JP2009108033A - Hepatic fibrosis-suppressing agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide hepatic fibrosis-suppressing agents, to provide a screening method of the agents, to provide a suppressing method of hepatic fibrosis, and to provide a treating or preventing method of hepatic fibrosis diseases, based on the suppressing method. <P>SOLUTION: The hepatic fibrosis-suppressing agents suitable for treating or preventing hepatic fibrosis diseases such as cirrhosis, are provided, which agents comprise as an active ingredient, chondroitinase ABC of a chondroitin sulfate proteoglycan-degrading enzyme, or a substance that inhibits the production or accumulation of chondroitin sulfate proteoglycans including ADAMTS-4, and an expression suppressant of C4ST-1, C6ST-1 or C6ST-2 of a sulfotransferase for chondroitin sulfate proteoglycans, such as siRNA, and a screening method of the agent is provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a liver fibrosis inhibitor, a method for suppressing liver fibrosis, and a method for treating or preventing a liver fibrosis disease based on the method.

  Chronic fibrosis of the liver is a general term for the terminal picture of a chronic inflammatory state that can develop in the liver, and is accompanied by excessive tissue fibrosis. Chronic fibrosis of the liver is a serious disease group often resulting in death through progressive liver dysfunction, but there is still no definitive treatment. Currently, liver cirrhosis is being treated for liver cirrhosis, but these treatments not only significantly inhibit the patient's quality of life (QOL), but the transplanted organ falls into fibrosis again. There are many. Recently, TGF-β inhibitors and angiotensin inhibitors are expected as novel therapeutic agents for fibrotic diseases, but their effectiveness has not been established. For this reason, development of the new medicine which can improve a patient's QOL and can prevent or delay progress of liver fibrosis is earnestly desired.

  Currently, as a candidate treatment for fibrosis of the liver, corticosteroids that suppress chronic inflammation, colchicine, IL-10, etc .; TGF-β inhibitor that activates fibroblasts, HGF (suppresses the action of TGF-β ), Endothelin inhibitors, angiotensin inhibitors, etc .; MMP (matrix metalloproteinase) that removes collagen is expected, but none of them has been established as a treatment (Non-patent document 1 [latest Review], and Non-Patent Documents 2 and 3).

  Proteoglycan (PG) is a molecule having a structure in which one or more glycosaminoglycan (GAG) chains are covalently bonded to a protein called a core protein. It is thought that the specific sugar chain structure of GAG chain is responsible for various functions of PG. Based on the type of GAG chain, PG is roughly divided into four types: chondroitin sulfate proteoglycan (CSPG), dermatan sulfate proteoglycan (DSPG), heparan sulfate proteoglycan (HSPG), and ketalan sulfate proteoglycan (KSPG) 4 and 5 [Review]). In particular, HSPG has been well studied because it binds to various cytokines and chemokines and greatly modifies their functions.

  On the other hand, chondroitin sulfate proteoglycan (CSPG) is an essential molecule in the embryonic period, and is abundant in each organ but is known to gradually decrease with birth, growth and aging. Surprisingly, however, the in vivo function of CSPG has not yet been clarified because of its versatility. On the other hand, there are reports that CSPG is increased in diseased organs such as cirrhosis and pulmonary fibrosis (Non-patent Documents 6 and 7). However, the significance of increasing CSPG in these diseases is not clear at all. In recent years, it has been found that mice genetically deficient in CSPG fall into embryonic lethality and organ dysplasia, and thus recognition of CSPG as an indispensable molecule in the living body has increased.

  Chondroitinase is known as a bacterial enzyme having an activity of degrading chondroitin sulfate (chondroitin sulfate proteoglycan). As a therapeutic agent using chondroitinase, a therapeutic agent for disc herniation using chondroitinase ABC (Patent Document 1), chondroitinase AC or chondroitinase B wound healing promoter (Patent Document 2), chondroitinase A therapeutic agent for scleroderma, psoriasis, keloid, and pulmonary fibrosis using AC or chondroitinase B (Patent Document 3) is known.

US Pat. No. 6,001,630 US Pat. No. 5,997,863 International Publication No. 2001/039795 Pamphlet Bataller R & Brenner DA, J. Clin. Invest. (2005) 115: 209-218 Iredale JP, BMJ (2004) 327: 143-147 Bissell DM, Exp. Mol. Med. (2001) 33: 179-190 Lin X, Development (2004) 131: 6009-6021 Hacker U et al. Nature Rev. Cell Biol. (2005) 6: 530-541 Kovalski I et al., Orv Hetil. (1993) 134: p.2019-2026 Westergren-Thorsson G et al., J Clin Invest. (1993) 92: p.632-637

  The present invention relates to a drug capable of suppressing fibrosis in liver tissue, a therapeutic agent for liver fibrosis disease containing the drug as an active ingredient, and a liver fibrosis inhibitor useful for the treatment or prevention of liver fibrosis disease. One object is to provide a screening method.

  As the inventors of the present invention have conducted extensive research to develop such a drug, excessive accumulation of chondroitin sulfate proteoglycan (CSPG), which has not been attributed to the pathogenesis of liver fibrosis, has occurred in liver tissues. I thought that it might promote fibrosis. As a result of researches based on this idea, the present inventors have found that chondroitinase ABC (chondroitinase) ABC, which is a chondroitin sulfate proteoglycan degrading enzyme, can efficiently degrade CSPG accumulated in fibrotic liver, and chondroit It was found that fibrosis of liver tissue can be remarkably suppressed by administering in vivo ABC. Furthermore, the same therapeutic effect can be achieved by administering a recombinant peptide of ADAMTS-4 (A disintegrin and metalloproteinase with thrombospondin motifs), which has the function of cleaving the core protein of versican, one of the chondroitin sulfate proteoglycans. I was able to win. That is, it was demonstrated that liver fibrosis can be suppressed by inhibiting the accumulation or biosynthesis of chondroitin sulfate proteoglycan, and the present invention was completed.

