuacuuauguuag -5’(配列番号:26)
[定量PCR Primer配列]
*mouse GalNac4S-6ST(タカラバイオ社製)
forward : 5’-GTGAGTTCTGCTGCGGTCCA-3’(配列番号:27)
reverse : 5’-AGTCCATGCTGATGCCCAGAG-3’(配列番号:28)
*mouse procollagen Type 1 alpha 2(タカラバイオ社製)
forward : 5’-ACCCGATGGCAACAATGGA-3’(配列番号:29)
reverse : 5’-ACCAGCAGGGCCTTGTTCAC-3’(配列番号:30)
*mouse α-SMA(タカラバイオ社製)
forward : 5’-CATCCGTAAAGACCTCTATGCCAAC-3’(配列番号:31)
reverse : 5’-ATGGAGCCACCGATCCACA-3’(配列番号:32)
*mouse rリボゾーム18S(タカラバイオ社製)
forward : 5’- TTCTGGCCAACGGTCTAGACAAC-3’(配列番号:33)
reverse : 5’- CCAGTGGTCTTGGTGTGCTGA-3’(配列番号:34)
本発明の薬剤は、例えば、心筋線維形成抑制剤として有用である。
この実施例では心筋症モデルマウスの心重量(mg)と体重(g)を測定し、心肥大の指標となる心重量・体重比を算出し、GalNAc4S-6ST (GalNac) si RNA心肥大抑制効果について比較検討を行った。心肥大は組織の線維性変化を示す指標でもある。
図1は、siRNA治療群(n=4)及び未治療群(n=4)の心重量(mg)・体重(g)比について算出した結果について示している。この結果、未治療群での比の値は、6.376±0.484で、siRNA治療群の値は5.442±0.203であった。この結果、未治療群に比べてsiRNA治療群では、有意に値の減少が見られた(p<0.05:t検定)。これは、GalNac 4S-6ST siRNAが病的な心肥大を抑制する効果を持つこと示した結果である。
本発明の薬剤は、例えば、心肥大抑制剤(心肥大治療剤)として有用である。
この実施例では、心筋症モデルマウスの心臓サンプルを使用してGalNac 4S-6ST siRNAのI型コラーゲン沈着(線維形成の指標)の抑制効果について比較検討を行った。実施例1と同じマウスより採取された心臓の組織サンプルを、凍結用包埋剤OCTコンパウンド(Miles社製)に包埋し、クライオスタット(カールツァイス社製)により薄切し、得られた切片をアセトン(シグマアルドリッチジャパン社製)で10分間固定後、リン酸緩衝液で洗浄し、さらに一次抗体として抗I型コラーゲンウサギ抗血清(ウサギポリクローナル抗体、1:2000希釈;LSL社製)を添加し、室温で一時間反応させた。続いて、二次抗体であるペルオキシダーゼ標識ヤギ由来抗ウサギIgG(1:200希釈;cappel社製)を添加し、室温で30分反応させた。反応後のサンプルにDAB基質(ニチレイ社製)を添加した。この標本を光学顕微鏡(ライカ社製)用いて観察した。
組織所見を図3に示した。未治療群においては、心筋線維間に非常に強いI型コラーゲンの陽性シグナルが確認出来た。これに対して、siRNA治療群では、I型コラーゲンの陽性シグナルは未治療群に比べて遥かに弱かった。以上のI型コラーゲンの免疫染色の結果を考察すると、GalNac 4S-6ST siRNAが、心筋組織におけるI型コラーゲンの過剰沈着に対して抑制効果をもつことが明らかとなった。この結果は、実施例1に示した定量PCRの結果と相関するものであった。
本発明の薬剤は、例えば、心筋組織におけるI型コラーゲン沈着抑制剤として有用である。
この実施例では、心筋症モデルマウスの心臓サンプルを使用してGalNac 4S-6ST siRNAのIII型コラーゲン沈着(線維形成の活動性の指標)の抑制効果について比較検討を行った。実施例3と同様の方法で得られた組織切片を、アセトン(シグマアルドリッチジャパン社製)で10分間固定後、リン酸緩衝液で洗浄し、さらに一次抗体として抗III型コラーゲンウサギ抗血清(ウサギポリクローナル抗体、1:2000希釈;LSL社製)を添加し、室温で一時間反応させた。続いて、二次抗体であるペルオキシダーゼ標識ヤギ由来抗ウサギIgG(1:200希釈;cappel社製)を添加し、室温で30分反応させた。反応後のサンプルにDAB基質(ニチレイ社製)を添加した。この標本を光学顕微鏡(ライカ社製)用いて観察した。
組織所見を図4に示した。未治療群においては、心筋線維間にやや強めのIII型コラーゲンの陽性シグナルが確認出来た。これに対して、siRNA治療群では、III型コラーゲンの陽性シグナルは対照群と同程度であった。以上のIII型コラーゲンの免疫染色の結果を考察すると、GalNac 4S-6ST siRNAが、心筋組織におけるIII型コラーゲンの沈着を抑制する効果、つまり活動的なコラーゲンの沈着に対しても有効的な抑制効果もつことが明らかとなった。
本発明の薬剤は、例えば、心筋組織におけるIII型コラーゲン沈着抑制剤として有用である。
この実施例では、DOX投与により、心筋症モデルマウスの心筋組織内に浸潤してくる線維芽細胞の動態に対するGalNAc 4S-6ST siRNAの薬理効果について検討した。実施例3と同様の方法で得られた組織切片を、アセトン(シグマアルドリッチジャパン社製)で10分間固定後、リン酸緩衝液で洗浄し、さらに一次抗体として抗マウス線維芽細胞抗体(ER-TR7:ラットモノクローナル抗体、1:400希釈;BMAバイオメディカルズ社製)を添加し、室温で一時間反応させた。続いて、二次抗体であるペルオキシダーゼ標識ヤギ由来抗ラット免疫グロブリン抗体(1:200希釈;バイオソース社製)を添加し、室温で30分反応させた。反応後のサンプルにDAB基質(ニチレイ社製)を添加した。この標本を光学顕微鏡(ライカ社製)用いて観察した。
組織所見を図5に示した。写真は心室隔壁部分フォーカスしたものである。未治療群においては、対照群に比べて多数の線維芽細胞の浸潤を認めた。これに対して、siRNA治療群では、未治療群に比べて、線維芽細胞の浸潤の程度を少なかった。この結果は、GalNac 4S-6ST siRNAが心筋組織における線維芽細胞の浸潤を抑制する薬理効果をもち、この効果が抗線維形成作用の一端である事を示すものであった。
本発明の薬剤は、例えば、心筋組織における線維芽細胞浸潤抑制剤として有用である。
実施例6:マウス腸管線維形成モデルにおけるGalNAc4S-6STの臨床的線維形成抑制効果
C57BL/6Jマウス(♀、6週齢、日本クレア社製)にデキストラン硫酸ナトリウム(DSS; 和光社製)を3%含有する高塩素水を8日間自由飲水させることにより大腸炎モデルを作成した。本DSS誘導性大腸炎モデルは再現性に優れており、マウス潰瘍性大腸炎やクローン病という炎症性腸疾患の標準的実験モデルとして広く用いられている他、腸管狭窄の組織学的特徴である全層性の炎症・線維性変化ならびに筋層の肥厚を呈するモデルでもある(Sasaki N, J Inflamm. 2005 2: 13、総説:Pucilowska JB et al. Am J Physiol Gastroenterol Liver Physiol. 279:G653-G659, 2000)。したがって、炎症性腸疾患に加え、組織上の腸管狭窄を呈する病態、すなわち、腸管ベーチェット(単純性潰瘍)、過敏性腸症候群、虚血性腸炎、薬剤性腸炎、放射線性腸炎、食道アカラシア、強皮症に伴う食道狭窄、SLE(全身性エリテマトーデス:systemic lupus erythematosus)に伴う腸管狭窄、ヒルス・シュプリング病、腸管摘出後の狭窄(術後狭窄)、消化管系の癌(舌癌、上咽頭癌、喉頭癌、食道癌、胃癌、小腸癌、大腸癌、直腸癌)に対する内視鏡的粘膜切除術後の腸管狭窄、腸閉塞、という疾病に幅広く共通する組織学的所見である。
さらに、5日目にマウス屠殺後に大腸長を測定したところ、GalNAc4S-6ST siRNA投与群で有意(p<0.005、t検定)に大腸の短縮が抑制されていた(図6)。大腸長は、腸管線維形成あるいは狭窄を反映する決定的な指標である。したがって、GalNAc4S-6ST siRNA投与群では、臨床的にも腸管線維性変化を強く抑制することが明らかになった。
本発明の薬剤は、例えば、腸管線維性変化抑制剤として有用である。
この実施例では、GalNAc4S-6ST siRNA投与による大腸局所の線維形成関連遺伝子発現をリアルタイム定量PCR法により検討した。
実施例6と同様の方法で腸管線維形成モデルを作成し、7日目にマウスを屠殺した。採取した大腸の一部を1.5 mlチューブにとりわけ、液体窒素で凍結した。実施例1と同様の方法でcDNA合成を行ない、定量的PCRを行なった。プライマーの配列、PCRサイクルも同様である。
結果を図7に示す。本モデルではGalNAc4S-6ST遺伝子の発現が増強してくるが、GalNAc4S-6ST siRNA治療によって有意(p<0.001、t検定)にノックダウンされていることが確認できた。また、線維形成の指標であるtype I collagenならびにα-SMAの発現増強も、GalNAc4S-6ST siRNAにより有意(ともにp<0.001、t検定)に抑制した。本結果は、GalNAc4S-6ST の発現を抑制することにより、大腸の線維性変化の増強を効果的に抑制できることを示している。
本発明の薬剤は、例えば、大腸における線維性変化抑制剤として有用である。
実施例6と同様の方法で腸管線維形成モデルを作成し、7日目にマウスを屠殺した。採取した大腸を実施例3と同様の方法で凍結ブロックならびに、組織切片を作成した。大腸組織切片のマッソン染色像を図8に示す。マッソン染色はコラーゲン繊維を視覚化する、組織の線維性変化を評価するための指標である。GalNAc4S-6ST siRNA投与群においては、対照群に比較し、全層性(粘膜固有層、粘膜下層、筋層)のコラーゲン繊維の沈着が明確に抑制されていた。
本発明の薬剤は、例えば、全層性(粘膜固有層、粘膜下層、筋層)のコラーゲン繊維沈着抑制剤として有用である。
実施例6と同様の方法で腸管線維形成モデルを作成し、7日目にマウスを屠殺した。採取した大腸を実施例3と同様の方法で凍結ブロックならびに、組織切片を作成した。得られた切片をアセトン(和光社製)で10分間固定後、リン酸緩衝液で洗浄し、一次抗体として抗ER-TR7抗体(ラットモノクローナル抗体、1μg/ml、BMA社製)を添加し、室温で1時間反応させた。続いて、ペルオキシダーゼ標識抗ラットIgG(1:200希釈)を用いて二次抗体反応を行った後、DAB基質(ニチレイバイオサイエンス社製)を添加し発色させた。その後リリー・マイヤーヘマトキシリン(武藤化学社製)により核染色を行い、光学顕微鏡(ライカ社製)下で試料を観察し、茶色のシグナルで可視化された抗体結合を観察した。
その結果、GalNAc4S-6ST siRNA治療群では対照群に比し、線維芽細胞の全層性の浸潤が明確に抑制されていた(図9)。したがって、GalNAc4S-6ST遺伝子発現抑制により、組織病変局所の線維芽細胞の浸潤及び定着を抑制することで、線維性変化の増強を軽減していることが判明した。
本発明の薬剤は、例えば、線維芽細胞浸潤抑制剤もしくは線維芽細胞定着抑制剤として有用である。
実施例6と同様の方法で腸管線維形成モデルを作成し、7日目にマウスを屠殺した。採取した大腸を実施例3と同様の方法で凍結ブロックならびに、組織切片を作成した。得られた切片をアセトン(和光社製)で10分間固定後、リン酸緩衝液で洗浄し、一次抗体として抗F4/80抗体(クローンA3-1、ラットモノクローナル抗体、2μg/ml:CALTAG LABORATORIES社製)を添加し、室温で1時間反応させた。続いて、ペルオキシダーゼ標識抗ラットIgG(1:200希釈)を用いて二次抗体反応を行った後、DAB基質(ニチレイバイオサイエンス社製)を添加し発色させた。その後リリー・マイヤーヘマトキシリン(武藤化学社製)により核染色を行い、光学顕微鏡(ライカ社製)下で試料を観察し、茶色のシグナルで可視化された抗体結合を観察した。
その結果、GalNAc4S-6ST siRNA治療群では対照群に比し、マクロファージの全層性の浸潤が明確に抑制されていた(図10)。したがって、GalNAc4S-6ST遺伝子発現抑制によって、マクロファージと線維芽細胞という線維性変化を持続・増強させる責任細胞群の浸潤を抑制し、総合的に組織の線維性変化を抑制していることが判明した。
本発明の薬剤は、例えば、マクロファージもしくは線維芽細胞の浸潤抑制剤として有用である。
GalNAc4S-6STは4位が硫酸化されたNアセチルガラクトサミンの6位に硫酸基を転移する酵素である。本実施例では、4位硫酸転移酵素であるGalNAc-4ST1、GalNAc-4ST2というファミリーの役割に検討を加えた。実施例6と同様の方法で腸管線維形成モデルを作成した。7日目にマウスを屠殺した。本実施例では、GalNAc 4S-6ST siRNAのみならず、GalNAc 4ST-1とGalNAc 4ST-2(Gene World社製)を混合してマウス1匹あたり1μgを、媒体である1%アテノコラーゲン(高研社製)200μLを混合したものを腹腔内投与した。本siRNA投与群をGalNAc ST siRNA投与群と表現する。対照群は実施例6と同様である。本実施例に用いたGalNAc 4S-6ST siRNA、GalNAc 4ST-1、GalNAc 4ST-2の塩基配列について以下に示す。配列は必ずしも本例のみに限定されない。
[GalNAc4ST-1 siRNAカクテル配列](GenBank accession number NM_175140)
(Gene world社製)
5’-ACCCCCAACTCGGAACGATGCGGCT-3’(配列番号:35)
5’-TGCATGTTCTCGTCCATCCTGCTG-3’(配列番号:36)
5’-CGCCACCGTGTACTGTACTGTGAAGT-3’(配列番号:37)
5’-AGGCT GCTCCAACTG GAAGAGGGTG-3’(配列番号:38)
[GalNAc4ST-2 siRNAカクテル配列](GenBank accession number NM_199055)
(Gene world社製)
5’-ATATAGTATCTAGGATATATGTAG-3’(配列番号:39)
5’-GAAGTACCAAAAGCTGGCTGCTCTA-3’(配列番号:40)
5’-TTCTATCACTTGGACTATTTGATGTT-3’(配列番号:41)
5’-TACACAACTCCACATTTGTAATTTG-3’(配列番号:42)
[GALNAC4S-6ST siRNAカクテル配列](GenBank accession number NM_029935)
(Gene world社製)
5’-CCAGAAGCCAAGCTCATTGTTATG-3’(配列番号:43)
5’-CTGTGGAGAGGTTGTACTCAGACTA-3’(配列番号:44)
5’-ATTTGCCTGGAAGACAACGTGAGAGC-3’(配列番号:45)
5’-GTCCCTTCTGCAGAAGCTGGGCCCACT-3’(配列番号:46)
マウス腸管線維形成モデルにおいては実施例6から10で詳細に検討した如く、組織の線維性変化が大腸長に集約されるため、本実施例においては7日目における大腸長を決定的な評価ポイントとして示す。GalNAc ST siRNA投与群においては、対照群に比較し有意(p<0.01、t検定)に大腸の短縮を抑制した(図11)。したがって、GalNAc 4ST-1、GalNAc 4ST-2遺伝子の発現を抑制することで、腸管線維形成が抑制されることが判明した。
本発明の薬剤は、例えば、腸管線維形成抑制剤として有用である。
実施例12:マウス肺気腫モデルにおけるC6ST-1 siRNAの肺胞間質における効果
この実施例では、基本的な肺気腫マウスモデルであるブタ膵臓由来エラスターゼ(PPE)気管内投与モデルを使用した。このマウスモデルは古典的ではあるが再現性にすぐれ、簡便であることより肺気腫モデルとして広く使用されている。組織学的には肺胞間質への炎症細胞浸潤が認められるため、肺気腫や慢性気管支炎等の慢性閉塞肺疾患(COPD:chronic obstructive pulmonary disease)に加えて、特発性間質性肺炎(IIPs:idiopathic interstitial pneumonias)、塵肺、肺結核後遺症など、慢性呼吸不全に至る疾患の組織所見に共通する(Karlinsky JB et al Am Rev Respir Dis 1978;117:1109-1133.、Otto-Verberne CJ et al Protective effect of pulmonary surfactant on elastase-induced emphysema in mice. Eur Respir J 1992;5:1223-1230.、Janoff A et al Prevention of elastase-induced experimental emphysema by oral administration of a synthetic elastase inhibitor. Am Rev Respir Dis 1980;121:1025-1029.、Christensen TG, et al. Irreversible bronchial goblet cell metaplasia in hamsters with elastase-induced panacinar emphysema. J Clin Invest 1977;59:397-404.、Lucey EC, et al. Remodeling of alveolar walls after elastase treatment of hamsters: results of elastin and collagen mRNA in situ hybridization. Am J Respir Crit Care Med 1998;158:555-564.、Snider GL, Lucey EC, Stone PJ. Animal models of emphysema. Am Rev Respir Dis 1986;133:149-169.)。
