JP2009234916A - Insulin resistance reducer based on chondroitin sulfate proteoglycan accumulation regulation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an insulin resistance reducer containing a substance inhibiting the production or accumulation of chondroitin sulfate proteoglycan as an active ingredient, and a method for screening the reducer. <P>SOLUTION: The versican siRNA containing a core site sequence of the versican which is one of chondroitin sulfate proteoglycans and the siRNA of C4ST1, C4ST2 or C4ST3 which is an enzyme for transferring the sulfate group of acetylgalactosamine which is a side chain of the chondroitin sulfate proteoglycan, are provided, and the insulin resistance reducer suitable for the gene treatment or prevention of diseases caused by the increase of the insulin resistance such as type 2 diabetes (NIDDM) is also provided. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an insulin resistance inhibitor based on control of chondroitin sulfate proteoglycan (CSPG) accumulation, a method for suppressing insulin resistance, and an insulin-independent type 2 diabetes mellitus disease (NIDDM) based on the method. The present invention relates to a method for treating or preventing insulin resistance diseases such as the above.

  Diabetes is a disease caused by a relative deficiency of insulin, a hormone that lowers blood glucose, and symptoms vary from person to person, but type 1 (IDDM), which is largely insulin-dependent, and type 2, which is insulin-independent ( NIDDM). Type 1 diabetes (IDDM) refers to a state in which pancreatic β cells that inherently produce insulin are destroyed by autoimmunity or the like. On the other hand, type 2 diabetes mellitus (NIDDM) is well-known as a lifestyle-related disease that has developed due to the addition of environmental factors such as obesity and lack of exercise to genetic factors. Current diabetes symptomatic treatment and pharmacotherapy are sufficient diet, keeping balance of nutrients (sugar, lipid, protein) and not to be deficient in minerals and vitamins, more than 10 minutes, more than 30 minutes if possible Exercise therapy that performs continuous whole body exercise, etc., nateglinide, which is a drug targeting insulin-related enzymes, insulin receptors as drug therapy, α-glucosidase inhibitor, sulfonylurea agent (SU agent), biguanide agent (BG agent), Oral diabetes drugs such as thiazolidine drugs are used in clinical settings. (See Non-Patent Documents 1 to 5)

  However, it has not been able to improve the patient's quality of life and effectively prevent or delay the decrease in insulin secretion. According to a recent research report by the Ministry of Health and Welfare, the existence of non-insulin dependent diabetes mellitus (NIDDM) It is clear that the prevalence rate is about 10% of the population over 40 years old, reaching about 6 million people in Japan as a whole. Lifestyle changes such as westernization of meals and lack of exercise are becoming an aging society. Accelerates the increase in diabetes, and continues to increase, and the development of new drugs that can be effectively prevented or delayed with a completely different viewpoint and mechanism that are safe and effective. Has been.

  Currently, diabetes is being studied around the world as a target after cancer. Among various drug discovery, recently, “pancreatic β-cell regeneration” is expected as one of the fundamental treatments. (See Non-Patent Documents 6-12)

  A proteoglycan is a molecule that has a structure in which one or more glycosaminoglycan (GAG) chains are covalently bound to a protein called a core protein, and the specific sugar chain structure of the GAG chain is responsible for the various functions of proteoglycans. Based on the type of GAG chain, proteoglycans are roughly classified into four groups: chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, heparan sulfate proteoglycan, and ketalan sulfate proteoglycan. (See Non-Patent Documents 13-19)

  Among these, heparan sulfate proteoglycans have been extensively studied to bind to various cytokines, adhesion molecules, and chemokines and greatly modify their functions.

On the other hand, chondroitin sulfate proteoglycan is an essential molecule in the embryonic period and is abundant in each organ, but it is known that it decreases with birth, growth and aging. Not yet clear.
So far, chondroitin sulfate proteoglycan (CSPG), heparan sulfate proteoglycan (HSPG), dermatan sulfate proteoglycan (DSPG), basic fibroblast growth factor (bFGF), decorin, biglycan, lumican, fibromodulin, etc. Has been reported to be expressed and accumulated in the glomerular basement membrane, pancreas, kidney and liver. (See Non-Patent Documents 20-26)

  However, the significance of CSPG increase in these diseases has not been clarified at all, and in recent years, it has been found that mice genetically deficient in CSPG fall into embryonic lethality and organ dysplasia. There is a growing recognition that it is an essential molecule.

  Versican (aka PG-M), a chondroitin sulfate proteoglycan, has a hyaluronic acid-binding domain near the N-terminus, a glycosaminoglycan addition domain in the middle, an EGF-like domain, a C-type lectin-like domain, and a complement. It has a body regulatory protein-like domain. The human Versican gene is composed of 15 exons and, as a result of alternative splicing, Versican has different glycosaminoglycan addition domain lengths V0, V1, V2, and V3 4 without glycosaminoglycan addition domains. Takes two different molecular types. Versican binds hyaluronic acid with high affinity via the hyaluronic acid binding domain, and binds to sulfated glycolipids and extracellular matrix components tenascin-R and fibrin-1 via the C-type lectin domain. In addition, Versican has been reported to promote cell proliferation by binding to the EGF receptor via the EGF-like domain, and also has cell adhesion inhibitory activity via the chondroitin sulfate chain. . (See Non-Patent Documents 27-33)

  Currently, sulfonylurea drugs (SU agents), biguanide drugs, insulin resistance improvers, α-glucosidase inhibitors, fast-acting insulin secretagogues, etc. are used as antidiabetic drugs, but are still difficult to treat As a research,

  To date, Versican is one of the chondroitin sulfate proteoglycans (CSPG) and is known as a protein that corresponds to the core protein. Regarding chondroitin sulfate proteoglycan, neural stem cell differentiation inducer (Patent Document 1), nerve regeneration using human / bone morphogenetic protein (Patent Document 2), glycosyl transfer agent (Patent Document 3), treatment of central nervous system damage (Patent Document 4), human, nerve regeneration using bone morphogenetic protein (Patent Document 5), treatment inhibitor of vascular smooth muscle cells (Patent Document 6), etc. are known. Regarding Versican, prooteoglycan Known generation promoter (Patent Document 7), hair growth composition (Patent Document 8), ADAMTS4 function inhibitor (Patent Document 9), method of suppressing scar tissue generation by implantable medical device (Patent Document 10), etc. It has been.

  In addition, US PATENT has patents related to PAT.NO.5,180,808 (patent document 11) related to the structure, nucleobase sequence, etc. and cardiovascular diseases such as PAT.NO.6,579,682 (patent document 12). Application, also in PubMedline, reports on structure, cardiovascular system, reports on Versican and metabolic abnormalities are related to lipid metabolism system (Non-Patent Document J Lipid Res. 2005 Sep; 46 (9): 1999-2006. Epub 2005 Jul 1. (Non-Patent Document 34) has been reported, but not only humans and rats, but also Versican siRNA, C4ST-1 siRNA, C4ST-2 siRNA, C4ST using mouse type 2 diabetes (NIDDM) model -3 Application examples and efficacy studies with siRNA administration have not been conducted so far.

Patent Publication 2005-278641 Patent Publication 2005-007196 Patent Publication 2004-024208 Patent publication 2005-526740 Patent Publication No. 09-501932 Patent Publication 08-510209 Patent Publication 2006-028071 Patent Publication 2005-200383 Patent Publication 2004-244339 Patent Publication 07-024053 U.S. Patent No. 5,180,808 U.S. Patent No. 6,579,682 Fujita, T et al. Biochemical Pharmacology (1996); 52: 407-411 Tsukuda, K et al. Horm Metab Res (1998); 30: 42-49 Fujitani, S et al. Metabolism (1996); 45: 184-189 Spiegelman et al. J Clin Invest (1997); 100: 1863-1869 Olefsky et al. Diabetes (1997); 46: 1678-1683 Glueck C.J et al. Curr Diab Rep. (2003) Aug; 3 (4): 303-312 Juntunen KS et al. Am J ClinNutr. (2003) Feb; 77 (2): 385-391 Van Lerberghe S et al. Diabeets Metab. (2002) Feb; 28 (1): 33-38 Buchanan TA. J Clin Endocrinol Metab. (2001) Mar; 86 (3): 989-993 Fallucca F et al. Diabetes Res Clin Pract. (1989) Nov 6; 7 (4): 277-284 Ostenson CG et al. Exp Clin Endocrinol. (1989) May; 93 (2-3): 241-247 Ward WK et al. J Clin Endocrinol Metab. (1985) Dec; 61 (6): 1039-1045 Lindahl, U et al. (1972) In Glycoproteins (Gottschalk, A. ed) pp. 491-517, Elsevier, New York. Oegema, T et al. J. Biol. Chem. (1984); 259: 1720-1726 Sugahara, K et al. J. Biol. Chem. (1988); 263: 10168-10174 Sugahara, K et al. J. Biol. Chem. (1992); 267: 6027-6035 De Waard et al. J. Biol. Chem. (1992); 267: 6036-6043 Moses, J, Oldberg et al. Eur. J. Biol. (1992); 248: 521-526 Yamada, S et al. Trends in Glycoscience and Glycotechnology,. (1998); 10: 95-123 Schaefer L et al. FASEB J. (2001) Mar; 15 (3): 559-561 McCarthy K.J et al. J Histochem Cytochem. (1994) Apr; 42 (4): 473-484 Hanneken A et al. Arch Ophthalmol. (1991) Jul; 109 (7): 1005-1011 Fushimi H et al. J Intern Med. (1989) Dec; 226 (6): 409-416 Klein D.J et al Diabetes. (1989) Jan; 38 (1): 130-139 Klein D.J et al. Diabetes. (1986) Oct; 35 (10): 1130-1142 Rohrbach DH et al. J Biol Chem. (1983) Oct 10; 258 (19): 11672-11677 Krusius T et al. J Biol Chem. (1987) Sep 25; 262 (27): 13120-13125 Naso MF et al. J Biol Chem. (1994) Dec 30; 269 (52): 32999-33008 Ito K et al. J Biol Chem. (1995) Jan 13; 270 (2): 958-965 Shinomura T et al. J Biol Chem. (1995) Apr 28; 270 (17): 10328-10333 Zako M et al. J Biol Chem. (1995) Feb 24; 270 (8): 3914-3918 Yang BL et al. J Cell Biochem. (1999) Feb 1; 72 (2): 210-220 Aspberg A et al. J Biol Chem. (1999) Jul 16; 274 (29): 20444-20449 Davidsson P et al. J Lipid Res. (2005) Sep; 46 (9): 1999-2006

  An object of the present invention is to provide an insulin resistance inhibitor, a therapeutic agent for an insulin resistance disease containing the drug as an active ingredient, and a screening method for the insulin resistance inhibitor.

  One object of the present invention is to provide a drug capable of suppressing chondroitin sulfate proteoglycan (CSPG) accumulation in pancreatic β cells, useful for the treatment or prevention of non-insulin dependent diabetes mellitus (NIDDM) based on chondroitin sulfate proteoglycan (CSPG) accumulation. Objective.

  As the inventors of the present invention have conducted extensive research to develop such a drug, the excessive accumulation of chondroitin sulfate proteoglycan (CSPG), which has not been considered as the etiology of insulin resistance disease until now, is caused by pancreatic β cells. I thought that it might promote insulin resistance.

  As a result of conducting research based on this idea, the present inventors have found that one of chondroitin sulfate proteoglycans (CSPG), Versican, one of chondroitin sulfate proteoglycan (CSPG) sulfotransferases, C4ST-1, C4ST- 2. Treating the C4ST-3 gene with siRNA suppresses CSPG expression in pancreatic β cells, increases insulin positive β cell expression, decreases Amyloid precursor protein expression, and can significantly suppress insulin resistance. I found it. That is, it was demonstrated that insulin resistance can be suppressed by inhibiting the accumulation or biosynthesis of chondroitin sulfate proteoglycan, and the present invention was completed. Substances that inhibit the production or accumulation of chondroitin sulfate proteoglycans are useful as insulin resistance inhibitors. In addition, the drug becomes 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, and a screening method for an insulin resistance inhibitor, more specifically,
[1] an insulin resistance inhibitor comprising as an active ingredient a substance that inhibits the production or accumulation of chondroitin sulfate proteoglycans,
[2] The drug according to [1], wherein the substance is a substance having a chondroitin sulfate proteoglycan degradation promoting action,
[3] The drug according to [1], wherein the substance has a chondroitin sulfate proteoglycan synthesis inhibitory action,
[4] The drug according to [1], wherein the substance is a substance having a chondroitin sulfate proteoglycan desulfation effect,
[5] The drug according to [1], wherein the substance has a chondroitin sulfate proteoglycan sulfation inhibitory action.
[6] The drug according to any one of [1] to [5], wherein production or accumulation of chondroitin sulfate proteoglycan is inhibited in pancreatic β cells,
[7] The drug according to any one of [1] to [6] for treatment or prevention of insulin resistance disease,
[8] The drug according to [7], wherein the insulin resistance disease is an insulin resistance disease associated with metabolic syndrome,
[9] The drug according to [7], wherein the insulin resistance disease is non-insulin dependent (type 2) diabetes,
[10] A screening method for an insulin resistance inhibitor, comprising selecting a substance having an action of inhibiting the production or accumulation of chondroitin sulfate proteoglycan from a test sample,
[11] The screening method according to [10], including a step of selecting a substance having the action according to any one of (a) to (d) below:
(A) Degradation promoting action of chondroitin sulfate proteoglycan (b) Synthesis inhibitory action of chondroitin sulfate proteoglycan (c) Desulfation action of chondroitin sulfate proteoglycan (d) Sulfation inhibitory action of chondroitin sulfate proteoglycan [12] Insulin resistance inhibitor The method for screening according to [10] or [11], wherein the screening method is for treatment or prevention of insulin resistance disease,
Is provided.
The present invention further relates to the following.
[13] Use of the drug according to any one of [1] to [9] in the production of an insulin resistance inhibitor,
[14] A method for treating insulin resistance, comprising a step of administering the drug according to any one of [1] to [9] to an individual (patient or the like),
[15] A composition comprising the drug according to any one of [1] to [9] and a pharmaceutically acceptable carrier.

  According to the present invention, it has been revealed that chondroitin sulfate proteoglycan generation and accumulation are related to the development of insulin resistance. It was shown that insulin resistance is suppressed by inhibiting the production and accumulation of chondroitin sulfate proteoglycans. 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 is also important for industry and industry.

Hereinafter, the present invention will be described in detail.
As a pathological condition associated with diabetes which is one of typical metabolic syndromes (visceral fat syndrome), there are an insulin secretion abnormality in pancreatic β cells and a deficiency of insulin action including glucose uptake in an insulin target tissue. The present inventors paid attention to the function of chondroitin sulfate proteoglycan in order to make improvement of abnormal insulin secretion in pancreatic β cells one of the effective methods for treating diabetes. A state in which chondroitin sulfate proteoglycan accumulation was suppressed in a diabetic model mouse was analyzed in detail. As a result, many cells with improved accumulation of chondroitin sulfate proteoglycan compared to wild-type pancreatic β cells were observed. In addition, the pathological condition of diabetes such as blood sugar level was improved. That is, it has been found that inhibition of chondroitin sulfate proteoglycan production or accumulation promotes improvement of the abnormal accumulation state of chondroitin sulfate proteoglycan in pancreatic β cells that are deeply involved in diabetes, leading to improvement of insulin resistance.

