WO2008012932A1 - Réducteur de la résistance à l'insuline - Google Patents

Réducteur de la résistance à l'insuline Download PDF

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WO2008012932A1
WO2008012932A1 PCT/JP2006/323657 JP2006323657W WO2008012932A1 WO 2008012932 A1 WO2008012932 A1 WO 2008012932A1 JP 2006323657 W JP2006323657 W JP 2006323657W WO 2008012932 A1 WO2008012932 A1 WO 2008012932A1
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Prior art keywords
chondroitin sulfate
sirna
protein
gene
proteodarican
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PCT/JP2006/323657
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English (en)
Japanese (ja)
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Hiroyuki Yoneyama
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Stelic Institute Of Regenerative Medicine, Stelic Institute & Co.
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Publication of WO2008012932A1 publication Critical patent/WO2008012932A1/fr

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    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • 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
    • 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
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

  • the present invention relates to an insulin resistance inhibitor based on control of accumulation of chondroitin sulfate proteoglycan (CSPG), a method for suppressing insulin resistance, and insulin-independent type 2 diabetes based on the method
  • CSPG chondroitin sulfate proteoglycan
  • NIDDM insulin resistance diseases
  • Type 1 diabetes is a condition in which the splenic j8 cells that inherently produce insulin are destroyed by autoimmunity.
  • type 2 diabetes mellitus is a well-known lifestyle-related disease that has developed due to the addition of environmental factors such as obesity and lack of exercise to genetic factors.
  • a proteodalycan is a molecule having a structure in which one or more glycosaminoglycan (GAG) chains are covalently bonded to a protein called a core protein, and the specific sugar chain structure of the GAG chain is a proteoglycan.
  • GAG glycosaminoglycan
  • Proteodarican is based on the type of GAG chain, and chondroitin sulfate proteodarican, dermatan sulfate proteodarican, henoran sulfate proteodarican, and ketalan sulfate proteodarican. Broadly divided into four. (See Non-Patent Documents 13-19)
  • chondroitin sulfate proteodarican is an essential molecule during the embryonic period and is abundant in each organ, but is known to decrease with birth, growth, and aging. Kang's in vivo function has not yet become clear.
  • chondroitin sulfate proteoglycan CSPG
  • HSPG heparan sulfate proteoglycan
  • DSPG dermatan sulfate proteoglycan
  • bFu basic fibroblast growtn factor
  • Versican (aka PG-M), a chondroitin sulfate proteodarican, has a hyaluronic acid binding domain near the N-terminal, a glycosaminodarlican addition domain in the middle, an EGF-like domain in the C-terminal, C It has a type lectin-like domain and a complement regulatory protein-like domain.
  • the human Versican gene is composed of 15 exons, and as a result of alternative splicing, Ve rsican takes four different molecular types: V0, VI, V2 with different glycosaminodalican-capped domain lengths, and V3 without glycosaminoglycan-capped domain.
  • Versican binds hyaluronic acid with high affinity via the hyaluronic acid-binding domain, and binds to sulfated glycolipids and extracellular matrix components tenascin-Ryafibrin-1 via the C-type lectin domain.
  • Versican has been reported to promote cell proliferation at least partially by binding to the EGF receptor via the EGF-like domain, and also reported to have cell adhesion inhibitory activity via the chondroitin sulfate chain. Speak. (See Non-Patent Documents 27-33)
  • sulfo-urea drugs SU agents
  • biguanide drugs biguanide drugs
  • insulin resistance improvers insulin resistance improvers
  • anti-darcosidase inhibitors anti-darcosidase inhibitors
  • fast-acting insulin secretagogues etc.
  • research is still ongoing as a pathological field that is difficult to treat.
  • Versican is one of the chondroitin sulfate proteoglycans (CSPG) and is known as a protein corresponding to the core protein.
  • CSPG chondroitin sulfate proteoglycans
  • Patent Document 1 neural stem cell differentiation inducer
  • Patent Document 2 nerve regeneration using human Z bone morphogenetic protein
  • Patent Document 3 glycosyl transfer agent
  • Patent Document 4 central nervous system damage
  • Patent document 5 nerve regeneration using human, bone morphogenetic protein
  • therapeutic inhibitor of vascular smooth muscle cells patent document 6
  • Patent Literature 7 Darican growth promoter
  • Patent Literature 8 hair growth composition
  • ADAMTS4 function inhibitor Patent Literature 9
  • Patent Literature 10 a method for suppressing scar tissue formation using an implantable medical device
  • Non-patent Document 34 Not limited to Versican siRNA, C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA administration examples and efficacy studies using the mouse type 2 diabetes (NIDDM) model. .
  • Patent Document 1 Patent Publication 2005- 278641
  • Patent Document 2 Patent Publication 2005-007196
  • Patent Document 3 Patent Publication 2004-024208
  • Patent Document 4 Patent Publication 2005-526740
  • Patent Document 5 Patent Publication No. 09-501932
  • Patent Document 6 Patent Publication 08-510209
  • Patent Document 7 Patent Publication 2006-028071
  • Patent Document 8 Patent Publication 2005-200383
  • Patent Document 9 Patent Publication 2004-244339
  • Patent Literature 10 Patent Publication No. 07-024053
  • Patent Document 11 US Pat. No. 5,180,808
  • Patent Document 12 US Pat. No. 6,579,682
  • Non-patent literature l Fujita, T et al. Biochemical Pharmacology (1996); 52: 407-411
  • Non-patent literature 2 Tsukuda, K et al. Horm Metab Res (1998); 30: 42-49
  • Non-Patent Document 3 Fujitani, S et al. Metabolism (1996); 45: 184-189
  • Non-Patent Document 4 Spiegelman et al. J Clin Invest (1997); 100: 1863-1869
  • Non-Patent Document 5 01efsky et al. Diabetes (1997); 46: 1678-1683
  • Non-patent literature 6 Glueck CJ et al. Curr Diab Rep. (2003) Aug; 3 (4): 303-312
  • Non-patent literature 7 Juntunen KS et al. Am J ClinNutr. (2003) Feb; 77 (2) : 385-391
  • Non-Patent Document 8 Van Lerberghe S et al. Diabeets Metab. (2002) Feb; 28 (1): 33-38
  • Non-Patent Document 9 Buchanan TA. J Clin Endocrinol Metab. (2001) Mar; 86 (3): 989-993
  • Non-Patent Document 10 Fallucca F et al. Diabetes Res Clin Pract. (1989) Nov 6; 7 (4): 277-2
  • Non Patent Literature l l Ostenson CG et al. Exp Clin Endocrinol. (1989) May; 93 (2-3): 241- 247
  • Non-Patent Document 12 Ward WK et al. J Clin Endocrinol Metab. (1985) Dec; 61 (6): 1039-10 45
  • Non-Patent Document 13 Lindahl, U et al. (1972) In Glycoproteins (Gottschalk, A. ed) pp. 491 —517, Elsevier, New York.
  • Non-Patent Document 14 Oegema, T et al. J. Biol. Chem. (1984); 259: 1720-1726
  • Non-patent literature 15 Sugahara, K et al. J. Biol. Chem. (1988); 263: 10168-10174
  • Non-patent literature 16 Sugahara, K et al. J. Biol. Chem. (1992); 267: 6027 -6035
  • Non-Patent Document 17 De Waard et al. J. Biol. Chem. (1992); 267: 6036-6043
  • Non-Patent Document 18 Moses, J, Oldberg et al. Eur. J. Biol. (1992); 248: 521-526
  • Non-Patent Document 19 Yamada, S et al. Trends in Glycoscience and Glycotechnology,. (199
  • Non-Patent Document 20 Schaefer L et al. FASEB J. (2001) Mar; 15 (3): 559-561
  • Non-Patent Document 21 McCarthy K.J et al. J Histochem Cytochem. (1994) Apr; 42 (4): 473-48
  • Non-Patent Document 22 Hanneken A et al. Arch Ophthalmol. (1991) Jul; 109 (7): 1005-1011
  • Non-Patent Document 23 Fushimi H et al. J Intern Med. (1989) Dec; 226 (6): 409-416
  • Non-Patent Document 24 Klein DJ et al Diabetes. (1989) Jan; 38 (1): 130-139
  • Non-Patent Document 25 Klein D.J et al. Diabetes. (1986) Oct; 35 (10): 1130-1142
  • Non-Patent Document 26 Rohrbach DH et al. J Biol Chem. (1983) Oct 10; 258 (19): 11672-1167
  • Non-Patent Document 27 Krusius T et al. J Biol Chem. (1987) Sep 25; 262 (27): 13120-13125
  • Non-Patent Document 28 Naso MF et al. J Biol Chem. (1994) Dec 30; 269 ( 52): 32999-33008
  • Non-patent document 29 Ito K et al. J Biol Chem. (1995) Jan 13; 270 (2): 958-965
  • Non-Patent Document 30 Shinomura T et al. J Biol Chem. (1995) Apr 28; 270 (17): 10328-103
  • Non-patent document 31 Zako M et al. J Biol Chem. (1995) Feb 24; 270 (8): 3914-3918
  • Non-patent document 32 Yang BL et al. J Cell Biochem. (1999) Feb 1; 72 ( 2): 210-220
  • Non-patent literature 33 Aspberg A et al. J Biol Chem. (1999) Jul 16; 274 (29): 20444-20449
  • Non-patent literature 34 Davidsson P et al. J Lipid Res. (2005 ) Sep; 46 (9): 1999-2006 Disclosure of the Invention
  • the present invention imposes provision of an insulin resistance inhibitor, a therapeutic agent for an insulin resistance disease containing the drug as an active ingredient, and a method for screening an insulin resistance inhibitor.
  • the present invention relates to an agent capable of suppressing chondroitin sulfate proteodarican (CSPG) accumulation in spleen ⁇ cells, useful for the treatment or prevention of non-insulin dependent diabetes mellitus (NIDDM) based on chondroitin sulfate proteodarican (CSPG) accumulation
  • NIDDM non-insulin dependent diabetes mellitus
  • CSPG chondroitin sulfate proteodarican
  • C SPG chondroitin sulfate proteodarican
  • insulin resistance can be suppressed by inhibiting the accumulation or biosynthesis of chondroitin sulfate proteodarican, and the present invention was completed.
  • Substances that inhibit the production or accumulation of chondroitin sulfate proteodalycan are useful as insulin resistance inhibitors.
  • the drug is a drug for treating or preventing an insulin resistant disease.
  • the present invention relates to an insulin resistance inhibitor, a therapeutic agent for an insulin resistance disease containing the drug as an active ingredient, a screening method for an insulin resistance inhibitor, and the like, more specifically,
  • an insulin resistance inhibitor comprising as an active ingredient a substance that inhibits the production or accumulation of chondroitin sulfate proteodarican,
  • a screening method for an insulin resistance inhibitor which comprises selecting a substance having an action of inhibiting the production or accumulation of chondroitin sulfate proteodarican from a test sample,
  • the present invention further relates to the following.
  • composition comprising the drug according to any one of [1] to [9] and a pharmaceutically acceptable carrier.
  • the present invention has revealed that the development and accumulation of chondroitin sulfate proteodalycan is related to the development of insulin resistance. It was shown that insulin resistance is suppressed by inhibiting the production and accumulation of chondroitin sulfate proteoglycan. It will be possible to provide a new concept of therapeutic drugs for insulin resistance. Insulin resistance in particular is closely related to metabolic syndrome (visceral fat syndrome) such as diabetes 'hyperlipidemia, obesity' arteriosclerosis, and the number of patients is increasing in modern society. It has significant medical and industrial significance.