最初にマウスモデルの作製についてであるが、C57BL6/J マウス(メス、5~6週齢、日本クレア社製)に、PPE(4単位;Calbiochem-Novabiochem社製)を気管内投与する。投与後3週間飼育し肺組織を採取した。対照群は同様のマウスでPPE未投与のものを用いた。
C6ST-1 siRNAの投与については実施例1と同様に行なった。PPE投与後より1週間ごとにC6ST-1 siRNA 1μg(ambion社製)とsiRNAの媒体である1%アテノコラーゲン(高研社製)を混合したもの腹腔内投与した。投与量は200μL/ mouseで行った。
*[C6ST-1 siRNAカクテル配列]
(GenBank accession number NM_016803)
5’-gcgccccctctccccatggagaaag-3’(配列番号:47)
5’-gctttgcctcaggatttccgggacc-3’(配列番号:48)
5’-ggttcagccttggtctaccgtgatgtc-3’(配列番号:49)
5’-gcagttgttgctatgcgacctgtat-3’(配列番号:50)
採取した右肺組織を凍結用包埋剤OCTコンパウンド(Miles社製)に包埋し、液体窒素を用いて凍結ブロックを作製した。その凍結ブロックからクライオスタット(Microm社製)を用いて厚さ6μmの切片を作製した。
得られた切片を1%グルタールアルデヒド(ナカライテスク社製)で10分間固定後、さらにホルモールカルシウム液で10分間固定した。リン酸緩衝液で洗浄後、リリマイヤーヘマトキシリン液(シグマアルドリッチジャパン社製)で室温、5分間染色し脱色液(0.5% HCL含有70%エタノール、いずれもナカライテスク社製試薬を用いて作製)にて軽く洗浄した。その後10分間水洗いを行った。エオジンアルコールで室温、5分間染色し10分間水洗いを行った。100%エタノールで軽く洗浄後、3分間静置した。さらにキシレン(ナカライテスク社製)軽く洗浄後、10分間静置した。この標本を光学顕微鏡(ライカ社製)用いて観察し組織の様子を確認した。
得られた組織図を図12に示した。対照群(PPE未投与マウス)の所見では蜂の巣様の肺胞隔壁が特徴的な正常な肺実質の組織所見が確認出来る。しかしながら、中央の写真が示す未治療群(PPE投与、C6ST-1 siRNA未投与)では、肺気腫に特徴的な所見である肺胞隔壁の破壊消失と気腔の拡大に伴う気腫性病変が観察出来る。これに対して酵素治療群(PPE投与、C6ST-1 siRNA投与)では若干の肺胞隔壁の消失と気腫性病変が見られるものの、その程度は大幅に改善されていることが観察出来る所見であった。
この実施例では、C6ST-1 siRNA投与による肺胞間質の線維化関連遺伝子発現をリアルタイム定量PCR法により検討した。
実施例12と同様の方法でCOPDモデルを作成した。採取した肺組織の一部を1.5 mlチューブにとりわけ、液体窒素で凍結した。実施例1と同様の方法でcDNA合成を行ない、定量的PCRを行なった。I型コラーゲンとα-SMAのプライマーの配列、PCRサイクルは同様である。C6ST-1のプライマーの配列を以下に示す。
[定量PCR Primer配列]
*mouse C6ST-1(タカラバイオ社製)
forward : 5’-TGTTCCTGGCATTTGTGGTCATA -3’(配列番号:51)
reverse : 5’-CCAACTC GCTCAGGGACAAGA-3’(配列番号:52)
結果を図13に示す。本モデルではC6ST-1遺伝子の発現が増強してくるが、C6ST-1 siRNA治療によって有意(p<0.01、t検定)にノックダウンされていることが確認できた。また、線維化の指標であるtype I collagenならびにα-SMAの発現増強も、C6ST-1 siRNAにより有意(ともにp<0.01、t検定)に抑制した。本結果は、C6ST-1 の発現を抑制することにより、肺間質の線維性変化の増強を効果的に抑制できることを示している。
本発明の薬剤は、例えば、肺間質の線維性変化抑制剤として有用である。
実施例12と同様の方法で腸管線維形成モデルを作成した。採取した肺組織を実施例3と同様の方法で凍結ブロックならびに、組織切片を作成した。得られた切片をアセトン(和光社製)で10分間固定後、リン酸緩衝液で洗浄し、一次抗体として抗ER-TR7抗体(ラットモノクローナル抗体、1μg/ml、BMA社製)を添加し、室温で1時間反応させた。続いて、ペルオキシダーゼ標識抗ラットIgG(1:200希釈)を用いて二次抗体反応を行った後、DAB基質(ニチレイバイオサイエンス社製)を添加し発色させた。その後リリー・マイヤーヘマトキシリン(武藤化学社製)により核染色を行い、光学顕微鏡(ライカ社製)下で試料を観察し、茶色のシグナルで可視化された抗体結合を観察した。
その結果、対照群では肺胞隔壁の破壊した間質に線維芽細胞の強い集積を認めた(図14、対照群の図左)。また、肺胞隔壁が破壊されつつある部位の間質においても多数の集積を認めた(図14、対照群の図右)ことから、COPDのような病態において、線維芽細胞の過剰集積から肺胞壁の破壊過程に至るという予想外の結果を得た。一方、C6ST-1 siRNA治療群では、線維芽細胞の肺胞組織間質への浸潤が明確に抑制されていた(図14)。したがって、C6ST-1 遺伝子発現抑制により、肺胞組織間質の線維芽細胞の浸潤及び定着を抑制することで、線維性変化の増強を軽減していることが判明した。
本発明の薬剤は、例えば、肺胞組織間質の線維芽細胞浸潤もしくは定着抑制剤として有用である。
実施例12と同様の方法でCOPDモデルを作成した。採取した肺組織を実施例3と同様の方法で凍結ブロックならびに、組織切片を作成した。得られた切片をアセトン(和光社製)で10分間固定後、リン酸緩衝液で洗浄し、一次抗体として抗F4/80抗体(クローンA3-1、ラットモノクローナル抗体、2μg/ml:CALTAG LABORATORIES社製)を添加し、室温で1時間反応させた。続いて、ペルオキシダーゼ標識抗ラットIgG(1:200希釈)を用いて二次抗体反応を行った後、DAB基質(ニチレイバイオサイエンス社製)を添加し発色させた。その後リリー・マイヤーヘマトキシリン(武藤化学社製)により核染色を行い、光学顕微鏡(ライカ社製)下で試料を観察し、茶色のシグナルで可視化された抗体結合を観察した。
その結果、C6ST-1 siRNA治療群では対照群に比し、マクロファージの肺胞間質への浸潤が明確に抑制されていた(図15)。したがって、C6ST-1遺伝子発現抑制によって、マクロファージと線維芽細胞という線維性変化を持続・増強させる責任細胞群の浸潤を抑制し、総合的に組織の線維性変化を抑制していることが判明した。
本発明の薬剤は、例えば、肺胞間質へのマクロファージ浸潤抑制剤として有用である。
本実施例では、C6ST-1 siRNAの肺気腫モデルマウスにおける臨床効果を検討するために静肺コンプライアンス(statistic compliance: Cst)を指標として呼吸機能に与える影響について評価した。Cstは肺組織の伸びやすさを表しているが、肺胞域の組織破壊を伴う疾患である肺気腫はCstが上昇する。
実施例12で示してきた方法と同様の手順で肺気腫モデルマウスを作製し、C6ST-1 siRNAで治療を行った。それらのマウスの自発呼吸を麻酔薬にて停止させたのちFlexiVent(SCIREQ社製)呼吸機能解析装置のP-V roop modeを用いてCstを測定した。マウスとFlexiVentとの接続はマウスの自発呼吸停止後に正中切開を行い、気管内に専用カニューレを挿管し気管支周囲を結紮する方法にて行った。
本実施例の結果について図16に示した。対照群では42.62±2.25μL/cmH2Oであったが、未治療群で51.22±5.2μL/cmH2O(vs対照群P=0.03 t検定)と統計学的に有意な増加を認めた。また、C6ST-1 siRNA投与群におけるCstは、42.92±1.82μL/cmH2O(vs未治療群P=0.03 t検定)と、未治療群に対して有意に低下していた。
したがって、C6ST-1 siRNA投与群では、PPEの気管内投与により誘導される肺気腫に伴うCstの増加を有意に抑制することが明らかになった。また、本実施例に示された結果は、C6ST-1 の遺伝子発現を抑制することにより、肺組織の間質線維性変化に伴う肺胞破壊を抑制するだけでなく実際の臨床症状(呼吸状態)を改善する効果を有することを示唆するものであった。
本発明の薬剤は、例えば、肺胞破壊抑制剤として有用である。
肺胞の気腔拡大を特徴的な病理像として示す肺気腫では、気腫性病変の進行に従い肺容量が増加する。本実施例ではC6ST-1 siRNAの治療効果が、細胞レベルでの破壊を抑制するだけでなく臓器の形態維持・保存の効果にまで及ぶものであることを立証することを目的として行われた。
本実施例で使用した肺組織は、実施例12で使用した肺組織の右肺を使用して行っている。マウスより摘出した肺組織をリン酸緩衝液で軽く洗浄後、同じくガラス容器に満たしたリン酸緩衝液につけた。リン酸緩衝液を満たしたガラス容器の重量を予め測定しておき、肺組織を容器に加えた後に増加した重量を液量に換算し肺容量とした。
本実施例の結果について図17に示した。対照群における肺容量は277.5±61.85μLであったが、未治療群で413.33±77.67μL(vs対照群P=0.024 t検定)と統計学的に有意な肺容量の増加を認めた。また、C6ST-1 siRNA治療群における肺容量は、292.5±51.23μL(vs未治療群P=0.027 t検定)と未治療群に対して有意に肺容量の低下を認めた。
これらの結果をまとめると、C6ST-1の遺伝子発現を抑制することにより、PPEの気管内投与により誘導される肺気腫に伴う肺容量の増加を効果的に抑制することが明らかになった。したがって、細胞レベルでの破壊を抑制するだけでなく、臓器の形態維持・保存の効果、もしくは破壊された組織の修復効果を持ち合わせていることを示すものであった。
本発明の薬剤は、例えば、肺気腫に伴う肺容量増加抑制剤として有用である。
4位および6位硫酸化が重要であることをもう一つの実施例で示す。本実施例では、実施例11と同様の方法で、GalNAcST siRNA投与による肺胞間質の線維化関連遺伝子発現をリアルタイム定量PCR法により検討した。siRNAの配列は実施例11と同様である。
実施例12と同様の方法で肺気腫モデルを作成した。採取した肺組織の一部を1.5 mlチューブにとりわけ、液体窒素で凍結した。実施例1と同様の方法でcDNA合成を行ない、定量的PCRを行なった。プライマーの配列、PCRサイクルは実施例1ならびに13と同様である。TGF-βの配列は以下に示す。
[定量PCR Primer配列]
*mouse TGF-β(タカラバイオ社製)
forward : 5’-GTGTGGAGCAACATGTGGAACTCTA -3’(配列番号:53)
reverse : 5’-TTGGTTCAGCCACTGCCGTA -3’(配列番号:54)
結果を図18に示す。本実施例においても、線維化の指標であるtype I collagen、α-SMA、ならびにTGF-βの発現増強が、GalNAcST siRNAにより有意(ともにp<0.01、t検定)に抑制した。本結果は、GAlNAc4ST-1、GAlNAc4ST-2ならびにGAlNAc4S-6STの発現を抑制することにより、肺間質の線維性変化の増強を効果的に抑制できることを示している。
本発明の薬剤は、例えば、肺間質の線維性変化抑制剤として有用である。
本実施例では、GAlNAcST siRNAの肺気腫モデルマウスにおける臨床効果を検討するために静肺コンプライアンス(statistic compliance: Cst)を指標として呼吸機能に与える影響について評価した。
実施例12で示してきた方法と同様の手順で肺気腫モデルマウスを作製し、GAlNAcST siRNAで治療を行った。それらのマウスの自発呼吸を麻酔薬にて停止させたのちFlexiVent(SCIREQ社製)呼吸機能解析装置のP-V roop modeを用いてCstを測定した。マウスとFlexiVentとの接続はマウスの自発呼吸停止後に正中切開を行い、気管内に専用カニューレを挿管し気管支周囲を結紮する方法にて行った。
本実施例の結果について図18に示した。対照群では42.62±2.25μL/cmH2Oであったが、未治療群で51.22±5.2μL/cmH2O(vs対照群P=0.03 t検定)と統計学的に有意な増加を認めた。また、C6ST-1 siRNA投与群におけるCstは、44.15±2.29μL/cmH2O(vs未治療群P=0.0018 t検定)と、未治療群に対して有意に低下していた。
したがって、GalNAcST siRNA投与群では、PPEの気管内投与により誘導される肺気腫に伴うCstの増加を有意に抑制することが明らかになった。また、本実施例に示された結果は、GAlNAc4ST-1、GAlNAc4ST-2ならびにGAlNAc4S-6STの遺伝子発現を抑制することにより、肺組織の間質線維性変化に伴う肺胞破壊を抑制するだけでなく実際の臨床症状(呼吸状態)を改善する効果を有することを示唆するものであった。
本発明の薬剤は、例えば、肺胞破壊抑制剤もしくは呼吸状態改善剤として有用である。
本実施例ではGalNAcST siRNAの治療効果が、細胞レベルでの破壊を抑制するだけでなく臓器の形態維持・保存の効果にまで及ぶものであることを立証することを目的として行われた。
本実施例で使用した肺組織は、実施例12で使用した肺組織の右肺を使用して行っている。マウスより摘出した肺組織をリン酸緩衝液で軽く洗浄後、同じくガラス容器に満たしたリン酸緩衝液につけた。リン酸緩衝液を満たしたガラス容器の重量を予め測定しておき、肺組織を容器に加えた後に増加した重量を液量に換算し肺容量とした。
本実施例の結果について図20に示した。対照群における肺容量は277.5±61.85μLであったが、未治療群で413.33±77.67μL(vs対照群P=0.024 t検定)と統計学的に有意な肺容量の増加を認めた。また、GalNAcST siRNA治療群における肺容量は、315±51.96μL(vs未治療群P=0.049 t検定)と未治療群に対して有意に肺容量の低下を認めた。
これらの結果をまとめると、GAlNAc4ST-1、GAlNAc4ST-2ならびにGAlNAc4S-6STの遺伝子発現を抑制することにより、PPEの気管内投与により誘導される肺気腫に伴う肺容量の増加を効果的に抑制することが明らかになった。したがって、細胞レベルでの破壊を抑制するだけでなく、臓器の形態維持・保存の効果、もしくは破壊された組織の修復効果を持ち合わせていることを示すものであった。
本発明の薬剤は、例えば、肺の形態維持剤、もしくは肺の形態保存剤として有用である。
以下の実施例では生後2日目C57BL/6JcLマウス(雌、日本クレア社製)のStreptozotocin投与により2型糖尿病モデルを作製し、C4ST-1(Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 1)siRNA、C4ST-2(Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 2)siRNA、C4ST-3(Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 3)siRNA処置による体重変動、血糖値変動、遺伝子発現や膵臓組織における線維性変化を検討した。各siRNAの投与法については、実施例1と同様の方法で行なった。配列を以下に示す。
*[C4ST-1 siRNAカクテル配列]
C4ST1(Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 1)
(GenBank accession number NM_021439)
5’-ACAAAGCCATGAAGCCGGCGCTGCTGGAAGTGATGAGGATGAACAGAATT-3’(配列番号:55)
5’-CAACCTGAAGACCCTTAACCAGTACA-3’(配列番号:56)
5’-GCATCCCAGAGATCAACCACCGCTTG-3’(配列番号:57)
*[C4ST-2 siRNAカクテル配列]
C4ST2(Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 2)
(GenBank accession number NM_021528)
5’-GCCAGGAGTGGGCCCAGCCCAGGGC -3’(配列番号:58)
5’-ATGACCAAGCCGCGGCTCTTCCGGCTG -3’(配列番号:59)
5’-AGAGCCTGCTGGACCGGGGCAGCCCCTA -3’(配列番号:60)
5’-GAGACCCCCTGGACATCCCCCGGGAACA-3’(配列番号:61)
*[C4ST-3 siRNAカクテル配列]
C4ST3(Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 3)
(GenBank accession number XM_355798)
5’-ATGACTGTCGCCTGCCACGCGTGCCA -3’(配列番号:62)
5’-CAGCATGGGAAGACGCTCCTGTTGCA -3’(配列番号:63)
5’-TCCAAGCGCAATCCCTGCGCACGAGGCG -3’(配列番号:64)
5’-GCCTGGCCTGCTGCCCTCGCTGGCC -3’(配列番号:65)
まず、サンプル調製するため以下の様に行った。
実施例21:Streptozocin誘発性 C57BL/6JcL 2型糖尿病モデルマウスによるC4ST-1 siRNA、C4ST-2 siRNA、C4ST-3 siRNA処置における抗肥満効果の検討