  The present invention relates to an insulin resistance-suppressing agent comprising, as an active ingredient, a substance that inhibits the production or accumulation of chondroitin sulfate proteoglycans.

  The “chondroitin sulfate proteoglycan” of the present invention is one of proteoglycans and is a generic name for a covalently bonded compound of chondroitin sulfate / dermatan sulfate, which is a typical sulfated mucopolysaccharide, and a protein (core protein). The “chondroitin sulfate proteoglycan” in the present invention is preferably a human chondroitin sulfate proteoglycan, but the species from which the chondroitin sulfate proteoglycan is derived is not particularly limited, and is a protein equivalent to a chondroitin sulfate proteoglycan in a non-human organism (such as a homolog or ortholog). ) Is also included in the “chondroitin sulfate proteoglycan” in the present invention. For example, the present invention can be carried out if the organism has a protein corresponding to human chondroitin sulfate proteoglycan and has a protein equivalent to human chondroitin sulfate proteoglycan. The chondroitin sulfate proteoglycan in the present invention includes a so-called part-time proteoglycan in which a glycosaminoglycan (GAG) chain is temporarily bound due to inflammation or the like to become a proteoglycan.

  In the following description, examples of chondroitin sulfate proteoglycans include aggrican, versican, neurocan, brevican, βglycan, Decorin, Biglycan, Fibromodulin, and PG-Lb. The chondroitin sulfate proteoglycan in the present invention is not limited to these, and any substance having activity as a chondroitin sulfate proteoglycan may be used. Here, the activity of chondroitin sulfate proteoglycan includes, for example, cell adhesion ability or cell growth promotion. A person skilled in the art can evaluate the activity as a chondroitin sulfate proteoglycan by the following method. Protein containing a partial region of chondroitin sulfate proteoglycan amino acid sequence, or high homology with a partial region (usually 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more) Measure proliferation of tumor cells (eg, Caco-2, HT-29 cells, etc.) in the presence of proteins with A protein having an effect of promoting mitotic proliferation can be determined as a protein having chondroitin sulfate proteoglycan activity (Int J Exp Pathol. 2005 Aug; 86 (4): 219-29 and Histochem Cell Biol. 2005 Aug; 124 (2): 139-49). Here, the high homology means 50% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more (for example, 95% or more, further 96%, 97%, 98% or 99%). % Or higher) homology. This homology is determined by the mBLAST algorithm (Altschul et al. (1990) Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-7 ) Can be determined.

  “Insulin resistance” in the present invention refers to a state in which the presence of insulin in a living body is abnormal. Examples include, but are not limited to, a decrease in insulin secretion from pancreatic β cells and a decrease in insulin action in skeletal muscle, liver, and adipose tissues, which are target tissues of insulin.

  Examples of `` inhibiting production or accumulation '' of chondroitin sulfate proteoglycans in the present invention include `` promotion of degradation '', `` synthesis inhibition '', `` desulfation '', `` sulfation inhibition '' and the like of chondroitin sulfate proteoglycans, However, the present invention is not limited to this, and it means that the abundance, function or activity of chondroitin sulfate proteoglycan is reduced or eliminated as compared with the comparison target. In the present invention, the `` substance that inhibits production or accumulation '' of chondroitin sulfate proteoglycan is not particularly limited, but preferably `` substance that has an action of promoting degradation of chondroitin sulfate proteoglycan '', `` substance that has an inhibitory effect on synthesis '', `` “Substance having desulfation action” or “substance having sulfation inhibitory action”.

  “Promoting degradation” of chondroitin sulfate proteoglycan includes, for example, inhibition of expression / reduction of the protein serving as the core of chondroitin sulfate proteoglycan. Here, the “protein that becomes the core of chondroitin sulfate proteoglycan” includes, for example, core proteins such as aggrican, versican, neurocan, and brevican if they are matrix type chondroitin sulfate proteoglycans. Examples of membrane-type chondroitin sulfate proteoglycans include core proteins such as βglycan, Decorin, Biglycan, Fibromodulin, and PG-Lb. These are only examples, and are not limited to these, and may be any protein that can be a core of chondroitin sulfate proteoglycans.

  “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 proteoglycan” means that transcription and translation of the gene encoding the protein that is the core of chondroitin sulfate proteoglycan occurs, or the protein that is the core of chondroitin sulfate proteoglycan by these transcription and translation Is generated. Examples of the “function of the protein serving as the core of chondroitin sulfate proteoglycan” include, for example, the function of the protein binding to chondroitin sulfate and the binding to other components in the cell. The various functions described above can be appropriately evaluated (measured) by those skilled in the art using general techniques. Specifically, the method described in the examples described later, or the method can be appropriately modified and carried out.

  Furthermore, “promotion of degradation” of chondroitin sulfate proteoglycan may be an increase in expression of an enzyme that cleaves or degrades chondroitin sulfate proteoglycan or an enzyme related thereto. Examples of these enzymes include, but are not limited to, metalloproteinases (for example, ADAM-TS1, ADAM-TS4, ADAM-TS5, etc.), chondroitinase, and Calpain I. “Acceleration of degradation” may be a decrease in the abundance of chondroitin sulfate proteoglycan caused by administration of these enzymes or a part of the enzymes.

  “Degradation promotion” may be caused by administration of a substance that promotes suppression of chondroitin sulfate proteoglycan expression. Examples of these substances include, but are not limited to, n-butylate, Diethylcarbamazepine, Tunicamycin, non-steroidal estrogen, and cyclofenil deiphenol.

Preferable embodiments of the “substance having a decomposition promoting action” include, for example, compounds (nucleic acids) selected from the group consisting of the following (a) to (c).
(A) An antisense nucleic acid for a transcription product of a gene encoding a core protein of chondroitin sulfate proteoglycan or a part thereof (b) A ribozyme activity that specifically cleaves a transcription product of a gene encoding the core protein of chondroitin sulfate proteoglycan Nucleic acid (c) Nucleic acid having an action of inhibiting the expression of the gene encoding the core protein of chondroitin sulfate proteoglycan by RNAi effect

Examples of the “substance having a decomposition promoting action” include compounds selected from the group consisting of the following (a) to (c).
(A) an antibody that binds to the core protein of chondroitin sulfate proteoglycan (b) a chondroitin sulfate proteoglycan variant having a dominant negative property to the core protein of chondroitin sulfate proteoglycan (c) a small molecule that binds to the core protein of chondroitin sulfate proteoglycan Compound

  “Synthesis inhibition” of chondroitin sulfate proteoglycan includes, for example, inhibition of glycosaminoglycan biosynthesis, inhibition of enzymes involved in chondroitin sulfate proteoglycan synthesis, but is not limited thereto, and chondroitin sulfate proteoglycan is synthesized. Refers to inhibiting any of the processes.

  As substances that inhibit the synthesis of chondroitin sulfate proteoglycans, examples of substances that inhibit the biosynthesis of glycosaminoglycans include β-D-xyloside, 2-deoxy-D-glucose (2-DG), ethane-1- Examples include hydroxy-1,1-diphosphonate (ETDP) and 5-hexyl-2-deoxyuridine (HUdR). These and other substances inhibit the biosynthesis of glycosaminoglycans and inhibit the synthesis of chondroitin sulfate proteoglycans.

  On the other hand, examples of enzymes involved in chondroitin synthesis include GalNac4ST-1, GalNac4ST-2, GalNac4-6ST, UA2OST, GalT-I, GalT-II, GlcAT-I, and XylosylT. By inhibiting these and other enzymes and suppressing their expression, the synthesis of chondroitin sulfate proteoglycans is inhibited.

Preferable embodiments of the “substance having a synthesis inhibitory action” include, for example, compounds (nucleic acids) selected from the group consisting of the following (a) to (c).
(A) an antisense nucleic acid for a transcription product of a gene encoding chondroitin sulfate proteoglycan synthase or a part thereof (b) a nucleic acid having a ribozyme activity that specifically cleaves a transcription product of a gene encoding chondroitin sulfate proteoglycan synthase ( c) Nucleic acid having an action of inhibiting the expression of a gene encoding chondroitin sulfate proteoglycan synthase by RNAi effect

Examples of the “substance having a synthetic inhibitory action” include compounds selected from the group consisting of the following (a) to (c).
(A) an antibody that binds to chondroitin sulfate proteoglycan synthase (b) a chondroitin sulfate proteoglycan synthase variant having a dominant negative property with respect to chondroitin sulfate proteoglycan synthase (c) a low molecular compound that binds to chondroitin sulfate proteoglycan synthase

  “Desulfation” of chondroitin sulfate proteoglycans refers to removal of sulfate groups in chondroitin sulfate proteoglycans, such as desulfation by endogenous or externally administered desulfating enzymes, or sulfate by a compound that suppresses sulfation. Examples include, but are not limited to, the process of removing sulfate groups.

  Examples of desulfating enzymes include Chondroitin-4-sulfatase and Chondroitin-6-sulfatease. Examples of the compound that suppresses sulfation include Chlorate and EGF receptor antagnist.

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).
(A) an antisense nucleic acid for a transcription product of a gene encoding a chondroitin sulfate proteoglycan desulfase inhibitor protein or a part thereof (b) a transcript of a gene encoding a chondroitin sulfate proteoglycan desulfase inhibitor protein is specifically cleaved Nucleic acid having ribozyme activity (c) Nucleic acid having an action of inhibiting the expression of a gene encoding a chondroitin sulfate proteoglycan desulfase inhibitor protein by the RNAi effect

Examples of the “substance having desulfating action” include compounds selected from the group consisting of the following (a) to (c).
(A) an antibody that binds to a chondroitin sulfate proteoglycan desulfase inhibitor compound (b) a chondroitin sulfate proteoglycan desulfation inhibitor protein variant that has dominant negative properties against the chondroitin sulfate proteoglycan desulfase inhibitor protein (c) chondroitin sulfate Low molecular weight compounds that bind to proteoglycan desulfase inhibitor compounds

  Here, the “desulfation-inhibiting compound” is not limited to proteins, and includes non-protein compounds such as coenzymes, for example.

  The “sulfation inhibitory action” of chondroitin sulfate proteoglycan includes, for example, inhibition of sulfate group transferase, but is not limited to this, and refers to inhibition of sulfation that occurs during the synthesis of chondroitin sulfate proteoglycan. .

  Examples of the sulfotransferase include C4ST-1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1), C4ST-2 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2), C4ST-3 (Chondroitin DN- Examples thereof include acetylgalactosamine-4-O-sulfotransferase 3), D4ST, C6ST-1, and C6ST-2.

Preferable embodiments of “substances having a sulfation inhibiting action” include, for example, compounds (nucleic acids) selected from the group consisting of the following (a) to (c).
(A) An antisense nucleic acid for a transcript of a gene encoding chondroitin sulfate proteoglycan sulfate transferase or a part thereof (b) A ribozyme activity that specifically cleaves a transcript of a gene encoding chondroitin sulfate proteoglycan sulfate transferase (C) a nucleic acid having an action of inhibiting the expression of a gene encoding chondroitin sulfate proteoglycan sulfate transferase by the RNAi effect

Examples of the “substance having sulfation inhibitory action” include compounds selected from the group consisting of the following (a) to (c).
(A) an antibody that binds to chondroitin sulfate proteoglycan sulfate group (b) a chondroitin sulfate proteoglycan sulfate group variant (c) a low molecular weight compound that binds to chondroitin sulfate proteoglycan sulfate group

  The enzymes exemplified above include not only one enzyme corresponding to one gene but also an enzyme group sharing certain characteristics. For example, chondroitinase is a generic term for enzymes such as ABC, AC, and B that share the characteristics of mucopolysaccharide-degrading enzymes but differ in substrate specificity. For example, chondroitinase AC I cleaves the N-acetylhexosaminide bond of chondroitin sulfates (A, C or E), chondroitin, chondroitin sulfate-dermatan sulfate hybrid type and hyaluronic acid. An oligosaccharide having a Δ4-glucuronic acid residue at the non-reducing end is generated. This enzyme does not act on dermatan sulfate (chondroitin sulfate B, having hexuronic acid L-iduronic acid), keratan sulfate, heparan sulfate, and heparin. In addition, chondroitinase AC II cleaves the N-acetylhexosaminide bond of chondroitin, chondroitin sulfate A and chondroitin sulfate C in a desorption reaction, and produces Δ4-unsaturated disaccharides (ΔDi-0S, ΔDi- 4S and ΔDi-6S). This enzyme also works well on hyaluronic acid. It does not act on dermatan sulfate (chondroitin sulfate B) and becomes a competitive inhibitor of this enzyme. Chondroitinase B (dermatanase) is an oligosaccharide having a Δ4-hexuronic acid residue at the non-reducing end by cleaving the N-acetylgalactosaminide bond bound to L-iduronic acid of dermatan sulfate by elimination reaction. Disaccharide and tetrasaccharide). This enzyme does not act on chondroitin sulfate A and chondroitin sulfate C that do not contain L-iduronic acid. Dermatan, a derivative obtained by removing the sulfate group of dermatan sulfate, does not serve as a substrate for this enzyme. The site where the second position of the L-iduronic acid unit of dermatan sulfate is sulfated is better cleaved by this enzyme. Chondroitinase ABC cleaves the N-acetylhexosaminide bond of chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate, chondroitin and hyaluronic acid in a reactive manner, resulting in a Δ4-hexuronic acid residue at the non-reducing end Mainly produces disaccharides with This enzyme does not act on keratan sulfate, heparin and heparan sulfate. Chondroitinase is a generic name for such enzymes that have different properties but have a common property called mucopolysaccharide-degrading enzyme. Chondroitinase ACI, Chondroitinase AC II, Chondrotinase B, and Chondrotinase ABC exemplified here Not limited.

  Moreover, the enzyme group which shares such a characteristic does not necessarily correspond to one gene on genomic DNA. For example, chondroitin-4-sulfatase and chondroitin-6-sulfatase are sequences referred to by a plurality of accession numbers on the genome database (for example, Genbank accession number NT_039500 (part of which is accession number CAAA01098429 (SEQ ID NO: 91)), and NT_078575, NT_039353, NW_001030904, NW_001030811, NW_001030796, NW_000349) are searched on the public gene database Genbank.