  • metabolic syndrome visceral fat syndrome
  • FIG. L Diagram showing changes in body weight during 14 days in a control group and a Versican siRNA-treated group in a type 2 diabetes (NIDDM) model mouse (female) induced by Streptozotocin. is there.
  • the vertical axis represents weight (g) and the horizontal axis represents days.
  • FIG.2 Versican siRNA-treated model in type 2 diabetes (NIDDM) model mice (female) induced by Streptozotocin! After insulin tolerance test, 0 min, 15 min, It is a figure which shows the fluctuation
  • the vertical axis represents the blood glucose level (mg / dL), and the horizontal axis represents the blood glucose measurement time (min) after the insulin load test.
  • FIG. 3 GAPDH, Versican (Versican) by RT-PCR on the 14th day (last day) in the control group, Versican (Versican) siRNA treatment group in type 2 diabetes (NIDDM) model mice induced by Streptozotocin ). Versican (Versican) expression is suppressed in the siRNA-treated group!
  • FIG. 4 Reduced deposition of Amyloid precursor protein (APP) in Versi can siRNA-treated mice in type 2 diabetes (NIDDM) model mice induced by Streptozotocin ( (Upper), a photograph showing detection results (red brown signal, blue signal) of insulin positive spleen ⁇ -cell rise (lower). Magnification 500 times (upper), 200 times (lower).
  • NIDDM type 2 diabetes
  • FIG. 5 Detection of chondroitin sulfate proteodarican (CSPG) in the control group, Versican siRNA treated group in type 2 diabetes (NIDDM) model mice induced by Streptozotocin Versican siRNA It is a photograph showing the reduction effect of chondroitin sulfate proteodarican (CSPG) (purple signal) in the treatment group. 400x magnification. The name of the antibody clone used for detection is shown below the photograph.
  • FIG. 6 14-day body weight changes in the control group, C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA treatment group in type 2 diabetes (NIDDM) model mice induced by Streptozotocin
  • the vertical axis represents body weight (g), and the horizontal axis represents days.
  • FIG. 6 is a graph showing changes in blood glucose levels in the Control group, C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA treatment group after 60 minutes.
  • the vertical axis represents blood glucose level (mg / dL), and the horizontal axis represents blood glucose measurement time (min) after the insulin tolerance test.
  • FIG. 8 Day 14 (last day) in the control group, C4ST-1 siRNA, C4ST-2 siRNA, and C4ST-3 siRNA treatment group in type 2 diabetes (NIDDM) model mice induced by Streptozotocin
  • FIG. 5 shows the expression of GAPDH and Versican by RT-PCR.
  • C4ST-1 siRNA, C4ST-2 siRNA, C4ST-3 This shows that C4ST-1, C4ST-2, and C4ST-3 expression was suppressed in the siRNA treatment group! /.
  • FIG. 10 Chondroitin sulfate protease in control group and C4ST-1 siRNA treatment group in type 2 diabetes (NIDDM) model mice induced by Streptozotocin It is a photograph showing the reduction effect of chondroitin sulfate proteoglycan (CSPG) (purple signal) in the C4ST-1 siRNA treatment group as a result of detection of oglycan (CSPG). 400x magnification. The name of the antibody clone used for detection is shown below the photo.
  • CSPG chondroitin sulfate proteoglycan
  • the pathological conditions associated with diabetes include abnormal insulin secretion in splenic j8 cells and impaired insulin action, including glucose uptake in insulin target tissues.
  • the present inventors have paid attention to the function of chondroitin sulfate proteodalycan in order to improve the abnormal secretion of insulin in splenic j8 cells as one of the effective methods for treating diabetes. Then, a state in which accumulation of chondroitin sulfate proteodarican was suppressed in a diabetic model mouse was created and analyzed in detail. As a result, the accumulation of chondroitin sulfate proteodarican was improved compared to wild-type spleen j8 cells.
  • the present invention relates to an insulin resistance inhibitor comprising as an active ingredient a substance that inhibits the production or accumulation of chondroitin sulfate proteodalycan.
  • the "chondroitin sulfate proteodarican” of the present invention is one of the proteodaricans, and is a covalent bond between chondroitin sulfate Z dermatan sulfate, which is a typical sulfated mucopolysaccharide, and protein (coprotein).
  • the “chondroitin sulfate proteodarican” in the present invention is preferably a human chondroitin sulfate proteodarican, but the species from which it is derived is not particularly limited. Proteins equivalent to can (such as homologs and orthologs) are also included in “chondroitin sulfate proteodaricans” in the present invention.
  • the present invention can be carried out as long as the organism has a protein corresponding to human chondroitin sulfate proteodalycan and has a protein equivalent to human chondroitin sulfate proteoglycan.
  • the chondroitin sulfate proteodarican in the present invention is one of the causes of inflammation. Occasionally, glycosaminodarlican (GAG) chains are combined to become proteodaricans, so-called part-time proteodaricans.
  • GAG glycosaminodarlican
  • examples of chondroitin sulfate proteoglycans include aggrican, versican, neurocan, brevican, ⁇ -glycan, Decorm, Biglycan, Fibromodulin, and PG-Lb.
  • the chondroitin sulfate proteodarican in the present invention is not limited to these, and any substance having activity as a chondroitin sulfate proteodarican can be used.
  • the activity of chondroitin sulfate proteodalycan includes, for example, cell adhesion ability or cell growth promotion.
  • a person skilled in the art can evaluate the activity as chondroitin sulfate proteodalycan by the following method.
  • a protein containing a partial region of chondroitin sulfate proteodarican amino acid sequence, or a high homology with a partial region usually 70% or more, preferably 80% or more, more preferably 90% or more, most preferably Measure the divisional proliferation of tumor cells (eg Caco-2, HT-29 cells, etc.) in the presence of proteins with greater than 95%).
  • Proteins that have the effect of promoting mitotic proliferation can be determined as proteins with chondroitin sulfate proteodarican activity (Int J Exp Pathol. 2005 Aug; 86 (4): 219-29 and Histochem Cell Biol. 2005 Aug; 124 (2): 139-49).
  • high homology means 50% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more (e.g., 95% or more, further 96%, 97%, 98% or 99% or higher) homology.
  • This homology is determined by the mBLAST algorithm (Altschul et al. (1990) Proc. Natl. Acad. Sci. USA 8 7: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873— 7).
  • Insulin resistance in the present invention refers to a state in which the presence of insulin in a living body is abnormal.
  • the ability to reduce insulin secretion such as ⁇ -cell force in the spleen and the action of insulin in the target tissues of skeletal muscle, liver, and adipose tissue are not limited to these.
  • “inhibiting the production or accumulation of chondroitin sulfate proteodarican” means, for example, “promotion of degradation”, “synthesis inhibition”, “desulfation”, “sulfate sulfate” But not limited to them, the abundance, function or activity of chondroitin sulfate proteodarican is reduced or eliminated compared to the comparison target. That means.
  • the “substance that inhibits the production or accumulation” of chondroitin sulfate proteodarican is not particularly limited, but preferably the “substance that has an activity of promoting degradation of chondroitin sulfate proteoglycan” and “the substance has an inhibitory effect on synthesis”. “Substance”, “Substance with desulfurization and oxidation”, or “Substance with sulfation-inhibiting action”.
  • Protein that is the core of chondroitin sulfate proteodarican includes, for example, core proteins such as aggrican, versican, neurocan, and b revican in the case of matri X type chondroitin sulfate proteoglycan.
  • membrane-type chondroitin sulfate proteodlicans include core proteins such as j8 glycan, Decorin, Biglycan, Fibromodulin, and PG-Lb. These are only examples, and are not limited to these, and may be any protein that is widely used as the core of chondroitin sulfate proteodalycan.
  • “Expression” includes “transcription” from a gene or “translation” into a polypeptide and “degradation inhibition” of a protein. “Expression of the protein that is the core of chondroitin sulfate proteodarican” refers to the transcription and translation of the gene that encodes the protein that is the core of chondroitin sulfate proteodarican, or the chondroitin sulfate proteo This means that the protein that forms the core of Darican is produced.
  • “the function of the protein serving as the core of chondroitin sulfate proteodarican” includes, for example, the function of the protein binding to chondroitin sulfate and the binding to other components in the cell.
  • degradation promotion of chondroitin sulfate proteodarican may be an increase in the expression of an enzyme that cleaves or degrades chondroitin sulfate proteodarican or an enzyme related thereto.
  • these enzymes include, but are not limited to, meta-oral proteinases (for example, AD AMTS-1, ADAMTS-4, ADAMTS-5, etc.) chondroitinase, Calpain I, and the like.
  • “Acceleration of degradation” is a decrease in the abundance of chondroitin sulfate proteodarican caused by administration of these enzymes or a part of them. A little.
  • Degradation promotion may be caused by administration of a substance that promotes suppression of chondroitin sulfate proteodarican expression.
  • substances include, for example, n-butylate, Diethyl carbamazepine, i'unicamycin, non-steroidal estrogen, and cyclofenil deiphenol.
  • Preferable embodiments of the "substance having a decomposition promoting action” include, for example, a compound (nucleic acid) selected from the group consisting of the following (a) to (c).
  • nucleic acid having a ribozyme activity that specifically cleaves the transcription product of the gene encoding the core protein of chondroitin sulfate proteodarican
  • examples of the “substance having a decomposition promoting action” include compounds selected from the following groups (a) to (c).
  • chondroitin sulfate proteodarican Low molecular weight compound that binds to the core protein of chondroitin sulfate proteodarican
  • “Synthetic inhibition” of chondroitin sulfate proteodarican means, for example, inhibition of glycosaminodarlican biosynthesis, chondroitin sulfate proteodarican synthesis Inhibition of enzymes involved in the above, but is not necessarily limited to these, it refers to inhibiting any of the processes in which chondroitin sulfate proteodarican is synthesized.
  • Examples of substances that inhibit the synthesis of chondroitin sulfate proteodarican include those that inhibit glycosaminoglycan biosynthesis, such as j8-D-xyloside, 2-deoxy-D-glucose (2-D (J), ethane-1-hydroxy-1, 1-diphosphonate (ETDP), 5-hexoxy 2-aeoxyundine (H UdR), etc.
  • glycosaminoglycan biosynthesis such as j8-D-xyloside, 2-deoxy-D-glucose (2-D (J), ethane-1-hydroxy-1, 1-diphosphonate (ETDP), 5-hexoxy 2-aeoxyundine (H UdR), etc.
  • the biosynthesis of glycosaminodarlicans can be achieved with these and other substances. Inhibited and the synthesis of chondroitin sulfate proteodalycan is inhibited.
  • examples of enzymes involved in chondroitin synthesis include GalNAc4ST-1, GalNAc4 ST-2, GALNAC4S-6ST, UA20ST, GalT-I, GalT-II, GlcAT-I, and XylosylT.
  • Preferable embodiments of the "substance having a synthesis inhibitory action” include, for example, a compound (nucleic acid) selected from the group consisting of the following (a) to (c).
  • examples of the "substance having a synthesis inhibitory action” include compounds selected from the following groups (a) to (c).
  • Desulfation of chondroitin sulfate proteodarican refers to removal of sulfate groups in chondroitin sulfate proteodarican, for example, desulfation or sulfation by a desulfase enzyme that is administered with endogenous or external force. Examples include, but are not limited to, suppression of sulfation by a compound that suppresses sulfation.