妊娠14日目 C57BL/6JcLマウス(日本クレア社製)を飼育、出産させ、出生後2日令 C57BL/6JcLマウス雌、各々にStreptozocin 10 mg/mL(SIGMA社製)20μL/headに皮下注射し、4週令までCE-2(日本クレア社製)の飼料、滅菌水を与え飼育し、4週令よりHigh Fat Diet食(日本クレア社製)、滅菌水を与え、2週間飼育させた。2週間目にsiRNA C4ST-1(Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 1)、C4ST-2(Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 2)、C4ST-3(Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 3)1μg(GeneWorld社製)をsiRNA媒体である1% Atelocollagen(高研社製)と混合させたものを1回/1週間 200μL 腹腔内投与(1回/1週間)の2回(2週間)処置を行った。実験14日目にBrdU 5 mg/mL(ZyMED Laboratory.Inc社製)100μLを尾静脈内投与処置し、1時間後、解剖し、肝臓、膵臓、腎臓、精巣、卵巣、筋肉を摘出し、免疫染色用サンプル、遺伝子発現解析用サンプルを得た。体重変動に関しては、実施例期間中、14日間、経時的に測定を行った。
図21に結果を示した。縦軸に体重(g)を横軸に測定日数(days)を示した。図21よりControl群と比較してC4ST-1 siRNA処置群、C4ST-2 siRNA処置群、C4ST-3 siRNA処置群において処置10日目以降、体重増加が抑えられる傾向を示した。C4ST-2 siRNA処置群、C4ST-3 siRNA処置群においては、処置18日目に有意に(ともにp<0.05)体重増加が抑制させた。本結果はC4ST-1、C4ST-2、C4ST-3の発現を抑制することにより、2型糖尿病に付随する肥満を抑制できることを示している。
本発明の薬剤は、例えば、体重増加抑制剤、もしくは2型糖尿病に付随する肥満抑制剤として有用である。
解剖前日にインスリン抵抗性テスト(Insulin-tolerance test)としてHuman crystalline insulin(0.75 U/kg)を腹腔内投与処置し、0分後、15分後、60分後の血糖値をグルテストエース血糖値測定器(ボンビックス薬品社製)を用い測定を行い評価を実施した。siRNA処置後、0分後、15分後、60分後の血糖値の変動を図22に示した。縦軸に血糖値(mg/dL)、横軸にInsulin-tolerance test後、0分後、15分後、60分後を示した。
図22よりControl群と比較してC4ST-1 siRNA処置群、C4ST-2 siRNA処置群、C4ST-3 siRNA処置群において処置後、0分後、15分後においては有意な血糖値の減少は認められなかったが、60分後においてC4ST-1 siRNA処置群、C4ST-2 siRNA処置群、C4ST-3 siRNA処置群各々で有意な血糖値の減少が認められた。本結果は、C4ST-1、C4ST-2、C4ST-3の発現抑制により、2型糖尿病の本質的な機能障害であるインスリン抵抗性を効果的に改善できることを示している。
本発明は、例えば、2型糖尿病のインスリン抵抗性改善剤として有用である。
Streptozocin誘発性C57BL/6JcLマウス、雌より摘出した臓器(膵臓)50 mg当たりに対し、実施例1と同様にcDNAを調整し、以下の組成でPCR反応を行った。
PCR Buffer 2μL [組成:166 mM (NH4)2SO4(Sigma Aldrich Japan社製)、670 mM Tris pH8.8(Invitrogen社製)、67 mM MgCl2・6H2O(Sigma Aldrich Japan製)、100 mM 2-mercaptoethanol](WAKO社製)]、25 mM dNTP mix 0.8μL(Invitrogen社製)、DMSO 0.6μL(Sigma Aldrich Japan社製)、Primer Forward 0.2μL(GeneWorld社製)、Primer Reverse 0.2μL(GeneWorld社製)、大塚蒸留水 15.7μL(大塚製薬社製)、Taq polymerase 0.1μL(Perkin Elmer社製)、上記より得られたcDNA 1μLを混合させAuthorized Thermal Cycler(eppendorf社製)により94℃ 45second、56℃ 45second、72℃ 60second 35cycle反応させた。反応終了後、得られたPCR産物に2μL Loading Dye(Invitrogen社製)を加え、1.5% UltraPure Agarose(Invitrogen社製)ゲルを作製し、Mupid-2 plus(ADVANCE社製)により100 V、20分間 電気泳動を行った。泳動後、1×LoTE[組成:3 mM Tris-HCl(pH7.5)(Invitrogen社製)、0.2 mM EDTA(pH7.5)(Sigma Aldrich Japan社製)]にて10000倍希釈Ethydium Bromide(Invitrogen社製)染色液中で20分間~30分間振とうさせた後、I-Scope WD(ADVANCE製)に設置したEXILIM(CASIO社製)にてゲル撮影し遺伝子発現の確認を行った。
今回、用いたPrimer(Forward、Reverse)(GeneWorld社製)を以下に列記する。
[Primer配列]
*GAPDH
Forward : 5’-CTGCCAAGTATGACATCA -3’(配列番号:66)
Reverse : 5’-TACTCCTTGGAGGCCATGTAG -3’(配列番号:67)
*C4ST1(Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 1)
Forward : 5’-gtggatgaggaccacgaact-3’(配列番号:68)
Reverse : 5’-cttttcaagcggtggttgat-3’(配列番号:69)
*C4ST2(Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 2)
Forward : 5’-acctcctagacccacacacg-3’(配列番号:70)
Reverse : 5’-ggatgttggcaaaccagtct-3’(配列番号:71)
*C4ST3(Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 3)
Forward : 5’-atgagcccttcaacgaacac-3’(配列番号:72)
Reverse : 5’-tggtagaaggggctgatgtc-3’(配列番号:73)
本実施例では、2型糖尿病モデルマウスの膵臓組織サンプルを使用して、C4ST-1 siRNA、C4ST-2 siRNA、C4ST-3 siRNAのアミロイド前駆タンパク質(APP:Amyloid precursor protein)沈着抑制効果について比較検討を行った。β細胞の存在するisletにおけるAPP、アミロイド線維の沈着は、2型糖尿病の重要な病理組織所見であることは古くから知られているが、近年ではアルツハイマー病との関わりも判明している(Johnson KH et al. N Eng J Med 321:513, 1989.、Rhodes CJ. Science 307:380, 2005.、Haan MN. Nat Clin Pract Neurol 3:159, 2006.、Prentki M et al. J Clin Invest 116:1802, 2006.)。
得られた組織サンプル切片を、実施例3と同様の方法で抗アミロイド前駆体タンパクヤギ抗体(calbiochem社)を用いて染色を行い、組織レベルの発現を評価、検討した。図24に正常マウス(Normal)群、Control群、C4ST-2 siRNA処置群の組織画像を示した。2型糖尿病モデルマウスのisletにおいてはAPPの沈着が増強してくるが、Control群と比較してC4ST-2 siRNA処置群では明確に抑えられていることが確認された。
本発明の薬剤は、例えば、アミロイド線維沈着抑制剤として有用である。
採取した膵臓組織を実施例3と同様の方法で凍結ブロックならびに、組織切片を作成した。得られた切片をアセトン(和光社製)で10分間固定後、リン酸緩衝液で洗浄し、一次抗体として抗ER-TR7抗体(ラットモノクローナル抗体、1μg/ml:BMA社製)を添加し、室温で1時間反応させた。続いて、ペルオキシダーゼ標識抗ラットIgG(1:200希釈)を用いて二次抗体反応を行った後、DAB基質(ニチレイバイオサイエンス社製)を添加し発色させた。その後リリー・マイヤーヘマトキシリン(武藤化学社製)により核染色を行い、光学顕微鏡(ライカ社製)下で試料を観察し、茶色のシグナルで可視化された抗体結合を観察した。
その結果、C4ST-1 siRNA、C4ST-2 siRNA、C4ST-3 siRNA各治療群では対照群に比し、線維芽細胞の膵臓isletへの浸潤が明確に抑制されていた(図25)。したがって、C4ST-1、C4ST-2、C4ST-3遺伝子発現抑制によって、β細胞の局在するisletへの線維芽細胞浸潤と定着を抑制することで、組織の線維性変化の増強を軽減していることが判明した。
本発明の薬剤は、例えば、isletへの線維芽細胞浸潤抑制剤として有用である。
採取した膵臓組織を実施例3と同様の方法で凍結ブロックならびに、組織切片を作成した。得られた切片をアセトン(和光社製)で10分間固定後、リン酸緩衝液で洗浄し、一次抗体として抗F4/80抗体(クローンA3-1、ラットモノクローナル抗体、2μg/ml:CALTAG LABORATORIES社製)を添加し、室温で1時間反応させた。続いて、ペルオキシダーゼ標識抗ラットIgG(1:200希釈)を用いて二次抗体反応を行った後、DAB基質(ニチレイバイオサイエンス社製)を添加し発色させた。その後リリー・マイヤーヘマトキシリン(武藤化学社製)により核染色を行い、光学顕微鏡(ライカ社製)下で試料を観察し、茶色のシグナルで可視化された抗体結合を観察した。
その結果、C4ST-1 siRNA、C4ST-2 siRNA、C4ST-3 siRNA各治療群では対照群に比し、マクロファージの肺胞間質への浸潤が明確に抑制されていた(図26)。したがって、C6ST-1遺伝子発現抑制によって、マクロファージと線維芽細胞という線維性変化を持続・増強させる責任細胞群の浸潤を抑制し、総合的に組織の線維性変化を抑制していることが判明した。
本発明の薬剤は、例えば、肺胞間質へのマクロファージ浸潤抑制剤として有用である。
4位および6位硫酸化が重要であることを示すためのもう一つの実施例を示す。本実施例では、実施例11、実施例18と同様の方法でGalNAcST siRNA投与によるインスリン抵抗性の改善を検討した。実施例21と同様の方法で2型糖尿病モデルを作成し、実施例22と同様の方法でインスリン抵抗性を検査した。結果を図27に示す。
GalNAcST siRNA投与によりインスリン負荷後の良好な血糖降下作用が認められた。本結果は、GalNAc4ST-1、GalNAc4ST-2、GalNAc4S-6STの発現を抑制することにより、膵臓組織isletの線維性変化に伴うインスリン抵抗性を効果的に改善できることを示している。
本発明の薬剤は、例えば、血糖降下剤、もしくは膵臓組織のインスリン抵抗性改善剤として有用である。
腎臓組織の線維性変化は、あらゆる腎疾患のエンドポイントと考えられている。古典的には1)間質性腎疾患(tubulointerstitial disease)が腎線維形成の代表的疾患とされてきた。これには薬剤性、感染性、放射線性、重金属性間質性腎障害の他に、シェーグレン症候群や移植拒絶反応(chronic allograft nephropathyなど)、移植片対宿主反応(Graft-versus-host diseaseなど)なども含まれる。次に、2)血管性腎疾患が挙げられる。いわゆる高血圧に伴う腎硬化症の他、近年では動脈硬化症、メタボリック症候群に伴う線維形成も含まれる。さらには、糸球体原発疾患でも、蛋白尿などの結果、間質の線維形成を来たし慢性腎不全につながることから、3)原発性糸球体疾患、ならびに4)続発性糸球体疾患における腎線維性変化がある。3)にはIgA腎症、微小変化型ネフローゼ症候群、膜性腎症、膜性増殖性腎炎、巣状糸球体硬化症(FGFS:focal segmental glomerulosclerosis)が、4)には糖尿病性腎症、SLE(systemic lupus erythematosus)に伴うループス腎炎、慢性関節リウマチに伴う腎症、アミロイド腎症、B型肝炎やC型肝炎に伴う腎症などが含まれる。最後に、5)尿路閉塞に伴う間質性腎疾患が挙げられ、尿路結石、腫瘍、神経因性膀胱などが含まれる。
近年、腎機能の程度により分類・診断される臨床的な疾患概念として、これらの疾患群を慢性腎疾患(CKD:chronic kidney disease)と総称するようになった。CKDは間質の線維性変化により徐々に進行し、慢性腎不全、ESRD(end-stage renal disease)に至る。従来、ESRDに対しては人工透析しか決定的な治療法がなかった。アンギオテンシン系の降圧剤の使用によりESRDへの進行を遅らせる可能性も示唆されているが、腎線維形成そのものを標的とした治療法は確立されていない。CKDに対する新規治療が切に望まれるが、その病態進行プロセスから、線維性変化を治療標的とすることが、原発疾患の種類に関わらず共通した治療戦略として決定的な意義を持つ。CKDは全世界で5億人の患者が存在すると報告されていること、生活習慣の変化に伴い今後も一層患者数の増加が予測されること、更には、人工透析に至るまでに心血管障害で死亡するリスクが極めて高いことが大規模試験で明らかになったこと、から、CKDを21世紀型疾患として捉え、積極的な治療を行なうことが医療界の重大な課題となっている(Sergio A et al. Hypertension 38:635, 2001.、Weiner DE et al. JASN 15:1307, 2004.、Anavekar NS et al. N Eng J Med 351:1285, 2004.、Remuzzi G et al. J Clin Invest 116:288, 2006.、Tonelli M et al. BMJ 332:1426, 2006.、Khwaja A et al. Kidney International doi:10.1038/sj.ki.5002489)。
本実施例では、腎間質の組織学的な線維性変化に対する、4位及び6位硫酸基を修飾する糖鎖遺伝子阻害による変化を検討した。
実施例28と同様の方法で2型糖尿病モデルを作成した。妊娠14日目 C57BL/6JcLマウス(日本クレア社製)を飼育、出産させ、出生後2日齢C57BL/6JcLマウスにstreptozotocin(STZ:シグマ社製)を投与し、STZ誘発糖尿病モデルを作成した。マウスにSTZ(10 mg/ml)20μlを皮下に注射し、これを2日間で計3回行い、合計60μl投与した。これを4週齢まで親マウスと共に通常餌で飼育し、満4週齢で離乳させた後よりhigh fat diet(日本クレア社製)を給餌し、2週間飼育させた。2週間目にC4ST-1(Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 1)siRNA 1μg(GeneWorld社製)をsiRNA媒体である0.1% Atelocollagen(高研社製)と混合させたものを1回/1週間200μL腹腔内投与(1shot/1週間)の2回(2週間)処置を行った。実験14日目に解剖し、腎臓を摘出し、免疫染色用サンプルを得た。遺伝子発現や体重、インスリン抵抗性に関する効果は、実施例21、22、23に記載の通りである。
得られた腎臓組織サンプル切片をアセトン(和光社製)で10分間固定後、リン酸緩衝液で洗浄し、一次抗体として抗ER-TR7抗体(ラットモノクローナル抗体、1μg/ml、BMA社製)を添加し、室温で1時間反応させた。続いて、ペルオキシダーゼ標識抗ラットIgG(1:200希釈)を用いて二次抗体反応を行った後、DAB基質(ニチレイバイオサイエンス社製)を添加し発色させた。その後リリー・マイヤーヘマトキシリン(武藤化学社製)により核染色を行い、光学顕微鏡(ライカ社製)下で試料を観察し、茶色のシグナルで可視化された抗体結合を観察した。
その結果、C4ST-1 siRNA治療群では対照群に比べて腎皮質及び髄質における線維芽細胞(ER-TR7陽性細胞)の浸潤が少なかった(図28、原図はカラー)。また炎症性細胞の線維形成を定量化するため、ER-TR7陽性細胞をカウントした。それぞれの標本を顕微鏡下で400倍にて10視野観察し、陽性細胞数をカウントし対照群とC4ST-1 siRNA投与群とを比較した。その結果、対照群と比較しC4ST-1 siRNA治療群ではER-TR7の陽性率が有意に低下した(p<0.001)。
得られた腎臓組織サンプル切片をアセトン(和光社製)で10分間固定後、リン酸緩衝液で洗浄し、一次抗体として抗ER-TR7抗体(ラットモノクローナル抗体、1μg/ml、BMA社製)を添加し、室温で1時間反応させた。続いて、ペルオキシダーゼ標識抗ラットIgG(1:200希釈)を用いて二次抗体反応を行った後、DAB基質(ニチレイバイオサイエンス社製)を添加し発色させた。その後リリー・マイヤーヘマトキシリン(武藤化学社製)により核染色を行い、光学顕微鏡(ライカ社製)下で試料を観察し、茶色のシグナルで可視化された抗体結合を観察した。
その結果、C4ST-1 siRNA治療群では対照群に比べて腎皮質及び髄質におけるマクロファージ(F4/80陽性細胞)の浸潤が少なかった(図29、原図はカラー)。また、実施例29と同様に組織病変を定量化すると、対照群と比較し、C4ST-1 siRNA治療群ではマクロファージの陽性率が有意に低下していた(p<0.001)。
腎臓組織サンプル切片をアセトン(和光社製)で10分間固定後、リン酸緩衝液で洗浄し、一次抗体として抗ヒト平滑筋線維アクチン抗体(αSMA : マウスモノクローナル抗体、1:100、DACO社製)を添加し、室温で1時間反応させた。続いて、ヒストファインマウスステインキット(ニチレイバイオサイエンス社製)を用いて二次抗体反応を行った後、DAB基質(ニチレイバイオサイエンス社製)を添加し発色させた。その後リリー・マイヤーヘマトキシリン(武藤化学社製)により核染色を行い、光学顕微鏡(ライカ社製)下で試料を観察し、茶色のシグナルで可視化された抗体結合を観察した。