The present invention relates to a liver fibrosis inhibitor, a therapeutic agent for liver fibrosis using the drug as an active ingredient, a screening method for a liver fibrosis inhibitor, and more specifically,
[1] A liver fibrosis inhibitor comprising a substance that inhibits the production or accumulation of chondroitin sulfate proteoglycan as an active ingredient,
[2] The drug according to [1], wherein the substance has 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 the liver,
[7] The drug according to any one of [1] to [6] for treatment or prevention of liver fibrosis disease,
[8] The drug according to [7], wherein the liver fibrosis disease is chronic liver disease,
[9] The drug according to [7], wherein the liver fibrosis disease is chronic hepatitis, liver fibrosis, cirrhosis, liver failure, or liver cancer,
[10] A screening method for a liver fibrosis 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], comprising a step of selecting a substance having the action according to any one of the following (a) to (d):
(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] Inhibitor of fibrosis of liver The method for screening according to [10] or [11], wherein the screening method is for the treatment or prevention of a liver fibrosis disease,
Is provided.
Moreover, this invention includes the following.
[13] A liver fibrosis inhibitor comprising chondroitinase ABC as an active ingredient.
[14] The liver fibrosis inhibitor as described in [13] above, which is used for treatment or prevention of chronic liver disease.
[15] The liver fibrosis inhibitor as described in [14] above, wherein the chronic liver disease is cirrhosis.
[16] The liver fibrosis inhibitor according to any one of [13] to [15] above, wherein chondroitinase ABC is derived from Proteus vulgaris.
The present invention further relates to the following.
[17] Use of the drug according to any one of [1] to [9] in the production of a liver fibrosis inhibitor,
[18] A method for treating liver fibrosis, comprising a step of administering the agent according to any one of [1] to [9] to an individual (patient or the like),
[19] 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 clarified that the production and accumulation of chondroitin sulfate proteoglycan is related to the onset of cirrhosis. It was shown that liver fibrosis is suppressed by inhibiting the production and accumulation of chondroitin sulfate proteoglycans. It will be possible to provide a therapeutic drug for liver fibrosis that has never been seen before. In particular, liver fibrosis is closely related to chronic liver diseases such as chronic hepatitis, liver fibrosis, liver cirrhosis, liver failure, and liver cancer. It also has important industrial significance.

Hereinafter, the present invention will be described in more detail.
Fibrosis in the liver is a disease state associated with cirrhosis, which is one of typical chronic liver diseases. The present inventors paid attention to the function of chondroitin sulfate proteoglycan in order to improve the fibrosis state in the liver as one of the effective methods for treating cirrhosis. In addition, a state in which chondroitin sulfate proteoglycan accumulation is suppressed in a cirrhosis model mouse has been created and analyzed in detail. As a result, many cells with improved accumulation of chondroitin sulfate proteoglycan compared to wild-type liver are observed. The pathology of cirrhosis was improved. That is, it has been found that inhibiting the production or accumulation of chondroitin sulfate proteoglycan promotes improvement of abnormal accumulation state of chondroitin sulfate proteoglycan in the liver, which is deeply involved in cirrhosis, and leads to improvement of liver fibrosis.

The present invention relates to a liver fibrosis inhibitor 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 biological species derived from the chondroitin sulfate proteoglycan is not particularly limited. ) 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.

The “liver fibrosis” in the present invention refers to a fibrosis state in liver tissue. Examples include, but are not limited to, accumulation of extracellular matrix such as collagen in liver tissue and activation of fibroblasts.
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 a decomposition promoting action '', `` substance that has a synthesis inhibitory action '', “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 (eg, ADAMTS-1, ADAMTS-4, ADAMTS-5, etc.), chondroitinase, Calpain I, and the like. “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, GALNAC4S-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-sulfatase. Examples of the compound that suppresses sulfation include Chlorate and EGF receptor antagonist.

Preferable embodiments of the “substance having desulfating action” include, for example, compounds (nucleic acids) selected from the group consisting of the following (a) to (c).
(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 having a dominant negative property against a chondroitin sulfate proteoglycan desulfase inhibitor protein (c) chondroitin sulfate Low molecular weight compound that binds to proteoglycan desulfase inhibitor compound Here, the “desulfate inhibitor compound” is not limited to a protein, and includes, for example, a non-protein compound such as a coenzyme.