  • Examples of the desulfating enzyme include Chondroitin-4-sulfatase and Chondroitin-6-sulfatase.
  • Examples of the compound that suppresses sulfation include Chlorate and EGF receptor antagonist.
  • Preferable embodiments of the "substance having desulfating action” include, for example, compounds (nucleic acids) selected from the group consisting of the following (a) to (c).
  • nucleic acid having a ribozyme activity that specifically cleaves a transcript of a gene encoding a chondroitin sulfate proteodarican desulfating enzyme inhibitory protein
  • (C) a nucleic acid having an action of inhibiting the expression of a gene encoding a chondroitin sulfate proteodarican desulfating enzyme inhibitory protein by the RNAi effect
  • Examples of the "substance having a desulfating action” include compounds selected from the following groups (a) to (c).
  • the “desulfation-inhibiting compound” is not limited to a protein, and includes non-protein compounds such as coenzymes, for example.
  • the "sulfate inhibitory action" of chondroitin sulfate proteodarican includes, for example, inhibition of sulfate group transfer enzyme, but is not limited to this, and occurs in the process of chondroitin sulfate proteodarican synthesis. It refers to inhibition of sulfation.
  • Examples of the sulfotransferase include C4ST-l (Chondroitin D-N-acetylgalactosamine
  • Preferable embodiments of the "substance having a sulfate inhibitory effect” include, for example, compounds (nucleic acids) selected from the following groups (a) to (c).
  • nucleic acid having a ribozyme activity that specifically cleaves a transcript of a gene encoding chondroitin sulfate proteodarican sulfate transferase.
  • examples of the “substance having a sulfation inhibiting action” include compounds selected from the group consisting of the following (a) to (c).
  • the enzymes exemplified above include not only one enzyme corresponding to one gene but also an enzyme group sharing certain characteristics.
  • chondroitinase is a collective term for enzymes such as ABC, AC, and B that share the characteristics of mucopolysaccharide-degrading enzymes but differ in substrate specificity.
  • chondroitinase AC I cleaves the chondroitin sulfates (A, C or E), chondroitin, chondroitin sulfate-dermatan sulfate hybrid type and hyaluronic acid N-acetylhexoxide binding bond.
  • an oligosaccharide having a ⁇ 4-glucuronic acid residue at the non-reducing end is generated.
  • This enzyme does not act on dermatan sulfate (chondroitin sulfate B, which has L-iduronic acid as hexuronic acid), ketalan sulfate, heparan sulfate and heparin.
  • chondroitinase AC II cleaves the N-acetyl hexosaminide bond of chondroitin, chondroitin sulfate A and chondroitin sulfate C in an elimination reaction, resulting in ⁇ 4-unsaturated disaccharide (0 ⁇ 03, AD 4S and A Di-6S). This enzyme also works well on hyaluronic acid.
  • Chondroitinase B dermatanase
  • Produces oligosaccharides disaccharides and tetrasaccharides). This enzyme does not contain L-iduronic acid! /, Does not act on chondroitin sulfate A and chondroitin sulfate C.
  • Dermatan a derivative of dermatan sulfate with the sulfate group removed, is not a substrate for this enzyme.
  • the site where the second position of the L-iduronic acid unit of dermatan sulfate is sulfated is more cleaved by this enzyme.
  • Chondroitinase ABC cleaves the N-acetyl hexosaminide bond of chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, chondroitin and hyaluronic acid in an elimination reaction, and ⁇ 4- Mainly produces disaccharides with hexuronic acid residues.
  • Chondroitinase is a collective term for these enzymes that have different properties but share the same properties of mucopolysaccharide-degrading enzymes, and are not limited to one row and seven rows of Cnondroitinase ACI, then hondroitinase A and II, and then hondrotinase. Not limited to B, Chond roitinase ABC.
  • Enzymes that share such characteristics do not necessarily correspond to one gene on genomic DNA.
  • the ⁇ column is chondroitin—4—sulfatase, chondroitin—6—sulfatase, and sequences referenced by multiple accession numbers in the genome database (eg Gen bank accession number NT_039500 (some of which are (Accession number CAAA01098429 (SEQ ID NO: 91)), NT_078575, NT_039353, NW_001030904, NW_0 01030811, NW_001030796, NW_000349) are searched on the public gene database Genbank.
  • chondroitin sulfate proteodaricans include aggrican, versican, neurocan, brevican ⁇ ⁇ glycan, Decorm, Biglycan, Fibromodulin, P — Lb, an enzyme that cleaves or degrades chondroitin sulfate proteodarican, and these.
  • ADAMTS-1, ADAMTS-4, ADAMTS-5, Calpain I examples of enzymes involved in chondroitin synthesis, GalNAc4ST-1, GalNAc4ST-2, GALNAC4 S-6ST, UA20ST, GalT-1 C4ST-1, C4ST-2, C4ST-3, D4ST, C6ST-1, and C6ST-2, public genes of genes encoding in humans, exemplified as GalT-II, GlcAT-1, XylosylT, and sulfotransferase
  • the accession number, base sequence and amino acid sequence in the database Genbank are as follows.
  • aggrican (Accession number NM—007424, SEQ ID NO: 1 for nucleotide sequence, SEQ ID NO: 2 for amino acid sequence)
  • neurocan accesion number NM—010875, nucleotide sequence SEQ ID NO: 5, amino acid sequence SEQ ID NO: 6) brevican (Accession number NM_007529, nucleotide sequence number: 7, amino acid sequence number: 8)
  • jS glycan (Accession number AF039601, nucleotide sequence number: 9, amino acid sequence number: 10)
  • Biglycan (Accession number BC057185, SEQ ID NO: 13 for nucleotide sequence, SEQ ID NO: 14 for amino acid sequence)
  • Fibromodulin (Accession number NM—021355, nucleotide sequence number: 15, amino acid sequence number: 16)
  • PG-Lb (Accession number NM—007884, nucleotide sequence number: 17; amino acid sequence number: 18)
  • ADAMTS-1 (Accession number NM_009621, nucleotide sequence SEQ ID NO: 19, amino acid sequence SEQ ID NO: 20)
  • ADAMTS-4 (Accession number NM—172845, SEQ ID NO: 21 of nucleotide sequence, SEQ ID NO: 22 of amino acid sequence)
  • ADAMTS-5 (Accession number AF140673, nucleotide sequence SEQ ID NO: 23, amino acid sequence SEQ ID NO: 24)
  • Calpain I (Accession number NM—007600, nucleotide sequence number: 25, amino acid sequence number: 26)
  • GalNAc4ST-l accession number NM—175140, nucleotide sequence SEQ ID NO: 27, amino acid sequence SEQ ID NO: 28)
  • GalNAc4ST-2 (Accession number NM—199055, nucleotide sequence number: 29, amino acid sequence number: 30)
  • GALNAC4S-6ST (Accession number NM_029935, nucleotide sequence SEQ ID NO: 31, amino acid sequence SEQ ID NO: 32)
  • GalT-I (Accession number NM_016769, nucleotide sequence number: 35, amino acid sequence number: 36)
  • GalT-11 accession number BC064767, nucleotide sequence number: 37, amino acid sequence number: 38
  • GlcAT-I (Accession No. BC058082, nucleotide sequence SEQ ID NO: 39, amino acid sequence SEQ ID NO: 40, or accession number NM_024256, nucleotide sequence SEQ ID NO: 41, amino acid sequence SEQ ID NO: 42 )
  • XylosylT (Accession number NM—145828, nucleotide sequence number: 43, amino acid sequence number: 44)
  • C4ST-1 (Accession number NM— 021439, nucleotide sequence number: 45, amino acid sequence number: 46)
  • C4ST-2 (Accession number NM—021528, nucleotide sequence SEQ ID NO: 47, amino acid sequence SEQ ID NO: 48)
  • C4ST-3 (Accession No. XM—355798, nucleotide sequence SEQ ID NO: 49, amino acid sequence SEQ ID NO: 50)
  • D4ST accession number NM_028117, nucleotide sequence SEQ ID NO: 51, amino acid sequence SEQ ID NO: 52
  • C6ST-1 (Accession number NM—016803, SEQ ID NO: 53 of the nucleotide sequence, SEQ ID NO: 54 of the amino acid sequence)
  • C6ST-2 (Accession number AB046929, nucleotide sequence number: 55, amino acid sequence number: 56)
  • proteins other than those described 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.
  • a protein having the function of the protein for example, a function of binding to a structural component in a cell is included in the protein of the present invention.
  • the above-mentioned protein is, if f row, IJ number: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56 Amino acid sequence with one or more amino acids added, deleted, substituted or inserted
  • a protein consisting of a sequence, wherein the number of normally changing amino acids is within 30 amino acids, preferably within 10 amino acids, more preferably within 5 amino acids, most preferably within 3 amino acids.
  • Examples of the gene in the present invention include, for example, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, or 55. Etc.).
  • High homology means 50% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more (e.g., 95% or more, further 96%, 97%, 98% or 99% or more). It means the homology of (above). This homology was determined by the mBLAST algorithm (Altschul et al. (1990) Proc. Natl. Acad. Sci.
  • stringent conditions include, for example, “2 X SSC, 0.1% SDS, 50.C”, “2 X SSC, 0.1% SDS, 42 ° Cj,“ 1 X SSC, 0.1% SDS, 37 ⁇ ° C '', more stringent conditions as ⁇ 2 X SSC, 0.1% SDS, 65 ° C '', ⁇ 0.5 X SSC, 0.1% SDS, 42 ° C '' and ⁇ 0.2 X SSC, 0.1% SDS, 65 ° C '' Can be mentioned.
  • a person skilled in the art can convert a protein functionally equivalent to the above protein from the above highly homologous protein into an action of promoting the degradation of chondroitin sulfate proteodarican, a synthetic inhibitory action, a desulfation action, or It can be suitably obtained by using a method for measuring the activity of sulfate inhibitory action.
  • a specific activity measuring method is the screen in the present invention described later. It is described in the section of the Jung method.
  • those skilled in the art can appropriately obtain an endogenous gene corresponding to the above gene in another organism based on the base sequence of the above gene.
  • the above-mentioned proteins and genes corresponding to the above-mentioned proteins and genes in organisms other than humans, or the above-mentioned proteins and genes functionally equivalent to the above-mentioned proteins and genes are also simply referred to as It may be described in.
  • the protein of the present invention can be prepared not only as a natural protein but also as a recombinant protein using a gene recombination technique.
  • a natural protein for example, it can be prepared by a method using affinity chromatography using an antibody against the above protein against a cell (tissue) extract that is thought to express the above protein. It is.
  • a recombinant protein can be prepared, for example, by culturing cells transformed with DNA encoding the protein.
  • the above-mentioned protein of the present invention is suitably used, for example, in the screening method described later.
  • Nucleic acid in the present invention means RNA or DNA. Chemically synthesized nucleic acid analogs such as so-called PNA (peptide nucleic acid) are also included in the nucleic acid of the present invention. PNA replaces the pentose / phosphate skeleton, which is the basic skeleton structure of nucleic acid, with a polyamide skeleton with glycine as a unit, and has a three-dimensional structure very similar to nucleic acid.
  • PNA peptide nucleic acid
  • antisense nucleic acids inhibit the expression of target genes by inhibiting various processes such as transcription, splicing or translation (Hirashima and Inoue, Shinsei Kagaku Kenkyusho 2 Nucleic acid IV gene replication and expression, Japan Biochemical Society, Tokyo Chemical Doujin, 1993, 319-347.).