その結果、C4ST-1 siRNA治療群では対照群に比べて傍糸球体及び間質におけるαSMA陽性細胞の定着が明確に減少していた(図30、原図はカラー)。αSMAは線維芽細胞活性化の機能的なマーカーであり、本結果は、C4ST-1の遺伝子発現を抑制することで組織に浸潤している線維芽細胞の活性化が抑制していることを示している。
本発明の薬剤は、例えば、線維芽細胞活性化抑制剤として有用である。
実施例28と同様の方法で糖尿病性腎症モデルを作成し、GalNAc4S-6ST siRNAの効果を検討した。本実施例では経過をより長期に渡って観察した。GalNAc4S-6ST siRNAの配列は実施例1と同様であり、8週齢ならびに9週齢の時点で各1回腹腔内投与を施した。さらに、比較対象としてアンギオテンシンII受容体阻害剤(ARB)であるValsartanを30 mg/kgの容量で経口投与を、同一スケジュールで行なった。10週齢で免疫組織学的検査と遺伝子発現検査のための腎臓組織を採取した。実施例1と同様の方法で腎臓組織の定量的PCRを行い、遺伝子発現を検討した。本実施例では内部コントロールとして36B4を使用しており、その配列を以下に示す。
[定量PCR Primer配列]
*mouse 36B4(タカラバイオ社製)
forward : 5’-TTCCAGGCTTTGGGCATCA -3’(配列番号:74)
reverse : 5’-ATGTTCAGCATGTTCAGCAGTGTG -3’(配列番号:75)
結果を図31に示す(なお、GalNAc4S-6STは図中ではG#1と表記する)。糖尿病性腎症モデルにおいては、腎臓組織のGalNAc4S-6STの発現が増強するが、GalNAc4S-6ST siRNAの投与により、腎臓組織でのGalNAc4S-6ST遺伝子発現が有意に抑制されていた。さらに、線維化のマーカーであるαSMAとTGFβの発現増強が有意に抑制されていた。ARBと治療効果を比較検討すると、TGFβに関しては両群とも抑制効果を示し、有意差は出なかったが、αSMAに関してはGalNAc4S-6ST siRNA投与群がARB投与群に比較し有意な抑制効果を示した。本結果は、GalNAc4S-6STの遺伝子発現を抑制することにより、腎臓組織における線維性変化を示すマーカーが抑制できたことを示している。更には、線維芽細胞の活性化(αSMAの増強)に関しては、GalNAc4S-6ST siRNAの効果が際立って優れていることも示された。
線維芽細胞の腎組織内浸潤の程度は、実施例28と同様の方法で免疫組織学的検討を加え、定量的評価を行った。結果を図32に示す。GalNAc4S-6ST siRNA投与により、線維芽細胞の腎間質内浸潤は有意に抑制された。間質全体的な定量的検索ではARBとの有意差を認めなかったが、傍糸球体領域に浸潤する線維芽細胞に限定した検討では、GalNAc4S-6ST siRNA投与群が図の如く明確な抑制効果を示した。
本発明の薬剤は、例えば、線維芽細胞の腎間質内浸潤抑制剤として有用である。
線維芽細胞の腎組織内浸潤の程度は、実施例29と同様の方法で免疫組織学的検討を加え、定量的評価を行った。結果を図33に示す。GalNAc4S-6ST siRNA投与により、マクロファージの腎間質内浸潤は有意に抑制された。間質全体的な定量的検索ではARBとの有意差を認めなかったが、傍糸球体領域に浸潤するマクロファージに限定した検討では、GalNAc4S-6ST siRNA投与群が図の如く明確な抑制効果を示した。
本発明の薬剤は、例えば、マクロファージの腎間質内浸潤抑制剤として有用である。
実施例32、33と同様の方法で、タイプIVコラーゲンの免疫染色を行った。一次抗体として抗マウスIV型コラーゲンウサギ抗血清(エル・エス・エル社製)を添加し、室温で1時間反応させた。続いて、ペルオキシダーゼ標識抗ラビットIgG(1:200希釈)を用いて二次抗体反応を行った後、DAB基質(ニチレイバイオサイエンス社製)を添加し発色させた。その後リリー・マイヤーヘマトキシリン(武藤化学社製)により核染色を行い、光学顕微鏡(ライカ社製)下で試料を観察し、茶色のシグナルで可視化された抗体結合を観察した。
IVコラーゲンの蓄積を定量化するため、糸球体周囲に茶色のシグナルで可視化されたコラーゲンの厚みを測定した。それぞれの標本を糸球体15~20個において糸球体周囲で最も肥厚している部分の厚みをモニター上でノギスを用いて計測した。その結果、コントロール群に比較し、GalNAc4S-6ST siRNA投与群で有意に糸球体基底膜(GBM)の肥厚が抑制されていた(図34)。ARBと比較した場合、GalNAc4S-6ST siRNA投与群で肥厚をより優勢に抑制する傾向を示した。
本発明の薬剤は、例えば、糸球体基底膜肥厚抑制剤として有用である。
糖尿病性腎症の線維形成においてはアンギオテンシンIIの関与が報告されている。本実施例では腎臓組織の定量的PCRでアンギオテンシン経路の検討を行った。本モデルにおいてはアンギオテンシノーゲン及びアンギオテンシン変換酵素(ACE)の発現に増強が認められ(図35)、これが腎局所のアンギオテンシンIIを増強させる要因と考えられた。GalNAc4S-6ST siRNA投与群においてはアンギオテンシノーゲンとACEの抑制効果を認めた(図35)。本結果により、GalNAc4S-6STの遺伝子抑制によって線維芽細胞の浸潤・活性を抑制することにより、アンギオテンシン経路においても改善効果を示すことが判明した。
本発明の薬剤は、例えば、アンギオテンシノーゲン発現抑制剤、もしくはアンギオテンシン変換酵素抑制剤として有用である。
糖尿病性腎症からESRDに至る過程で、腎機能の低下に伴い血清クレアチニン濃度が上昇するため、臨床的に最も汎用される腎機能マーカーである。しかしながら、血清クレアチニンが上昇した時点では既に機能的ネフロンの50%の機能低下を来している事が明らかになっており、如何にクレアチニン上昇前に腎機能を保護するかが臨床的には大きい課題である。
本モデルにおいても既に組織学的線維形成が進行した18週齢においてようやく血清クレアチニン濃度の上昇を認めた(図36)。コントロール群に比較して、GalNAc4S-6ST siRNA投与群においては、血清クレアチニンの上昇を抑制した(図36)。本結果は、GalNAc4S-6STの遺伝子抑制により、腎組織の線維性変化を抑制する事で、血清クレアチニンの上昇すなわち腎機能の低下を抑制するという、極めて有効な腎保護作用を呈する事を示している。
本発明の薬剤は、例えば、腎機能低下抑制剤、もしくは腎保護剤として有用である。
4位及び6位硫酸化が重要である事をもう一つの実施例で示す。実施例11と同様の方法で、GalNAcST siRNA投与による腎間質の線維性変化を検討した。GalNAcST siRNAの投与スケジュールは実施例28と同様である。実施例31と同様の方法で、腎臓組織を用いた定量的PCRを行った。本実施例では内部コントロールにβアクチンを使用しており、その配列を以下に示す。
[定量PCR Primer配列]
*mouse βアクチン(タカラバイオ社製)
forward : 5’-CATCCGTAAAGACCTCTATGCCAAC -3’ (配列番号:76)
reverse : 5’-ATGGAGCCACCGATCCACA -3’ (配列番号:77)
図37に示す通り、糖尿病性腎症モデルの腎組織においては、GalNAc4ST-1、GalNAc4ST-2、GalNAc4S-6STの発現増強を認めたが、GalNAcST siRNA投与により、全ての遺伝子発現が有意に抑制された。
GalNAcST siRNA投与群においては、対照群と比較して、腎組織における線維形成のマーカーであるαSMA、TGFβ、CTGFの発現増強も有意に抑制された(図38)。更には、ACEの発現増強も有意に抑制された。本結果は、GalNAc4ST-1、GalNAc4ST-2、GalNAc4S-6STの発現を抑制する事によって、腎組織の線維形成を抑制し、腎保護に働く事を示している。実施例35の結果と併せると、いずれの遺伝子を抑制してもACEの低下が顕著である事から、血圧低下作用を有する事もまた明らかである。
本発明の薬剤は、例えば、降圧剤として有用である。
本実施例では、典型的な薬剤性間質性腎炎におけるGalNAc4S-6ST siRNAの効果を検討した。最初に、マウスモデルの作製についてであるが、C57BL6/Jマウス(オス、8週齢、日本クレア社製)に、アドリアマイシン(15 mg/kg;協和発酵社製)を腹腔内投与する。投与後1週間飼育し、腎臓組織を採取した。対照群は、同様のマウスでアドリアマイシンを投与せず、同時期に購入・飼育したものを用いた。
GalNac 4S-6ST siRNAは、実施例1と同様の方法で、アドリアマイシン投与の24時間前に、マウス1匹あたり、GalNac 4S-6ST siRNA 1μg(北海道システムサイエンス社製)と、媒体である1%アテノコラーゲン(高研社製)200μLを混合したもの腹腔内投与した。本実施例における典型的な薬剤性間質性腎炎のモデルにおいても、腎臓組織におけるGalNac 4S-6STの発現増強を認め(図39)、GalNac 4S-6ST siRNA投与によりその発現は有意に抑制された。
本実施例では、実施例34と同様の方法でタイプIコラーゲンの免疫染色を行った。一次抗体として抗ラットI型コラーゲンウサギ抗血清(エル・エス・エル社製)を添加し、室温で1時間反応させた。続いて、ペルオキシダーゼ標識抗ラビットIgG(1:200希釈)を用いて二次抗体反応を行った後、DAB基質(ニチレイバイオサイエンス社製)を添加し発色させた。その後リリー・マイヤーヘマトキシリン(武藤化学社製)により核染色を行い、光学顕微鏡(ライカ社製)下で試料を観察し、茶色のシグナルで可視化された抗体結合を観察した。
結果を図40に示す。コントロール群では傍糸球体から間質にかけて広くタイプIコラーゲンの沈着を認めるのに対し、GalNac 4S-6ST投与群では顕著に沈着が抑制されていた。本結果は、薬剤性間質性腎炎においても、GalNac 4S-6STの発現を抑制する事で、腎組織の線維性変化を抑制できる事を示している。
本発明の薬剤は、例えば、腎組織の線維性変化抑制剤として有用である。
まず、マウス腎線維化モデルの作成に関してであるが、C57BL/6JcLマウス(♀、8週齢、日本クレア社製)に片側尿管結紮術(UUO)を行い、腎線維化モデルを作成した。この腎線維症モデルマウスは再現性に優れており、マウスの腎線維症の実験系で広く用いられている(American journal of pathology 2003 163;4 1261-1273)。マウスをケタラール/キシラジン麻酔下で開腹し、尿管を露出させ、右尿管を4-0号手術用糸で2カ所結紮し、1-0号手術用糸にて腹膜、皮膚を縫合した。
腎線維化モデルマウスの典型例として片側尿管結紮術(UUO)による腎線維化モデルマウスを用いてC6ST siRNA投与によるC6ST発現の抑制効果をPCR法により確認した。C57BL/6JcLマウス(♀、8週齢、日本クレア社製)にUUOを施術し腎線維化モデルを作成した。また、UUOを施術する直前に、マウスにはC6ST-1 siRNA、C6ST-2 siRNA混合物(1μg/匹、ジーンワールド社製)、またはPBSをsiRNA媒体である0.1% Atelocollagen(高研社製)に混合し200μlを腹腔内に注射した。この処置を行ったマウス群をC6ST siRNA群、対照群と名付けた。実験8日目に解剖し、UUO施術腎を摘出し、免疫染色用サンプル、遺伝子発現解析用サンプルを得た。実施例1と同様の方法で定量的PCRを行なった。
今回、用いたC6ST-1 siRNA、C6ST-2 Primer(Forward、Reverse)(GeneWorld社製)を以下に示す。
*C6ST1(Chondroitin 6-sulfotransferase-1)
Forward : 5’-tgtgtggacacacctcccta-3’(配列番号:78)
Reverse : 5’-cttcaaaggtccccttcctc-3’(配列番号:79)
*C6ST2(Chondroitin 6-sulfotransferase-2)
Forward : 5’-cagcttgagccatttcaaca-3’(配列番号:80)
Reverse : 5’-gggtgaggcctttaggaaac-3’(配列番号:81)
[C6ST-1カクテル配列](Gene Bank accession number NM_016803)
(Gene world社製)
5’-gcgccccctctccccatggagaaag-3’(配列番号:82)
5’-gctttgcctcaggatttccgggacc-3’(配列番号:83)
5’-ggttcagccttggtctaccgtgatgtc-3’(配列番号:84)
5’-gcagttgttgctatgcgacctgtat-3’(配列番号:85)
[C6ST-2カクテル配列](Gene Bank accession number NM_021715)
(Gene world社製)
5’-tggggagagtgaggattcggtgaa-3’(配列番号:86)
5’-cggacgtgggactcgtcgaggacaaag-3’(配列番号:87)
5’-cgaaagtacctgcccgcccgtttcgc-3’(配列番号:88)
その結果、腎臓におけるC6ST-2(G#10)の発現増強が認められた(図41、C6ST-2をG#10と表記した)。C6ST siRNA治療群では発現減少が認められ、Atellocollagen媒体C6ST siRNA投与によりC6ST-2遺伝子のノックダウンが確認された(図41)。また、TGFβ、αSMA、タイプIコラーゲン、CTGFといった線維形成マーカーの発現増強も、C6ST siRNA投与により有意に抑制された。
摘出した組織を凍結用包埋剤OCTコンパウンド(サクラ社製)にて包埋し、液体窒素で凍結ブロックを作成し、クリオスタット(マイクロエッジ社製)を用いて厚さ6μmの切片を作成した。得られた切片を、実施例28と同様の方法で抗ER-TR7抗体を用いて免疫染色を行なった。光学顕微鏡(ライカ社製)下で試料を観察し、茶色のシグナルで可視化された抗体結合を観察し、定量化を行った。
その結果、UUO施術腎において、C6ST siRNA治療群では対照群に比べて腎間質における線維芽細胞の集積が有意に抑制されていた(図42)。
本発明の薬剤は、例えば、腎間質における線維芽細胞集積抑制剤として有用である。
実施例42と同様に、抗F4/80抗体で免疫染色、定量化を行った。その結果、UUO施術腎において、C6ST siRNA治療群では対照群に比べて腎間質におけるマクロファージの集積が有意に抑制されていた(図43)。
本発明の薬剤は、例えば、腎間質におけるマクロファージ集積抑制剤として有用である。
実施例42と同様に、抗タイプIVコラーゲン抗体で免疫染色を行い、実施例34と同様に定量化を行った。その結果、UUO施術腎において、C6ST siRNA治療群では対照群に比べて糸球体基底膜の線維性肥厚が有意に抑制されていた(図44)。このことから、C6ST-2の遺伝子発現を抑制する事により、炎症性細胞の浸潤、線維形成を抑制しうる事が認められた。
本発明の薬剤は、例えば、炎症性細胞浸潤抑制剤として有用である。
実施例30と同様に抗αSMA抗体で免疫染色を行った。その結果、UUO施術腎において、C6ST siRNA治療群では対照群に比べて、間質とりわけ傍糸球体領域におけるαSMA陽性細胞の明らかな減少を認めた(図45)。本結果は、C6ST-1ならびにC6ST-2の遺伝子発現を抑制する事により、腎間質に集積している線維芽細胞の活性化が抑制される事を示している。
本発明の薬剤は、例えば、腎間質における線維芽細胞活性化抑制剤として有用である。
同様に、抗ヒトACEウサギ抗体(サンタクルーズ社製)を用いて免疫染色を行なった。その結果、UUO施術腎において、C6ST siRNA治療群では対照群に比べて、間質とりわけ傍糸球体領域におけるACE陽性細胞の明らかな減少を認めた(図46)。本結果は、C6ST-2の遺伝子発現を抑制する事により、腎間質に集積している線維芽細胞の活性化が抑制され、それに伴ってACEの発現も抑制される事を示している。ACE抑制は血圧低下作用を有するため、本結果においても血圧降下あるいは動脈硬化抑制作用を有するものと強く考えられた。
本発明の薬剤は、例えば、動脈硬化抑制剤として有用である。
〔眼組織〕
眼組織においても他臓器と同様に、種々の原因による侵襲に対し、線維性変化が生じ、視力の低下・損失を招く。主要な疾患として、糖尿病網膜症、網膜静脈閉塞症、未熟児網膜症、加齢黄斑変性症、網膜色素変性症などが挙げられるが、角膜の炎症に伴って生じる線維形成、緑内障に伴う線維形成、白内障に伴う線維形成も含まれる(総説:Fiedlander M. J Clin Invest 117:576-586, 2007、Harada T et al. Genes and Dev. 21:367-378, 2007)。病理組織学的には線維形成に伴う視細胞の損傷・減少が視力低下の大きな要因となるため、眼組織の線維形成を抑制する事は、あらゆる眼疾患の視力低下を防ぐための新たな治療戦略として期待されている。
本実施例では糖尿病性網膜症のモデルにおける、網膜の線維形成とそれに伴う視細胞の損失を中心に検討を加えた。
実施例47:マウス糖尿病性網膜症モデルにおけるGalNAc4S-6ST (G#1) siRNAのコラーゲン集積における検討:
妊娠14日目 C57BL6J/JcLマウス(日本クレア社製)を飼育、出産させ、出生後2日令 C57BL6J/JcLマウス雌(日本クレア社製)、各々にStreptozocin 10 mg/mL(SIGMA社製) 20μL/head に皮下注射し、4週令までCE-2(日本クレア社製)の飼料、滅菌水を与え飼育し、4週令よりHigh Fat Diet食(日本クレア社製)、滅菌水を与え、2週間飼育させた。8週間目と9週間目に実施例11と同様の方法で、GalNac4S-6ST (G#1)siRNA(北海道システムサイエンス社製)をマウス1匹あたり1μgを、媒体である1%アテノコラーゲン(高研社製)200μLを混合したもの腹腔内投与した。18週間目にGalNac4S-6ST (G#1)siRNAの効果を検討するため、両群マウスの眼球を摘出し、免疫組織学的検討を加えた。