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), ketalan 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 ketalan 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: 68)), 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, ADAMTS-1, ADAMTS-4, ADAMTS-5, Calpain I, exemplified as enzymes involved in chondroitin synthesis, GalNAc4ST-1, GalNAc4ST-2, GALNAC4S-6ST, UA2OST, GalT-I, GalT-II, GlcAT-I, XylosylT, exemplified as sulfate group transferase, C4ST-1, C4ST-2, C4ST-3, D4ST, C6ST-1, C6ST-2 gene gene public accession number and nucleotide sequence in the public gene database Genbank 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)
ADAMTS-1 (accession number NM_009621, base sequence SEQ ID NO: 19 and amino acid sequence SEQ ID NO: 20)
ADAMTS-4 (Accession number NM_172845, SEQ ID NO: 21 of nucleotide sequence, SEQ ID NO: 22 of amino acid sequence)
ADAMTS-5 (Accession number AF140673, nucleotide sequence SEQ ID NO: 23, amino acid sequence SEQ ID NO: 24)
Calpain I (accession number NM_007600, nucleotide sequence 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)
GALNAC4S-6ST (accession number NM_029935, base 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), C4ST-1 (GenBank accession number NM_021439, SEQ ID NO: 45), C4ST -2 (GenBank accession number NM_021528, SEQ ID NO: 47), C4ST-3 (GenBank accession number XM_355798, SEQ ID NO: 49), and the like.

  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 GALNAC4S-6ST, and the like.

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: 57 to 65), for example, the above-mentioned chondroitin sulfate proteoglycan core protein even if it is a double-stranded RNA having a structure in which one or a small number of RNAs are added or deleted 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 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: 57. That is, on the basis of the base sequence set forth in SEQ ID NO: 57, selecting an arbitrary continuous RNA region of mRNA that is a transcription product of the sequence, and preparing a double-stranded RNA corresponding to this region, For those skilled in the art, it can be appropriately performed within the range of normal trials. In addition, selection of siRNA sequences having a stronger RNAi effect from mRNA sequences that are transcripts of the sequences can also be appropriately performed by those skilled in the art by known methods. If one strand is known, those skilled in the art can easily know the base sequence of the other strand (complementary strand). The siRNA can be appropriately prepared by those skilled in the art using a commercially available nucleic acid synthesizer. In addition, for synthesis of a desired RNA, a general synthesis contract service can be used.

  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 a human, and particularly preferably a protein derived from a human. Human-derived proteins can be appropriately obtained by those skilled in the art using the gene sequences or amino acid sequences disclosed in the present specification.

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 tissue or cells that inhibit the production or accumulation of chondroitin sulfate proteoglycan are not particularly limited, but are preferably liver tissue.
Compounds that inhibit the production or accumulation of chondroitin sulfate proteoglycans are expected to be drugs for the treatment or prevention of liver fibrosis diseases. Here, “treatment or prevention” does not necessarily need to have a complete therapeutic or prophylactic effect on tissues or cells that exhibit liver fibrosis, but may have a partial effect.

In the present invention, the liver fibrosis disease is not particularly limited as long as it is a disease accompanied by liver fibrosis, and preferable examples include chronic hepatitis such as chronic hepatitis, liver fibrosis, cirrhosis, liver failure, and liver cancer. Complications of these diseases are also included in the liver fibrosis disease of the present invention.
The liver fibrosis inhibitor of the present invention has an action of suppressing liver fibrosis by inhibiting the production or accumulation of chondroitin sulfate proteoglycan which is the cause of liver fibrosis. Accordingly, as a preferred embodiment of the present invention, for example, a therapeutic agent for chronic liver disease or a therapeutic agent for cirrhosis is provided, which comprises the liver fibrosis inhibitor of the present invention as an active ingredient.

The “liver fibrosis inhibitor” of the present invention can also be expressed as “hepatic fibrosis therapeutic agent”, “liver fibrosis 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 preventive effect that can suppress the occurrence of liver fibrosis in advance. Moreover, it is not necessarily limited to having a complete therapeutic effect on hepatic fibrosis-expressing cells (tissue), and may have 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 agent, a dosage form such as an injection, an infusion, an external medicine, an inhalant, 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 drug of the present invention is used as a gene therapy agent, a method of directly administering the drug of the present invention by injection and a method of administering a vector in which a nucleic acid has been incorporated 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 liver fibrosis 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 expressing liver fibrosis. For example, for chronic hepatitis, liver fibrosis, cirrhosis, liver failure, liver cancer, etc. Applied. The above-mentioned diseases may be accompanied with other diseases.

Moreover, as a preferable aspect of the present invention, it relates to a liver fibrosis inhibitor containing chondroitinase ABC as an active ingredient.
Here, chondroitinase ABC is a lyase classified by enzyme number EC 4.2.2.4 (chondroitin ABC lyase). Chondroitinase ABC is a polysaccharide that contains 1,4-β-D-hexosaminide linkages and 1,3-β-D-glucuronosyl linkages or 1,3-α-L-iduronosyl linkages. It has an activity of decomposing and removing it into a disaccharide containing -D-gluc-4-enuronosyl group. Chondroitinase ABC acts on chondroitin sulfate A (chondroitin-4-sulfate), chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C (chondroitin-6-sulfate), and hyaluronic acid to decompose them.

  Examples of chondroitinase ABC used in the present invention include, but are not limited to, for example, Proteus bacteria such as Proteus vulgaris, and Bacteroides stercoris such as Bacteroides stercoris. A) derived from genus bacteria is preferred. The chondroitinase ABC used in the present invention is more preferably derived from Proteus vulgaris. The chondroitinase ABC of the present invention may be purified from an extract of Proteus vulgaris by ammonium sulfate precipitation and DEAE-cellulose chromatography (Yamagata, T. et al., J. Biol Chem., (1968) 243, p. 1523-1535). Examples of chondroitinase ABC derived from Proteus vulgaris of the present invention include a protein having the amino acid sequence represented by SEQ ID NO: 9 disclosed in JP-A-06-098769, DDBJ / EMBL / GenBank Registered with accession number E07183 in an international nucleotide sequence database (for example, accessible from the DNA Data Bank of Japan (DDBJ) website http://www.ddbj.nig.ac.jp/Welcome-j.html) And a protein encoded by the base sequence. The chondroitinase ABC of the present invention has at least an activity of degrading chondroitin sulfate A (chondroitin-4-sulfate), chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C (chondroitin-6-sulfate) and hyaluronic acid. Is preferred.