Among those exemplified above, those corresponding to individual genes are shown as follows. That is, exemplified as the above chondroitin sulfate proteoglycan, aggrican, versican, neurocan, brevican, βglycan, Decorin, Biglycan, Fibromodulin, PG-Lb, an enzyme that cleaves or degrades chondroitin sulfate proteoglycan, or an enzyme related thereto, ADAM-TS1, ADAM-TS4, ADAM-TS5, Calpain I, exemplified as enzymes involved in chondroitin synthesis, GalNac4ST-1, GalNac4ST-2, GalNac4-6ST, UA2OST, GalT-I, GalT-II, GlcAT-I, XylosylT, exemplified by sulfate group transferase, C4ST-1, C4ST-2, C4ST-3, D4ST, C6ST-1, C6ST-2 gene gene public accession number and nucleotide sequence in public gene database The amino acid sequence is as follows.
aggrican (accession number NM_007424, nucleotide sequence SEQ ID NO: 1, amino acid sequence SEQ ID NO: 2)
versican (Accession number BC096495, nucleotide sequence SEQ ID NO: 3, amino acid sequence SEQ ID NO: 4)
neurocan (accession number NM_010875, nucleotide sequence SEQ ID NO: 5, amino acid sequence SEQ ID NO: 6)
brevican (accession number NM_007529, base sequence SEQ ID NO: 7, amino acid sequence SEQ ID NO: 8)
βglycan (accession number AF039601, nucleotide sequence SEQ ID NO: 9, amino acid sequence SEQ ID NO: 10)
Decorin (Accession number NM_007833, nucleotide sequence SEQ ID NO: 11, amino acid sequence SEQ ID NO: 12)
Biglycan (Accession number BC057185, base sequence SEQ ID NO: 13; amino acid sequence SEQ ID NO: 14)
Fibromodulin (accession number NM_021355, nucleotide sequence SEQ ID NO: 15 and amino acid sequence SEQ ID NO: 16)
PG-Lb (accession number NM_007884, base sequence SEQ ID NO: 17, amino acid sequence SEQ ID NO: 18)
ADAM-TS1 (accession number NM_009621, base sequence SEQ ID NO: 19 and amino acid sequence SEQ ID NO: 20)
ADAM-TS4 (Accession number NM_172845, SEQ ID NO: 21 of the nucleotide sequence, SEQ ID NO: 22 of the amino acid sequence)
ADAM-TS5 (accession number AF140673, base sequence SEQ ID NO: 23, amino acid sequence SEQ ID NO: 24)
Calpain I (accession number NM_007600, nucleotide sequence SEQ ID NO: 25, amino acid sequence SEQ ID NO: 26)
GalNac4ST-1 (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 SEQ ID NO: 29, amino acid sequence SEQ ID NO: 30)
GalNac4-6ST (Accession number NM_029935, nucleotide sequence SEQ ID NO: 31, amino acid sequence SEQ ID NO: 32)
UA2OST (accession number NM_177387, base sequence SEQ ID NO: 33, amino acid sequence SEQ ID NO: 34)
GalT-I (accession number NM_016769, base sequence SEQ ID NO: 35, amino acid sequence SEQ ID NO: 36)
GalT-II (accession number BC064767, base sequence SEQ ID NO: 37, amino acid sequence SEQ ID NO: 38)
GlcAT-I (accession number BC058082, base sequence SEQ ID NO: 39, amino acid sequence SEQ ID NO: 40, or accession number NM_024256, base sequence SEQ ID NO: 41, amino acid sequence SEQ ID NO: 42)
XylosylT (accession number NM_145828, nucleotide sequence SEQ ID NO: 43, amino acid sequence SEQ ID NO: 44)
C4ST-1 (Accession number NM_021439, nucleotide sequence SEQ ID NO: 45, amino acid sequence SEQ ID NO: 46)
C4ST-2 (Accession number NM_021528, nucleotide sequence SEQ ID NO: 47, amino acid sequence SEQ ID NO: 48)
C4ST-3 (accession number XM_355798, base 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, nucleotide sequence SEQ ID NO: 53, amino acid sequence SEQ ID NO: 54)
C6ST-2 (accession number AB046929, nucleotide sequence SEQ ID NO: 55, amino acid sequence SEQ ID NO: 56)

  Even proteins other than the above have high homology (usually 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more) with the sequences described in the sequence listing, for example. And the protein which has the function (For example, the function etc. which couple | bond with the structural component in a cell) which the said protein has is contained in the said protein of this invention. The protein is, for example, SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, A protein consisting of an amino acid sequence according to any one of 42, 44, 46, 48, 50, 52, 54, and 56, wherein one or more amino acids are added, deleted, substituted, or inserted, and The number of amino acids to be changed is within 30 amino acids, preferably within 10 amino acids, more preferably within 5 amino acids, and most preferably within 3 amino acids.

  Examples of the gene in the present invention include 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, 55, an endogenous gene (such as a homolog of the above-mentioned human gene) in another organism corresponding to the DNA comprising the base sequence described in any one of included.

  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, and 55, the endogenous DNA of other organisms corresponding to the DNA consisting of the base sequence described in any one of 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, 55 It has high homology with the DNA described in 1. High homology means 50% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more (for example, 95% or more, or 96%, 97%, 98% or 99% or more). ) Homology. This homology is determined by the mBLAST algorithm (Altschul et al. (1990) Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-7 ) Can be determined. When the DNA is isolated from a living body, 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, and 55 are considered to hybridize under stringent conditions. Here, as “stringent conditions”, for example, “2 × SSC, 0.1% SDS, 50 ° C.”, “2 × SSC, 0.1% SDS, 42 ° C.”, “1 × SSC, 0.1% SDS, 37 ° C.” More stringent conditions include “2 × SSC, 0.1% SDS, 65 ° C.”, “0.5 × SSC, 0.1% SDS, 42 ° C.” and “0.2 × SSC, 0.1% SDS, 65 ° C.” Can do.

  A person skilled in the art is able to measure a protein functionally equivalent to the above protein from the above highly homologous proteins by measuring the activity of the chondroitin sulfate proteoglycan degradation promoting action, synthesis inhibiting action, desulfating action or sulfation inhibiting action. Can be obtained as appropriate. A specific activity measurement method is described in the section of the screening method in the present invention described later. Further, 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. In the present specification, the protein and gene corresponding to the protein and gene in organisms other than humans, or the protein and gene functionally equivalent to the protein and gene described above are also simply described with the above names. There is a case.

  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. The natural protein can be prepared, for example, by a method using affinity chromatography using an antibody against the protein against an extract of a cell (tissue) considered to express the protein. On the other hand, the recombinant protein can be prepared, for example, by culturing cells transformed with DNA encoding the protein. The above protein of the present invention is suitably used, for example, in the screening method described below.

  The “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 is obtained by replacing the pentose / phosphate skeleton, which is the basic skeleton structure of nucleic acids, with a polyamide skeleton having glycine as a unit, and has a three-dimensional structure very similar to nucleic acids.

  As a method for inhibiting the expression of a specific endogenous gene, a method using an antisense technique is well known to those skilled in the art. There are several factors as described below for the action of the antisense nucleic acid to inhibit the expression of the target gene. Inhibition of transcription initiation by triplex formation, inhibition of transcription by hybridization with a site where an open loop structure was locally created by RNA polymerase, inhibition of transcription by hybridization with RNA undergoing synthesis, intron and exon Splicing inhibition by hybridization at splice point, splicing inhibition by hybridization with spliceosome formation site, inhibition of translocation from nucleus to cytoplasm by hybridization with mRNA, hybridization with capping site and poly (A) addition site Inhibition of splicing by RNA, inhibition of translation initiation by hybridization with a translation initiation factor binding site, translation inhibition by hybridization with a ribosome binding site near the initiation codon, peptide by hybridization with an mRNA translation region or polysome binding site Elongation inhibition, and the like inhibiting gene expression by hybrid formation with the site of interaction between nucleic acids and proteins. In this way, antisense nucleic acids inhibit the expression of target genes by inhibiting various processes such as transcription, splicing or translation (Hirashima and Inoue, Shinsei Kagaku Kenkyu 2 Lecture and Expression of Nucleic Acid IV Gene, Japan Biochemical) (Academic Society, Tokyo Chemical Doujin, 1993, 319-347).

  The antisense nucleic acid used in the present invention is capable of expressing a gene encoding any one of the above-mentioned chondroitin sulfate proteoglycan core protein, synthase, desulfase inhibitor protein, and sulfotransferase, and / or Or you may inhibit a function. As one embodiment, antisense complementary to the untranslated region near the 5 ′ end of mRNA of the gene encoding chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfase inhibitor protein, or sulfotransferase described above If the sequence is designed, it is considered effective for inhibiting translation of the gene. In addition, a sequence complementary to the coding region or the 3 ′ untranslated region can also be used. Thus, the nucleic acid containing the above-mentioned chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfase inhibitor protein, or the translation region of the gene encoding the sulfotransferase, and the antisense sequence of the sequence of the untranslated region Are also included in the antisense nucleic acid utilized 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) using a known method. The sequence of the antisense nucleic acid is a gene encoding a core protein, a synthetic enzyme, a desulfating enzyme inhibitory protein, or a sulfotransferase, or a part thereof, which is contained in an endogenous chondroitin sulfate proteoglycan possessed by an animal (cell) to be transformed. Although it is preferably a complementary sequence, it may not be completely complementary as long as gene expression can be effectively suppressed. The transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcript of the target gene. In order to effectively inhibit the expression of a target gene using an antisense nucleic acid, the length of the antisense nucleic acid is preferably at least 15 bases and less than 25 bases, but the antisense nucleic acid of the present invention is not necessarily of this length. For example, it may be 100 bases or more, or 500 bases or more.

  The antisense nucleic acid of the present invention is not particularly limited. For example, the base sequence of Versican gene (GenBank accession number BC096495, SEQ ID NO: 3), the base sequence of C4ST-1 gene (GenBank accession number NM_021439, SEQ ID NO: 45), C4ST-2 gene base sequence (GenBank accession number NM_021528, SEQ ID NO: 47), C4ST-3 gene base sequence (GenBank accession number XM_355798, SEQ ID NO: 49), etc. can do.

  Inhibition of the expression of the above chondroitin sulfate proteoglycan core protein, synthase, desulfase inhibitor protein, or sulfate-encoding gene can also be carried out using ribozyme or ribozyme-encoding DNA. is there. A ribozyme refers to an RNA molecule having catalytic activity. Some ribozymes have a variety of activities, but research focusing on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes that cleave RNA in a site-specific manner. Some ribozymes have a size of 400 nucleotides or more, such as group I intron type and M1 RNA contained in RNase P, but some have an active domain of about 40 nucleotides called hammerhead type or hairpin type. (Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 1990, 35, 2191.)

  For example, the self-cleaving domain of the hammerhead ribozyme cleaves 3 ′ of C15 in the sequence G13U14C15, but base pairing between U14 and A9 is important for its activity, and instead of C15, A15 or U15 However, it has been shown that it can be cleaved (Koizumi, M. et al., FEBS Lett, 1988, 228, 228.). By designing a ribozyme whose substrate binding site is complementary to the RNA sequence in the vicinity of the target site, it is possible to create a restriction RNA-cleaving ribozyme that recognizes the sequence UC, UU or UA in the target RNA (Koizumi, M. et al., FEBS Lett, 1988, 239, 285., Makoto Koizumi and Eiko Otsuka, Protein Nucleic Acid Enzymes, 1990, 35, 2191., Koizumi, M. et al., Nucl Acids Res, 1989, 17, 7059 .).

  Hairpin ribozymes are also useful for the purposes of the present invention. This ribozyme is found, for example, in the minus strand of tobacco ring spot virus satellite RNA (Buzayan, JM., Nature, 1986, 323, 349.). It has been shown that hairpin ribozymes can also produce target-specific RNA-cleaving ribozymes (Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751., Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, 112.). In this way, by specifically cleaving the transcript of the gene encoding the above chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfase inhibitor protein, or sulfotransferase using ribozyme, Expression can be inhibited.

  Inhibition of endogenous gene expression can also be carried out by RNA interference (RNA interference, hereinafter abbreviated as “RNAi”) using double-stranded RNA having the same or similar sequence as the target gene sequence.

  With the completion of genome projects in recent years, the entire human base sequence has been deciphered and many disease-related genes have been actively identified. Currently, therapeutic methods and drug development targeting specific genes are being actively implemented. Of particular interest is the application of small interfering RNA (siRNA), which exhibits specific post-transcriptional repression effects, to gene therapy. RNAi is a technique that is currently attracting attention because when a double-stranded RNA (dsRNA) is directly taken into a cell, expression of a gene having a sequence homologous to this dsRNA is suppressed. In mammalian cells, it is possible to induce RNAi by using short dsRNA (siRNA). RNAi is more stable, easier to experiment, and less expensive than knockout mice. Has many advantages.

  A nucleic acid having an inhibitory action by the RNAi effect is generally also called siRNA or shRNA. RNAi is a short double-stranded RNA (hereinafter abbreviated as “dsRNA”) consisting of a sense RNA consisting of a sequence homologous to the mRNA of the target gene and an antisense RNA consisting of a complementary sequence to the cell. This is a phenomenon that induces destruction by specifically and selectively binding to the target gene mRNA, and efficiently inhibiting (suppressing) the expression of the target gene by cleaving the target gene. For example, when dsRNA is introduced into a cell, expression of a gene having a homologous sequence with that RNA is suppressed (knocked down). Since RNAi can suppress the expression of target genes in this way, it can be used as a simple gene knockout method to replace conventional complicated and low efficiency homologous recombination methods, or a method applicable to gene therapy. It is attracting attention as. The RNA used for RNAi is not necessarily completely identical to the above-mentioned chondroitin sulfate proteoglycan core protein, synthase, desulfase inhibitor protein, or a gene encoding a sulfotransferase, or a partial region of the gene. , Preferably having complete homology.

  In designing siRNA, the target is not particularly limited as long as it is a gene encoding the above-mentioned chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfase inhibitor protein, or sulfate transferase, and any region can be used. Can be all target candidates. For example, the base sequence of the Versican gene (SEQ ID NO: 3), the base sequence of the C4ST-1 gene (SEQ ID NO: 45), the base sequence of the C4ST-2 gene (SEQ ID NO: 47), the base sequence of the C4ST-3 gene ( It can be created based on SEQ ID NO: 49) and the like. More specifically, a partial region of the sequence can be a target candidate. For example, a partial region of the base sequence of the Versican gene (SEQ ID NO: 57), the base sequence of the C4ST-1 gene Partial region (SEQ ID NO: 58), partial region of the C4ST-2 gene base sequence (SEQ ID NO: 59), partial region of the C4ST-3 gene base sequence (SEQ ID NO: 60), C6ST-1 gene A partial region of the base sequence (SEQ ID NO: 61), a partial region of the base sequence of the C6ST-2 gene (SEQ ID NO: 62), a partial region of the base sequence of the GalNAc4ST-1 gene (SEQ ID NO: 63), It can be prepared based on a partial region (SEQ ID NO: 64) of the base sequence of GalNAc4ST-2 gene, a partial region (SEQ ID NO: 65) of the base sequence of GalNAc4-6ST, and the like. More specifically, siRNA targeting the DNA sequences specifically shown by the present specification (SEQ ID NOs: 71 to 73 and 80 to 90) can be exemplified.

  In order to introduce siRNA into a cell, a method in which siRNA synthesized in vitro is linked to plasmid DNA and introduced into the cell, a method in which two RNAs are annealed, or the like can be employed.

  The two RNA molecules may be molecules having a structure in which one end is closed, for example, an siRNA (shRNA) having a hairpin structure. shRNA is an RNA molecule called a short hairpin RNA (short hairpin RNA), which has a stem-loop structure so that a part of a single strand forms a complementary strand with another region. That is, molecules capable of forming a double-stranded RNA structure within the molecule are also included in the siRNA of the present invention.