摘出眼で凍結ブロック・切片を作成した。切片をアセトン(シグマアルドリッチジャパン社製)で10分間固定後、リン酸緩衝液で洗浄し、さらに一次抗体としてウサギ由来抗IV型コラーゲン抗血清(1:2000希釈;LSL社製)を添加し、室温で一時間反応させた。続いて、二次抗体であるペルオキシダーゼ標識抗ウサギIgG(1: 25希釈;Cappel社製)を添加し、室温で30分反応させた後、DAB基質を(ニチレイ社製)添加し酵素色素反応を行った。この標本を光学顕微鏡(ライカ社製)用いて観察した。
得られた網膜の組織像を図47に示した。対照群では視力に重要な視神経節細胞層(GCL:ganglion cell layer)から内顆粒層(INL:inner nuclear layer)にかけてタイプIVコラーゲンの沈着が増加していたのに比較し、GalNac4S-6ST (G#1)siRNA投与群では、とりわけGCLにおけるコラーゲン増生が顕著に抑制されていた。
本発明の薬剤は、例えば、視神経節細胞層におけるコラーゲン増生抑制剤として有用である。
実施例47と同様の方法で網膜の免疫組織学的検討を行った。一次抗体として抗コンドロイチン硫酸プロテオグリカン(CSPG)抗体(クローンCS56、マウスモノクローナル抗体、1:100;生化学工業社製)を添加し、室温で一時間反応させた。続いて、ヒストファインマウスステインキット(ニチレイ社製;マウスモノクローナル抗体に対して使用)を用いて二次抗体反応を行った。
図48に示すように、対照群においては、網膜のGCLならびに外顆粒層(ONL: outer nuclear layer)から網膜色素上皮細胞層にかけてのセグメントにCS56陽性所見の著しい増強を認めた。これに対して、GalNac4S-6ST (G#1)siRNA投与群ではCS56強度が顕著に減弱していた。それに伴って、GCL層における神経節細胞ならびに網膜色素上皮細胞の形態が極めて良好に維持されていた。
以上より、本モデルマウスにおいて誘導される網膜組織におけるCSPGの沈着は、GalNac4S-6ST (G#1)siRNAの生体内投与により顕著に抑制されることが明らかになった。CSPGはコラーゲンとともに線維形成病変形成に重要であると考えられる他、神経節細胞が軸索を伸張する過程を阻害すると報告されている(Brittis PA et al. Science 255:733, 1992)。既報ではin vitroの実験結果ならびに発生過程の報告しかなく、生体内病変での役割は全く未知であったが、本結果により初めて病変組織における役割が示唆された。
網膜損傷時における生体防御機構の一環として、視神経の再生が報告されてきたが、そのような傷害後再生に関わる視神経前駆細胞はグリア細胞である事が報告されている(Fischer AJ et al. Nature neuroscience 4:247, 2001、Ooto S et al. PNAS 101:13645, 2004)。本実施例ではグリア細胞のマーカーとして抗GFAPヤギ抗体(サンタクルーズ社製)を用い、実施例47と同様の方法で免疫染色を行なった。
結果を図49に示す。正常群、対照群ではGFAP陽性グリア細胞数に変化はなかったが、GalNac4S-6ST (G#1)siRNA投与群では、INLからGCLにかけて顕著な細胞数の増加を認めた。視神経の再生はINLからGCLに向かって起こると報告されている事から、本結果は、GalNac4S-6ST (G#1)siRNAにより、視神経の再生が活発に行なわれている過程を示すと考えられる。
本発明の薬剤は、例えば、視神経再生剤として有用である。
実施例47に作製したサンプルを用いて、神経節細胞の数を定量化した。その結果、糖尿病性網膜症モデルにおいてもGCLにおける神経節細胞の減少が認められたが、GalNac4S-6ST (G#1)siRNA投与によりこのような損失が有意に回復する事が明らかになった(図50)。
実施例1と同様の方法で眼組織のRNAを抽出し、定量的PCRを行なった。ミューラー細胞のマーカーであるglutamate synthetase(GS)の発現の変化を解析する事により、視神経再生能力に検討を加えた。PCRプライマー配列を以下に記す。
[定量PCR Primer配列]
*mouse GS(タカラバイオ社製)
forward : 5’-CTGTGAGCCCAAGTGTGTGGA -3’(配列番号:89)
reverse : 5’-GTCTCGAAACATGGCAACAGGA -3’(配列番号:90)
結果を図51に示す。GalNac4S-6ST (G#1)siRNA投与により、眼組織のGalNAc4S-6STの発現増強が有意に抑制できた。これに伴い、GalNac4S-6ST (G#1)siRNA投与群では、眼組織におけるGSの発現が有意に上昇していた。本結果は、GalNac4S-6ST (G#1)siRNA投与によりミューラー細胞の再生すなわち視神経の回復を導く事を示している。
本実施例により、GalNAc4S-6STの遺伝子発現を抑制する事で、網膜組織の線維性変化を抑制でき、結果として神経節細胞の再生に基づく視細胞損失の回避につながる事が判明した。このような組織学的所見は緑内障や糖尿病性網膜症を始め、幅広い眼線維化病変に共通する所見である。
本発明の薬剤は、例えば、ミューラー細胞再生剤、もしくは、神経節細胞再生剤として有用である。
肝臓における組織の線維性変化はあらゆる肝疾患の進行像あるいは終末像である。ウイルス性肝炎(A型肝炎ウイルス、B型肝炎ウイルス、C型肝炎ウイルス、D型肝炎ウイルス、E型肝炎ウイルス、G型肝炎ウイルス)由来の肝線維形成のみならず、アルコール性肝障害、非アルコール性脂肪性肝障害(NAFLD及びNASH)、代謝性肝障害、薬剤性肝障害、特発性門脈圧亢進症、バッド・キアリ症候群、自己免疫性肝炎、原発性胆汁性肝硬変、原発性硬化性胆管炎、胆道系疾患(胆道閉鎖症や胆道拡張症を含む)、腫瘍等の膵疾患による胆道閉鎖、移植片対宿主反応、慢性拒絶反応など、多岐にわたる(Bataller R et al. J Clin Invest 115:209, 2005. Iredale JP. J Clin Invest 117:539, 2007)。本実施例では、肝臓組織レベルの線維性変化の検討を行った。
妊娠14日目 C57BL/6JcLマウス(日本クレア社製)を飼育、出産させ、出生後2日齢C57BL/6JcLマウスにstreptozotocin(STZ:シグマ社製)を投与した。マウスにSTZ(10 mg/ml)20μlを皮下に注射し、これを2日間で計3回行い、合計60μl投与した。これを4週齢まで親マウスと共に通常餌で飼育し、満4週齢で離乳させた後よりhigh fat diet(日本クレア社製)を給餌し、2週間飼育させた。2週間目に、実施例11に記載のGalNAcST siRNAを1回/1週間200μL腹腔内投与(1 shot/1週間)の2回(2週間)処置を行った。実験14日目に解剖し、肝臓を摘出し、遺伝子発現解析ならびに免疫染色用サンプルを得た。
本実施例では、実施例1と同様の方法で、肝臓組織の定量的PCRを行なった。結果を図52に示す。本モデルの肝臓組織ではGalNAc4S-6STの発現が増強するが、GalNAcST siRNA投与により有意に抑制された。
実施例52と同様の方法で、肝臓組織の線維形成マーカーの発現を検討した。本モデルにおいては、タイプIコラーゲンならびにαSMAの発現が有意に上昇した(図53)。したがって、肝臓組織における線維性変化が増強している事が示された。これに対し、GalNAcST siRNA投与により、これらの線維形成マーカーの増強が有意に抑制された。
次に、実施例3と同様の方法で肝臓組織の免疫染色を行なった。一次抗体としてラット抗マウス線維芽細胞抗体(クローンER-TR7;1:500希釈;BMA社製)を添加し、室温で1時間反応させた。続いて、二次抗体としてペルオキシダーゼ標識抗ラットIgG(1:200希釈;バイオソース社製)を添加して、室温で30分更に反応させた後、DAB基質(ニチレイ社製)を添加した。光学顕微鏡(ライカ社製)下で試料を観察し、茶色のシグナルで可視化された抗体結合を確認した。
得られた肝組織の免疫染色像の例を図53に示す。対照群では線維芽細胞の集積が明確であり、ブリッジングを形成している組織像を認めた。これに比較し、GalNAcST siRNA投与群では線維芽細胞の集積をほとんど認めなかった。
実施例54で行った免疫組織学的染色に基づき、各試料の肝線維形成の程度を既報(Dai K, et al. World J Gactroenterol. 31:4822-4826, 2005、Hillebrandt S, et al. Nature Genetics 37:835-843, 2005)に従って、線維形成度数で評価した。線維形成度数の評価基準は以下の通りである。0度:正常、1度:中心静脈からの少量のコラーゲン伸長が認められる、2度:中心静脈からの明白なコラーゲン伸長が認められるが、肝臓全体を取り囲まない程度である、3度:中心静脈からの明白なコラーゲン伸長が認められ、肝臓全体を取り囲む程度である、4度:肝臓全体の瀰漫性コラーゲン伸長が認められ、偽小葉を形成している。
結果を図55にグラフで示す。各バーは、各群における線維形成度数の平均値±標準偏差を示している。対照群と比較すると、GalNAcST siRNA投与群(p<0.01、t検定)で統計学的に有意に線維形成が軽減されていた。以上より、GalNAcST siRNAの投与は、臨床的にも優れた肝線維形成抑制効果を発揮することが示された。
次に、実施例54と同様の方法で肝臓組織の免疫染色を行なった。一次抗体としてラット抗マウスF4/80抗体(クローンA3-1;2μg/ml;CALTAG LABORATORIES社製)を添加し、室温で1時間反応させた。続いて、二次抗体としてペルオキシダーゼ標識抗ラットIgG(1:200希釈;バイオソース社製)を添加して、室温で30分更に反応させた後、DAB基質(ニチレイ社製)を添加した。光学顕微鏡(ライカ社製)下で試料を観察し、茶色のシグナルで可視化された抗体結合を確認した。
得られた肝組織の免疫染色像の例を図56に示す。対照群ではマクロファージの集積が明確であり、炎症集簇層を形成している組織像を認めた。これに比較し、GalNAcST siRNA投与群ではマクロファージの過剰集積を認めなかった。
実施例52と同様の方法で肝臓の脂質代謝関連遺伝子の発現を検討した。ChREBP(carbohydrate response element-binding protein)ならびにACC2(Acetyl-CoA carxylase-2)の発現が増強していたが、GalNAcST siRNA投与群で有意に抑制された(図57)。本結果は、GAlNAc4ST-1、GalNAc4ST-2ならびにGalNAc4S-6STの遺伝子発現を抑制する事によって、肝臓組織の線維性変化が抑制でき、結果として糖脂質代謝が改善出来る事を示している。
本発明の薬剤は、例えば、糖脂質代謝改善剤として有用である。
実施例52と同様の方法で脂肪性肝障害モデルを作成し、同様の投与プロトコールで、実施例21に記載のC4ST-1 siRNA、C4ST-2 siRNA、C4ST-3 siRNAを投与し、肝臓組織を採取した。
次に、実施例3と同様の方法で肝臓組織の免疫染色を行なった。一次抗体としてラット抗マウス線維芽細胞抗体(クローンER-TR7;1:500希釈;BMA社製)を添加し、室温で1時間反応させた。続いて、二次抗体としてペルオキシダーゼ標識抗ラットIgG(1:200希釈;バイオソース社製)を添加して、室温で30分更に反応させた後、DAB基質(ニチレイ社製)を添加した。光学顕微鏡(ライカ社製)下で試料を観察し、茶色のシグナルで可視化された抗体結合を確認した。
得られた肝組織の免疫染色像の例を図58に示す。対照群では線維芽細胞の集積が明確であり、ブリッジングを形成している組織像を認めた。これに比較し、C4ST-1 siRNA、C4ST-2 siRNA、C4ST-3 siRNA投与群では全ての群において線維芽細胞の集積を認めなかった。
実施例58で行った免疫組織学的染色に基づき、各試料の肝線維形成の程度を線維形成度数で評価した。結果を図59に示す。各バーは、各群における線維形成度数の平均値±標準偏差を示している。対照群と比較すると、C4ST-1 siRNA、C4ST-2 siRNA、C4ST-3 siRNA投与群(p<0.001、t検定)で全てに統計学的に有意に線維形成が軽減されていた。以上より、C4ST-1 siRNA、C4ST-2 siRNA、C4ST-3 siRNAの投与は、臨床的にも優れた肝線維形成抑制効果を発揮することが示された。
実施例58のプロトコールでマウス屠殺時に採血を施し、血清中ALT(アラニントランスフェラーゼ)値をSRL社に委託し、測定した。結果を図60に示す。血清ALT値は肝細胞の破壊を示す臨床的に最も汎用される指標である。対照群では血清ALT値の上昇を認めたのに比較し、C4ST-1 siRNA、C4ST-2 siRNA、C4ST-3 siRNA投与群では平均値で50%以下の低下を認めた(図60)。本結果は、C4ST-1、C4ST-2、C4ST-3の遺伝子発現を抑制する事により、肝臓組織の線維性変化を抑制でき、結果として肝細胞障害を軽減し得る事を示している。
本発明の薬剤は、例えば、肝細胞障害軽減剤として有用である。
本実施例では、実験的に最も汎用されるマウス四塩化炭素誘導肝硬変モデルを用いて実験を行なった。まずマウスモデルの作成であるが、C57BL6/Jマウス(雌、5~6週齢、日本クレア社製)に、四塩化炭素(25μL/100 g体重;Sigma-Aldrich社製)を、週2回ずつ4週間にわたり(8回)、腹腔内に注射し、肝線維症を惹起した。さらに四塩化炭素を週2回ずつ2週間にわたって追加投与し(総計12回)、肝硬変を誘導した。肝硬変が誘導されたマウスを屠殺し、その肝臓を採取した(硬変肝)。一方、対照実験では、四塩化炭素の投与を行っていない同週齢のC57BL6/Jマウス(雌、日本クレア社製)から肝臓を採取した(正常肝)。
四塩化炭素の追加投与(9回目から12回目までの合計4回)と同様に、合計4回、実施例41と同一のC6ST siRNAとそのコントロールを腹腔内投与した。追加投与終了後、両群のマウスを屠殺し、肝臓組織切片を作成、免疫組織学的検討を行った。一次抗体としてラット抗マウス線維芽細胞抗体(クローンER-TR7;1:500希釈;BMA社製)を添加し、室温で1時間反応させた。続いて、二次抗体としてペルオキシダーゼ標識抗ラットIgG(1:200希釈;バイオソース社製)を添加して、室温で30分更に反応させた後、DAB基質(ニチレイ社製)を添加した。光学顕微鏡(ライカ社製)下で試料を観察し、茶色のシグナルで可視化された抗体結合を確認した。
得られた肝組織の免疫染色像の例を図61に示す。対照群では線維芽細胞の集積が明確であり、ブリッジングを形成している組織像を認めた。これに比較し、C6ST siRNA投与群では全ての群において線維芽細胞の集積を認めなかった。
実施例61で行った免疫組織学的染色に基づき、各試料の肝線維形成の程度を線維形成度数で評価した。結果を図62に示す。各バーは、各群における線維形成度数の平均値±標準偏差を示している。対照群と比較すると、C6ST siRNA投与群(p<0.05、t検定)で全てに統計学的に有意に線維形成が軽減されていた。以上より、C4ST-1 siRNA、C4ST-2 siRNA、C4ST-3 siRNAの投与は、臨床的にも優れた肝線維形成抑制効果を発揮することが示された。
実施例1と同様の方法で肝臓組織よりRNAを抽出し、定量的PCRを行なった。結果を図63に示す。対照群では線維形成マーカーであるαSMA、タイプIコラーゲン、CTGF、TGFβの発現増強を認めたが、C6ST siRNA投与群においては、有意に抑制されていた。本結果は、C6ST-1、C6ST-2の遺伝子発現抑制により、肝臓組織の線維性変化が抑制できる事を示している。
〔脳神経組織〕
本実施例ではパーキンソン病マウスモデルとして基本的な、MPTP誘導パーキンソン病モデルを使用した。このモデルは古典的であるが再現性にすぐれ、簡便であることよりパーキンソン病モデルとして汎用されている。組織学的には脳実質組織内への炎症細胞の浸潤と、ドーパミン産生ニューロンの減少を特徴とする。このような病変におけるニューロンの減少の原因として、古典的には神経原線維性変化が考えられている。
したがって、神経が障害される病態、すなわち、パーキンソン病、進行性核上性麻痺、大脳皮質基底核変性症、アルツハイマー病、ポリグルタミン病、筋萎縮性側索硬化症(ALS)、脊髄性進行性筋萎縮症、球脊髄性筋萎縮症、ハンチントン病、多発性硬化症などの代表的な神経変性疾患のみならず、多系統萎縮症(線条体黒質変性症、オリーブ橋小脳萎縮症、シャイ・ドレーガー症候群)、副腎白質ジストロフィー、ギラン・バレー症候群、重症筋無力症、フィッシャー症候群、慢性炎症性脱随性多発神経炎、ルイス・サムナー症候群、クロウ・フカセ症候群、正常圧水頭症、脊髄空洞症、プリオン病(クロイツフェルト・ヤコブ病、ゲルストマン・ストロイスラー・シャインカー病、致死性家族性不眠症)、亜急性硬化性全脳炎(SSPE)、進行性多巣性白質脳症(PML)、脊髄小脳変性症などを含む。更には、脳外傷後遺症、脳血管障害(脳梗塞、脳出血)後遺症、ウイルス性脳炎後遺症、細菌性髄膜炎後遺症、脊髄損傷後遺症や、脊髄神経、末梢神経、聴神経、視神経などにおける神経原線維性変化も含まれる。とりわけ上記の後遺症は抑うつ状態などの精神疾患の基盤となるものとの考えが古くからあるため、神経原線維性変化は精神症状の原因としても重要である。
本実施例では1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine(MPTP)によりドーパミンニューロンを選択的に変性させたパーキンソン病のマウスモデルを作成(Amende et al. (2005)Journal of NeuroEngineering and Rehabilitation 2(20) 1-13)してGalNAc4S-6ST siRNAを投与し、治療後における遺伝子発現と組織の様子を比較検討した。