  As the chondroitinase ABC of the present invention, commercially available products such as chondroitinase ABC (Proteus vulgaris) (Seikagaku Corporation, Tokyo, Japan), or chondroitinase ABC (Proteus vulgaris) (Sigma-Aldrich, Saint Louis, Missouri, USA). Furthermore, the chondroitinase ABC of the present invention can be expressed by a person skilled in the art by expressing a protein from a DNA encoding chondroitinase ABC (for example, having a base sequence registered under accession number E07183) by a recombinant method. By making it, it can manufacture easily. For details on conventional methods of molecular biology such as recombination, see, for example, Molecular Cloning: A Laboratory Manual / Volume 3. Joseph Sambrook and David W Russell. 3rd edition New York: Cold Spring Harbor Laboratory Press, 2001. Has been.

  The liver fibrosis inhibitor containing the chondroitinase ABC of the present invention as an active ingredient can effectively suppress fibrosis of the liver tissue (liver fibrosis). In the present invention, “suppressing fibrosis” means reducing or eliminating fibrotic lesions in a tissue in which fibrosis has occurred, or delaying or preventing the progression of further fibrosis (increasing fibrotic lesions). Means).

  The degree of fibrosis in liver tissue can be evaluated by various methods known to those skilled in the art. The most basic method involves fibrotic findings in liver biopsy, eg, fibrotic tissue image enhanced by special staining (aniline blue, trichrome, or silver staining) of a liver biopsy sample. Should be evaluated. A specific assessment of fibrosis can be performed, for example, according to Dai K, et al. World J Gactroenterol. 31: 4822-4826, 2005, Hillebrandt S, et al. Nature Genetics 37: 835-843, 2005. This can be done by expressing the liver fibrosis level of each sample in terms of fibrosis based on staining. In addition, the degree of liver fibrosis in liver tissue may be more simply evaluated using liver fibrosis markers such as collagen such as hyaluronic acid, type I, type III, and type IV, fibroblasts, and macrophages. A test based on counting platelet count that is simple but sensitively reflects the degree of liver fibrosis can also be used conveniently. The degree of liver fibrosis can also be roughly predicted by liver image diagnosis such as abdominal ultrasonography. In addition, using a measuring instrument (such as Fibro Scan502) for non-invasive liver fibrosis measurement based on the trans-end elastography technology recently developed by EcoSense (EcoSence, France) It can also be evaluated. The level of suppression of liver fibrosis by the liver fibrosis inhibitor containing chondroitinase ABC of the present invention as an active ingredient may be determined based on the evaluation of the degree of liver fibrosis obtained by these methods.

  The liver fibrosis inhibitory effect by the liver fibrosis inhibitor containing the chondroitinase ABC of the present invention as an active ingredient is, in particular, suppression of type III collagen elongation, type I collagen deposition, and fibroblast accumulation in liver tissue. , And at least one of suppression of macrophage accumulation, and more preferably all of them. The level of liver fibrosis suppression by a liver fibrosis inhibitor containing chondroitinase ABC of the present invention as an active ingredient may be determined using one or more of these inhibitory effects as an index.

  The present invention shows that chondroitinase ABC, which is an active ingredient of the liver fibrosis inhibitor containing chondroitinase ABC of the present invention as an active ingredient, has very high liver fibrosis as demonstrated in the examples described later. This is based on a completely new finding that it has an inhibitory effect. The effect of suppressing chondroitinase ABC in liver fibrosis is significantly superior to other chondroitinases. The excellent effect of chondroitinase ABC is that fibrotic liver tissue accumulates a kind or amount of chondroitin sulfate that is difficult to cleave with chondroitinase AC or chondroitinase B. It can also be attributed to being cut well. In recent years, the existence of various types of chondroitin sulfate such as those with different sulfation grades and those with different addition positions of sulfate groups has been reported, so this hypothesis can be said to be quite convincing. However, the present invention is not limited by such a hypothesis.

In the present invention, the degree of fibrosis in the liver tissue can be reduced by adding a liver fibrosis inhibitor containing chondroitinase ABC of the present invention as an active ingredient to the liver tissue sample for reaction.
Moreover, in this invention, the fibrosis of the liver tissue of a subject can be effectively suppressed by administering to the subject a liver fibrosis inhibitor containing chondroitinase ABC of the present invention as an active ingredient. The administration route of the liver fibrosis inhibitor containing chondroitinase ABC as an active ingredient is not limited, but a parenteral route is preferable. Suitable examples of parenteral routes include intravenous, intraarterial, intraperitoneal, intrahepatic, intrarectal, and subcutaneous routes. The liver fibrosis inhibitor containing the chondroitinase ABC of the present invention as an active ingredient may be administered systemically or locally (for example, directly administered to the fibrotic tissue of the liver).