  • the antisense nucleic acid used in the present invention encodes any one of the above-described chondroitin sulfate proteodarican core protein, synthase, desulfase-inhibiting protein, and sulfotransferase by any of the above-described actions. Gene expression and Z or function may be inhibited.
  • the above-mentioned chondroitin sulfate proteodarican core protein, synthase, desulfase inhibitor protein, or a gene encoding a sulfate transfer enzyme is complementary to the untranslated region near the 5 'end of the mRNA. If an antisense sequence is designed, it would be effective to inhibit gene translation.
  • a sequence complementary to the coding region or the 3 ′ untranslated region can also be used.
  • the anti-translation region of the anti-translation region consisting of the core protein, the synthase, the desulfation enzyme inhibitory protein, or the gene encoding the sulfotransferase as described above is not limited to the anti-translation region.
  • a nucleic acid containing a sense sequence is also included in the antisense nucleic acid used in the present invention.
  • the antisense nucleic acid to be used is linked downstream of an appropriate 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) by using a known method.
  • the sequence of the antisense nucleic acid is the gene encoding the endogenous chondroitin sulfate proteodarican core protein, synthase, desulfase inhibitor protein, or sulfotransferase of the animal (cell) to be transformed or one of them. It is preferable that the sequence is complementary to the region, but it may not be completely complementary as long as the expression of the gene can be effectively suppressed.
  • the transcribed RNA has a complementarity of preferably 90% or more, and most preferably 95% or more, to the target gene transcript.
  • 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 indispensable.
  • the length is not limited to this length, and may be, for example, 100 bases or more, or 500 bases or more.
  • the antisense nucleic acid of the present invention is not particularly limited.
  • the base sequence of Versican gene (GenBank accession number BC096495, SEQ ID NO: 3)
  • the base sequence of C4ST-1 gene (GenBank accession) No. NM—021439, SEQ ID NO: 45)
  • C4ST-2 gene base sequence (GenBank accession number NM—0221528, SEQ ID NO: 47)
  • C4ST-3 gene base sequence GenBank accession) No. XM-355798, SEQ ID NO: 49).
  • Ribozyme refers to an RNA molecule that has catalytic activity.
  • ribozymes having various activities, research focusing on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes that cleave RNA site-specifically.
  • Some ribozymes have a size of 400 nucleotides or more, such as the group I intron type and Ml RNA contained in RNase P, and some have an active domain of about 40 nucleotides called hammerhead type or hairpin type ( Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 19 90, 35, 2191.).
  • the self-cleaving domain of the hammerhead ribozyme has the ability to cleave 3 'of C15 in the sequence G13U14C15.
  • base pairing between U14 and A9 is important. It has been shown that A15 or U15 can also be cleaved (Koizumi, M. et al., FEBS Lett, 1988, 228, 228.) 0 Designing a ribozyme whose substrate binding site is complementary to the RNA sequence near the target site
  • a restriction enzyme-like RNA cleavage ribozyme that recognizes the sequence UC, UU, or UA in the target RNA can be generated (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 cleavage ribozymes can also be generated from hairpin ribozymes (Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751., Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, 112.).
  • the ribozyme is used to specifically cleave the above-mentioned chondroitin sulfate proteodarican core protein, synthase, desulfase inhibitor protein, or transcript of a gene encoding a sulfotransferase. Gene expression can be inhibited.
  • RNA interference (hereinafter abbreviated as "RNAi") using double-stranded RNA having the same or similar sequence as the target gene sequence. It can be carried out.
  • RNAi small interfering RNA
  • siRNA small interfering RNA
  • dsRNA double-stranded RNA
  • RNAi can be induced by using short dsRNA (siRNA). RNAi is more stable, easier to experiment, and less expensive than knockout mice. It has many advantages.
  • RNAi is a sense RNA that has a sequence power that is homologous to the mRNA of the target gene, a complementary double-stranded antisense RNA, and a short double-stranded RNA that is powerful (hereinafter abbreviated as “dsRNA”).
  • dsRNA a short double-stranded RNA that is powerful
  • RNAi can suppress the expression of target genes in this way, it can be applied as a simple gene knockout method instead of the conventional complicated and low-efficiency gene disruption method by homologous recombination, or for gene therapy. It is attracting attention as a method.
  • the RNA used for RNAi is the above chondroit It does not have to be completely identical to the gene encoding the core protein, synthetic enzyme, desulfurase inhibitor protein, or sulfotransferase, or a partial region of this gene. It is preferable to have.
  • the target is not particularly limited as long as it is a gene encoding the above-mentioned chondroitin sulfate proteoglycan core protein, synthase, desulfase inhibitor protein, or sulfate transferase. It is possible to make any arbitrary region as a target candidate.
  • the base sequence of Versican gene SEQ ID NO: 3
  • the base sequence of C4ST-1 gene SEQ ID NO: 45
  • the base sequence of C4ST-2 gene SEQ ID NO: 47
  • the base sequence of C4ST-3 gene It can be created based on SEQ ID NO: 49. More specifically, a partial region of the sequence can be a target candidate.
  • a partial region of the Versican gene base sequence (SEQ ID NO: 57), the base of the C4ST-1 gene Partial region of the sequence (SEQ ID NO: 58), partial region of the base sequence of the C4ST-2 gene (SEQ ID NO: 59), partial region of the base sequence of the C4ST-3 gene (SEQ ID NO: 60), C6ST -Partial region of the base sequence of 1 gene (SEQ ID NO: 61), partial region of the base sequence of C6ST-2 gene (SEQ ID NO: 62), partial region of the base sequence of GalNAc4ST-l gene (SEQ ID NO: 6 3), a partial region of the base sequence of the GalNAc4ST-2 gene (SEQ ID NO: 64), a partial region of the base sequence of GALNAC4S-6ST (SEQ ID NO: 65), and the like. More specifically, siRNA targeting the DNA sequences specifically shown by this specification (SEQ ID NOs: 71 to 73 and 80-90) can be exemplified.
  • siRNA synthesized in vitro is linked to plasmid DNA and introduced into the cell
  • a method of annealing two RNAs, 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 called short hairpin RNA, and is an RNA molecule 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 in the molecule are also included in the siRNA of the present invention.
  • preferred embodiments of the present invention include Versican, C4ST-1, C4ST-2, C4ST-3 and the like.
  • siRNA targeting the DNA sequence SEQ ID NOs: 71 to 73 and 80 to 90
  • RNAi effect for example, 1
  • a double-stranded RNA with a structure in which a small number of RNAs are added or deleted encodes the above-mentioned chondroitin sulfate proteodarican core protein, synthase, desulfurase oxidase inhibitor protein, or sulfate transferase
  • Any siRNA of the present invention may be used as long as it has a function of suppressing gene expression.
  • RNA used for RNAi does not have to be the same as the gene encoding the protein or a partial region of the gene. ) Preferred to have sex.
  • the force of DICER (a member of the RNase III nucleolytic enzyme family) contacts double-stranded RNA, and the double-stranded RNA is small iterfering RNA or It is thought to be broken down into small fragments called siRNA! /
  • the double-stranded RNA having the RNAi effect in the present invention includes double-stranded RNA before being digested by DICER as described above. That is, even a long-chain RNA that does not have an RNAi effect with the same length is expected to be decomposed into siRNA having an RNAi effect by the cell.
  • the length of the double stranded RNA is not particularly limited.
  • the strand RNA can be decomposed in advance with DICER, and the degradation product can be used as the agent of the present invention.
  • This degradation product is expected to contain double-stranded RNA molecules (siRNA) having the RNAi effect. According to this method, it is not necessary to particularly select a region on mRNA expected to have an RNAi effect. That is, the region on the mRNA of the above-mentioned gene of the present invention having an RNAi effect does not necessarily need to be accurately defined.
  • the above-mentioned "double-stranded RNA that can be suppressed by the RNAi effect" of the present invention means that, for those skilled in the art, the above-mentioned chondroitin sulfate proteoglycan core protein, synthase, and desulfase that are targets of the double-stranded RNA It can be appropriately prepared based on the base sequence of the gene encoding the inhibitory protein or sulfate transferase. For example, SEQ ID NO: 71
  • the double-stranded RNA of the present invention can be prepared based on the base sequence described in 1.
  • RNA RNA sequence set forth in SEQ ID NO: 71
  • an arbitrary continuous RNA region of mRNA that is a transcription product of the sequence is selected, and a double-stranded RNA corresponding to this region is prepared.
  • the person skilled in the art can appropriately carry out within the range of normal trials.
  • those skilled in the art can also appropriately select a siRNA sequence having a stronger RNAi effect from the mRNA sequence that is a transcription product of the sequence, by a known method.
  • a siRNA can be appropriately prepared by those skilled in the art using a commercially available nucleic acid synthesizer.
  • a general synthetic contract service can be used for synthesis of desired RNA.
  • the siRNA in the present invention may be a mixture of a plurality of sets of double-stranded RNAs for a region containing a target sequence, which need not necessarily be a set of double-stranded RNAs for 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. You can use the composite contract service.
  • the siRNA of the present invention includes so-called “cocktail siRNA”.
  • RNA ribonucleotides
  • one or more ribonucleotides constituting siRNA may be a corresponding deoxyribonucleotide.
  • This “corresponding” refers to the same base species (adenine, guanine, cytosine, thymine (uracil)) although the structures of the sugar moieties are different.
  • a deoxyribonucleotide corresponding to a ribonucleotide having adenine refers to a 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 preferable embodiment of the compound capable of suppressing the expression of the gene encoding the 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 includes the above-mentioned chondroitin sulfate proteodarican core protein, synthase, desulfase inhibitor protein, or expression regulatory region of a gene encoding a sulfotransferase (for example, Specific examples include the base sequence represented by SEQ ID NO: 66, which is the promoter region of PG-Lb.)
  • SEQ ID NO: 66 which is the promoter region of PG-Lb.
  • the compound is, for example, a promoter DNA fragment of a gene encoding the above chondroitin sulfate proteodarican core protein, synthase, desulfase inhibitor protein, or sulfotransferase, and binding activity to the DNA fragment It can be obtained by a screening method using as an index.
  • those skilled in the art will determine whether or not the desired compound inhibits the expression of the above-mentioned chondroitin sulfate-teododalican core protein, synthase, desulfase-inhibiting protein, or gene encoding sulfotransferase. The determination can be appropriately carried out by a known method such as a reporter assay method.
  • the DNA (vector) capable of expressing the RNA of the present invention is also the above-described core protein, synthetic enzyme, desulfase-inhibiting protein, or sulfate group of the chondroitin sulfate proteodalycan of the present invention.
  • Preferred embodiments of compounds capable of inhibiting the expression of genes encoding transferases are included in the embodiments.
  • the DNA (beta) capable of expressing the double-stranded RNA of the present invention is a DNA that encodes one strand of the double-stranded RNA and a DNA force that encodes the other strand of the double-stranded RNA, respectively.
  • DNA having a structure linked to a promoter so that it can be expressed.
  • the above-mentioned DNA of the present invention can be appropriately prepared by those skilled in the art using a general genetic technique. More specifically, the expression vector of the present invention is inserted by appropriately inserting DNA encoding the RNA of the present invention into various known expression vectors. It is possible to produce a kuta.
  • a preferred embodiment of the vector of the present invention is a vector that expresses RNA (siRNA) capable of suppressing the expression of Versican, C4ST-1, C4ST-2, C4ST-3, and the like by the RNAi effect. it can.