妊娠14日目 C57BL/6JcLマウス(日本クレア社製)を飼育、出産させ、8週令 C57BL/6JcLマウス雌(日本クレア社製)、各々に実施例1と同一のGalNAc4S-6ST siRNA 1μg(GeneWorld社製)をsiRNA媒体である1% Atelocollagen(高研社製)と混合させたもの200μL腹腔内投与した。その後、二日、三日そして四日後の三度にわたりドーパミンニューロンのみを選択的に破壊するMPTP(Sigma Aldrich Japan社製)を30 mg/kgで体内へ投与したものを飼育した。実験8日目にBrdU 5 mg/mL(ZyMED Laboratory.Inc社製)100μlを尾静脈内投与処置し、1時間後、解剖し、脳を摘出し、免疫染色用サンプル、遺伝子発現解析用サンプルを得た。
遺伝子発現は実施例1と同様の方法で、定量的に検討を行った。結果を図64に示す。本パーキンソン病モデルにおいては、脳組織のGalNAc4S-6ST ならびに線維形成マーカーであるTGFβ、タイプIコラーゲン、αSMAの発現が増強するが、GalNAc4S-6ST siRNA投与により、これらの発現が有意に抑制された。したがって、GalNAc4S-6STの発現を抑制する事により、脳組織における線維性変化を抑制できる事が明らかになった。
本発明の薬剤は、例えば、脳組織における線維性変化抑制剤として有用である。
実施例3と同様に脳組織のサンプルを用いて、脳内の神経細胞における線維形成に対してGalNAc4S-6ST生体内投与により得られる組織所見を比較した。得られた切片を4%PFAリン酸緩衝液(ナカライテスク社製)で10分間固定後、脱イオン水で洗浄し、一次抗体として線維芽細胞抗体(ER-TR7; 1:100希釈;BMA社製)を添加し、4℃で一晩反応させた。次に二次抗体であるAlexa488標識抗ラットIgGヤギ抗体(1:200希釈;Invitrogen社)を添加し、室温で30分反応させた。
以上の手法にて得られた組織像を図65に示した。未治療群での強い陽性シグナルは脳梁膨大後部顆粒皮質付近にて対照群より脳内における線維芽細胞の浸潤を示している結果であった。さらに、GalNAc4S-6ST siRNA治療群においては、線維芽細胞の陽性所見は激減していた。以上の結果より、パーキンソン病マウスモデルの誘導される脳組織におけるER-TR7の陽性シグナルの所見は、GalNAc4S-6ST siRNA生体内投与により顕著に抑制されることが明らかになった。
次に、上記の線維形成が神経細胞の減少に伴っているかを調べるために、実施例64と同様の方法で、神経再生関連遺伝子の脳組織における発現を定量化した。ドーパミン神経の生存と分化を制御し、再生を促進する因子GDNF、ならびにドーパミン神経を形成する因子であるNurr1の発現が、GalNAc4S-6ST siRNA投与により増強していた(図66)。本結果は、GalNAc4S-6STの発現を抑制する事により、脳組織におけるドーパミン神経の再生を促進できる事を示している。
本発明の薬剤は、例えば、脳組織におけるドーパミン神経再生促進剤として有用である。
以上の実施例で示された結果を最終的に明らかにするために得られた組織サンプル切片をドーパミンニューロンのマーカーとする抗チロシンハイドロキシラーゼ抗体にてドーパミンニューロンを染め、組織所見を比較検討した。このチロシンハイドロキシラーゼ(TH: thyrosine hydroxylase)はドーパミン前駆体をドーパミンへ転換する酵素である。実施例64と同様の方法で得られた切片を4%PFAリン酸緩衝液(ナカライテスク社製)で10分間固定後、脱イオン水で洗浄し、一次抗体としてラビットポリクローナル抗チロシンハイドロキシラーゼ抗体(1:50希釈;Calbiochem社)を添加し、室温で1時間反応させた。次に二次抗体であるAlexa488標識抗ラビット-ロバ抗体(1:200希釈;Invitrogen社)を添加し、室温で30分反応させた。
図67に対照群、未治療群、GalNAc4S-6ST siRNA、処置群各々の組織画像を示した(原図はカラーである)。中脳上丘付近において、対照群では正常にチロシンハイドロキシラーゼが発現していることが確認できるが未治療群においては陰性のシグナルとして現れた。これは、MPTPにより選択的にドーパミンニューロンが破壊されたことが示唆される所見である。一方、GalNAc4S-6ST siRNA処置を行った群におけるシグナルは未治療群に比べて強く現れていることが確認できた。つまり、GalNAc4S-6ST siRNAの生体内投与によって線維性変化の抑制に伴い、ドーパミンニューロンの機能回復が期待できる結論が得られた。
本発明の薬剤は、例えば、ドーパミンニューロン機能回復剤として有用である。
実施例64と同様に、妊娠14日目C57BL/6JcLマウス(日本クレア社製)を飼育、出産させ、8週令C57BL/6JcLマウス雌(日本クレア社製)、各々にGalNAcST(GalNAc4ST-1、GalNAc4ST-2とGALNAC4S-6STのカクテル配列を混合したもの)siRNA 1μg(GeneWorld社製)をsiRNA媒体である1% Atelocollagen(高研社製)と混合させたもの200μL腹腔内投与した。その後、二日、三日そして四日後の三度にわたりドーパミンニューロンのみを選択的に破壊するMPTP(Sigma Aldrich Japan社製)を30 mg/kgで体内へ投与したものを飼育した。実験8日目にBrdU 5 mg/mL(ZyMED Laboratory.Inc社製)100μlを尾静脈内投与処置し、1時間後、解剖し、脳を摘出し、免疫染色用サンプル、遺伝子発現解析用サンプルを得た。
実施例67と同様に、得られた切片を抗TH抗体で免疫染色を行なった。結果を図68に示す。GalNAcST siRNA処置を行った群においては、TH陽性ドーパミン神経細胞の減少が抑制されていることが確認できた。つまり、GalNAc4ST-1、GalNAc4ST-2、GalNAc4S-6STの発現抑制によって、実施例67と同様に、神経原線維性変化を抑制し、ドーパミンニューロンの機能回復が期待できる結論が得られた。
本発明の薬剤は、例えば、神経原線維性変化抑制剤として有用である。
妊娠14日目 C57BL/6JcLマウス(日本クレア社製)を飼育、出産させ、出生後2日令 C57BL/6JcLマウス 雌(日本クレア社製)、各々にStreptozocin 10mg/mL(SIGMA社製) 20μL/head に皮下注射し、4週令までCE-2(日本クレア社製)の飼料、滅菌水を与え飼育し、4週令よりHigh Fat Diet食(日本クレア社製)、滅菌水を与え、2週間飼育させた。2週間目にコンドロ-4-脱硫酸化酵素(C4-サルファターゼ)(20単位/ml;生化学工業社製)を4単位/ a headで、ならびに溶媒(リン酸緩衝液)を2回/1週間、腹腔内投与(1shot/1週間)の4回(2週間)処置を行った。14日目にC4-サルファターゼの効果を検討するため、両群マウスの眼球を摘出し、免疫組織学的検討を加えた。
摘出眼で凍結ブロック・切片を作成した。切片をアセトン(シグマアルドリッチジャパン社製)で10分間固定後、リン酸緩衝液で洗浄し、さらに一次抗体として抗コンドロイチン硫酸プロテオグリカン(CSPG)抗体(クローンCS56、マウスモノクローナル抗体、10μg/mL;生化学工業社製)を添加し、室温で一時間反応させた。続いて、ヒストファインマウスステインキット(ニチレイ社製;マウスモノクローナル抗体に対して使用)を用いて二次抗体反応を行った後、DAB基質を(ニチレイ社製)添加し酵素色素反応を行った。この標本を光学顕微鏡(ライカ社製)用いて観察した。
得られた組織像を図69に示した(原図はカラーである)。未治療群では網膜の硝子体側に新規にCS56陽性所見を認めてくる。これに対して酵素治療群ではCS56強度が減弱している。CS56は、硫酸基を認識する抗体であり、この減弱は4位硫酸基の減弱を反映しているものと示唆される。以上より、本モデルマウスにおいて誘導される網膜組織におけるCSPGの沈着は、C4-サルファターゼの生体内投与により修飾を受け抑制されることが明らかになった。
C4-サルファターゼは、GalNAcの4位の硫酸基を脱硫酸化する酵素である。従って、GalNAcの4位の硫酸化を阻害することによって、生体レベルにおいて組織の線維形成を抑制できることが示された。即ち、GalNAcの4位の硫酸基の脱硫酸化酵素は、組織線維形成抑制剤として有用である。
実施例69と同様の方法で得られた切片をアセトン(シグマアルドリッチジャパン社製)で10分間固定後、リン酸緩衝液で洗浄し、一次抗体としてラット由来抗血管内皮細胞抗体(CD31;1:200希釈;ファーミンジェン社製)を添加し、室温で1時間反応させた。次に二次抗体であるペルオキシダーゼ標識ロバ由来抗ラットIgG(1:200希釈;バイオソース社製)を添加し、室温で30分反応させた。反応後のサンプルにDAB基質(ニチレイ社製)を添加した。この標本を光学顕微鏡(ライカ社製)用いて観察した。得られた組織像を図70に示した(原図はカラーである)。未治療群では網膜硝子体側におけるCD31陽性細胞の数が増加しており、一部硝子体に突出している像も認めた。これは、本モデルが糖尿病性網膜症の前増殖網膜症の段階を反映していると考えられ、モデルの有効性を示すものである。これに対して酵素治療群では、同部位のCD31陽性細胞の数が明らかに減少していた。以上の結果から、2型糖尿病モデルにおいては網膜硝子体側の血管内皮細胞数が増加してくるが、C4-サルファターゼ投与により、そのような血管増生が抑制させることが示唆された。
即ち、GalNAcの4位の硫酸基の脱硫酸化酵素は、血管増生抑制剤として有用である。
実施例69と同様の方法で得られた切片をアセトン(シグマアルドリッチジャパン社製)で10分間固定後、リン酸緩衝液で洗浄し、一次抗体としてウサギ由来抗IV型コラーゲン抗体(1:250希釈;Sigma社製)を添加し、室温で1時間反応させた。次に二次抗体であるペルオキシダーゼ標識抗ウサギIgG(1:200希釈;Jackson ImmunoResearch社製)を添加し、室温で30分反応させた。反応後のサンプルにDAB基質(ニチレイ社製)を添加した。この標本を光学顕微鏡(ライカ社製)用いて観察した。
得られた組織像を図71に示した(原図はカラーである)。未治療群では網膜硝子体側におけるIV型コラーゲン陽性所見の増加とともに、網膜内境界膜に平行して連なる所見を認めた。これは、静脈形態の異常と共に、コラーゲンの増生、すなわち線維化変化を示唆するものである。これに対して酵素治療群では、同部位のIV型コラーゲンの増生が顕著に抑制されていた。以上の結果から、2型糖尿病モデルにおいては網膜コラーゲン増生を認めるが、C4-サルファターゼ投与により抑制させることが明らかになった。
即ち、GalNAcの4位の硫酸基の脱硫酸化酵素は、2型糖尿病性網膜症の治療剤として有用である。
実施例72~74では、前述の(実施例69~71)2型糖尿病モデルマウスより採取した肝臓を用いて、C4-サルファターゼの肝臓における、抗線維化効果(線維芽細胞の組織浸潤を指標)について比較・検討した。凍結ブロックの作製、免疫染色の手法などは、全て前述の方法で行った。図72に示すように、未治療群においては、多数の線維芽細胞の浸潤を認めた。これに対して、C4-サルファターゼ治療群では、未治療群に比べて、線維芽細胞の浸潤の程度は少なかった。この結果は、C4-サルファターゼが線維芽細胞の浸潤を抑制する薬理作用をもち、この効果が抗線維化作用の一端である事を示すものであった。
即ち、GalNAcの4位の硫酸基の脱硫酸化酵素は、線維芽細胞浸潤抑制剤として有用である。
実施例73では、前述の(実施例69~71)2型糖尿病モデルマウスより採取した肝臓を用いてC4-サルファターゼの肝臓における、抗炎症効果(マクロファージの組織浸潤を指標)について比較・検討した。凍結ブロックの作製、免疫染色の手法などは、全て前述の方法で行った。図73に示すように、未治療群においては、スポットの形成を伴う、多数のマクロファージの浸潤を認めた。これに対して、C4-サルファターゼ治療群では、未治療群に比べて、マクロファージの浸潤の程度は少なかった。この結果は、C4-サルファターゼがマクロファージの浸潤を抑制する薬理作用をもち、この効果が抗炎症作用の一端である事を示すものであった。
即ち、GalNAcの4位の硫酸基の脱硫酸化酵素は、マクロファージ浸潤抑制剤、あるいは抗炎症剤として有用である。
実施例74は、実施例72と実施例73の結果を補足する目的で解析を行った。本実施例では、前述の(実施例69~71)2型糖尿病モデルマウスより採取した血清を用いて、肝機能の指標であるアスパラギン酸アミノ基転移酵素(AST)、アラニンアミノ基転移酵素(ALT)、脂質代謝の指標であるトリアシルグリセロール(TG)を測定した。図74には、その結果を示している。生化学検査は、検査受託業者に測定を依頼した。未治療群(unt)では、対照群(nor)に比べて、AST、ALT、TGのいずれの値も上昇傾向であった。一方、C4-サルファターゼ治療群(C4sul)では、AST、ALT、TGの検査値の上昇は、未治療群(unt)に比べて抑制される傾向にあった。これらの結果は、実施例72と実施例73の結果を支持するものであり、C4-サルファターゼの抗線維化効果と、抗炎症効果によって肝機能が保たれていることを示している。
即ち、GalNAcの4位の硫酸基の脱硫酸化酵素は、肝機能疾患に対する治療剤として有用である。
本実施例では1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine(MPTP)の代謝産物であるMPDPを用いてモデルを作成した、本実験を遂行した。
8週令 C57BL/6JcLマウス雌(日本クレア社製)、各々にC4-サルファターゼ(生化学工業社製)4 U/ml 100μl a headで、day 0、day 2にて腹腔内投与し、siRNA については、GALNAC4S-6ST siRNA(北海道システムサイエンス社製) 1μg/ 1% Atelocollagen/ 200μlをday 0で前投与した。day 2、day 3、day 4にて、MPDP (Sigma Aldrich Japan社製)を(30mg/ kg)で連日投与を行った。
本実施例に用いたmouse GalNac 4S-6ST siRNA薬の塩基配列について以下に示す。配列は必ずしも本例のみに限定されない。
[mouse GalNac4-6STsiRNA](Gene Bank accession number NM_029935)
(北海道システムサイエンス社製)
5’- gcagcccagcaagaugaauaagauc-ag -3’(配列番号:91)
3’-ua-cgucgggucguucuacuuauucuag -5’ (配列番号:92)
次に、RT reaction(cDNA合成)を行うため以下の手技を行った。算出して得られたRNAサンプルを500 ng/20μlの濃度に調整し、68℃、3分間、BLOCK INCUBATOR(ASTEC製)にて加温し、10分間、氷冷した。氷冷後、予め調製していたRT Pre Mix 液(組成:25 mM MgCl2 18.64μl(Invitrogen社製)、5×Buffer 20μl(Invitrogen社製)、0.1 M DTT 6.6μl(Invitrogen社製)、10 mM dNTP mix 10μl(Invitrogen社製)、RNase Inhibitor 2μl(Invitrogen社製)、MMLV Reverse transcriptase 1.2μl(Invitrogen社製)、Random primer 2μl(Invitrogen社製)、滅菌蒸留水19.56μl(大塚蒸留水:大塚製薬社製)を80μl加えBLOCK INCUBATOR(ASTEC社製)にて42℃、1時間、加温反応させ、1時間後、BLOCK INCUBATOR(ASTEC社製)にて99℃、5分間、加熱した後、氷冷し求めるcDNA 100μlを作製し、合成して得られたcDNAを用いて、以下の組成でPCR反応を行った。
MPDP誘導性パーキンソンモデルにおけるCSPG発現について、CS-56抗体(抗CSPG抗体:生化学工業社製 1: 100希釈)を用いた免疫染色にて検討した結果について図75に示す。図75に示したように、未治療群では、CSPG陽性のシグナルが強く発現している。これに対して、C4-サルファターゼ治療群、遺伝子治療群では陽性のシグナルの発現は低下していた。
C4-サルファターゼと、GalNAc4S-6ST siRNAのドーパミン作動性神経に対する薬理効果を明らかにする目的で、MPDP誘導性パーキンソンモデルにおけるドーパミン作動性神経の局在を、抗チロシン水酸化酵素(tyrosine hydroxylase, TH 1: 20希釈)抗体、二次抗体はAlexa-488標識抗ウサギIgG抗体(Invitrogen社製1: 200希釈)を用いた蛍光免疫染色を用いて解析を行った。図76に示すように、脳組織にドーパン作動性神経の局在は、未治療群では他の群に比べて、陽性のシグナルが弱く発現している。これに対して、C4-サルファターゼ治療群、遺伝子治療群では陽性のシグナルの発現は、対照群と大きな差異は見られなかった。C4-サルファターゼ投与、siRNA投与によるドーパミン作動性神経の保護作用、もしくは再生・修復促進作用が示される結果となった。
即ち、GalNAcの4位もしくは6位のインヒビター(GalNAcの4位の硫酸基の脱硫酸化酵素、およびGalNAc4S-6ST siRNA)はドーパミン作動性神経保護剤、もしくは再生・修復促進剤として有用である。
C4-サルファターゼと、GalNAc siRNAの抗炎症効果を比較する目的で、実施例75、実施例76で使用した組織切片と同様のサンプルから前述の方法で抽出したtotal RNAを用いてTNF-αの発現について定量PCR法で解析した。定量PCRについては、SYBR premix kit(タカラバイオ社製)とReal-time PCR thermal cycler DICE(タカラバイオ社製)を用いて行った。PCR反応の条件は、95℃を10秒、95℃を5秒と60℃を30秒の40サイクルとし、最後に融解曲線分析を行った。用いたプライマーの塩基配列について以下に示す。
mouse βアクチン(タカラバイオ社製)