The present invention also relates to a method for suppressing fibrosis in the liver of a subject by administering to the subject a liver fibrosis inhibitor comprising chondroitinase ABC of the present invention as an active ingredient.
Furthermore, since the hepatic fibrosis inhibitor containing chondroitinase ABC of the present invention as an active ingredient can effectively suppress fibrosis of liver tissue, liver fibrosis disease (disease with excessive fibrosis of liver tissue) It can be used advantageously for treatment and prevention. An example of a more suitable liver fibrosis disease in the present invention is, but not limited to, a chronic liver disease. Chronic liver disease is generally accompanied by liver fibrosis. Specific examples of the chronic liver disease according to the present invention include chronic hepatitis (chronic hepatitis B, chronic hepatitis C, etc.), liver fibrosis, cirrhosis, liver failure, liver cancer and the like. The liver fibrosis inhibitor comprising chondroitinase ABC of the present invention as an active ingredient is particularly useful for the treatment and prevention of cirrhosis. In the present invention, “cirrhosis” refers to a state of the liver in which extensive fibrosis occurs in the liver and the normal liver lobule structure is lost and remodeled to an abnormal structure (pseudolobule / regenerative nodule). . Cirrhosis is classified into many types according to its etiology and pseudolobular characteristics. The liver in such a state is called cirrhosis liver. On the other hand, “liver fibrosis” is a state in which abnormal fibrosis is present but liver lobule is not remodeled, and can generally be said to be a pre-stage of cirrhosis. Specific examples of cirrhosis in the present invention include viral cirrhosis, parasitic cirrhosis, toxic cirrhosis, malnutrition cirrhosis, alcoholic cirrhosis, congestive cirrhosis, hepatic sclerosis, Charcot cirrhosis, Todd cirrhosis, primary biliary Cirrhosis, secondary biliary cirrhosis, monolobic cirrhosis, cirrhosis transferred from chronic non-suppurative destructive cholangitis, obstructive cirrhosis, biliary cirrhosis, biliary cirrhosis, atrophic cirrhosis, postnecrotic cirrhosis, post-hepatic cirrhosis Nodular cirrhosis, mixed cirrhosis, small nodular cirrhosis, compensatory cirrhosis, decompensated cirrhosis, major nodular cirrhosis, septal cirrhosis, idiopathic cirrhosis, periportal cirrhosis, portal cirrhosis, etc. However, it is not limited to these.

  As a subject to which a liver fibrosis inhibitor containing chondroitinase ABC of the present invention as an active ingredient is to be administered, mammals including humans, domestic animals, pets, laboratory animals and the like are preferable. In particular, mammals (patients) suffering from liver fibrosis diseases such as cirrhosis and liver fibrosis are preferred as subjects according to the present invention. In another embodiment, a mammal (patient) suffering from a liver fibrosis disease (eg, chronic non-suppurative destructive cholangitis, etc.) that is likely to shift to cirrhosis in the future is also preferred as a subject. In the present invention, the subject is administered with a liver fibrosis inhibitor containing the chondroitinase ABC of the present invention as an active ingredient, thereby reducing the degree of liver fibrosis or delaying or preventing the progression of fibrosis. As a result, it is possible to treat or prevent a liver fibrosis disease by reducing liver damage due to fibrosis and maintaining or improving liver function at a higher level.

The dose of the hepatic fibrosis inhibitor containing the chondroitinase ABC of the present invention as an active ingredient can be appropriately determined by those skilled in the art. Generally, the dose of chondroitinase ABC is 10 μg to 1 mg per kg body weight. It is.
The present invention thus provides a liver fibrosis disease such as chronic liver disease by administering a liver fibrosis inhibitor comprising the chondroitinase ABC of the present invention as an active ingredient to a subject (for example, a patient). In particular, it also relates to a method for treating or preventing cirrhosis or the like).

  The hepatic fibrosis inhibitor containing chondroitinase ABC of the present invention as an active ingredient is pharmaceutically acceptable (pharmaceutically inactive) carrier, excipient, and / or dilution in addition to chondroitinase ABC. It may be a pharmaceutical composition optionally containing an agent. Examples of such carriers, excipients and / or diluents include water, saline, phosphate buffered buffer, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, pectin, xanthan gum , Gum arabic, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose and the like. The pharmaceutical composition of the present invention may further contain additives such as preservatives. The pharmaceutical composition of the present invention may further contain other pharmacological components.

The pharmaceutical composition of the present invention may be provided as an oral preparation or a parenteral preparation, but is more preferably provided as a parenteral preparation. As parenteral preparations, liquid preparations such as liquids and suspensions are preferable, and injections are particularly preferable.
The present invention also provides a method for screening a liver fibrosis inhibitor characterized by 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, a liver fibrosis inhibitor or a candidate compound for a liver fibrosis inhibitor can be efficiently obtained.