  • siRNA RNA
  • chondroitin sulfate proteodarican core protein, synthase, desulfation enzyme inhibitor compound, or antibody that binds to a sulfotransferase can be prepared by methods known to those skilled in the art.
  • a polyclonal antibody can be obtained, for example, as follows. Serum is obtained by immunizing small animals such as rabbits with recombinant (recombinant) protein expressed in microorganisms as a fusion protein with the above-mentioned natural protein or GST, or a partial peptide thereof.
  • ammonium sulfate precipitation protein A, protein G column, DEAE ion exchange chromatography, core protein of the above chondroitin sulfate proteodarican, synthase, desulfase inhibitor compound, or sulfate transferase Or by purification using a utility column coupled with a synthetic peptide.
  • a monoclonal antibody for example, the above-mentioned chondroitin sulfate proteodarican core protein, synthetic enzyme, desulfase inhibitor compound, or sulfotransferase or its partial peptide is immunized to a small animal such as a mouse.
  • the spleen is removed from the mouse, ground and separated to separate the cells, and the cells and mouse myeloma cells are fused using a reagent such as polyethylene glycol, and the resulting fused cells (hybridoma)
  • a reagent such as polyethylene glycol
  • a clone that produces an antibody that binds to the above chondroitin sulfate proteoglycan coprotein, synthase, desulfase inhibitor compound, or sulfotransferase is selected.
  • the obtained noci / 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, It can be prepared by purification using the above-mentioned chondroitin sulfate proteodarican core protein, synthetic enzyme, desulfating enzyme inhibitory compound, or a protein column coupled with a sulfotransferase protein or a synthetic peptide. Is possible.
  • the antibody of the present invention binds to the above-described chondroitin sulfate proteodarican core protein of the present invention, a synthase, a desulfase inhibitor compound, or a sulfotransferase.
  • a human antibody, a humanized antibody by genetic recombination, an antibody fragment thereof, or an antibody modification product thereof may be used.
  • the protein of the present invention used as a sensitizing antigen for obtaining an antibody is not limited with respect to the animal species from which it is derived, but a protein derived from a mammal such as a mouse is preferred, and a protein derived from a human is particularly preferred.
  • a human-derived protein can be appropriately obtained by those skilled in the art using the gene sequence or amino acid sequence 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 the protein include an amino group (N) terminal fragment and a carboxy (C) terminal fragment of the 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 in vitro with proteins, protein-expressing cells or lysates thereof. And fusion of sensitized lymphocytes with human-derived permanent mitotic cells, such as U266, to produce a hyperidoma that produces the desired human antibody with protein-binding activity. .
  • chondroitin sulfate proteodarican core protein, synthase, desulfase inhibitor compound, or antibody to sulfate group transferase of the present invention binds to the protein to thereby regulate the expression or function of the protein. An inhibiting effect is expected.
  • a human antibody or a humanized antibody is preferred in order to reduce immunogenicity.
  • the present invention relates to the above chondroitin sulfate proteodarican as a substance capable of inhibiting the function of the core protein, synthase, desulfase inhibitor, or sulfotransferase of the above chondroitin sulfate proteodarican. It also contains a low molecular weight substance (low molecular weight compound) that binds to a core protein, a synthetic enzyme, a desulfurizing oxidase inhibiting compound, or a sulfotransferase.
  • the low molecular weight substance may be a natural or artificial compound. Through 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.
  • the above-mentioned chondroitin sulfate proteodarican core protein synthetic enzyme, desulfase inhibitor protein, or sulfotransferase of the present invention
  • the above-mentioned chondroitin sulfate proteodarican And a mutant having a dominant negative property (dominant negative protein) with respect to a core protein, a synthase, a desulfase inhibitor protein, or a sulfotransferase.
  • the chondroitin sulfate proteodarican core protein, synthase, desulfurase inhibitor protein, or the protein variant having a dominant negative property to sulfate group refers to the core of chondroitin sulfate proteodarican. It refers to a protein having a function of eliminating or reducing the activity of an endogenous wild-type protein by expressing a gene encoding a protein, a synthase, a desulfase-inhibiting protein, or a sulfotransferase.
  • Examples of such dominant negative proteins include Versican core protein mutants that competitively inhibit the binding to chondroitin sulfate with the wild-type Versican core protein.
  • the organ, tissue, or cell that inhibits the production or accumulation of chondroitin sulfate proteodarican is not particularly limited, but is preferably an organ, tissue, or cell that secretes insulin, or an organ that acts on insulin.
  • a compound that inhibits the production or accumulation of chondroitin sulfate proteodarican is expected to be a drug for the treatment or prevention of insulin resistance diseases.
  • treatment or prevention refers to a case where it is not necessary to have a complete therapeutic or preventive effect on an organ, tissue, or cell that exhibits insulin resistance. It's okay.
  • the insulin resistance disease is not particularly limited as long as it is a disease associated with insulin resistance, but preferably a disease exhibiting insulin resistance associated with metabolic syndrome (visceral fat syndrome), Non-insulin dependent (type 2) diabetes and the like.
  • metabolic syndrome such as obesity, hyperlipidemia, arteriosclerosis, and complications such as fatty liver disorder and visceral fat are also included in the insulin resistance disease of the present invention.
  • the insulin resistance inhibitor of the present invention has an action of suppressing insulin resistance by inhibiting the production or accumulation of chondroitin sulfate proteodalycan, which is the cause of insulin resistance. Therefore, as a preferred embodiment of the present invention, for example, a metabolic syndrome therapeutic agent or a non-insulin dependent (type 2) diabetes therapeutic agent comprising the insulin resistance inhibitor of the present invention as an active ingredient is provided.
  • the “insulin resistance inhibitor” of the present invention can also be expressed as “insulin resistance therapeutic agent”, “insulin resistance improving agent” or the like.
  • the “inhibitor” can also be expressed as “medicine”, “pharmaceutical composition”, “therapeutic drug”, and the like.
  • the “treatment” in the present invention includes a preventive effect that can suppress the occurrence of insulin resistance in advance.
  • it is not necessarily limited to the case where the insulin resistance-expressing cell (tissue) has a complete therapeutic effect, and may be a case where it has a partial effect.
  • the drug of the present invention can be mixed with a physiologically acceptable carrier, excipient, diluent or the like, and can be administered orally or parenterally as a pharmaceutical composition.
  • a physiologically acceptable carrier such as granules, powders, tablets, capsules, solvents, emulsions or suspensions
  • parenteral preparation a dosage form such as an injection, a drip infusion, an external medicine, or a suppository can be selected. Examples of injections include subcutaneous injections, intramuscular injections, and intraperitoneal injections.
  • the topical drug may be a nasal agent or an ointment.
  • the preparation technique of the above 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, and mixing and compression-molding.
  • an excipient lactose, starch, mannitol or the like is generally used.
  • disintegrant calcium carbonate or carboxymethyl cellulose calcium is generally used.
  • binder gum arabic, carboxymethylcellulose, or polyvinylpyrrolidone is used.
  • talc magnesium stearate and the like are known.
  • the tablet containing the drug of the present invention can be subjected to a known coating for masking or enteric preparation.
  • the coating agent ethyl cellulose, polyoxyethylene glycol or the like can be used.
  • an injection can be obtained by dissolving the agent of the present invention as a main component together with an appropriate dispersant, or dissolving or dispersing in a dispersion medium.
  • aqueous solvent distilled water, physiological saline, Ringer's solution, or the like is used as a dispersion medium.
  • oil-based solvents various vegetable oils such as propylene glycol are used as dispersion media.
  • a preservative such as paraben can be added as necessary.
  • a known isotonic agent such as sodium chloride or glucose can be added.
  • a soothing agent such as salt benzalcoum 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, polybutyl alcohol, acrylic acid, methacrylic acid, polyvinylpyrrolidone, or the like is generally used.
  • a preservative such as salt benzalkonium.
  • a suppository can also be obtained by combining an oily base material such as cacao butter or an aqueous gel base material such as cellulose derivative as a carrier.
  • a method of administering a vector incorporating a nucleic acid in addition to the method of directly administering the drug of the present invention by injection, a method of administering a vector incorporating a nucleic acid can be mentioned.
  • the above-mentioned vectors include adenovirus vectors, adeno-associated virus vectors, herpes vinores vectors, vaccinia winores betaters, retro winores betaters, and lentivirus vectors. Can be invested well.
  • a phospholipid vesicle such as a ribosome
  • Endoplasmic reticulum with siRNA or shRNA retained in the lipofusion method Introduce into a predetermined cell.
  • the obtained cells are then administered systemically, for example, intravenously or intraarterially. It can also be administered locally to an insulin resistant tissue or the like.
  • siRNA has a very excellent specific post-transcriptional repressive effect in vitro.In vivo, it is rapidly degraded by the nuclease activity in serum and is therefore more optimal and effective due to its limited duration. Development of a new delivery system has been demanded.
  • the biocompatible material atelocollagen is mixed with nucleic acid from Ochiya, T et al. Nature Med., 5: 707-710, 1999, Curr. Gene Ther., 1: 31-52, 2001.
  • nucleic acid from Ochiya, T et al. Nature Med., 5: 707-710, 1999, Curr. Gene Ther., 1: 31-52, 2001.
  • degrading enzymes in the living body also protects nucleic acids and is a very suitable carrier for siRNA. This is not a limitation.
  • the necessary amount (effective amount) of the drug of the present invention is administered to mammals including humans within the range of safe doses.
  • 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 power varies depending on age, sex, symptoms, administration route, number of administrations, and dosage forms.
  • the dose in the case of adenovirus is about 10 6 to 10 13 per day, 1 week to 8 It is administered at weekly intervals.
  • RNA introduction kit for example, Adeno Express: Clontech
  • the application site or the type of the disease is not particularly limited as long as it is a disease that expresses insulin resistance.
  • a disease that expresses insulin resistance For example, diabetes, obesity, hyperlipidemia, arteriosclerosis, hypertension, etc. Applies to The above diseases may be concurrent with other diseases.
  • the present invention also provides a method for screening an insulin resistance inhibitor, which comprises selecting a substance having an action of inhibiting the production or accumulation of test sample chondroitin sulfate proteodarican.
  • a candidate compound for an insulin resistance inhibitor or an insulin resistance inhibitor can be efficiently obtained.
  • a preferred embodiment of the screening method of the present invention is any of the following (a) to (d): A method for screening an insulin resistance inhibitor, comprising a step of selecting a substance having the above action.
  • CSPG Chondroitin sulfate proteodarican
  • GAG glycosaminoglycan
  • Test compounds for example, huge compound libraries owned by pharmaceutical companies
  • chondroitin sulfate proteodarican, synthase, desulfase inhibitor compound, sulfate transferase, degradation promoting enzyme, and desulfase used are derived from human, mouse, Forces derived from rats and the like are not particularly limited to those derived from these.
  • the part of chondroitin sulfate proteodalycan is a component such as a glycosaminodarican chain, a core protein, or a part thereof, and is not particularly limited.
  • 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, a compound library
  • Examples include gene library expression products, cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, and the like.
  • the "contact" to the test compound in the embodiment described below is usually chondroitin sulfate proteodarican, a part thereof, a synthase, a desulfase inhibitor compound, a sulfotransferase, a degradation promoting enzyme. Or by mixing desulfating enzyme with test compound iS Not limited to this method.