forward : 5’-CATCCGTAAAGACCTCTATGCCAAC -3’(配列番号:93)
reverse : 5’-ATGGAGCCACCGATCCACA -3’ (配列番号:94)
Tumor Necrosis Factor (TNF-α):(タカラバイオ社製)
forward : 5’-CAGGAGGGAGAACAGAAACTCCA-3(配列番号:95)
reverse: 5’-CCTGGTTGGCTGCTTGCTT-3’ (配列番号:96)
図77に示すように、未治療群と比較し、C4-サルファターゼ治療群ではTNF-αの発現が抑制されていた。遺伝子治療群では、発現抑制の傾向を示すに留まった。これらの結果より、C4-サルファターゼが、炎症を抑制する作用を持つ事が示された。
即ち、GalNAcの4位の硫酸基の脱硫酸化酵素は抗炎症剤として有用である。
この実施例では、実施例73の結果を補足する目的で、ドーパミン作動性神経の発現に関与する遺伝子であるNurr1をマーカーとして検討を行った。C4-サルファターゼとGalNAc4S-6STが及ぼす影響について、前述の定量PCR法を用いて検討した結果について示す。用いたプライマーの塩基配列について以下に示す。
Nuclea receptor subfamily 4 GroupA member2 (Nurr1) :(タカラバイオ社製)
forward : 5’-CTGCCCTGGCTATGGTCACA-3’ (配列番号:97)
reverse : 5’-AGACAGGTAGTTGGGTCGGTTCA-3’ (配列番号:98)
図78に示すように、C4-サルファターゼ治療群、遺伝子治療群では、未治療群に比べてNurr1の遺伝子発現が有意(P <0.001)に増加していた。この結果は、実施例76の結果を支持するものであり、C4-サルファターゼ投与、siRNA投与によるドーパミン作動性神経の保護作用、もしくは再生・修復促進作用が示される結果となった。
即ち、GalNAcの4位もしくは6位のインヒビター(GalNAcの4位の硫酸基の脱硫酸化酵素、およびGalNAc4S-6ST siRNA)はドーパミン作動性神経保護剤、もしくは再生・修復促進剤として有用である。
慢性の組織線維形成(線維形成性組織変化)は全身臓器に起こり得るものであり、臓器機能不全により個体を死に至らしめる疾患群の総称である(総説:Wynn TA, J. Clin. Invest. 117:524-529, 2007)。
線維形成性疾患は慢性炎症の終末像と考えられ、全身の臓器に起こり当該臓器の機能不全さらには個体を死に至らしめる疾患の総称である。心血管疾患、脳血管疾患など従来死亡率の高かった疾患の本質は、組織レベルでの線維形成とそれに伴う機能不全であるという認識がなされつつあり、欧米死因の45%は線維形成に起因するとの見解がある。この新しい見解によれば、個体の病的な死亡は、癌、感染症、線維形成、という3つに集約される事になる。従来の概念である肝硬変、肺線維症、腎硬化症のみに留まらず、癌・感染症以外の極めて多彩な、主には慢性疾患が「線維形成性疾患」と定義され得る。とりわけ近年の生活習慣の変化(俗にいう欧米化)に伴い、NASH(非アルコール性脂肪性肝炎)やCKD(慢性腎疾患)という生命を脅かす新しい疾患(疾患概念)が急増しており、これらが組織の線維形成に起因するという知見は、「線維形成性疾患」治療剤確立の緊急性を示している。しかしながら、現在、移植や人工臓器しか対応がなく、根本的な治療方法が無いという決定的な課題を抱えていた。
臨床的にも難治性機能不全を呈する線維形成性疾患は死亡の決定的要因になるばかりでなく、日常のQOL(quality of life)を著しく阻害する疾患でもある。組織線維形成抑制剤の確立はそれゆえ極めて重要であるが、現在市場に出ている薬剤はない。実験的にもTGF-β阻害剤を中心に、アンギオテンシン阻害剤、炎症性サイトカイン阻害剤、TLR阻害剤、MMP阻害剤など精力的に研究が進められているが、未だ有効性が確立した方法とは言えない(総説:Wynn TA, J. Clin. Invest. 117:524-529, 2007)。
線維形成性疾患の確認は、決定的には病理像(線維芽細胞やコラーゲンの免疫染色、マッソン染色を含む)で行った。治療効果(線維形成阻害効果)は病理像の他、臨床症状、各臓器におけるコラーゲン発現量で決定した。
技術的手法としては、糖鎖遺伝子に対する核酸医薬(siRNA)で直接的な遺伝子ノックダウン法により治療効果を証明した。
Claims (12)
- N-アセチルガラクトサミンの4位または6位の硫酸化インヒビターを成分とする、組織線維形成抑制剤。
- 生体組織の線維形成に対して抑制効果を有することを特徴とする、請求項1に記載の薬剤。
- 前記インヒビターがN-アセチルガラクトサミンの4位または6位の硫酸基転移酵素の機能を阻害する活性を有する、請求項1または2に記載の薬剤。
- 前記インヒビターが、N-アセチルガラクトサミンの4位または6位の硫酸基転移酵素遺伝子の発現を抑えるsiRNAである、請求項3に記載の薬剤。
- 前記インヒビターがN-アセチルガラクトサミンの4位または6位の硫酸基の脱硫酸化酵素である、請求項1または2に記載の薬剤。
- 線維形成性疾患の治療用または予防用の、請求項1~5のいずれかに記載の薬剤。
- 糖鎖を構成するN-アセチルガラクトサミンの4位または6位の硫酸化を阻害する化合物を選択する工程を含む、組織線維形成抑制剤のスクリーニング方法。
- 以下の工程(a)~(c)を含む、組織線維形成抑制剤のスクリーニング方法。
(a)N-アセチルガラクトサミンまたはN-アセチルガラクトサミンを含有する糖鎖と、被検化合物を接触させる工程
(b)N-アセチルガラクトサミンの4位または6位について硫酸化の程度を測定する工程
(c)被検化合物を接触させない場合と比較して、硫酸化の程度を低下させる化合物を選択する工程 - 以下の工程(a)~(c)を含む、組織線維形成抑制剤のスクリーニング方法。
(a)N-アセチルガラクトサミンの4位または6位の硫酸基転移酵素と、被検化合物を接触させる工程
(b)前記酵素の硫酸基転移活性を測定する工程
(c)被検化合物を接触させない場合と比較して、前記活性を低下させる化合物を選択する工程 - 以下の工程(a)~(c)を含む、組織線維形成抑制剤のスクリーニング方法。
(a)N-アセチルガラクトサミンの4位または6位の硫酸基転移酵素をコードする遺伝子を発現する細胞に、被検化合物を接触させる工程
(b)前記細胞における遺伝子の発現量を測定する工程
(c)被検化合物を接触させない場合と比較して、前記遺伝子の発現量を低下させる化合物を選択する工程 - 以下の工程(a)~(c)を含む、組織線維形成抑制剤のスクリーニング方法。
(a)N-アセチルガラクトサミンの4位または6位の硫酸基転移酵素をコードする遺伝子の転写調節領域とレポーター遺伝子とが機能的に結合した構造を有するDNAを含む細胞または細胞抽出液と、被検化合物を接触させる工程
(b)前記レポーター遺伝子の発現量を測定する工程
(c)被検化合物を接触させない場合と比較して、前記レポーター遺伝子の発現量を低下させる化合物を選択する工程 - 以下の工程(a)および(b)を含む、線維形成性疾患の治療または予防のための医薬組成物の製造方法。
(a)被検試料から、請求項7~11のいずれかに記載の方法により、組織線維形成抑制剤を選択する工程
(b)前記薬剤と医薬的に許容される担体とを混合する工程
Priority Applications (16)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN200880127570XA CN101965196A (zh) | 2007-12-27 | 2008-12-26 | 糖链相关基因及其利用 |
JP2009547912A JP4585611B2 (ja) | 2007-12-27 | 2008-12-26 | 糖鎖関連遺伝子、およびその利用 |
KR1020157037089A KR20160007671A (ko) | 2007-12-27 | 2008-12-26 | 당쇄 관련 유전자, 및 그의 이용 |
KR1020197037818A KR102244872B1 (ko) | 2007-12-27 | 2008-12-26 | 당쇄 관련 유전자, 및 그의 이용 |
EP20151386.8A EP3673910B1 (en) | 2007-12-27 | 2008-12-26 | Sugar chain-related gene and use thereof |
KR1020167022756A KR20160103554A (ko) | 2007-12-27 | 2008-12-26 | 당쇄 관련 유전자, 및 그의 이용 |
KR1020187034630A KR20180132162A (ko) | 2007-12-27 | 2008-12-26 | 당쇄 관련 유전자, 및 그의 이용 |
KR1020177023558A KR101926292B1 (ko) | 2007-12-27 | 2008-12-26 | 당쇄 관련 유전자, 및 그의 이용 |
US12/809,969 US20110027248A1 (en) | 2007-12-27 | 2008-12-26 | Sugar Chain-Related Gene and Use Thereof |
EP08866254A EP2238987A4 (en) | 2007-12-27 | 2008-12-26 | SUGAR CHAIN RELATED GENE AND ITS USE |
EP18151424.1A EP3360556B1 (en) | 2007-12-27 | 2008-12-26 | Sugar chain-related gene and use thereof |
US14/075,919 US20140128452A1 (en) | 2007-12-27 | 2013-11-08 | Sugar chain-related gene and use thereof |
US14/704,333 US20150290238A1 (en) | 2007-12-27 | 2015-05-05 | Sugar chain-related gene and use thereof |
US15/216,231 US20170067058A1 (en) | 2007-12-27 | 2016-07-21 | Sugar chain-related gene and use thereof |
US16/237,875 US20190330637A1 (en) | 2007-12-27 | 2019-01-02 | Sugar chain-related gene and use thereof |
US17/805,290 US20220340910A1 (en) | 2007-12-27 | 2022-06-03 | Sugar chain-related gene and use thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2007-336518 | 2007-12-27 | ||
JP2007336518 | 2007-12-27 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/809,969 A-371-Of-International US20110027248A1 (en) | 2007-12-27 | 2008-12-26 | Sugar Chain-Related Gene and Use Thereof |
US14/075,919 Continuation US20140128452A1 (en) | 2007-12-27 | 2013-11-08 | Sugar chain-related gene and use thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2009084232A1 true WO2009084232A1 (ja) | 2009-07-09 |
Family
ID=40823968
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2008/004025 WO2009084232A1 (ja) | 2007-12-27 | 2008-12-26 | 糖鎖関連遺伝子、およびその利用 |
Country Status (7)
Country | Link |
---|---|
US (6) | US20110027248A1 (ja) |
EP (4) | EP3045178A1 (ja) |
JP (3) | JP4585611B2 (ja) |
KR (6) | KR102244872B1 (ja) |
CN (2) | CN103656644A (ja) |
SG (2) | SG10202001748VA (ja) |
WO (1) | WO2009084232A1 (ja) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2460403A1 (en) * | 2009-07-31 | 2012-06-06 | Stelic Institute Of Regenerative Medicine, Stelic Institute & Co. | Steatohepatitis-liver cancer model animal |
WO2013073667A1 (ja) | 2011-11-18 | 2013-05-23 | 日東電工株式会社 | 腸管線維症処置剤 |
WO2014013535A1 (ja) * | 2012-07-17 | 2014-01-23 | 株式会社ステリック再生医科学研究所 | 粘膜治癒促進剤 |
JP2015017070A (ja) * | 2013-07-12 | 2015-01-29 | 株式会社エンザミン研究所 | インスリン抵抗性改善剤およびインスリン抵抗性疾患予防用の健康補助食品 |
WO2018105708A1 (ja) | 2016-12-07 | 2018-06-14 | 株式会社ステリック再生医科学研究所 | 慢性疾患の治療及び予防用医薬組成物 |
US10646579B2 (en) | 2015-11-04 | 2020-05-12 | Tme Therapeutics Inc. | Complex comprising RNAi molecule and N-acetylated chitosan |
EP3711755A1 (en) * | 2007-06-29 | 2020-09-23 | Stelic Institute Of Regenerative Medicine, Stelic Institute & Co. | Method of fixing and expressing physiologically active substance |
WO2020225871A1 (ja) * | 2019-05-08 | 2020-11-12 | 株式会社Tмeセラピューティックス | 食道狭窄抑制剤 |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP6387084B2 (ja) * | 2013-05-01 | 2018-09-05 | アイオーニス ファーマシューティカルズ, インコーポレーテッドIonis Pharmaceuticals,Inc. | アポリポタンパク質c−iiiの発現を調節するための組成物および方法 |
HUE051749T2 (hu) | 2015-03-02 | 2021-03-29 | Conagen Inc | Labirintusgomba mikroorganizmusokból származó szabályozó elemek |
US10633454B2 (en) | 2016-11-01 | 2020-04-28 | Conagen Inc. | Expression of modified glycoproteins and glycopeptides |
EP4252629A3 (en) | 2016-12-07 | 2023-12-27 | Biora Therapeutics, Inc. | Gastrointestinal tract detection methods, devices and systems |
WO2018183934A1 (en) | 2017-03-30 | 2018-10-04 | Progenity Inc. | Treatment of a disease of the gastrointestinal tract with a chst15 inhibitor |
EP3762485A4 (en) * | 2018-03-05 | 2021-12-08 | Conagen Inc. | ORGANISMS AND PROCESSES FOR THE PRODUCTION OF LOW SULPHATION GLYCOMOLECULES |
CN109897812B (zh) * | 2019-03-19 | 2021-09-03 | 江南大学 | 一种表达软骨素4-硫酸转移酶基因的重组菌及其应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2004504262A (ja) * | 1999-12-02 | 2004-02-12 | アイベックス テクノロジーズ, インコーポレイテッド | 線維芽細胞の増殖の弱毒化 |
WO2007049424A1 (ja) * | 2005-10-27 | 2007-05-03 | Stelic Institute Of Regenerative Medicine, Stelic Institute & Co. | 肝線維化抑制剤 |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP4571242B2 (ja) * | 1996-07-24 | 2010-10-27 | 生化学工業株式会社 | ヒト由来のグリコサミノグリカンスルホトランスフェラーゼのポリペプチド及びそれをコードするdna |