A preferred embodiment of the screening method of the present invention is a method for screening a liver fibrosis 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, GALNAC4S-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 ADAMTS-1, ADAMTS-4, ADAMTS-5, Chondroitinase ABC (ChABC), Chondroitinase AC, Chondroitinase B, and Calpain I. Examples of “desulfating enzyme” include Chondroitin-4-sulfatase, Chondroitin-6-sulfatase, 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 liver fibrosis.
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. If chondroitinase ABC is added and the CS-GAG chain is degraded, WFA lectin cannot be bound, so the principle is used. More specifically, FITC-labeled WFA lectin (such as EY) is added to the CS-coated well before and after mixing the test compound, and CS-GAG is decomposed to change the FITC fluorescence intensity in the well. 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. Similar to WFA lectin, FITC-labeled anti-CS antibody can be added to CS-coated wells, and large-scale screening can be performed in a very short time and simply if changes in fluorescence values are observed.
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 Step of selecting In the above method, first, a test compound is contacted with a group of substances containing a chondroitin sulfate proteoglycan or a part thereof, a cell extract, or an enzyme and a substrate constituting the chondroitin sulfate proteoglycan synthesis process. Let
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, for example, 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 the treatment of liver fibrosis.
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 by the standard method of separating and culturing mononuclear cells after collecting peripheral blood from healthy individuals (Uhlin-Hansen L et al., Blood 82: 2880, 1993.). More simply, known cell lines such as fibroblast cell line NIH3T3 (Phillip HA, et al. J. Biol. Chem. 279: 48640, 2004, 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 step of selecting a substance that reduces the amount that has been subjected to sulfation as compared with the case of measuring in step 1 In the above method, first, a test compound is contacted 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 liver fibrosis.
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 The step of measuring the sulfation activity of chondroitin sulfate proteoglycan in a cell extract or substance group (c) The step of selecting a compound that decreases the activity compared to the case where the test compound is not contacted In the above method, 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 liver fibrosis.
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 a liver fibrosis inhibitor comprising the following steps (a) to (d), wherein a compound that increases the expression level of an enzyme or a desulfating enzyme 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 liver fibrosis or a candidate compound for treating liver fibrosis.

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 liver fibrosis or a candidate compound for treating liver fibrosis.
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 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 a 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.

  “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. “A cell extract containing DNA” refers to, for example, a chondroitin sulfate proteoglycan core protein, a synthase, a desulfase-inhibiting protein, or a sulfotransferase in a cell extract contained in a commercially available in vitro transcription / translation kit. Examples include those to which DNA having a structure in which a transcriptional regulatory region of a gene and a reporter gene are functionally linked is added.

  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 liver fibrosis or a candidate compound for treating liver fibrosis.
In addition, when the reporter gene is functionally bound to the chondroitin sulfate proteoglycan degradation promoting enzyme or desulfation enzyme, the expression level of the reporter gene is increased (enhanced) compared to the control. Select a compound. The compound to be increased (enhanced) becomes a drug for suppressing liver fibrosis or a candidate compound for treating liver fibrosis.

The liver fibrosis inhibitor found in the screening method of the present invention is preferably for treating or preventing a liver fibrosis 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, the present invention is a method for screening a liver fibrosis inhibitor comprising 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 (anti-chondroitin sulfate proteoglycan antibody) can also be included in the components of the kit for screening for a fibrosis inhibitor 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 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 a liver fibrosis disease, comprising the step of administering the agent of the present invention to an individual (eg, a patient).
The subject of the prevention or treatment method of the present invention is not particularly limited as long as it is an organism capable of developing a liver fibrosis 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 a liver fibrosis inhibitor.
It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.

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.
Example 1 Increased Chondroitin Sulfate Proteoglycan (CSPG) Expression in a Mouse Cirrhosis Model In this example, a mouse liver fibrosis model induced by carbon tetrachloride (CCl ₄ ) is used as a typical example of a chronic fibrotic disease model mouse. A mouse cirrhosis model was prepared, and the state of proteoglycan accumulation was examined by immunohistological staining. This mouse liver fibrosis model has excellent reproducibility and is widely used as an experimental system for cirrhosis models (Sakaida I, et al. Hepatology 40: 1304-1311, 2004).
First, carbon tetrachloride (25 μL / 100 g body weight; Sigma-Aldrich) was added to C57BL / 6JcL mice (female, 5-6 weeks old, manufactured by CLEA Japan) twice a week for 4 weeks (8 times) Injected intraperitoneally, hepatic fibrosis was induced. Further, carbon tetrachloride was administered twice a week for 2 weeks (12 times in total) to induce cirrhosis. The mice in which cirrhosis was induced were sacrificed, and the livers were collected (cirrhosis liver). On the other hand, in a control experiment, livers were collected from normal-age C57BL / 6JcL mice (female, manufactured by CLEA Japan, Inc.) not receiving carbon tetrachloride (normal liver).
A part of the collected second lobe of the liver was embedded in a freezing embedding agent OCT compound (manufactured by Miles), and a frozen block was prepared with liquid nitrogen. From the frozen block, a 6 μm-thick section was prepared using a cryostat (manufactured by Microm).
The obtained sections were fixed with acetone (manufactured by Wako) for 10 minutes, washed with a phosphate buffer, and further anti-chondroitin sulfate proteoglycan (CSPG) antibody (clone CS56, mouse monoclonal antibody, 10 μg / mL; Chemical Industries), anti-chondroitin-4-sulfate proteoglycan (C4SPG) antibody (clone LY111, mouse monoclonal antibody, 10 μg / mL; manufactured by Seikagaku Corporation), anti-chondroitin-6-sulfate proteoglycan (C6SPG) antibody (clone MC21C) Mouse monoclonal antibody, 10 μg / mL; manufactured by Seikagaku Corporation), or anti-heparan sulfate proteoglycan (HSPG) / Perlecan antibody (rat monoclonal antibody, 10 μg / mL; manufactured by Dainippon Pharmaceutical Co., Ltd.), room temperature For 1 hour. Subsequently, using a histofine mouse stain kit (manufactured by Nichirei; used for mouse monoclonal antibody) or peroxidase-labeled anti-rat IgG (manufactured by Biosource; 1: 200 dilution; used for rat monoclonal antibody) After performing the secondary antibody reaction, DAB substrate (manufactured by Nichirei) was added. The sample was observed under an optical microscope (manufactured by Leica), and antibody binding visualized with a brown signal was confirmed.