  Further, as a preferred embodiment of the present invention, RNA (siRNA) capable of suppressing the expression of Versican, C4ST-1, C4ST-2, C4ST-3 and the like by the RNAi effect, which is specifically shown by the present specification In the siRNA targeting the DNA sequence (SEQ ID NOs: 71 to 73 and 80 to 90), for example, even if it is a double-stranded RNA having a structure in which one or a small number of RNAs are added or deleted, the above chondroitin sulfate Any siRNA of the present invention may be used as long as it has a function of suppressing the expression of a gene encoding a proteoglycan core protein, a synthase, a desulfase inhibitor protein, or a sulfotransferase.

  RNA used for RNAi (siRNA) does not have to be completely identical (homologous) to the gene encoding the protein or a partial region of the gene, but may have perfect identity (homology) preferable.

  The details of the RNAi mechanism are still unknown, but an enzyme called DICER (a member of the RNase III nuclease family) contacts double-stranded RNA, and the double-stranded RNA is called a small interfering RNA or siRNA. It is thought to be broken down into pieces. The double-stranded RNA having the RNAi effect in the present invention includes a double-stranded RNA before being degraded by DICER as described above. That is, it is expected that even a long-chain RNA that does not have an RNAi effect with the same length is decomposed into siRNA having an RNAi effect in a cell. The length is not particularly limited.

  For example, a long double-stranded RNA corresponding to the full-length or almost full-length region of the above-mentioned chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfase-inhibiting protein, or gene encoding a sulfotransferase, It is possible to decompose with DICER in advance and use the degradation product as the drug of the present invention. This degradation product is expected to contain a double-stranded RNA molecule (siRNA) having an RNAi effect. According to this method, a region on mRNA that is expected to have an RNAi effect need not be selected. That is, the region on the mRNA of the above-mentioned gene of the present invention having the RNAi effect does not necessarily need to be accurately defined.

  The above-mentioned “double-stranded RNA that can be suppressed by the RNAi effect” of the present invention means, in those skilled in the art, the above-mentioned chondroitin sulfate proteoglycan core protein, synthase, desulfase inhibitor protein, which is a target of the double-stranded RNA, Or it can produce suitably based on the base sequence of the gene which codes a sulfotransferase. For example, the double-stranded RNA of the present invention can be prepared based on the base sequence shown in SEQ ID NO: 71. That is, based on the base sequence set forth in SEQ ID NO: 71, selecting any continuous RNA region of mRNA that is a transcription product of the sequence, and preparing a double-stranded RNA corresponding to this region, For those skilled in the art, it can be appropriately performed within the range of normal trials. In addition, selection of siRNA sequences having a stronger RNAi effect from mRNA sequences that are transcripts of the sequences can also be appropriately performed by those skilled in the art by known methods. If one strand is known, those skilled in the art can easily know the base sequence of the other strand (complementary strand). The siRNA can be appropriately prepared by those skilled in the art using a commercially available nucleic acid synthesizer. In addition, for synthesis of a desired RNA, a general synthesis contract service can be used.

  In addition, the siRNA in the present invention is not necessarily a set of double-stranded RNAs for the target sequence, and may be a mixture of a plurality of sets of double-stranded RNA for the region containing the target sequence. Here, siRNA as a nucleic acid mixture corresponding to the target sequence can be appropriately prepared by a person skilled in the art using a commercially available nucleic acid synthesizer and a DICER enzyme. Synthetic contract service can be used. The siRNA of the present invention includes so-called “cocktail siRNA”.

  In the siRNA of the present invention, not all nucleotides are necessarily ribonucleotides (RNA). That is, in the present invention, the one or more ribonucleotides constituting the siRNA may be corresponding deoxyribonucleotides. This “corresponding” refers to the same base species (adenine, guanine, cytosine, thymine (uracil)) although the structures of the sugar moieties are different. For example, deoxyribonucleotide corresponding to ribonucleotide having adenine refers to deoxyribonucleotide having adenine. The “plurality” is not particularly limited, but preferably refers to a small number of about 2 to 5.

  Furthermore, a DNA (vector) capable of expressing the RNA of the present invention is also included in a preferred embodiment of the compound capable of suppressing the expression of the gene encoding the above-described protein of the present invention. For example, 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. Those skilled in the art can appropriately prepare the above DNA of the present invention by general genetic engineering techniques. More specifically, 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.

  In addition, the expression inhibitory substance of the present invention includes the above-mentioned chondroitin sulfate proteoglycan core protein, synthase, desulfurase inhibitor protein, or an expression regulatory region of a gene encoding a sulfotransferase (for example, a promoter region. As an example, the above-mentioned chondroitin sulfate proteoglycan core protein, synthase, desulfase inhibitor can be suppressed by binding to the base sequence represented by SEQ ID NO: 66, which is the promoter region of PG-Lb. Compounds that inhibit the expression of a protein or gene encoding a sulfotransferase are included. The compound uses, for example, the above-mentioned chondroitin sulfate proteoglycan core protein, synthase, desulfase inhibitor protein, or promoter DNA fragment of a gene encoding a sulfotransferase, and the binding activity with the DNA fragment is used as an indicator. The screening method can be used. Further, those skilled in the art know whether or not to inhibit the expression of a gene encoding the above-mentioned chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfase inhibitor protein, or sulfotransferase, for a desired compound. This method can be appropriately carried out by, for example, a reporter assay method.

  Further, the DNA (vector) capable of expressing the RNA of the present invention also expresses the above-described chondroitin sulfate proteoglycan core protein, synthase, desulfase inhibitor protein, or sulfate transferase gene encoding the present invention. Are included in preferred embodiments of the compounds capable of inhibiting For example, the DNA (vector) capable of expressing the double-stranded RNA of the present invention is a DNA encoding one strand of the double-stranded RNA and a DNA encoding the other strand of the double-stranded RNA, respectively. It is a DNA having a structure linked to a promoter so that it can be expressed. Those skilled in the art can appropriately prepare the above DNA of the present invention by general genetic engineering techniques. More specifically, the expression vector of the present invention can be prepared by appropriately inserting DNA encoding the RNA of the present invention into various known expression vectors.

  A preferred embodiment of the vector of the present invention includes a vector that expresses RNA (siRNA) that can suppress the expression of Versican, C4ST-1, C4ST-2, C4ST-3, etc. by the RNAi effect.

  The above-mentioned chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfase inhibitor compound, or antibody that binds to a sulfotransferase can be prepared by methods known to those skilled in the art. If it is a polyclonal antibody, it can obtain as follows, for example. A small animal such as a rabbit is immunized with a recombinant (recombinant) protein expressed in a microorganism as a fusion protein with the above-mentioned natural protein or a GST, or a partial peptide thereof to obtain serum. Coupling this with, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, core protein of the above chondroitin sulfate proteoglycan, synthetic enzyme, desulfating enzyme inhibitory compound, or sulfotransferase or synthetic peptide It is prepared by purifying with an affinity column or the like. In the case of a monoclonal antibody, for example, the above chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfase inhibitor compound, or sulfotransferase or a partial peptide thereof is immunized to a small animal such as a mouse, and the spleen is obtained from the mouse. And the cells are separated by grinding, and the cells and mouse myeloma cells are fused using a reagent such as polyethylene glycol, and the above chondroitin sulfate proteoglycan is obtained from the fused cells (hybridoma) formed thereby. Clones that produce antibodies that bind to the core protein, synthetic enzyme, desulfase inhibitor compound, or sulfotransferase of Subsequently, the obtained hybridoma is transplanted into the abdominal cavity of the mouse, and ascites is collected from the mouse, and the obtained monoclonal antibody is obtained by, for example, ammonium sulfate precipitation, protein A, protein G column, DEAE ion exchange chromatography, the above chondroitin. It can be prepared by purification using an affinity column coupled with a proteoglycan sulfate core protein, a synthetic enzyme, a desulfating enzyme inhibitory compound, or a sulfotransferase protein or a synthetic peptide.

  The antibody of the present invention is not particularly limited as long as it binds to the above-described chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfase inhibitor compound, or sulfate group transferase of the present invention. In addition to the above, 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 antibody acquisition is not limited with respect to the animal species from which it is derived, but is preferably a protein derived from a mammal such as a mouse or human, and particularly preferably a protein derived from a human. Human-derived proteins can be appropriately obtained by those skilled in the art using the gene sequences or amino acid sequences disclosed in the present specification.

  In the present invention, the protein used as the sensitizing antigen may be a complete protein or a partial peptide of the protein. Examples of the partial peptide of protein include an amino group (N) terminal fragment and a carboxy (C) terminal fragment of protein. As used herein, “antibody” means an antibody that reacts with the full length or fragment of a protein.

  In addition to immunizing non-human animals with antigens to obtain the above hybridomas, human lymphocytes such as human lymphocytes infected with EB virus are sensitized with proteins, protein-expressing cells or lysates thereof in vitro. Lymphocytes can be fused with human-derived myeloma cells having permanent mitotic activity, such as U266, to obtain hybridomas that produce desired human antibodies having binding activity to proteins.

  The above-mentioned chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfase inhibitor compound, or antibody to sulfotransferase of the present invention is expected to inhibit the expression or function of the protein by binding to the protein. Is done. When the obtained antibody is used for the purpose of administering it to the human body (antibody treatment), a human antibody or a humanized antibody is preferred in order to reduce immunogenicity.

  Furthermore, the present invention provides the above chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfurization as a substance capable of inhibiting the function of the above chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfating enzyme inhibitory compound, or sulfotransferase. It also contains a low molecular weight substance (low molecular weight compound) that binds to an oxidase inhibiting compound or a sulfotransferase. The low molecular weight substance may be a natural or artificial compound. Usually, it is a compound that can be produced or obtained by using methods known to those skilled in the art. The compound of the present invention can also be obtained by the screening method described later.

  Furthermore, as a substance capable of inhibiting the expression or function of the above-mentioned chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfase inhibitor protein, or sulfotransferase of the present invention, the above chondroitin sulfate proteoglycan core protein, synthetic enzyme, Examples thereof include a desulfurase inhibitor protein or a mutant (dominant negative protein) having a dominant negative property with respect to a sulfotransferase. “The chondroitin sulfate proteoglycan core protein, synthase, desulfase-inhibiting protein, or the protein variant having a dominant negative property to sulfate group” refers to the chondroitin sulfate proteoglycan core protein, synthase, desulfurization It refers to a protein having a function of eliminating or reducing the activity of an endogenous wild-type protein by expressing an oxidase-inhibiting protein or a gene encoding 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.

  In the present invention, the organ, tissue or cell that inhibits the production or accumulation of chondroitin sulfate proteoglycan is not particularly limited, but is preferably an organ, tissue or cell that secretes insulin, or an organ, tissue or cell that acts on insulin. More preferably, pancreatic β cells.

  Compounds that inhibit the production or accumulation of chondroitin sulfate proteoglycans are expected to be drugs for the treatment or prevention of insulin resistance diseases. Here, “treatment or prevention” does not necessarily need to have a complete therapeutic or prophylactic effect on an organ, tissue, or cell exhibiting insulin resistance, but may have a partial effect.

  In the present invention, the insulin resistance disease is not particularly limited as long as it is a disease accompanied by insulin resistance, but preferably a disease exhibiting insulin resistance associated with metabolic syndrome (visceral fat syndrome), insulin-independent type (type 2) Diabetes and the like can be mentioned. Further, 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 proteoglycan which is the cause of insulin resistance. Therefore, as a preferred embodiment of the present invention, for example, a therapeutic agent for metabolic syndrome or a therapeutic agent for non-insulin dependent (type 2) diabetes 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. In the present invention, the “suppressor” can also be expressed as “medicine”, “pharmaceutical composition”, “therapeutic drug”, and the like.

  The “treatment” in the present invention includes a prophylactic effect that can suppress the occurrence of insulin resistance in advance. Moreover, it is not necessarily limited to having a complete therapeutic effect on insulin resistance-expressing cells (tissue), and may be a partial effect.

  The agent of the present invention can be mixed with a physiologically acceptable carrier, excipient, diluent or the like and administered orally or parenterally as a pharmaceutical composition. As oral preparations, dosage forms such as granules, powders, tablets, capsules, solvents, emulsions or suspensions can be used. As the parenteral preparation, a dosage form such as an injection, an infusion, an external medicine, or a suppository can be selected. Examples of injections include subcutaneous injections, intramuscular injections, intraperitoneal injections, and the like. Examples of the medicine for external use include a nasal administration agent or an ointment. The preparation technology for making the above-mentioned dosage form so as to include the drug of the present invention as the main component is known.

  For example, a tablet for oral administration can be produced by adding an excipient, a disintegrant, a binder, a lubricant, and the like to the drug of the present invention, mixing, and compressing and shaping. As the excipient, lactose, starch, mannitol or the like is generally used. As the disintegrant, calcium carbonate, carboxymethyl cellulose calcium and the like are generally used. As the binder, gum arabic, carboxymethylcellulose, or polyvinylpyrrolidone is used. As the lubricant, talc, magnesium stearate and the like are known.

  The tablet containing the drug of the present invention can be coated with a known coating for masking or enteric preparation. As the coating agent, ethyl cellulose, polyoxyethylene glycol, or the like can be used.

  An injection can be obtained by dissolving the drug of the present invention as a main component together with an appropriate dispersant, or dissolving or dispersing in a dispersion medium. Depending on the selection of the dispersion medium, either a water-based solvent or an oil-based solvent can be used. In order to use an aqueous solvent, distilled water, physiological saline, Ringer's solution, or the like is used as a dispersion medium. In the oil solvent, various vegetable oils and propylene glycol are used as a dispersion medium. At this time, a preservative such as paraben may be added as necessary. In addition, known isotonic agents such as sodium chloride and glucose can be added to the injection. Further, a soothing agent such as benzalkonium chloride or procaine hydrochloride can be added.

  Moreover, it can be set as an external preparation by making the chemical | medical agent of this invention into a solid, liquid, or semi-solid composition. About 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. As the solvent, water, ethyl alcohol, polyethylene glycol, or the like can be used. As the thickener, bentonite, polyvinyl alcohol, acrylic acid, methacrylic acid, or polyvinylpyrrolidone is generally used. A preservative such as benzalkonium chloride can be added to the composition. Also, a suppository can be obtained by combining an oily base material such as cacao butter or an aqueous gel base material such as a cellulose derivative as a carrier.

  When the agent of the present invention is used as a gene therapy agent, a method of administering a vector incorporating a nucleic acid in addition to a method of directly administering the agent of the present invention by injection can be mentioned. Examples of the vector include an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a vaccinia virus vector, a retrovirus vector, a lentivirus vector, and the like, and can be efficiently administered by using these virus vectors.