  • the above “contact” can be performed by contacting a cell expressing these proteins or a part thereof with a test compound.
  • the origin of the "cell” in the embodiments described below includes cells derived from humans, mice, rats, etc., but is not particularly limited to cells derived from these, and in each embodiment It is also possible to use microbial cells such as Escherichia coli and yeast transformed to express the protein to be used once.
  • microbial cells such as Escherichia coli and yeast transformed to express the protein to be used once.
  • a cell expressing a chondroitin sulfate proteodarican can be expressed as a cell that expresses an endogenous chondroitin sulfate proteodarican gene or an exogenous chondroitin sulfate proteodarican gene, Cells in which the gene is expressed can be used.
  • a cell in which an exogenous chondroitin sulfate proteodarican gene is expressed can be usually prepared by introducing an expression vector into which a chondroitin sulfate proteodarican gene is inserted into a host cell.
  • the expression vector can be produced by a general genetic engineering technique.
  • chondroitin sulfate proteodarican core protein means, for example, a matrix type chondroitin sulfate proteoglycan, a core protein such as aggrican, vers ican, neurocan, brevican, or a membrane type chondroitin sulfate.
  • Proteoglycans are core proteins such as Decorin, Biglycan, Fibromodulin, and PG-Lb.
  • Examples of the “synthetic enzyme” include GalNAc4ST-1, GalNAc4ST-2, GALNAC4S-6ST, UA20ST, GalT-1, GalT-II, GlcAT-1, and XylosylT.
  • “Sulfyltransferase” includes, for example, C4ST-l (Chondroitin D—N—acetylgalactosamine—4—0—sulfotransfer ase 1), C4b ⁇ —2 (and honaroitin D—N—acetylgalactosamine—4—0—sulfotransferase 2). ), C4 ST-3 (Chondroitin DN-acetylgalactosamine-4-0-sulfotransferase 3), D4ST, C6ST-1, C6ST-2, and the like.
  • Examples of the “degradation promoting enzyme” include ADAMTS-1, ADAMTS-4, ADAMTS-5, honaroitinase ABC (and hABC) ⁇ Cnondroitinase AC, Chondroitinase B, Calpain I and the like.
  • Examples of the “desulfating enzyme” include Chondroitin-4-sulfatase and nondroitm-sulfatase.
  • the screening method of the present invention there may be mentioned a method comprising a step of selecting a compound having an action of promoting the degradation of chondroitin sulfate proteodarican.
  • the above-described method has the following process power.
  • a test compound is brought into contact with chondroitin sulfate proteodarican or a part thereof.
  • the amount of chondroitin sulfate proteodarican or a part thereof is measured.
  • the measurement can be performed 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 antibody that binds to chondroitin sulfate proteodarican or a part thereof. It can also be detected using a chromatographic method or mass spectrometry.
  • a compound that reduces the abundance of the chondroitin sulfate proteodarican or a part thereof is then selected as compared with the case where the test compound is not contacted (control).
  • the compound that lowers becomes a drug for the treatment of insulin resistance.
  • CS-GAG includes chondroitin sulfate A (CS-A), CS-B, CS-C (Seikagaku Corporation, ICN, Sigma, etc.), human-derived proteodalycan (BGN, ISL, etc.), etc.
  • CS-A chondroitin sulfate A
  • CS-B CS-C
  • BGN human-derived proteodalycan
  • BGN human-derived proteodalycan
  • a WFA lectin (Nodafuji lectin) binding method can be mentioned as a simple method. Since WFA lectin binds to the GalNAc residue of CS-GAG chain, CS-GAG can be easily detected.
  • chondroitinase Chondroitinase
  • chondroitinase Use ABC. If the CS-GAG chain is degraded by chondroitinase ABC, the WFA lectin cannot be bound.
  • FITC-labeled WFA lectin such as EY
  • EY FITC-labeled WFA lectin
  • an anti-CS antibody (clone: CS56, manufactured by Seikagaku Corporation) that directly labels CS-GAG itself can be used.
  • FITC-labeled anti-CS antibody can be added to CS-coated wells so that mass screening can be performed in a very short time and simply if changes in fluorescence values are observed.
  • free GAG can be obtained by adding 2-AB (2-aminobenzamide) or 2-AP (2-aminopyridine, both of which are manufactured by LUD) to the plate before and after mixing of the test compound. More detailed analysis is possible by simply fluorescently labeling the reducing end of the chain and analyzing each type of sugar chain and the content of each type by HPLC, MALDI-MS, LC-MS, etc. . This is a method for the next stage of screening in which the properties of candidate compounds are examined in detail.
  • a method including a step of selecting a substance having an inhibitory action on chondroitin sulfate proteodarican synthesis can be mentioned.
  • the above-described method of the present invention also has the following process power, for example.
  • a test compound is brought into contact with a cell group expressing chondroitin sulfate proteodarican or a part thereof, a cell extract, or a substance group containing an enzyme and a substrate constituting the synthesis process of chondroitin sulfate proteodarican.
  • a test compound is brought into contact.
  • the synthesis amount of chondroitin sulfate proteodarican or an intermediate in the synthesis process is measured.
  • the measurement can be appropriately carried out by those skilled in the art by a known method, for example, a method using a labeled antibody, mass spectrometry, chromatography, or the like.
  • a compound that reduces (suppresses) the synthesis amount is selected as compared with the case where the test compound is not contacted!
  • Compounds that decrease (suppress) become drugs for the treatment of insulin resistance.
  • chondroitin sulfate is produced in 16 hours of cell culture by the standard method of collecting and culturing mononuclear cells after collecting peripheral blood from healthy individuals (Uhlin-Hansen L et al. , Blood 82: 2880, 1993.). More simply, known cell lines such as fibroblast cell line NIH3T3 (Phillip HA, et al. J. Biol. Chem. 279: 48640, 2004), renal tubule-derived cancer cell line ACHN (Kawashima H et al., J. Biol. Chem.
  • CS-GAG synthases such as GalNAc4ST-l and XylosylT Can be made into CHO cells and L cells by a well-known method, and a cell line in which the gene is constitutively expressed can be prepared. By using such a cell line that constantly synthesizes CS-GAG, it is possible to more clearly determine the candidate treatment compound.
  • a method including a step of selecting a substance having a desulfating action of chondroitin sulfate proteodarican can be mentioned.
  • the above-described method of the present invention also has the following process power, for example.
  • a test compound is brought into contact with chondroitin sulfate proteodarican or a part thereof.
  • the amount of chondroitin sulfate proteodarican or a part thereof that has received sulfate is measured.
  • the measurement can be performed 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 antibody that binds to the structure of desulfurization oxidation remaining in chondroitin sulfate proteodarican or a part thereof. It can also be detected using chromatography, mass spectrometry, and the like.
  • a compound that reduces the abundance of the chondroitin sulfate proteodarican or a part thereof is then selected as compared with the case where the test compound is not contacted (control).
  • the compound that lowers becomes a drug for the treatment of insulin resistance.
  • the detection method was carried out by desulfating the disaccharide structure of the desulfated fragment remaining on the core protein side of the proteodarican into the anti-proteodarican A di4S antibody (clone; 2-B-6, 4 Recognize the part that received sulfate at the position) or anti-proteodarican ⁇ di6S (clone; 3-B-3, recognize the part that received sulfate at position 6. By reacting with Kogyo Kogyo Co., Ltd., it is possible to easily detect the portion subjected to desulfurization.
  • FITC-labeled 2-B-6 and 3-B-3 antibodies can be reacted on the plate before and after mixed culture, and changes in fluorescence values can be easily detected.
  • a compound with increased fluorescence intensity before and after the reaction can be determined to be a substance that further promotes desulfation, and can be easily identified as a novel therapeutic candidate compound that satisfies this concept.
  • a method comprising a step of selecting a substance having a sulfation inhibitory action on chondroitin sulfate proteodalycan.
  • the above method of the present invention also has the following process power, for example.
  • ( a ) A step in which a test compound is contacted with a substance or a cell extract expressing chondroitin sulfate proteodarican or a part thereof, or a substance group including an enzyme, a substrate, or the like that constitutes a sulfated process of chondroitin sulfate proteodarican.
  • a test substance is brought into contact with chondroitin sulfate proteodarican or a part thereof.
  • the amount of chondroitin sulfate proteodarican or a part thereof that has received sulfate is measured.
  • the measurement can be performed by methods known to those skilled in the art.
  • chondroitin sulfate proteodarican or some of its sulfate Detection can be carried out by measuring the amount of label using a labeled compound or antibody that binds. Moreover, it can also detect using a chromatography method, a mass spectrometry, etc.
  • the cells and cell lines that promote the sulfation of chondroitin sulfate are the same as the cells and cell lines described in ( c ) above.
  • Various test compounds are mixed in the process of culturing such a cell line for a certain period of time, and the degree of sulfate before and after the culture is measured, for example, an antibody (clone; LY111 ) And antibodies that detect 6-position sulfation (clone; MC21C, also available from Seikagaku Corporation). Fluorescence-labeled antibodies may be used to compare fluorescence values before and after culture. Similarly to (c) above, detection methods using 2-B-6 and 3-B-3 antibodies may be performed before and after culture. Also good.
  • a cell line in which a gene for a sulfotransferase such as C4ST-1 or C6ST-1 is introduced into CHO cells or L cells by a well-known method and is expressed constantly. Can be created. By using such a cell line to which a sulfate group is constantly added, it is possible to more clearly determine a treatment candidate compound.
  • Another preferred embodiment of the present invention is a compound that decreases the expression level of the chondroitin sulfate proteodarican core protein, the synthase, the desulfase inhibitor protein, or the sulfotransferase gene of the present invention, A compound that increases the expression level of the chondroitin sulfate proteoglycan degradation-promoting enzyme or desulfating enzyme gene
  • a method for screening an insulin resistance inhibitor comprising the following steps (a) to (d).
  • test compound is brought into contact with a cell expressing a gene encoding a chondroitin sulfate proteodlican core protein, a synthetic enzyme, a desulfase inhibitor protein, a sulfotransferase, a degradation promoting enzyme, or a desulfase enzyme.
  • a gene encoding a chondroitin sulfate proteodalycan core protein, a synthetic enzyme, a desulfurase inhibitor protein, a sulfotransferase, a degradation promoting enzyme, or a desulfurase is selected.
  • a test compound is brought into contact with the cells to be expressed.
  • the expression level of the gene encoding chondroitin sulfate proteodlican core protein, synthetic enzyme, desulfase inhibitor protein, sulfate transferase, degradation promoting enzyme, or desulfase is measured.
  • gene expression includes both transcription and translation. The gene expression level can be measured by methods known to those skilled in the art.
  • cellular force mRNA that expresses any of the above proteins is extracted according to a standard method, and Northern hybridization method, RT-PCR method, DNA array method, etc. using this mRNA as a cage are performed. Thus, the amount of transcription of the gene can be measured. Also,
  • the protein By collecting protein fractions from cells expressing genes encoding any of the above proteins, and detecting the expression of any of the above proteins by electrophoresis such as SDS-PAGE, You can also measure the amount of translation. Further, by performing Western blotting using an antibody against any of the above-mentioned proteins, the protein can be obtained. It is also possible to measure the amount of translation of a gene by detecting the expression of the protein.
  • the antibody used for detecting 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 compared with the case where the test compound is not brought into contact with the test compound!
  • the gene is a chondroitin sulfate proteodarican core protein, a synthase, a desulfase inhibitor protein, or a sulfotransferase
  • the expression level of the gene is a control.