  An example of an immunostained image of the obtained liver tissue is shown in FIG. In FIG. 1, the left column indicates normal liver and the right column indicates cirrhosis liver. In normal liver, that is, in the liver of untreated same-aged mice, positive signals were observed in the liver sinusoid for CSPG (top row) and C4SPG (second row), but C6SPG (third row). Was not detected at all. On the other hand, in cirrhotic liver, that is, cirrhosis-like fibrosis induced by carbon tetrachloride, CSPG (top row), C4SPG (second row), C6SPG (third row) In the center, there was a wide range of positive signals with significantly enhanced signal intensity compared to normal liver. As for heparan sulfate proteoglycan (HSPG), a strong positive signal was detected in the sinusoidal center in normal liver, but no significant difference was observed in the intensity or distribution of signal in cirrhotic liver. Therefore, in this cirrhosis model, the increase in the amount of CSPG in the liver was found to be dramatic compared to HSPG.

Example 2: Cleaving ability of various chondroitinases to chondroitin sulfate accumulated in mouse cirrhotic liver In this example, the ability of each type of chondroitinase to cut chondroitin sulfate (CSPG) in tissue sections of cirrhotic liver It was investigated. As shown below, tissue sections of cirrhotic liver were treated with chondroitinase, and after the enzymatic reaction, the CSPG remaining in the tissue sections, that is, the CSPG that was not cut by chondroitinase treatment, was immunostained. Detected with.
First, C57BL / 6JcL mice (female, 5-6 weeks old, manufactured by CLEA Japan) were subjected to carbon tetrachloride (25 μL / 100 g body weight; manufactured by Sigma-Aldrich) twice a week for 4 weeks (8 times). Injection into the abdominal cavity caused liver fibrosis. Carbon tetrachloride was further administered twice a week for 2 weeks (total 12 times) to induce cirrhosis. The mice in which cirrhosis was induced were sacrificed, and the livers were collected (cirrhosis liver).
A part of the collected second lobe of the liver was embedded in a freezing embedding agent OCT compound (manufactured by Miles), and a frozen block was prepared with liquid nitrogen. From the frozen block, a 6 μm-thick section was prepared using a cryostat (manufactured by Microm).
The obtained section was fixed with acetone (manufactured by Wako) for 10 minutes, washed with phosphate buffer, and then incubated in Tris-HCL buffer (20 mM, pH 8.0) at 37 ° C. for 15 minutes. Chondroitinase ABC solution (Seikagaku Corporation; 5 U / mL), chondroitinase AC solution (Seikagaku Corporation; 5 U / mL), chondroitinase B solution (Seikagaku Corporation; 5 U / mL) ), Or buffer alone (chondroitinase (−)) was added at 100 μL per section to cover the sections, and further incubated at 37 ° C. for 1 hour.

After completion of the enzyme reaction, an anti-CSPG antibody (clone CS56, mouse monoclonal antibody, 10 μg / mL; manufactured by Seikagaku Corporation) was added and reacted at room temperature for 1 hour, and then histofine mouse staining kit (manufactured by Nichirei) Then, DAB substrate (manufactured by Nichirei) was added. The sample was observed under an optical microscope (manufactured by Leica), and antibody binding visualized with a brown signal was confirmed.
The results are shown in FIG. 2 (magnification 200 times). In sections treated only with the buffer solution (that is, sections not subjected to enzyme treatment), a CSPG positive signal (brown) was detected in a wide range with a remarkably high intensity comparable to that of cirrhotic liver in FIG. 1 (FIG. 2). Leftmost; chondroitinase (-)). On the other hand, in the section treated with chondroitinase AC, the CSPG positive signal was attenuated and the positive area was also decreased as compared with the chondroitinase (−) section (FIG. 2; second from the left; Chondroitinase AC). This indicated that chondroitinase AC can certainly cleave CSPG accumulated in cirrhotic liver tissue. However, CSPGs that remained uncut completely were still detected in this section treated with chondroitinase AC. Further, in the section treated with chondroitinase B, the CSPG positive signal was further attenuated and the positive area was further decreased as compared with the section treated with chondroitinase AC. This shows that chondroitinase B can cleave more CSPG accumulated in cirrhotic liver tissue. However, even in sections treated with chondroitinase B, CSPG remaining along the pseudolobular structure was detected. Thus, the treatment with chondroitinase AC or B could not completely cut the CSPG deposited in the highly fibrotic cirrhotic liver.

  In contrast to these results, the CSPG positive signal (brown) was hardly detected in the sections treated with chondroitinase ABC, and the disappearance of CSPG was clearly shown in comparison with the chondroitinase (-) sections. That is, it was shown that chondroitinase ABC can cleave CSPG deposited in highly fibrotic lesions such as cirrhosis very efficiently.