  It is also possible to introduce the drug of the present invention into a phospholipid vesicle such as a liposome and administer the vesicle. The endoplasmic reticulum holding siRNA or shRNA is introduced into a predetermined cell by the lipofection method. Then, the obtained cells are systemically administered, for example, intravenously or intraarterially. It can also be administered locally to an insulin resistant tissue or the like. siRNAs have excellent specific post-transcriptional repressive effects in vitro, but are more rapidly and optimally delivered in vivo due to their limited duration because they are rapidly degraded by nuclease activity in serum. System development has been demanded. As an example, Ochiya, T et al., Nature Med., 5: 707-710, 1999, Curr. Gene Ther., 1: 31-52, 2001, biocompatible material atelocollagen is mixed with nucleic acid and combined. When the body is formed, it has been reported that it is a carrier suitable for siRNA and has a function of protecting nucleic acid from degrading enzymes in the living body. Not limited.

The necessary amount (effective amount) of the drug of the present invention is administered to mammals including humans within the safe dose range. The dosage of the drug of the present invention can be appropriately determined finally based on the judgment of a doctor or veterinarian in consideration of the type of dosage form, administration method, patient age and weight, patient symptoms, and the like. For example, although it varies depending on age, sex, symptom, administration route, administration frequency, dosage form, for example, the dose in the case of adenovirus is about 10 ⁶ to 10 ¹³ per day, Administered at 8 week intervals.

  In order to introduce siRNA or shRNA into a target tissue or organ, a commercially available gene introduction kit (for example, Adeno Express: Clontech) can also be used.

  When the drug of the present invention is used, the application site or the type of the disease is not particularly limited as long as it is a disease that expresses insulin resistance. For example, for diabetes, obesity, hyperlipidemia, arteriosclerosis, hypertension, etc. Applied. The above-mentioned diseases may be accompanied 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 chondroitin sulfate proteoglycan from a test sample. By the screening method of the present invention, an insulin resistance inhibitor or a candidate compound for an insulin resistance inhibitor can be efficiently obtained.

A preferred embodiment of the screening method of the present invention is a method for screening an insulin resistance inhibitor comprising the step of selecting a substance having the action described in any of the following (a) to (d).
(A) Degradation promoting action of chondroitin sulfate proteoglycan (b) Synthesis inhibition action of chondroitin sulfate proteoglycan (c) Desulfation action of chondroitin sulfate proteoglycan (d) Sulfation inhibition action of chondroitin sulfate proteoglycan

As a basic principle of screening common to these, a typical example includes one including the following steps.
(1) Chondroitin sulfate proteoglycan (CSPG) itself / or glycosaminoglycan (GAG) chain / cells that synthesize (generate) CSPG or GAG chain (2) Test compounds (for example, enormous compound live of pharmaceutical companies Rally)
(3) Method of detecting cut section of chondroitin sulfate proteoglycan (CSPG) / chondroitin sulfate proteoglycan (CSPG) amount / free glycosaminoglycan (GAG) amount The above three tools are used. The procedure of mixing (1) and (2) in a test tube or on a culture dish and detecting the effect simply by (3) is desirable.

  Hereinafter, embodiments of the screening method of the present invention will be exemplified. In the embodiments described below, the chondroitin sulfate proteoglycan used, the synthetic enzyme, the desulfating enzyme inhibitory compound, the sulfotransferase, the degradation promoting enzyme, and the desulfating enzyme are derived from humans, mice, rats, etc. However, it is not particularly limited to those derived from these. The part of the chondroitin sulfate proteoglycan is a component such as a glycosaminoglycan chain, a core protein, or a part thereof, and is not particularly limited.

  In addition, the test compound used in the embodiments described below is not particularly limited. For example, a single compound such as a natural compound, an organic compound, an inorganic compound, a protein, and a peptide, and expression of a compound library and a gene library Examples include products, cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts and the like.

  In addition, the “contact” to the test compound in the embodiment described below usually involves chondroitin sulfate proteoglycan, a part thereof, a synthase, a desulfase inhibitor compound, a sulfotransferase, a degradation promoting enzyme, or a desulfase enzyme. Although it is performed by mixing with a test compound, it is not limited to this method. For example, the above “contact” can be performed by bringing a cell expressing these proteins or a part thereof into contact with a test compound.

  In addition, examples of the origin of the “cell” in the embodiments described below include cells derived from human, mouse, rat, etc., but are not particularly limited to cells derived from these, and express proteins used in each embodiment. It is also possible to use microbial cells such as Escherichia coli and yeast transformed as described above. For example, “cells expressing chondroitin sulfate proteoglycan” include cells expressing an endogenous chondroitin sulfate proteoglycan gene or cells into which an exogenous chondroitin sulfate proteoglycan gene has been introduced and expressed. can do. A cell in which an exogenous chondroitin sulfate proteoglycan gene is expressed can be usually prepared by introducing an expression vector into which a chondroitin sulfate proteoglycan gene is inserted into a host cell. The expression vector can be prepared by general genetic engineering techniques.

  In the following description, the term “chondroitin sulfate proteoglycan core protein” refers to, for example, a matrix type chondroitin sulfate proteoglycan, a core protein such as aggrican, versican, neurocan, brevican, or a membrane type chondroitin sulfate proteoglycan. It is a core protein such as Decorin, Biglycan, Fibromodulin, PG-Lb. “Synthetic enzymes” include, for example, GalNac4ST-1, GalNac4ST-2, GalNac4-6ST, UA2OST, GalT-I, GalT-II, GlcAT-I, XylosylT and the like. In addition, “sulfotransferase” includes, for example, C4ST-1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1), C4ST-2 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2), C4ST-3 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 3), D4ST, C6ST-1, C6ST-2 and the like. Examples of the “degradation promoting enzyme” include ADAM-TS1, ADAM-TS4, ADAM-TS5, Chondroitinase ABC (ChABC), Chondroitinase AC, Chondroitinase B, and Calpain I. Examples of “desulfating enzyme” include Chondroitin-4-sulfatase, Chondroitin-6-sulfatease, and the like.

As an aspect of the screening method of the present invention, a method including a step of selecting a compound having an action of promoting the degradation of chondroitin sulfate proteoglycan can be mentioned. The above method of the present invention comprises, for example, the following steps.
(A) a step of contacting a test compound with chondroitin sulfate proteoglycan or a part thereof (b) a step of measuring the abundance of chondroitin sulfate proteoglycan or a part thereof (c) when measured in the absence of the test compound The process of selecting substances that reduce the abundance in comparison

  In the above method, first, a test compound is brought into contact with chondroitin sulfate proteoglycan or a part thereof.

  In the present method, the amount of chondroitin sulfate proteoglycan or a part thereof is then 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 proteoglycan or a part thereof. Moreover, it can also detect using a chromatography method, a mass spectrometry, etc.

  In this method, a compound that reduces the abundance of the chondroitin sulfate proteoglycan 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.

  A simple example of a method and a specific example that can be evaluated (measured) as to whether or not the test compound has the activity of the above-mentioned (a) decomposition promoting action is shown below.

(A) Screening method embodiment relating to the degradation promoting action of chondroitin sulfate proteoglycan:
As CS-GAG, chondroitin sulfate A (CS-A), CS-B, CS-C (Seikagaku, ICN, Sigma, etc.), human-derived proteoglycan (BGN, ISL, etc.), etc. are prepared. The 96-well plate is coated at a concentration of 10 μg / mL (by a known method such as Kawashima H et al .; J. Biol. Chem. 277: 12921-12930, 2002.). Various test compounds are added to each well of the plate, and changes in CS-GAG are detected after 2 hours of reaction at 37 ° C.

  As a detection method, for example, 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 ABC is used as a positive control for the test compound. When CS-GAG chain is degraded by addition of chondroitinase ABC, WGA lectin cannot be bound, so the principle is used. More specifically, FITC-labeled WGA lectin (such as EY) is added to the CS-coated wells before and after mixing the test compound, and the CS-GAG is decomposed to change the FITC fluorescence intensity in the wells. Quantification and quantification can be performed very easily using a detection device such as a fluorescence plate reader or a fluorescence microscope. A compound with the lowest fluorescence value before and after mixing can be determined as a novel therapeutic candidate compound that satisfies this concept.

  As another detection method, an anti-CS antibody (clone: CS56, manufactured by Seikagaku Corporation) that directly labels CS-GAG itself can be used. As with the WGA lectin, FITC-labeled anti-CS antibodies can be added to CS-coated wells, and large-scale screening can be performed in an extremely short time and simply by looking at changes in fluorescence values.

  As a more detailed detection method, use the sGAG Assay Kit (manufactured by WIESLAB), Sulphanated Glycosaminoglycans, ELISA Kit (manufactured by FUNAKOSHI), etc. by using the plate before and after mixing of the test compound as it is, and accurately determine the GAG content. There are methods to quantify and quantify.

  More specifically, by adding 2-AB (2-aminobenzamide) or 2-AP (2-aminopyridine; both from LUD, etc.) to the plate before and after mixing the test compound, the reducing end of the free GAG chain is More detailed analysis is possible by simply fluorescently labeling 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 characteristics of candidate compounds are examined in detail.

As another aspect of the screening method of the present invention, there can be mentioned a method comprising a step of selecting a substance having an inhibitory action on chondroitin sulfate proteoglycan synthesis. The above method of the present invention comprises, for example, the following steps.
(A) contacting a test compound with a substance group containing a cell expressing chondroitin sulfate proteoglycan or a part thereof, the cell extract, or an enzyme and a substrate constituting the synthesis process of chondroitin sulfate proteoglycan (b) A step of measuring a synthesis amount of chondroitin sulfate proteoglycan or an intermediate in a synthesis process thereof in a cell, a cell extract or a substance group (c) a compound which decreases the synthesis amount compared to a case where a test compound is not contacted Process to select

  In the above method, first, a test compound is brought into contact with a substance group containing a cell expressing chondroitin sulfate proteoglycan or a part thereof, the cell extract, or an enzyme and a substrate constituting a synthesis process of chondroitin sulfate proteoglycan.

  Next, the synthesis amount of chondroitin sulfate proteoglycan or an intermediate in the synthesis process thereof is measured. The measurement can be appropriately performed by those skilled in the art by a known method such as a method using a labeled antibody, mass spectrometry, chromatography, or the like.

  Further, a compound that reduces (suppresses) the amount of synthesis is selected as compared with the case where the test compound is not contacted (control). A compound that lowers (suppresses) becomes a drug for treating insulin resistance.

  A simple example of a method and a specific example capable of evaluating (measuring) whether or not the test compound has the activity of the above-mentioned (b) synthesis inhibitory action is shown below.

(B) Screening method for the chondroitin sulfate proteoglycan synthesis inhibitory action:
Cells and cell lines that synthesize chondroitin sulfate are known to the investigator. In humans, for example, chondroitin sulfate is produced in 16 hours of cell culture using the standard method of separating and culturing mononuclear grades 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, etc.), renal tubule-derived cancer cell line ACHN (Kawashima H et al., J. Biol. Chem. 277: 12921, 2002), distal renal tubular cell line MDCK (Borges FT et al., Kidney Int. 68: 1630, 2005., etc.), vascular endothelial cell line Many examples include HUVEC (Schick BP et al., Blood 97: 449, 2001, etc.). In the process of culturing such a cell line for a certain period of time, various test compounds are mixed, and the change in the CS-GAG amount before and after the culture can be easily quantified by the method (a). A compound that suppresses an increase in the amount of CS-GAG after cell culture (ie, reflects the amount of CS-GAG synthesis) can be easily determined as a therapeutic candidate compound that satisfies this concept.

  Furthermore, more selectively, for example, a cell line in which CS-GAG synthase genes such as GalNac4ST-1 and XylosylT are introduced into CHO cells and L cells by a well-known method and expressed constantly can be prepared. it can. By using such a cell line that constantly synthesizes CS-GAG, it is possible to more clearly determine a therapeutic candidate compound.

As another aspect of the screening method of the present invention, there may be mentioned a method comprising a step of selecting a substance having a desulfating action of chondroitin sulfate proteoglycan. The above method of the present invention comprises, for example, the following steps.
(A) a step of contacting a test compound with chondroitin sulfate proteoglycan or a part thereof (b) a step of measuring the amount of chondroitin sulfate proteoglycan or a part thereof subjected to sulfation (c) in the absence of the test compound The process of selecting substances that reduce the amount of sulfation compared to when measured in

  In the above method, first, a test compound is brought into contact with chondroitin sulfate proteoglycan or a part thereof.

  In this method, the amount of chondroitin sulfate proteoglycan or a part thereof that has undergone sulfation is then 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 label using a labeled compound or antibody that binds to the desulfated structure remaining in chondroitin sulfate proteoglycan or a part thereof. Moreover, it can also detect using a chromatography method, a mass spectrometry, etc.

  In this method, a compound that reduces the abundance of the chondroitin sulfate proteoglycan 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.

  A simple example of a method and a specific example that can be evaluated (measured) as to whether or not the test compound has the activity (c) desulfation activity is shown below.

(C) Screening method aspect regarding desulfation action of chondroitin sulfate proteoglycan:
Basically, human-derived proteoglycan (BGN, ISL, etc.) is prepared in the same manner as in the above method (a) and coated on a 96-well plate at a concentration of 10 μg / mL (Kawashima H et al .; J. Biol Chem. 277: 12921-12930, 2002. etc.)). Various test compounds are added to each well of the plate, and changes in CS-GAG are detected after 2 hours of reaction at 37 ° C.

  The detection method is to desulfate the disaccharide structure of the desulfated fragment remaining on the core protein side of the proteoglycan into the anti-proteoglycan Δdi4S antibody (clone; 2-B-6, the portion that had been sulfated at position 4 Or a part that has undergone desulfation by reacting with anti-proteoglycan Δdi6S (clone; 3-B-3, which recognizes the part that was sulphated at position 6. Both are manufactured by Seikagaku Corporation) Can be easily detected. Therefore, FITC-labeled 2-B-6 and 3-B-3 antibodies are reacted on the plate before and after mixed culture, and the change in the fluorescence value 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.

As another aspect of the screening method of the present invention, there may be mentioned a method comprising a step of selecting a substance having a sulfation inhibitory action of chondroitin sulfate proteoglycan. The above method of the present invention comprises, for example, the following steps.
(A) a step of contacting a test compound with a substance group containing a chondroitin sulfate proteoglycan or a cell or cell extract expressing the chondroitin sulfate proteoglycan or an enzyme, a substrate, etc. constituting the sulfation process of chondroitin sulfate proteoglycan (b) , A step of measuring the sulfation activity of chondroitin sulfate proteoglycan in a cell extract or substance group (c) a step of selecting a compound that decreases the activity compared to the case where no test compound is contacted

  In the above method, first, a test substance is brought into contact with chondroitin sulfate proteoglycan or a part thereof.

  In this method, the amount of chondroitin sulfate proteoglycan or a part thereof that has undergone sulfation is then 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 proteoglycan or a sulfated structure of a part thereof. Moreover, it can also detect using a chromatography method, a mass spectrometry, etc.

  In this method, a compound that reduces the abundance of the chondroitin sulfate proteoglycan 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.

  A simple example of a method and a specific example capable of evaluating (measuring) whether or not the test compound has the above-mentioned (d) activity of inhibiting sulfation is shown below.