  • a compound that decreases (suppresses) becomes a drug for suppressing insulin resistance or a candidate compound for treating insulin resistance.
  • the gene is a chondroitin sulfate proteodarican degradation-promoting enzyme or a desulfurization enzyme
  • the expression level of the gene is increased (enhanced) compared to the control.
  • Select. Compounds that increase (enhance) become drugs for the inhibition of insulin resistance or candidate compounds for the treatment of insulin resistance.
  • the expression level of the chondroitin sulfate proteodarican core protein, synthetic enzyme, desulfase inhibitor protein, or sulfotransferase gene of the present invention is reduced.
  • This is a method of selecting a compound or a compound that increases the expression level of a chondroitin sulfate proteodarican degradation-promoting enzyme or desulfating enzyme gene using the expression of a reporter gene as an index.
  • the method of the present invention includes, for example, the following steps (a) to (d).
  • the transcriptional regulatory region of a gene encoding a chondroitin sulfate proteodarican core protein, a synthetic enzyme, a desulfase inhibitor protein, a sulfotransferase, a degradation-promoting enzyme, or a desulfase enzyme and the reporter gene are functional.
  • the reporter gene is a core protein of chondroitin sulfate proteodalycan
  • the reporter gene is A step of selecting a compound in which the expression level of the reporter gene is increased compared to the control when functionally bound to an enzyme that promotes the degradation of chondroitin sulfate proteodarican or a desulfurization enzyme
  • “functionally linked” refers to a gene encoding chondroitin sulfate proteodarican core protein, synthase, desulfase inhibitor protein, sulfotransferase, degradation promoting enzyme, or desulfase So that transcription factor binds to the transcriptional regulatory region of the protein and induces the expression of the reporter gene, chondroitin sulfate proteodalycan core protein, synthase, desulfase inhibitor protein, sulfate transferase, accelerated degradation It means that a transcriptional regulatory region of an enzyme or a gene encoding a desulfating enzyme is linked to a reporter gene.
  • the reporter gene is linked to other genes and forms a fusion protein with other gene products, chondroitin sulfate proteodlican core protein, synthase, desulfase inhibitor protein, sulfate If the expression of the fusion protein is induced by binding of a transcription factor to the transcriptional regulatory region of a gene encoding a transferase, a degradation promoting enzyme, or a desulfating enzyme, the above-mentioned "functionally bound" Is included.
  • the reporter gene used in the present method is particularly restricted if its expression is detectable. Examples include CAT gene, lacZ gene, luciferase gene, and GFP gene. “Chondroitin sulfate proteodalycan core protein, synthase
  • a cell containing DNA having a structure in which a transcriptional regulatory region of a gene encoding a desulfase inhibitor protein, a sulfotransferase, a degradation promoting enzyme, or a gene encoding a desulfase enzyme and a reporter gene are functionally linked for example, Examples include cells into which a vector having such a structure has been 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 a calcium phosphate precipitation method, an electric pulse perforation method, a lipofussion method, a microinjection method, or the like.
  • the “cell containing DNA having a” includes a cell in which the structure is inserted into a chromosome.
  • 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 utilizing homologous recombination.
  • Transcriptional regulatory region of a gene encoding a chondroitin sulfate proteodarican core protein, a synthetic enzyme, a desulfurase inhibitor protein, a sulfotransferase, a degradation promoting enzyme, or a desulfase, and a reporter gene are functional.
  • the cell extract containing DNA having a structure bound to is, for example, a cell extract contained in a commercially available in vitro transcription / translation kit, chondroitin sulfate proteolycan core protein, synthase, desulfate enzyme.
  • a DNA containing a structure in which a transcriptional regulatory region of a gene encoding a repressor protein, a sulfotransferase, a degradation promoting enzyme, or a desulfating enzyme and a reporter gene are functionally linked is included. it can.
  • contact refers to "transcriptional regulation of a gene encoding chondroitin sulfate proteodarican core protein, synthase, desulfase inhibitor protein, sulfotransferase, degradation-promoting enzyme, or desulfuric acid enzyme.
  • a test compound is added to the culture solution of ⁇ cells containing DNA having a structure in which a region and a reporter gene are functionally linked '', or a test compound is added to the above-described commercially available cell extract containing the DNA Can be done.
  • the test compound is a protein, for example, a DNA solid that expresses the protein It is also possible to carry out the introduction by introducing a cell into the cell.
  • the expression level of the reporter gene is measured in the following manner.
  • 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.
  • the reporter gene is a CAT gene
  • the expression level of the reporter gene can be measured by detecting the chloramfecole acetylene by the gene product.
  • the reporter gene is the lac Z gene
  • the expression level of the reporter gene can be measured by detecting the fluorescence of the GFP protein.
  • the measured expression level of the reporter gene is compared with that measured in the absence of the test compound (control).
  • the reporter gene is then functionally linked to a chondroitin sulfate proteodarican core protein, a synthase, a desulfase inhibitor protein, or a gene encoding a sulfate transferase.
  • a compound that lowers (suppresses) becomes a drug for suppressing insulin resistance or a candidate compound for treating insulin resistance.
  • the reporter gene when the reporter gene is functionally linked to a chondroitin sulfate proteodarican degradation-promoting enzyme or a desulfurization enzyme, the expression level of the reporter gene is higher than that of the control. Increase (enhance) the compound! A compound that increases (intensifies) becomes a drug for suppressing insulin resistance or a candidate compound for treating insulin resistance.
  • the insulin resistance inhibitor found in the screening method of the present invention is preferably for the treatment or prevention of an insulin resistance disease.
  • 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 the chondroitin sulfate proteodarican of the present invention as a constituent element.
  • the kit of the present invention can contain Sarako, various reagents and containers used in the method of the present invention.
  • an anti-chondroitin sulfate proteodarican 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.
  • a method for screening an insulin resistance inhibitor which comprises the step of detecting whether the production or accumulation of chondroitin sulfate proteodarican is inhibited. Therefore, in the screening method, for example, a probe for a gene encoding the core protein of chondroitin sulfate proteodarican that can be used for detection of chondroitin sulfate proteodarican, or a primer for amplifying an arbitrary region of the gene Oligonucleotides or antibodies that recognize chondroitin sulfate proteodarican (anti-chondroitin sulfate proteodarican antibody)
  • the oligonucleotide specifically hybridizes, for example, to the DNA of the Versican core protein gene of the present invention.
  • “specifically hybridize” means normal hybridization conditions, preferably stringent hybridization conditions (for example, Sambu Norec et al., Molecular Cloning. Cold Spring Harbor Laboratory Press, New York, In the USA, 2nd edition, 1989), which means that no significant cross-hybridization occurs with DNA encoding other proteins. If specific hybridization is possible, the oligonucleotide does not need to be completely complementary to the nucleotide sequence of the Versican core protein gene of the present invention! /.
  • hybridization conditions include, for example, “2 X SSC, 0.1% SDS, 50 ° C.”, “2 X SSC, 0.1% SDS, 42 ° C.”, “1 X SSC” , 0.1% SDS, 37 ° C '' and more stringent conditions as ⁇ 2 X SSC, 0.1% SDS, 65 ° C '', ⁇ 0.5 X SSC, 0.1% SDS, 42 ° C '' and ⁇ 0.2 X SSC '' , 0.1% SDS, 65 ° C. ”.
  • Rapid As a method using -hyb buffer (Amersham Life Science)
  • a probe after pre-hybridization at 68 ° C for 30 minutes or more, add a probe and keep at 68 ° C for 1 hour or more to form a hybrid.
  • Prehybridization Solution (CLONTECH)
  • a labeled probe and incubate at 37-55 ° C for 1 hour or more.
  • the temperature of the prehybridization and noble hybridization can be set to 60 ° C
  • the stringent condition can be set to 68 ° C.
  • the conditions such as the salt concentration and temperature of the buffer, as well as other conditions such as the probe concentration, probe length, probe base sequence composition, and reaction time. Can be set.
  • the oligonucleotide can be used as a probe or primer in the above-described screening kit of the present invention.
  • the length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp.
  • the primer is not particularly limited as long as it can amplify at least a part of the DNA of the gene of the present invention described above, for example, the force described in SEQ ID NO: 69 or 70!
  • the present invention also provides a method for treating or preventing an insulin resistant disease, which comprises the step of administering the agent of the present invention to an individual (eg, 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 an insulin resistance 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 dose 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 dose.
  • the present invention relates to the use of the agent of the present invention in the manufacture of an insulin resistance inhibitor.
  • Chondroitin D—N—acetylgalactosamine— Examples are given focusing on 4-0-sulfotransferase 3 (C4ST-1, C4ST-2, C4ST-3).
  • NIDD M power ⁇ type diabetes model
  • a type 2 diabetes model was prepared by Streptozotocin administration of C57BL / 6JcL mice (female, manufactured by CLEA Japan, Inc.) on the second day after birth.
  • Versican siRNA treatment caused body weight fluctuation, blood glucose level fluctuation, and gene expression by siRNA administration. investigated.
  • sample preparation was performed as follows.
  • Example 1 StreDtozotocin-induced C57BL / 6TcL Type 2 diabetes (NIDDM) model mouse body weight perturbation in Versican (siRNA) treatment
  • mice 14th day of pregnancy C57BL / 6JcL mice (CLEA Japan, Inc.) are bred and given birth, 2 days after birth
  • Example 2 StreDtozotocin induction C57BL / 6TcL type 2 diabetes mellitus (NIDDM) model mice in Versican (siRNA) treatment rfn.
  • FIG. 2 shows the changes in blood glucose level after 0, 15, and 60 minutes of intraperitoneal administration of human crystalline insulin (0.75 U / kg) in the Control group and Versican siRNA treatment group after Versican siRNA treatment.
  • the vertical axis represents the blood glucose level (mg / dL), and the horizontal axis represents the insulin tolerance test, 0 minutes, 15 minutes, and 60 minutes after.
  • Fig. 2 shows that no significant change was observed between the Control group and Versican siRNA treatment group at 0 and 15 minutes, and a significant change between the Control group and Versican siRNA treatment group after 60 minutes. Was recognized.
  • mice Streptozocin-induced C57BL / 6JcL mice, 50 mg of organ (spleen) isolated from females, 1 mL of RNA-Bee (TEL-TEST) is added and electric homogenizer (DIGITAL HO MOGENIZER, ASONE) After grinding, chloroform 200 L (manufactured by Sigma Aldrich Japan) is gently mixed, then ice-cooled for about 5 minutes, and centrifuged at 12,000 rpm at 4 ° C for 15 minutes. Centrifoge 5417R, eppendorf The product was centrifuged.
  • RNA concentration in the sample extracted with the plate reader was calculated.
  • RNA sample was adjusted to a concentration of 500 ng / 20 / ⁇ L, heated at 68 ° C. for 3 minutes with BL OCK INCUBATOR (manufactured by ASTEC), and cooled on ice for 10 minutes. Prepare in advance after cooling on ice! RT Pre Mix solution (Composition: 25 mM MgCl 18.64 ⁇ L (Invitrogen)), 5 X Buffer 20
  • PCR Buffer 2 ⁇ L Composition: 166 mM (NH 2) SO (Sigma Aldrich Japan), 670 mM T
  • IX LoTE Composition: 3 mM Tris—HCl (pH7.5) (Invitrogen), 0.2 mM EDTA (pH7.5) (Sigma Aldrich Japan)] Ethydium Bromide (Invitrogen)
  • EXILIM manufactured by CASIO
  • I-Scope WD manufactured by AD VANCE
  • Fig. 3 shows the gene expression results of GAPDH and Versican in the Control group and Versican siRNA treatment group by RT-PCR.