AU6897198A (en) * | 1997-04-11 | 1998-11-11 | Beth Israel Deaconess Medical Center | Use of chondroitinase in the manufacture of a medicament in the treatment and prevention of mucoid secretions |
EP1839671A3 (en) * | 1999-12-02 | 2007-10-31 | BioMarin Pharmaceutical Inc. | Attenuation of fibroblast proliferation |
US6713274B2 (en) * | 2002-05-13 | 2004-03-30 | The Regents Of The University Of California | Method for identifying modulators of sulfotransferase activity |
EP1541580B1 (en) * | 2002-07-10 | 2013-02-27 | Seikagaku Corporation | Sulfotransferase inhibitors |
PT1734970E (pt) * | 2004-03-12 | 2015-03-11 | Intercept Pharmaceuticals Inc | Tratamento de fibrose utilizando ligandos de rfx |
JP2009286695A (ja) * | 2006-09-08 | 2009-12-10 | Stelic Institute Of Regenerative Medicine | 眼線維性血管新生抑制剤 |
JP4147264B2 (ja) * | 2006-09-08 | 2008-09-10 | 株式会社ステリック再生医科学研究所 | 神経線維性変性抑制剤 |
EP3456357A1 (en) * | 2007-06-29 | 2019-03-20 | Stelic Institute Of Regenerative Medicine, Stelic Institute & Co. | Method of fixing and expressing physiologically active substance |
-
2008
- 2008-12-26 CN CN201310495268.2A patent/CN103656644A/zh active Pending
- 2008-12-26 WO PCT/JP2008/004025 patent/WO2009084232A1/ja active Application Filing
- 2008-12-26 SG SG10202001748VA patent/SG10202001748VA/en unknown
- 2008-12-26 CN CN200880127570XA patent/CN101965196A/zh active Pending
- 2008-12-26 US US12/809,969 patent/US20110027248A1/en not_active Abandoned
- 2008-12-26 EP EP15194114.3A patent/EP3045178A1/en not_active Withdrawn
- 2008-12-26 KR KR1020197037818A patent/KR102244872B1/ko active IP Right Grant
- 2008-12-26 KR KR1020167022756A patent/KR20160103554A/ko not_active Application Discontinuation
- 2008-12-26 SG SG10201402793PA patent/SG10201402793PA/en unknown
- 2008-12-26 KR KR1020157037089A patent/KR20160007671A/ko active Application Filing
- 2008-12-26 KR KR1020187034630A patent/KR20180132162A/ko not_active Application Discontinuation
- 2008-12-26 EP EP08866254A patent/EP2238987A4/en not_active Withdrawn
- 2008-12-26 KR KR1020107016694A patent/KR20100111282A/ko not_active Application Discontinuation
- 2008-12-26 EP EP18151424.1A patent/EP3360556B1/en active Active
- 2008-12-26 KR KR1020177023558A patent/KR101926292B1/ko active IP Right Grant
- 2008-12-26 JP JP2009547912A patent/JP4585611B2/ja active Active
- 2008-12-26 EP EP20151386.8A patent/EP3673910B1/en active Active
-
2010
- 2010-08-02 JP JP2010173610A patent/JP4751956B2/ja active Active
-
2011
- 2011-05-17 JP JP2011110047A patent/JP5481426B2/ja active Active
-
2013
- 2013-11-08 US US14/075,919 patent/US20140128452A1/en not_active Abandoned
-
2015
- 2015-05-05 US US14/704,333 patent/US20150290238A1/en not_active Abandoned
-
2016
- 2016-07-21 US US15/216,231 patent/US20170067058A1/en not_active Abandoned
-
2019
- 2019-01-02 US US16/237,875 patent/US20190330637A1/en not_active Abandoned
-
2022
- 2022-06-03 US US17/805,290 patent/US20220340910A1/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2004504262A (ja) * | 1999-12-02 | 2004-02-12 | アイベックス テクノロジーズ, インコーポレイテッド | 線維芽細胞の増殖の弱毒化 |
WO2007049424A1 (ja) * | 2005-10-27 | 2007-05-03 | Stelic Institute Of Regenerative Medicine, Stelic Institute & Co. | 肝線維化抑制剤 |
Non-Patent Citations (7)
Title |
---|
ANAVEKAR NS ET AL., N ENG J MED, vol. 351, 2004, pages 1285 |
KHWAJA A ET AL., KIDNEY INTERNATIONAL |
REMUZZI G ET AL., J CLIN INVEST, vol. 116, 2006, pages 288 |
See also references of EP2238987A4 |
SERGIO A ET AL., HYPERTENSION, vol. 38, 2001, pages 635 |
TONELLI M ET AL., BMJ, vol. 332, 2006, pages 1426 |
WEINER DE ET AL., JASN, vol. 15, 2004, pages 1307 |
Cited By (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3711755A1 (en) * | 2007-06-29 | 2020-09-23 | Stelic Institute Of Regenerative Medicine, Stelic Institute & Co. | Method of fixing and expressing physiologically active substance |
US9596834B2 (en) | 2009-07-31 | 2017-03-21 | Stelic Institute Of Regenerative Medicine, Stelic Institute & Co. | Steatohepatitis-liver cancer model animal |
EP2460403A4 (en) * | 2009-07-31 | 2013-11-20 | Stelic Inst Of Regenerative Medicine Stelic Inst & Co | ANIMAL MODEL OF LIVER CANCER WITH STATATO-HEPATITIS |
EP2460403A1 (en) * | 2009-07-31 | 2012-06-06 | Stelic Institute Of Regenerative Medicine, Stelic Institute & Co. | Steatohepatitis-liver cancer model animal |
CN102548393B (zh) * | 2009-07-31 | 2015-06-03 | 斯特里克再生医学学院,斯特里克学院事务所 | 脂肪性肝炎-肝癌模型动物 |
WO2013073667A1 (ja) | 2011-11-18 | 2013-05-23 | 日東電工株式会社 | 腸管線維症処置剤 |
KR20140097405A (ko) | 2011-11-18 | 2014-08-06 | 닛토덴코 가부시키가이샤 | 장 섬유증 치료제 |
EP3693462A1 (en) * | 2012-07-17 | 2020-08-12 | Stelic Institute&Co. | Mucosal healing promoter |
JPWO2014013535A1 (ja) * | 2012-07-17 | 2016-06-23 | 株式会社ステリック再生医科学研究所 | 粘膜治癒促進剤 |
WO2014013535A1 (ja) * | 2012-07-17 | 2014-01-23 | 株式会社ステリック再生医科学研究所 | 粘膜治癒促進剤 |
EP3878962A1 (en) * | 2012-07-17 | 2021-09-15 | Stelic Institute & Co. | Mucosal healing promoter |
JP2015017070A (ja) * | 2013-07-12 | 2015-01-29 | 株式会社エンザミン研究所 | インスリン抵抗性改善剤およびインスリン抵抗性疾患予防用の健康補助食品 |
US10646579B2 (en) | 2015-11-04 | 2020-05-12 | Tme Therapeutics Inc. | Complex comprising RNAi molecule and N-acetylated chitosan |
WO2018105708A1 (ja) | 2016-12-07 | 2018-06-14 | 株式会社ステリック再生医科学研究所 | 慢性疾患の治療及び予防用医薬組成物 |
US11911409B2 (en) | 2016-12-07 | 2024-02-27 | Stelic Institute & Co., Inc. | Pharmaceutical composition for treatment and prevention of chronic disease |
WO2020225871A1 (ja) * | 2019-05-08 | 2020-11-12 | 株式会社Tмeセラピューティックス | 食道狭窄抑制剤 |
EP3967313A4 (en) * | 2019-05-08 | 2022-11-16 | TME Therapeutics Inc. | MEDICINE TO ALLEVIATE A NARRATED ESOPHAGUS |
Also Published As
Publication number | Publication date |
---|---|
KR20180132162A (ko) | 2018-12-11 |
KR20190143485A (ko) | 2019-12-30 |
EP3360556A1 (en) | 2018-08-15 |
EP3360556B1 (en) | 2020-04-08 |
US20110027248A1 (en) | 2011-02-03 |
US20150290238A1 (en) | 2015-10-15 |
KR20160007671A (ko) | 2016-01-20 |
KR102244872B1 (ko) | 2021-04-27 |
SG10201402793PA (en) | 2014-10-30 |
EP3045178A1 (en) | 2016-07-20 |
JP4751956B2 (ja) | 2011-08-17 |
JPWO2009084232A1 (ja) | 2011-05-19 |
JP5481426B2 (ja) | 2014-04-23 |
KR20160103554A (ko) | 2016-09-01 |
JP4585611B2 (ja) | 2010-11-24 |
US20170067058A1 (en) | 2017-03-09 |
EP3673910A1 (en) | 2020-07-01 |
EP3673910B1 (en) | 2023-11-22 |
KR20170100064A (ko) | 2017-09-01 |
KR20100111282A (ko) | 2010-10-14 |
EP2238987A4 (en) | 2011-11-02 |
JP2011037847A (ja) | 2011-02-24 |
KR101926292B1 (ko) | 2018-12-06 |
US20220340910A1 (en) | 2022-10-27 |
CN103656644A (zh) | 2014-03-26 |
EP2238987A1 (en) | 2010-10-13 |
SG10202001748VA (en) | 2020-04-29 |
CN101965196A (zh) | 2011-02-02 |
US20140128452A1 (en) | 2014-05-08 |
JP2011162558A (ja) | 2011-08-25 |
US20190330637A1 (en) | 2019-10-31 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP4751956B2 (ja) | 糖鎖関連遺伝子、およびその利用 | |
KR101573009B1 (ko) | 세포막 재봉합을 조절하기 위한 조성물 및 방법 | |
JP4101276B2 (ja) | 慢性閉塞性肺疾患改善剤 | |
US20240011027A1 (en) | Methods and compositions for restoring stmn2 levels | |
JP4147264B2 (ja) | 神経線維性変性抑制剤 | |
JP2006213621A (ja) | Adoplinタンパク質、およびその利用 | |
JP2009292725A (ja) | 腎疾患改善剤 | |
JP2009286695A (ja) | 眼線維性血管新生抑制剤 | |
JPWO2008149980A1 (ja) | 線維化抑制剤 | |
Etteger | RNF125, a novel PPARγ target gene in adipogenesis | |
JP2009234916A (ja) | コンドロイチン硫酸プロテオグリカン蓄積制御に基づくインスリン抵抗性抑制剤 | |
WO2011140315A2 (en) | Method of reducing proteins misfolding and/or aggregation | |
WO2008029871A1 (fr) | Inhibiteur de la dégénérescence des fibres nerveuses | |
Burgess | Molecular and Functional Analysis of Best1 Mutations in Retinal Disease |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 200880127570.X Country of ref document: CN |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 08866254 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2009547912 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 20107016694 Country of ref document: KR Kind code of ref document: A |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2008866254 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 12809969 Country of ref document: US |