(D) Screening method aspect regarding the sulfation inhibitory action of chondroitin sulfate proteoglycan:
The cells and cell lines that promote sulfation of chondroitin sulfate match the cells and cell lines described in (c) above. In the process of culturing such cell lines for a certain period of time, various test compounds are mixed, and the degree of sulfation before and after culturing is measured, for example, the antibody that detects 4-position sulfation (clone: LY111) or the 6-position sulfation The antibody to be detected (clone; MC21C, both manufactured by Seikagaku Corporation) can be easily confirmed. 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. Compounds that suppress the increase in sulfation after cell culture (LY111 and MC21C fluorescence values increase), or promote the progress of desulfation after cell culture (2-B-6 and 3-B-3 fluorescence values increase) Can be easily determined as a therapeutic candidate compound that satisfies this concept.

  Furthermore, more selectively, for example, a cell line in which a sulfated transferase gene such as C4ST-1 or C6ST-1 is introduced into CHO cells or L cells by a well-known method and expressed constantly is prepared. Can do. By using such a cell line to which a sulfate group is constantly added, a treatment candidate compound can be determined more clearly.

Another preferred embodiment of the present invention is a compound that decreases the expression level of the chondroitin sulfate proteoglycan core protein, synthase, desulfase inhibitor protein, or sulfotransferase gene of the present invention, or promotes the degradation of chondroitin sulfate proteoglycan A method for screening an insulin resistance inhibitor comprising the following steps (a) to (d), wherein a compound that increases the expression level of an enzyme or a desulfase gene is selected.
(A) contacting a test compound with a cell expressing a gene encoding a chondroitin sulfate proteoglycan core protein, a synthetic enzyme, a desulfase inhibitor protein, a sulfotransferase, a degradation accelerating enzyme, or a desulfase enzyme (b) ) A step of measuring the expression level of a gene in the cell (c) a step of comparing the expression level with a case where the expression level is measured in the absence of a test compound (control) (d) a core protein of chondroitin sulfate proteoglycan, In the case of a synthetic enzyme, a desulfurase inhibitor protein, or a sulfotransferase, a compound in which the expression level of the gene is reduced compared to the control, the gene is a chondroitin sulfate proteoglycan degradation-promoting enzyme, or desulfurization In the case of an oxidase, the expression level of the gene is increased compared to the control. Selecting a compound that are

  In the above method, first, a test compound is brought into contact with a cell expressing a gene encoding a chondroitin sulfate proteoglycan core protein, a synthetic enzyme, a desulfase inhibitor protein, a sulfotransferase, a degradation promoting enzyme, or a desulfase enzyme. .

  Next, in the present method, the expression level of a gene encoding chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfase inhibitor protein, sulfotransferase, degradation promoting enzyme, or desulfase is measured. Here, “gene expression” includes both transcription and translation. The gene expression level can be measured by methods known to those skilled in the art.

  For example, mRNA is extracted from cells expressing any of the above proteins according to a conventional method, and the transcription amount of the gene is determined by performing Northern hybridization, RT-PCR, DNA array, etc. using this mRNA as a template. Measurements can be made. In addition, by collecting a protein fraction from a cell expressing a gene encoding any of the above proteins and detecting the expression of any of the above proteins by electrophoresis such as SDS-PAGE, the amount of translation of the gene can be reduced. Measurements can also be made. Furthermore, the amount of translation of a gene can be measured by detecting the expression of the protein by performing Western blotting using an antibody against any of the above proteins. The antibody used for detection of the protein is not particularly limited as long as it is a detectable antibody. For example, both a monoclonal antibody and a polyclonal antibody can be used.

  In this method, the expression level of the gene is then compared with the case where the test compound is not contacted (control).

  In this method, when the gene is a chondroitin sulfate proteoglycan core protein, a synthase, a desulfase-inhibiting protein, or a sulfotransferase, the expression level of the gene is decreased compared to the control ( Select the compound being suppressed. The compound that decreases (suppresses) becomes a drug for suppressing insulin resistance or a candidate compound for insulin resistance treatment.

  In addition, when the gene is a chondroitin sulfate proteoglycan degradation promoting enzyme or a desulfating enzyme, a compound whose expression level of the gene is increased (enhanced) compared to the control is selected. The compound to be increased (enhanced) becomes a drug for suppressing insulin resistance or a candidate compound for insulin resistance treatment.

In addition, as one aspect of the screening method of the present invention, the chondroitin sulfate proteoglycan core protein, the synthetic enzyme, the desulfase inhibitor protein, or the compound that decreases the expression level of the sulfotransferase gene, or chondroitin sulfate In this method, a compound that increases the expression level of a proteoglycan degradation-promoting enzyme or desulfating enzyme gene is selected 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).
(A) Structure in which a transcriptional regulatory region of a gene encoding chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfase inhibitor protein, sulfate transferase, degradation promoting enzyme, or desulfase is functionally linked to a reporter gene (B) a step of contacting a test compound with a cell or cell extract containing DNA having DNA (b) a step of measuring the expression level of the reporter gene (c) a step of comparing with a case where the test compound is not contacted (control) d) When the reporter gene is functionally linked to a chondroitin sulfate proteoglycan core protein, synthase, desulfase inhibitor protein, or sulfotransferase, the expression level of the reporter gene is compared to the control. Compound, the reporter gene is chondroit When operatively linked to degradation-promoting enzyme or desulfating enzymes, sulfate proteoglycans step of selecting compounds that expression levels of the reporter gene is increased as compared with the control

  In this method, first, a chondroitin sulfate proteoglycan core protein, a synthetic enzyme, a desulfase inhibitor protein, a sulfotransferase, a degradation promoting enzyme, or a transcriptional regulatory region of a gene encoding a desulfase, and a reporter gene are functionally functional. A test compound is brought into contact with a cell or cell extract containing DNA having a bound structure.

  Here, “functionally linked” means transcription into the transcriptional regulatory region of a gene encoding chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfase inhibitor protein, sulfotransferase, degradation promoting enzyme, or desulfase. A gene that encodes chondroitin sulfate proteoglycan core protein, synthase, desulfase inhibitor protein, sulphate transferase, degradation promoting enzyme, or desulfatase so that expression of the reporter gene is induced by binding of the factor The transcriptional regulatory region and the reporter gene. Therefore, even when the reporter gene is linked to other genes and forms a fusion protein with other gene products, chondroitin sulfate proteoglycan core protein, synthase, desulfase inhibitor protein, sulfate transferase 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 degradation-promoting enzyme or desulfating enzyme, the above-mentioned “functionally bound” means include. Based on the cDNA base sequence of the gene encoding chondroitin sulfate proteoglycan core protein, synthase, desulfase inhibitor protein, sulfate transferase, degradation-promoting enzyme, or desulfase, it exists in the genome for those skilled in the art It is possible to obtain a transcriptional regulatory region of a gene encoding chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfase inhibitor protein, sulfate transferase, degradation promoting enzyme, or desulfase, by a well-known method.

  The reporter gene used in this method is not particularly limited as long as its expression can be detected, and examples thereof include CAT gene, lacZ gene, luciferase gene, and GFP gene. “It has a structure in which a transcriptional regulatory region of a gene encoding chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfase inhibitor protein, sulfate transferase, degradation promoting enzyme, or desulfase is functionally linked to a reporter gene. Examples of the “cell containing DNA” include a cell into which a vector having such a structure inserted is introduced. Such vectors can be prepared by methods well known to those skilled in the art. Introduction of the vector into the cells can be performed by a general method such as calcium phosphate precipitation, electric pulse perforation, lipofection, microinjection and the like. “It has a structure in which a transcriptional regulatory region of a gene encoding chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfase inhibitor protein, sulfate transferase, degradation promoting enzyme, or desulfase is functionally linked to a reporter gene. The “cell containing DNA” also 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 using homologous recombination.

  “The chondroitin sulfate proteoglycan core protein, synthase, desulfase inhibitor protein, sulfate transferase, degradation-promoting enzyme, or transcriptional regulatory region of the gene encoding desulfase is functionally linked to the reporter gene. “Cell extract containing DNA” refers to, for example, a cell extract contained in a commercially available in vitro transcription / translation kit, chondroitin sulfate proteoglycan core protein, synthase, desulfase inhibitor protein, sulfate transferase, degradation promoting enzyme Or a DNA added with a DNA having a structure in which a transcriptional regulatory region of a gene encoding a desulfating enzyme and a reporter gene are functionally linked.

  Here, “contact” means that “the transcriptional regulatory region of a gene encoding chondroitin sulfate proteoglycan core protein, synthetic enzyme, desulfase inhibitor protein, sulfotransferase, degradation promoting enzyme, or desulfase enzyme and the reporter gene function. The test compound can be added to the culture solution of “cells containing DNA having a structure that has been chemically bound” or the test compound is added to the above-described commercially available cell extract containing the DNA. When the test compound is a protein, for example, it can be performed by introducing a DNA vector expressing the protein into the cell.

  In this method, the expression level of the reporter gene is then measured. The expression level of the reporter gene can be measured by methods known to those skilled in the art depending on the type of the reporter gene. For example, when the reporter gene is a CAT gene, the expression level of the reporter gene can be measured by detecting acetylation of chloramphenicol by the gene product. When the reporter gene is a lacZ gene, by detecting the color development of the dye compound catalyzed by the gene expression product, and when the reporter gene is a luciferase gene, the fluorescent compound catalyzed by the gene expression product By detecting fluorescence, and in the case of a GFP gene, the expression level of the reporter gene can be measured by detecting fluorescence due to the GFP protein.

  In this method, the expression level of the measured reporter gene is then compared with that measured in the absence of the test compound (control).

  In this method, when the reporter gene is functionally linked to a gene encoding chondroitin sulfate proteoglycan core protein, synthase, desulfase inhibitor protein, or sulfotransferase, then the reporter gene is used. A compound whose gene expression level is reduced (suppressed) compared to the control is selected. The compound that decreases (suppresses) becomes a drug for suppressing insulin resistance or a candidate compound for insulin resistance treatment.

  In addition, when the reporter gene is functionally linked to the chondroitin sulfate proteoglycan degradation-promoting enzyme or desulfating enzyme, the expression level of the reporter gene is increased (enhanced) compared to the control. Select. The compound to be increased (enhanced) becomes a drug for suppressing insulin resistance or a candidate compound for insulin resistance treatment.

  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. For example, the kit of the present invention can contain the chondroitin sulfate proteoglycan of the present invention as a constituent element. The kit of the present invention can further contain various reagents, containers and the like used in the method of the present invention. For example, an anti-chondroitin sulfate proteoglycan 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.

  In a preferred embodiment of the present invention, there is a method for screening an insulin resistance inhibitor, which comprises the step of detecting whether the production or accumulation of chondroitin sulfate proteoglycan is inhibited. Accordingly, in the screening method, for example, an oligonucleotide such as a probe for a gene encoding the core protein of chondroitin sulfate proteoglycan or a primer for amplifying an arbitrary region of the gene, which can be used when detecting chondroitin sulfate proteoglycan, Alternatively, an antibody that recognizes chondroitin sulfate proteoglycan (an anti-chondroitin sulfate proteoglycan antibody) can also be included in the component of the insulin resistance inhibitor screening kit of the present invention.

  For example, the oligonucleotide specifically hybridizes to the DNA of the Versican core protein gene of the present invention. Here, “specifically hybridize” means normal hybridization conditions, preferably stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, In the condition described in the second edition 1989), it means that cross-hybridization does not occur significantly with DNA encoding other proteins. If specific hybridization is possible, the oligonucleotide does not need to be completely complementary to the base sequence of the Versican core protein gene of the present invention.

  As hybridization conditions in the present invention, for example, “2 × SSC, 0.1% SDS, 50 ° C.”, “2 × SSC, 0.1% SDS, 42 ° C.”, “1 × SSC, 0.1% SDS, 37 ° C.” More stringent conditions include “2 × SSC, 0.1% SDS, 65 ° C.”, “0.5 × SSC, 0.1% SDS, 42 ° C.” and “0.2 × SSC, 0.1% SDS, 65 ° C.” Can do. More specifically, as a method using Rapid-hyb buffer (Amersham Life Science), prehybridization is performed at 68 ° C for 30 minutes or more, and then a probe is added and kept at 68 ° C for 1 hour or more for hybridization. Then wash 3 times for 20 minutes at room temperature in 2 x SSC, 0.1% SDS, then 3 times for 20 minutes at 37 ° C in 1 x SSC, 0.1% SDS, and finally 1 x SSC Washing can be performed twice in 0.1% SDS at 50 ° C for 20 minutes. In addition, for example, prehybridization in Expresshyb Hybridization Solution (CLONTECH) for 30 minutes or more at 55 ° C, add labeled probe, and incubate at 37-55 ° C for 1 hour or more, in 2 × SSC, 0.1% SDS In addition, washing for 20 minutes at room temperature can be performed three times, and washing for 20 minutes at 37 ° C. in 1 × SSC and 0.1% SDS can be performed once. Here, for example, by setting the temperature at the time of pre-hybridization, hybridization, or the second washing higher, the conditions can be made more stringent. For example, the prehybridization and hybridization temperatures can be 60 ° C., and the stringent conditions can be 68 ° C. Those skilled in the art can set conditions by considering other conditions such as probe concentration, probe length, probe base sequence configuration, reaction time, etc. in addition to such conditions as buffer salt concentration and temperature. can do.

  The oligonucleotide can be used as a probe or primer in the screening kit of the present invention. When the oligonucleotide is used as a primer, the length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp. The primer is, for example, the one described in SEQ ID NO: 69 or 70, but is not particularly limited as long as it can amplify at least a part of the DNA of the gene in the present invention.

  The present invention also provides a method for treating or preventing an insulin resistant disease, comprising the step of administering the agent of the present invention to an individual (for example, a patient).

  An individual subject to the prevention or treatment method of the present invention is not particularly limited as long as it is an organism capable of developing 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 dosage varies depending on the weight and age of the patient, the administration method, etc., but a person skilled in the art (such as a doctor, veterinarian, pharmacist, etc.) can appropriately select an appropriate dosage.

  Furthermore, the present invention relates to the use of the agent of the present invention in the manufacture of an insulin resistance inhibitor.

It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.

  There is of course no actual clinical treatment report that controls the accumulation of these proteoglycans and observed pathological changes, but this time, as an example, Versican containing the core protein, which is one of the chondroitin sulfate proteoglycans, and the helical side when elongated from the core protein. Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1, Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2, Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 3 (C4ST-1, Examples will be shown focusing on C4ST-2 and C4ST-3). Although various studies have been conducted so far, there is no example regarding the application of Versican and the like to the type 2 diabetes model (NIDDM), and it is expected as a therapeutic / preventive drug by a new mechanism.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

Experiment 1: Streptozotocin-induced C57BL6J / JcL Versican (siRNA) treatment in mice with type 2 diabetes (NIDDM) model mice, changes in body weight, blood glucose level, Versican gene expression, tissue level, examination In this example, C57BL6J on the second day after birth A type 2 diabetes model was prepared by administering Streptozotocin to / JcL mice (female, manufactured by CLEA Japan, Inc.), and body weight fluctuation, blood sugar level fluctuation by siRNA treatment, and gene expression by siRNA administration were examined.
First, the sample preparation was performed as follows.