  • Example 4 Streptozotocin-induced C57BL / 6.TcL Evaluation and examination of spleen tissue level by immunostaining (APP, Insulin) in Versican (siRNA) treatment with type 2 diabetes (NIDDM) model mice The obtained tissue sample sections were stained with anti-Amyloid precursor protein antibody and anti-insulin antibody to evaluate and examine the expression at the tissue level.
  • Fig. 4 shows tissue images of the Control group and Versic an siRNA treatment group.
  • Example 5 Streptozotocin-induced C57BL / 6.TcL Evaluation of spleen tissue level by immunostaining (CS56) in Versican (siRNA) treatment using type 2 diabetes (NIDDM) model mice CS56 ( Staining was performed using an antibody that stains chondroitin sulfate proteodarican itself, and the expression at the tissue level was evaluated and examined.
  • Fig. 5 shows the yarn and woven images of the Control group and Versican siRNA treatment group.
  • C2BL-1 Chodroitin DN-acetylgalactosam ine— 4—1
  • C57BL / 6JcL mice female, manufactured by CLEA Japan, Inc.
  • siRNA ⁇ and 4ST-2 (and then honaroitin D—N—acetylgalactosamine — 4— O—sulfotransferase 2) siRNA, C4ST—3 (Chondroitin D—N—acetylgalactosamine— 4— O-sulfotransferase 3)
  • siRNA ⁇ and 4ST-2 and then honaroitin D—N—acetylgalactosamine — 4— O—sulfotransferase 2
  • siRNA, C4ST—3 Chodroitin D—N—acetylgalactosamine— 4— O-sulfotransferase 3
  • Example 6 C4ST-1 (Chondroitin D—N—acetylgalactosamine—4—0—sulfotransferase 1) siRNA, Streptozodn-induced C57BL / 6.TcL NIDDM model mouse 4ST—2 (Shon Branin D—N — Acetylgalactosamine— 4—0—sulfotransferase 2) siRNA, 4a ⁇ —3 (Cnondroi tin D—N—acetylgalactosamine—4—0—sulfotransferase 3) Body weight fluctuation during siRNA treatment Day 14 of pregnancy C57BL / 6JcL mice (CLEA Japan) C57BL / 6JcL mice females were injected subcutaneously into Streptozocin 10 mg / mL (SIGMA) 20 L / head, and CE-2 (CLEA Japan) until 4 weeks of age.
  • SIGMA Streptozocin 10 mg / mL
  • siRNA and 4ST-1 and nondroitin D—N—acetylgalactosamine—4— ⁇ —sulfotransferase 1
  • 4 ST—2 Chodroitin D—N—acetylgalactosamine—4—0—sulfotransferase 2
  • C4ST—3 Cho ondroitin D—N—acetylgalactosamine—4-0—sulfotransferase 3) 1 ⁇ g (GeneWorld) mixed with 1% Atelocollagen (Koken), siRNA medium, once / week 200
  • Treatment was performed twice (2 weeks) by intraperitoneal administration of ⁇ L (once / week).
  • Example 7 Streptozocin-induced C57BL / 6.TcL NIDDM model mouse C4ST- Chondroitin D—N—acetylgalactosamine—4—0—sulfotransferase 1) siRNA, 4ST—2 (and hon Georgiain D—N—acetylgalactosamine) — 4—0—sulfotransferase 2) siRNA, 4a ⁇ —3 (Cnondroi tin D—N—acetylgalactosamine—4—0—sulfotransferase 3) Changes in blood glucose level during siRNA treatment.
  • Example 8 Streptozodn-induced C57BL / 6.TcL NIDDM model mouse C4ST-1 (C hondroitin D—N—acetylgalactosamine—4-0—sulfotransferase 1) siRNA, 4ST—2 (and hon sauin D—N — Acetylgalactosamine— 4—0—sulfotransferase 2) siRNA, 4a ⁇ —3 (Cnondroi tin D—N—acetylgalactosamine—4—0—sulfotransferase 3) izs child in siRNA treatment 3 ⁇ 4 Evaluation of current level
  • mice Streptozocin-induced C57BL / 6JcL mice, 50 mg of organ (spleen) isolated from females, 1 mL of RNA-Bee (TEL-TEST) is added and electric homogenizer (DIGITAL HO MOGENIZER, ASONE) After grinding, chloroform 200 L (manufactured by Sigma Aldrich Japan) is gently mixed, then ice-cooled for about 5 minutes, and centrifuged at 12,000 rpm at 4 ° C for 15 minutes. Centrifoge 5417R, eppendorf The product was centrifuged.
  • RNA concentration in the sample extracted with the plate reader was calculated.
  • RNA sample was adjusted to a concentration of 500 ng / 20 ⁇ L, and the mixture was calored at 68 ° C for 3 minutes using BLOCK INCUBATOR (ASTEC). Warm and cool on ice for 10 minutes.
  • RT Pre Mix solution composition: 25 mM MgCl 18.64 prepared in advance after ice cooling
  • PCR Buffer 2 ⁇ L Composition: 166 mM (NH 2) SO (Sigma Aldrich Japan), 670 mM T
  • Example 9 StreDtozodn Induction Case C57ST / 6 (Chondroitin D—N—acetylgalactosamine—4—0—sulfotransferase 1) siRNA, 4ST—2 (and hon sauin D—N—acetylgalactosamine) in C57BL / 6TcL NIDDM model mice — 4—0— sulfotransferase 2) siRNA, shi 4a ⁇ — 3 (Cnondroi tin DN-acetylgalactosamine-4-0-sulfotransferase 3) Evaluation of tissue level (APR Insulin) using immune 3 ⁇ 4 color in siRNA treatment
  • FIG. 9 shows tissue images of the Control group, the C4ST-1 siRNA treatment group, the C4ST-2 siRNA treatment group, and the C4ST-3 siRNA treatment group.
  • Example 10 StreDtozotocin-induced C57BL / 6.TcL type 2 diabetes (NIDDM) model mouse ⁇ Cydroitin D—N—acetylgalactosamine— 4— ⁇ —sulfotransferase l) siRN A, C4ST-2 (Chondroitin D—N—acetylgalactosamine— 4— ⁇ —sulfotransferase 2) siRNA, C4ST-3 (Chondroitin D —N— acetylgalactosamine— 4— ⁇ —sulfotransferase 3) Evaluation of knee tissue level by immunostaining (esse) in siRNA treatment,
  • FIG. 10 shows tissue images of the Control group, the C4ST-1 siRNA treatment group, the C4ST-2 siRNA treatment group, and the C4ST-3 siRNA treatment group.
  • chondroitin sulfate proteoglycan a side of a chondroitin sulfate proteoglycan containing a Versican core site sequence, which is one of the chondroitin sulfate proteoglycans.
  • C4ST1 Chondroitin D-N-acetylgala ctosamine- 4-O-sulfotransferase 1
  • C43 ⁇ 4 ⁇ 2 Cnondroitin DN-acetylgalactosamine- 4— O— sulfotransferase 2
  • C4ST3 Chondroitin D—N—acetylgalactosamine— 4—0— sulfot ransferase 3
  • siRNA suppresses chondroitin sulfate proteoglycan accumulation in splenic Langerhans islet j8 cells and suppresses insulin resistance to treat type 2 diabetes (NIDDM) Or it has an effect on prevention.
  • the insulin resistance inhibitor according to the present invention is a chondroitin sulfate proteo around the splenic Langerin's Islet j8 cells whose Versican expression is suppressed by administration of Versica siRNA, C4ST-1 siRNA, C4ST-2 siRNA, C4ST-3 siRNA. Since it has an inhibitory effect on glycan accumulation, it is very useful in suppressing insulin resistance of splenic Langerhans islet ⁇ cells.
  • Type 2 diabetes by administering an insulin resistance inhibitor having an inhibitory effect on chondroitin sulfate proteoglycan accumulation of the present invention (NIDDM) Disease treatment or prevention methods can be an excellent therapy useful for further improvement of patients' quality of life and medical care because the pathology can be effectively improved by an unprecedented mechanism of action and drug therapy. .
  • NIDDM chondroitin sulfate proteoglycan accumulation of the present invention

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Abstract

La présente invention, comme exemple de la recherche sur les effets de l'accumulation du protéoglycane chondroïtine sulfate (CSPG), concerne un siRNA versican contenant une séquence de site centrale de versican qui est l'un des protéoglycanes chondroïtine sulfate, un siRNA de C4ST1, C4ST2 ou C4ST3 qui est une enzyme transférant un groupe sulfate de l'acétylgalactosamine qui est une chaîne latérale du protéoglycane chondroïtine sulfate, et un réducteur de la résistance à l'insuline approprié pour une thérapie génique ou la prévention pour une maladie provoquée par une augmentation de la résistance à l'insuline, telle que le diabète de type II (NIDDM). L'invention concerne également un procédé permettant de réduire la résistance à l'insuline à partir de l'inhibition de l'accumulation du protéoglycane chondroïtine sulfate (CSPG) par l'administration d'un siRNA versican, un siRNA C4ST-1, un siRNA C4ST-2 ou un siRNA C4ST-3 à un sujet à tester. L'invention concerne également un procédé de traitement ou de prévention d'une maladie provoquée par une augmentation de la résistance à l'insuline, telle que le diabète de type II, à l'aide de ces derniers.
PCT/JP2006/323657 2006-07-24 2006-11-28 Réducteur de la résistance à l'insuline WO2008012932A1 (fr)

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JP2006201307A JP2009234916A (ja) 2006-07-24 2006-07-24 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

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012046453A (ja) * 2010-08-27 2012-03-08 Stelic Institute Of Regenerative Medicine 慢性炎症性疾患治療剤

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WO2024113004A1 (fr) * 2022-11-28 2024-06-06 The University Of Melbourne Combinaisons pharmaceutiques et leurs utilisations

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JPH03500168A (ja) * 1988-06-27 1991-01-17 ライナー　ローラント 医薬の製造の為のグリコースアミノグリカンの使用
JP2005198640A (ja) * 2003-11-25 2005-07-28 Biomarker Science:Kk 遺伝子発現方法
JP2005263688A (ja) * 2004-03-18 2005-09-29 Nippon Suisan Kaisha Ltd α−グルコシダーゼ阻害剤
WO2006035445A2 (fr) * 2004-09-29 2006-04-06 Insight Biopharmaceuticals Ltd. Procedes pour traiter des pathologies associees a un stress oxydatif

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Publication number Priority date Publication date Assignee Title
JPH03500168A (ja) * 1988-06-27 1991-01-17 ライナー　ローラント 医薬の製造の為のグリコースアミノグリカンの使用
JP2005198640A (ja) * 2003-11-25 2005-07-28 Biomarker Science:Kk 遺伝子発現方法
JP2005263688A (ja) * 2004-03-18 2005-09-29 Nippon Suisan Kaisha Ltd α−グルコシダーゼ阻害剤
WO2006035445A2 (fr) * 2004-09-29 2006-04-06 Insight Biopharmaceuticals Ltd. Procedes pour traiter des pathologies associees a un stress oxydatif

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012046453A (ja) * 2010-08-27 2012-03-08 Stelic Institute Of Regenerative Medicine 慢性炎症性疾患治療剤

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