Example 1 Streptozotocin-induced C57BL6J / JcL Type 2 diabetes mellitus (NIDDM) model mice treated with Versican (siRNA) in weight change 14th day of pregnancy C57BL6J / JcL mice (manufactured by CLEA Japan, Inc.) were raised and born, and 2 days after birth C57BL6J / JcL mice female (manufactured by Claire Japan), each injected subcutaneously into Streptozocin 10 mg / mL (manufactured by SIGMA) 20 μL / head, and CE-2 (manufactured by Claire Japan) feed and sterilized until 4 weeks of age The animals were fed with water, raised from 4 weeks old, fed with High Fat Diet (manufactured by CLEA Japan) and sterilized water, and raised for 2 weeks. In 2 weeks, siRNA Versican 1μg (GeneWorld) mixed with 1% Atelocollagen (Koken), siRNA medium, once / week 200μL intraperitoneally (1shot / week) Treatment was performed (2 weeks). On the 14th day of the experiment, BrdU 5 mg / mL (ZyMED Laboratory. Inc.) 100 μL was administered into the tail vein, and after 1 hour, dissection was performed, and the liver, pancreas, kidney, testis, ovary, and muscle were removed and immunized. Samples for staining and gene expression analysis were obtained. The mouse body weight was measured over time until the day of dissection in the Control group and the Versican siRNA treatment group, and is shown in FIG. The vertical axis represents body weight (g), the horizontal axis represents the experimental period (days), and comparison was made between the Control group (n = 6) and the Versican siRNA-treated group (n = 6). As shown in FIG. 1, a tendency to suppress weight gain was observed in the Versican siRNA-treated group as compared to the Control group.

Example 2 Change of blood glucose level in Versican (siRNA) treatment with Streptozotocin-induced C57BL6J / JcL type 2 diabetes (NIDDM) model mice Treatment with human crystalline insulin (0.75 U / kg) as an insulin-tolerance test on the day before dissection Then, after 0 minutes, 15 minutes, and 60 minutes, the blood glucose level was measured using a Glutest Ace blood glucose level measuring device (manufactured by Bonbix Pharmaceutical Co., Ltd.) and evaluated. FIG. 2 shows changes in blood glucose level after intraperitoneal administration of human crystalline insulin (0.75 U / kg) in the Control group and Versican siRNA treatment group after treatment with Versican siRNA, 0 minutes, 15 minutes, and 60 minutes. The vertical axis represents the blood glucose level (mg / dL), and the horizontal axis represents 0 minutes, 15 minutes, and 60 minutes after the insulin-tolerance test. From Fig. 2, there was no significant change between the Control group and the Versican siRNA treatment group at 0 minutes and 15 minutes, but a significant change between the Control group and the Versican siRNA treatment group after 60 minutes. Was recognized.

Example 3 Evaluation and Examination of Versican Gene Expression Level in Streptozotocin-Induced C57BL6J / JcL Type 2 Diabetes Mellitus (NIDDM) Model Mouse in Versican (siRNA) Treatment
Streptozocin-induced C57BL6J / JcL mice, 50 mg of organ (pancreas) extracted from females, 1 mL of RNA-Bee (TEL-TEST) is added and pulverized with an electric homogenizer (DIGITAL HOMOGENIZER, ASONE) After mixing, add chloroform 200μL (Sigma Aldrich Japan) and mix gently, then ice-cool for about 5 minutes and centrifuge using a centrifuge (Centrifuge 5417R, Eppendorf) for 15 minutes at 12,000 rpm and 4 ° C. Went. Transfer 500 μL of the supernatant after centrifugation to another Eppendorf tube, add 500 μL of isopropannol (Sigma Aldrich Japan) equivalent to the supernatant, mix, add 1 μL glycogen (Invitrogen), and cool on ice for 15 minutes. did. Centrifuge for 15 minutes at 12,000 rpm and 4 ° C after 15 minutes of ice cooling, and then wash the RNA precipitate with 75% Ethanol 1000 μL (Sigma Aldrich Japan) three times for 30 minutes to 1 hour. After drying, dissolve in 50 μL of Otsuka distilled water (Otsuka Pharmaceutical Co., Ltd.), further dilute 100 times with Otsuka distilled water (Otsuka Pharmaceutical Co., Ltd.), and plate reader (POWER Wave on UV plate (Corning Coaster)). The RNA concentration in the sample extracted by XS, manufactured by BIO-TEK was calculated.

Next, the following procedure was performed to perform RT reaction (cDNA synthesis).
The RNA sample obtained by the calculation was adjusted to a concentration of 500 ng / 20 μL, heated at 68 ° C. for 3 minutes with BLOCK INCUBATOR (manufactured by ASTEC), and ice-cooled for 10 minutes. After cooling with ice, RT Pre Mix solution (composition: 25 mM MgCl ₂ 18.64 μL (Invitrogen)), 5 × Buffer 20 μL (Invitrogen), 0.1 M DTT 6.6 μL (Invitrogen), 10 mM dNTP mix 10 μL (Invitrogen), RNase Inhibitor 2 μL (Invitrogen), MMLV Reverse transcriptase 1.2 μL (Invitrogen), Random primer 2 μL (Invitrogen), sterile distilled water 19.56 μL (Otsuka distilled water: Otsuka (Manufactured by Pharmaceutical Co., Ltd.) was added 80 μL and reacted at 42 ° C. for 1 hour with BLOCK INCUBATOR (manufactured by ASTEC), and after 1 hour, heated at 99 ° C. for 5 minutes with BLOCK INCUBATOR (manufactured by ASTEC), 100 μL of cDNA obtained by cooling with ice was prepared, and a PCR reaction was performed with the following composition using the synthesized cDNA.
PCR Buffer 2 μL [Composition: 166 mM (NH ₄ ) ₂ SO ₄ (Sigma Aldrich Japan),
670 mM Tris pH 8.8 (manufactured by Invitrogen), 67 mM MgCl ₂ · 6H ₂ O (manufactured by Sigma Aldrich Japan), 100 mM 2-mercaptoethanol (manufactured by WAKO)], 25 mM dNTP mix 0.8 μL (Invitrogen) DMSO 0.6 μL (Sigma Aldrich Japan), Primer Forward 0.2 μL (GeneWorld), Primer Reverse 0.2 μL (GeneWorld), Otsuka distilled water 15.7 μL (Otsuka Pharmaceutical), Taq polymerase 0.1 μL (Perkin Elmer) and 1 μL of the cDNA obtained from the above were mixed and reacted at 94 ° C. 45 second, 56 ° C. 45 second, 72 ° C. 60 second 35 cycles using Authorized Themal Cycler (eppendorf). After completion of the reaction, 2 μL Loading Dye (Invitrogen) is added to the obtained PCR product to prepare 1.5% UltraPure Agarose (Invitrogen) gel, and 100 V for 20 minutes using Mupid-2 plus (ADVANCE). Electrophoresis was performed. After electrophoresis, diluted 1: 10,000 with 1 × LoTE [Composition: 3 mM Tris-HCl (pH 7.5) (Invitrogen), 0.2 mM EDTA (pH 7.5) (Sigma Aldrich Japan)]] Ethydium Bromide (Invitrogen After shaking in staining solution for 20-30 minutes, gel expression was confirmed with EXILIM (CASIO) installed in I-Scope WD (ADVANCE) to confirm gene expression.

  FIG. 3 shows the gene expression results of GAPDH and Versican in the Control group and Versican siRNA treatment group by RT-PCR.

The Versican siRNA and Primer (Forward, Reverse) (GeneWorld) used here are shown below.
[Primer array]
* GAPDH (GeneWorld)
Forward: 5'-CTGCCAAGTATGACATCA -3 '(SEQ ID NO: 67)
Reverse: 5'-TACTCCTTGGAGGCCATGTAG -3 '(SEQ ID NO: 68)
* Versican (GeneWorld)
Forward: 5'-GACGACTGTCTTGGTGG-3 '(SEQ ID NO: 69)
Reverse: 5'-ATATCCAAACAAGCCTG-3 '(SEQ ID NO: 70)
[Versican siRNA cocktail sequence] (GenBank accession number BC096495)
(GeneWorld)
5'-ATGAAAGGCATCTTATGGATGTGCTCA-3 '(SEQ ID NO: 71)
5'-GGCAGCCACCAGCAGGTACACTCT-3 '(SEQ ID NO: 72)
5'-CTGCTCAACAGGCTTGTTTGGATAT-3 '(SEQ ID NO: 73)

  From FIG. 3, expression of GAPDH, which is a positive control, was observed by RT-PCR in both the Control group and the Versican (siRNA) -treated group. Furthermore, in Versican, expression was decreased in the Versican (siRNA) -treated group compared with the Control group, and knockdown of the Versican gene was confirmed by administration of Atellocollagen vehicle Versican siRNA.

Example 4 Streptozotocin-induced C57BL6J / JcL Type 2 diabetes mellitus (NIDDM) model mice were evaluated for pancreatic tissue level by immunostaining (APP, Insulin) in Versican (siRNA) treatment, and the obtained tissue sample sections were treated with anti-Amyloid precursor protein Staining was performed using antibodies and anti-insulin antibodies, and the expression at the tissue level was evaluated and examined. FIG. 4 shows tissue images of the Control group and the Versican siRNA treatment group.

  From FIG. 4, it was confirmed by immunohistochemical staining that the deposition of Amyloid precursor protein was suppressed in the Versican siRNA-treated group compared to the Control group. As a result of staining with anti-insulin antibody, it was observed that the expression of insulin-positive β cells in the pancreas was increased in the Versican siRNA-treated group as compared to the Control group.

Example 5 Streptozotocin-induced C57BL6J / JcL Type 2 diabetes mellitus (NIDDM) model mice were evaluated for pancreatic tissue level by immunostaining (CS56) in Versican (siRNA) treatment, and the obtained tissue sample sections were analyzed with CS56 (chondroitin sulfate proteoglycan itself And the expression at the tissue level was evaluated and examined. FIG. 5 shows tissue images of the Control group and the Versican siRNA treatment group.

  From FIG. 5, as a result of staining with CS56 (an antibody that stains chondroitin sulfate proteoglycan itself), deposition of chondroitin sulfate proteoglycan (CS56) was strongly observed in the Control group, whereas chondroitin sulfate proteoglycan (CS56) was observed in the Versican siRNA treatment group. It was observed that the deposition of CS56) was suppressed.

Experiment 2: Streptozocin-induced C57BL6J / JcL NIDDM model mouse
C4ST-1 (Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 1) siRNA,
C4ST-2 (Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 2) siRNA,
C4ST-3 (Chondroitin D-N-acetylgalactosamine-4-O-sulfotransferase 3) Body weight, blood glucose level change, gene expression, tissue level evaluation and examination in siRNA treatment

In this example, a type 2 diabetes model was prepared by Streptozotocin administration of C57BL6J / JcL mice (female, manufactured by Claire Japan) on the second day after birth,
C4ST-1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1) siRNA,
C4ST-2 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2) siRNA,
C4ST-3 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 3) The body weight fluctuation by the siRNA treatment, the blood sugar level fluctuation, and the gene expression by the siRNA administration were examined.
First, the sample preparation was performed as follows.

Example 6 Streptozocin-induced C57BL6J / JcL NIDDM model mouse
C4ST-1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1) siRNA,
C4ST-2 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2) siRNA,
C4ST-3 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 3) Body weight fluctuation in siRNA treatment 14th day of pregnancy C57BL6J / JcL mice (manufactured by CLEA Japan, Inc.) are bred and given birth, 2 days after birth C57BL6J / JcL mice Females are each injected subcutaneously into Streptozocin 10mg / mL (SIGMA) 20μL / head and fed with CE-2 (CLEA Japan) feed and sterilized water until 4 weeks of age. Diet diet (manufactured by CLEA Japan, Inc.) and sterilized water were given and reared for 2 weeks. In the second week, siRNA C4ST-1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1),
C4ST-2 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2),
C4ST-3 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 3)

  1μg (GeneWorld) mixed with 1% Atelocollagen (Koken) siRNA medium once / week 200μL intraperitoneal administration (once / week) twice (2 weeks) Went. On the 14th day of the experiment, BrdU 5 mg / mL (ZyMED Laboratory. Inc.) 100 μL was administered into the tail vein, and after 1 hour, dissection was performed, and the liver, pancreas, kidney, testis, ovary, and muscle were removed and immunized. Samples for staining and gene expression analysis were obtained. Regarding body weight fluctuation, measurement was performed over time for 14 days during the period of the examples. The results are shown in FIG. The vertical axis represents body weight (g), and the horizontal axis represents measurement days (days). As shown in FIG. 6, the C4ST-1 siRNA-treated group, C4ST-2 siRNA-treated group, and C4ST-3 siRNA-treated group showed a tendency to suppress weight gain after the 12th day of treatment.

Example 7 Streptozocin-induced C57BL6J / JcL NIDDM model mouse
C4ST-1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1) siRNA,
C4ST-2 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2) siRNA,
C4ST-3 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 3) Blood glucose level fluctuation in siRNA treatment Human crystalline insulin (0.75 U / kg) was administered intraperitoneally as an insulin-tolerance test the day before dissection, 0 minutes later, After 15 minutes, 60 minutes later, the blood glucose level was measured using a Glutest Ace blood glucose level measuring instrument (manufactured by Bonbics Pharmaceutical Co., Ltd.) and evaluated. FIG. 7 shows changes in blood glucose levels after 0 minutes, 15 minutes, and 60 minutes after siRNA treatment. The vertical axis represents the blood glucose level (mg / dL), and the horizontal axis represents 0 minutes, 15 minutes, and 60 minutes after the insulin-tolerance test. From FIG. 7, compared with the Control group, a significant decrease in blood glucose level was observed at 0 minutes and 15 minutes after treatment in the C4ST-1 siRNA treatment group, C4ST-2 siRNA treatment group, and C4ST-3 siRNA treatment group. However, after 60 minutes, a significant decrease in blood glucose level was observed in each of the C4ST-1 siRNA treatment group, the C4ST-2 siRNA treatment group and the C4ST-3 siRNA treatment group.

Example 8 Streptozocin-induced C57BL6J / JcL NIDDM model mouse
C4ST-1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1) siRNA,
C4ST-2 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2) siRNA,
C4ST-3 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 3) Evaluation and examination of gene expression level in siRNA treatment
Streptozocin-induced C57BL6J / JcL mice, 50 mg of organ (pancreas) extracted from females, 1 mL of RNA-Bee (TEL-TEST) is added and pulverized with an electric homogenizer (DIGITAL HOMOGENIZER, ASONE) After mixing, add chloroform 200μL (Sigma Aldrich Japan), mix gently, cool on ice for about 5 minutes, and centrifuge using a centrifuge (Centrifuge 5417R, Eppendorf) for 15 minutes at 12,000 rpm and 4 ° C. Went. Transfer 500 μL of the supernatant after centrifugation to another Eppendorf tube, add 500 μL of isopropannol (Sigma Aldrich Japan) equivalent to the supernatant, mix, add 1 μL glycogen (Invitrogen), and cool on ice for 15 minutes. did. Centrifuge for 15 minutes at 12,000 rpm and 4 ° C after 15 minutes of ice cooling, and then wash the RNA precipitate with 75% Ethanol 1000 μL (Sigma Aldrich Japan) three times for 30 minutes to 1 hour. After drying, dissolve in 50 μL of Otsuka distilled water (Otsuka Pharmaceutical Co., Ltd.), further dilute 100 times with Otsuka distilled water (Otsuka Pharmaceutical Co., Ltd.), and plate reader (POWER Wave on UV plate (Corning Coaster)). The RNA concentration in the sample extracted by XS, manufactured by BIO-TEK was calculated.

For RT reaction (cDNA synthesis), the calculated RNA sample was adjusted to a concentration of 500 ng / 20 μL, heated at 68 ° C for 3 minutes with BLOCK INCUBATOR (ASTEC), 10 minutes, Ice-cooled. After cooling with ice, RT Pre Mix solution (composition: 25 mM MgCl ₂ 18.64 μL (Invitrogen)), 5 × Buffer 20 μL (Invitrogen), 0.1 M DTT 6.6 μL (Invitrogen), 10 mM dNTP mix 10 μL (Invitrogen), RNase Inhibitor 2 μL (Invitrogen), MMLV Reverse transcriptase 1.2 μL (Invitrogen), Random primer 2 μL (Invitrogen), sterile distilled water 19.56 μL (Otsuka distilled water: Otsuka (Manufactured by Pharmaceutical Co., Ltd.) was added 80 μL and reacted at 42 ° C. for 1 hour with BLOCK INCUBATOR (manufactured by ASTEC), and after 1 hour, heated at 99 ° C. for 5 minutes with BLOCK INCUBATOR (manufactured by ASTEC), A PCR reaction was performed with the following composition using cDNA obtained by preparing 100 μL of cDNA to be cooled on ice.

PCR Buffer 2 μL [Composition: 166 mM (NH ₄ ) ₂ SO ₄ (Sigma Aldrich Japan), 670 mM Tris pH8.8 (Invitrogen), 67 mM MgCl ₂ · 6H ₂ O (Sigma Aldrich Japan)
, 100 mM 2-mercaptoethanol) (WAKO)], 25 mM dNTP mix 0.8 μL (Invitrogen), DMSO 0.6 μL (Sigma Aldrich Japan), Primer Forward 0.2 μL (GeneWorld), Primer Reverse 0.2 94 μL (manufactured by GeneWorld), 15.7 μL of Otsuka distilled water (manufactured by Otsuka Pharmaceutical), 0.1 μL of Taq polymerase (manufactured by Perkin Elmer), and 1 μL of cDNA obtained from the above were mixed by an Authorized Themal Cycler (manufactured by eppendorf). C. 45 second, 56.degree. C. 45 second, 72.degree. C. 60 second 35 cycles. After completion of the reaction, 2 μL Loading Dye (Invitrogen) is added to the obtained PCR product to prepare a 1.5% UltraPure Agarose (Invitrogen) gel, and 100 V for 20 minutes with Mupid-2 plus (ADVANCE). Electrophoresis was performed. After electrophoresis, diluted 1: 10,000 with 1 × LoTE [Composition: 3 mM Tris-HCl (pH 7.5) (Invitrogen), 0.2 mM EDTA (pH 7.5) (Sigma Aldrich Japan)]] Ethydium Bromide (Invitrogen After shaking for 20 to 30 minutes in a staining solution, gel photography was performed with EXILIM (manufactured by CASIO) installed in I-Scope WD (manufactured by ADVANCE) to confirm gene expression.
This time, Primer (Forward, Reverse) used (GeneWorld)
[Primer array]
* GAPDH
Forward: 5'-CTGCCAAGTATGACATCA -3 '(SEQ ID NO: 67)
Reverse: 5'-TACTCCTTGGAGGCCATGTAG -3 '(SEQ ID NO: 68)
* C4ST1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1)
Forward: 5'-gtggatgaggaccacgaact-3 '(SEQ ID NO: 74)
Reverse: 5'-cttttcaagcggtggttgat-3 '(SEQ ID NO: 75)
* C4ST2 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2)
Forward: 5'-acctcctagacccacacacg-3 '(SEQ ID NO: 76)
Reverse: 5'-ggatgttggcaaaccagtct-3 '(SEQ ID NO: 77)
* C4ST3 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 3)
Forward: 5'-atgagcccttcaacgaacac-3 '(SEQ ID NO: 78)
Reverse: 5'-tggtagaaggggctgatgtc-3 '(SEQ ID NO: 79)
* [C4ST-1 siRNA cocktail sequence]
C4ST1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1)
(GenBank accession number NM_021439)
5'-ACAAAGCCATGAAGCCGGCGCTGCTGGAAGTGATGAGGATGAACAGAATT-3 '(SEQ ID NO: 80)
5'-CAACCTGAAGACCCTTAACCAGTACA-3 '(SEQ ID NO: 81)
5'-GCATCCCAGAGATCAACCACCGCTTG-3 '(SEQ ID NO: 82)
* [C4ST-2 siRNA cocktail sequence]
C4ST2 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2)
(GenBank accession number NM_021528)
5'-GCCAGGAGTGGGCCCAGCCCAGGGC -3 '(SEQ ID NO: 83)
5'-ATGACCAAGCCGCGGCTCTTCCGGCTG-3 '(SEQ ID NO: 84)
5'-AGAGCCTGCTGGACCGGGGCAGCCCCTA -3 '(SEQ ID NO: 85)
5'-GAGACCCCCTGGACATCCCCCGGGAACA-3 '(SEQ ID NO: 86)
* [C4ST-3 siRNA cocktail sequence]
C4ST3 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 3)
(GenBank accession number XM_355798)
5'-ATGACTGTCGCCTGCCACGCGTGCCA -3 '(SEQ ID NO: 87)
5'-CAGCATGGGAAGACGCTCCTGTTGCA -3 '(SEQ ID NO: 88)
5'-TCCAAGCGCAATCCCTGCGCACGAGGCG -3 '(SEQ ID NO: 89)
5'-GCCTGGCCTGCTGCCCTCGCTGGCC -3 '(SEQ ID NO: 90)

The results are shown in FIG. Expression of GAPDH which is a positive control by RT-PCR is Control group,
C4ST1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1),
C4ST2 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2),
C4ST3 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 3)
C4ST1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1) siRNA,
C4ST2 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2) siRNA,
C4ST3 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 3) siRNA-treated group showed decreased expression, Atellocollagen medium
C4ST1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1) siRNA,
C4ST2 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2) siRNA,
C4ST3 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 3)
C4ST1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1),
C4ST2 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2),
Knockdown of the C4ST3 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 3) gene was confirmed.

Example 9 Streptozocin-induced C57BL6J / JcL NIDDM model mouse
C4ST-1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1) siRNA,
C4ST-2 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2) siRNA,
C4ST-3 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 3) Evaluation of tissue level (APP, Insulin) using immunostaining in siRNA treatment, and obtained tissue sample sections were treated with anti-Amyloid precursor protein antibody, anti-insulin Staining was performed using antibodies, and the expression at the tissue level was evaluated and examined. 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.

  From FIG. 9, it was confirmed that the deposition of Amyloid precursor protein was suppressed in the C4ST-1 siRNA-treated group, the C4ST-2 siRNA-treated group, and the C4ST-3 siRNA-treated group compared with the Control group in immunohistochemical staining. . As a result of staining with anti-insulin antibody, the expression of insulin-positive β cells in the pancreas is increased in the C4ST-1 siRNA-treated group, C4ST-2 siRNA-treated group, and C4ST-3 siRNA-treated group as compared with the Control group. It was observed.

Example 10 Streptozotocin-induced C57BL6J / JcL type 2 diabetes (NIDDM) model mouse
C4ST-1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1) siRNA,
C4ST-2 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2) siRNA,
C4ST-3 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 3) Evaluation of pancreatic tissue level by immunostaining (CS56) in siRNA treatment, the obtained tissue sample section CS56 (An antibody that stains chondroitin sulfate proteoglycan itself) Was used to evaluate and examine the expression at the tissue level. 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.

From FIG. 10, as a result of staining with CS56 (an antibody that stains chondroitin sulfate proteoglycan itself), deposition of chondroitin sulfate proteoglycan (CS56) was strongly observed in the Control group, whereas C4ST-1 siRNA treatment group, C4ST- 2 It was observed that deposition of chondroitin sulfate proteoglycan (CS56) was suppressed in the siRNA-treated group and the C4ST-3 siRNA-treated group.

Industrial applicability

As an example of examining the effect of accumulation of chondroitin sulfate proteoglycan (CSPG) according to the present invention, Versican siRNA containing the core site sequence of Versican which is one of chondroitin sulfate proteoglycans, acetylgalactosamine which is a side chain of chondroitin sulfate proteoglycan An enzyme that transfers sulfate groups
C4ST1 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 1),
C4ST2 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 2),
C4ST3 (Chondroitin DN-acetylgalactosamine-4-O-sulfotransferase 3) siRNA suppresses the accumulation of chondroitin sulfate proteoglycans in pancreatic Langerhans islet β cells and suppresses insulin resistance, thereby treating or preventing type 2 diabetes (NIDDM) Has an effect. Insulin resistance inhibitor according to the present invention suppresses the accumulation of chondroitin sulfate proteoglycan around the pancreatic Langerhans islet β cells by suppressing Versican expression by administration of Versican siRNA, C4ST-1 siRNA, C4ST-2 siRNA, C4ST-3 siRNA Since it has an effect, it is very useful in suppressing insulin resistance of pancreatic islets of Langerhans β cells. The method of treating or preventing type 2 diabetes mellitus (NIDDM) disease by administering an insulin resistance inhibitor having an inhibitory effect on chondroitin sulfate proteoglycan accumulation according to the present invention is effective in the pathology by an unprecedented mechanism of action and drug therapy. Can improve the patient's quality of life, and can be an excellent therapy useful for medical care.

All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

The fluctuation | variation of the body weight of 14 days in a Control (control) group in a type 2 diabetes (NIDDM) model mouse (female) induced by Streptozotocin and a Versican (Versican) siRNA treatment group is shown. The vertical axis represents body weight (g), and the horizontal axis represents the number of days. Control group after 0, 15, and 60 minutes after insulin tolerance test in a model after treatment with Versican siRNA in Streptozotocin-induced type 2 diabetes (NIDDM) model mice (female), (Versican) The blood glucose level change in the Versican siRNA-treated group is shown. The vertical axis represents blood glucose level (mg / dL), and the horizontal axis represents blood glucose measurement time (min) after the insulin load test. * p <0.05, ** P <0.01 (t-test) Examination of GAPDH and Versican expression by RT-PCR on the 14th day (final day) in the Control group and Versican siRNA treatment group in type 2 diabetes (NIDDM) model mice induced by Streptozotocin It shows that the expression of Versican (Versican) is suppressed in the group treated with Versican (Versican) siRNA. Detection results of decreased deposition of Amyloid precursor protein (APP) and elevated insulin-positive pancreatic β cells (lower) in Versican siRNA-treated groups in type 2 diabetes (NIDDM) model mice induced by Streptozotocin (lower) It is a photograph showing a reddish brown signal and a blue signal. Magnification 200 times (upper), 500 times (lower). Chondroitin sulfate in the Versican siRNA-treated group in the detection results of chondroitin sulfate proteglycan (CSPG) in the Control group and Versican siRNA-treated group in type 2 diabetes (NIDDM) model mice induced by Streptozotocin It is a photograph which shows the reduction effect of protein glycan (CSPG) (purple signal). 400x magnification. The name of the antibody clone used for detection is shown below the photo. The fluctuation | variation of the body weight of 14 days in a Control (control) group in a type 2 diabetes (NIDDM) model mouse induced | guided | derived with Streptozotocin, a C4ST-1 siRNA, a C4ST-2 siRNA, and a C4ST-3 siRNA treatment group is shown. The vertical axis represents body weight (g), and the horizontal axis represents the number of days. Streptozotocin-induced type 2 diabetes mellitus (NIDDM) model mice treated with C4ST-1 siRNA, C4ST-2 siRNA, C4ST-3 siRNA after 0, 15, and 60 minutes after insulin tolerance test The fluctuation | variation of the blood glucose level in a Control (control) group, a C4ST-1 siRNA, a C4ST-2 siRNA, and a C4ST-3 siRNA treatment group is shown. The vertical axis represents blood glucose level (mg / dL), and the horizontal axis represents blood glucose measurement time (min) after the insulin load test. * p <0.05, ** P <0.01 (t-test) GAPDH by RT-PCR on the 14th day (final 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 , Versican expression examined, showing that C4ST-1, C4ST-2, C4ST-3 expression was suppressed in the C4ST-1 siRNA, C4ST-2 siRNA, C4ST-3 siRNA treatment groups . Detection results of decreased deposition of Amyloid precursor protein (APP) in C4ST-1 siRNA treated group (upper) and elevated insulin-positive pancreatic β-cells (lower) in type 2 diabetes (NIDDM) model mice induced by Streptozotocin (red brown) (Signal, blue signal). Magnification 200 times (upper), 500 times (lower). Chondroitin sulfate proteinglycan in the C4ST-1 siRNA treatment group in the detection results of chondroitin sulfate proteinglycan (CSPG) in the control group and C4ST-1 siRNA treatment group in type 2 diabetes mellitus (NIDDM) model mice induced by Streptozotocin It is a photograph which shows the reduction effect of (CSPG) (purple signal). 400x magnification. The name of the antibody clone used for detection is shown below the photograph.

Claims (12)

  An insulin resistance inhibitor comprising as an active ingredient a substance that inhibits the production or accumulation of chondroitin sulfate proteoglycans.   The drug according to claim 1, wherein the substance has a chondroitin sulfate proteoglycan degradation promoting action.   The drug according to claim 1, wherein the substance has a chondroitin sulfate proteoglycan synthesis inhibitory action.   The drug according to claim 1, wherein the substance has a chondroitin sulfate proteoglycan desulfation effect.   The drug according to claim 1, wherein the substance is a substance having a chondroitin sulfate proteoglycan sulfation inhibitory action.   The agent according to any one of claims 1 to 5, wherein the production or accumulation of chondroitin sulfate proteoglycan is inhibited in pancreatic β cells.   The drug according to any one of claims 1 to 6, which is used for treatment or prevention of an insulin resistant disease.   The drug according to claim 7, wherein the insulin resistant disease is an insulin resistant disease associated with metabolic syndrome.   The drug according to claim 7, wherein the insulin resistant disease is non-insulin dependent (type 2) diabetes.   A method for screening an insulin resistance inhibitor, which comprises selecting a substance having an action of inhibiting the production or accumulation of chondroitin sulfate proteoglycan from a test sample. The screening method of Claim 10 including the process of selecting the substance which has the effect | action in any of the following (a)-(d).
(A) Degradation promoting action of chondroitin sulfate proteoglycan (b) Synthesis inhibition action of chondroitin sulfate proteoglycan (c) Desulfation action of chondroitin sulfate proteoglycan (d) Sulfation inhibition action of chondroitin sulfate proteoglycan   The screening method according to claim 10 or 11, wherein the insulin resistance inhibitor is used for treatment or prevention of an insulin resistance disease.