Example 3: Effect of chondroitinase ABC (Chase ABC) on liver fibrosis in a mouse cirrhosis model
C57BL / 6JcL mice (female, 5-6 weeks old, manufactured by CLEA Japan, Inc.) were treated with carbon tetrachloride (25 μL / 100 g body weight; manufactured by Sigma-Aldrich) twice a week for 4 weeks (8 times). And hepatic fibrosis was induced. Carbon tetrachloride was further administered twice a week for 2 weeks (12 times in total).
Prior to additional administration, the mice were preliminarily chondroitinase ABC (Chase ABC) (20 U / ml; manufactured by Sigma-Aldrich), chondroitinase AC (Chase AC) (20 U / ml; manufactured by Sigma-Aldrich), Chondroitinase B (Chase B) (20 U / ml; manufactured by Sigma-Aldrich) or an Alzette osmotic pump (Muromachi Kikai Co., Ltd.) into which 150 μl of PBS alone was injected was surgically implanted in the abdominal cavity. The groups of mice treated with this treatment were named Chase ABC enzyme group, Chase AC enzyme group, Chase B enzyme group, and control group, respectively. This treatment resulted in a constant volume of fluid being always infused from the implantable pump while both groups received an additional dose of carbon tetrachloride for 2 weeks.
After completing the additional administration of carbon tetrachloride, individual mice in each group were sacrificed and their livers were collected. A sample of a frozen section of the liver was prepared from the collected liver in the same manner as in Example 1, and immunohistological analysis was performed.

In this analysis, a rabbit anti-mouse type III collagen antibody (1: 250 dilution; manufactured by Santa Cruz) was added as a primary antibody to the sample and allowed to react at room temperature for 1 hour. Subsequently, alkaline phosphatase-labeled anti-rabbit IgG (1: 200 dilution; manufactured by Jackson ImmunoResearch) was added as a secondary antibody, and further reacted at room temperature for 30 minutes, followed by alkaline phosphatase chromogenic substrate (Vector Red; Vector Laboratory) Made) was added. The sample was observed under an optical microscope (manufactured by Leica), and antibody binding visualized with a red signal was confirmed.
An example of an immunostained image of the obtained liver tissue is shown in FIG. In the control group, type III collagen fibers detected by rabbit anti-mouse type III collagen antibody stretched not only from the general sinusoids but also from the central vein to the portal vein, and in some cases formed pseudolobules. (Left panel in FIG. 3). This was a typical image of cirrhosis. In contrast, in the Chase ABC enzyme group, the elongation of type III collagen was very mild (the right panel in FIG. 3).

Based on the immunohistological staining performed above, the degree of liver fibrosis in each sample has been reported (Dai K, et al. World J Gactroenterol. 31: 4822-4826, 2005, Hillebrandt S, et al. Nature Genetics 37 : 835-843, 2005). The evaluation criteria for the fibrosis frequency are as follows. 0 degree: normal, 1 degree: a small amount of collagen elongation from the central vein is observed, 2 degrees: obvious collagen elongation from the central vein is observed, but not to surround the entire liver, 3 degrees: central vein 4 degrees: diffuse collagen elongation of the entire liver is observed, forming a pseudolobule.
The results are shown graphically in FIG. Each bar shows the mean value ± standard deviation of the fibrosis frequency in each group. As shown in FIG. 4, the control group is 5.3 ± 0.4 (n = 6), whereas the Chase ABC enzyme group is 2.0 ± 0.6 (n = 6), and the Chase AC enzyme group is 2.9 ± 0.7 (n = 6), Chase B enzyme group was 2.6 ± 0.5 (n = 6). In comparison with the control group, fibrosis was statistically significantly reduced in the Chase ABC enzyme group (p = 0.00018, t test) and the Chase B enzyme group (p = 0.0029, t test). In particular, the Chase ABC enzyme group was statistically significantly more effective in suppressing fibrosis than the Chase AC enzyme group (p = 0.0019, t test) and the Chase B enzyme group (p = 0.018, t test). . On the other hand, there was no statistically significant difference between the Chase AC enzyme group and the Chase B enzyme group (p = 0.15, t test). A similar immunohistological analysis was performed on normal liver collected from normal mice to which carbon tetrachloride was not administered, but the fibrosis frequency was zero. Thus, it was shown that administration of chondroitinase ABC exhibits a clinically excellent liver fibrosis inhibitory effect.

Example 4: Inhibitory effect of chondroitinase ABC (Chase ABC) on type I collagen deposition in mouse cirrhosis model In fibrotic diseases including cirrhosis, an increase in type I collagen is the most important factor in the formation of lesion-constituting fibers. It is thought that there is. Thus, an immunostaining experiment for type I collagen was performed on the liver tissue samples derived from each group prepared in Example 3.
Rabbit anti-mouse type I collagen antibody as a primary antibody (1: 100 dilution; manufactured by Rockland) was used as a primary antibody to section samples derived from the control group, Chase ABC enzyme group, Chase AC enzyme group, and Chase B enzyme group prepared in Example 3. Was added and allowed to react for 1 hour at room temperature. Subsequently, alkaline phosphatase-labeled anti-rabbit IgG (1: 200 dilution; manufactured by Jackson ImmunoResearch) was added to the sample as a secondary antibody, and further reacted at room temperature for 30 minutes, and then an alkaline phosphatase chromogenic substrate (Vector Red; Vector) Laboratory product) was added. The sample was observed under an optical microscope (manufactured by Leica), and antibody binding visualized with a red signal was confirmed.
An example of an immunostained image of the obtained liver tissue is shown in FIG. As shown in FIG. 5, type I collagen was clearly detected along the pseudolobular structure in the control group, whereas it was only slightly seen in the Chase ABC enzyme group.

Therefore, based on the immunohistological staining performed above, the area positive for type I collagen was calculated using an unroof image analysis software (Win ROOF; Mitani Corporation). Measurements were made over a range of at least 15 mm ² on the liver section, and the results were expressed as a percentage of the positive area relative to the measured liver area. The results are shown graphically in FIG. As shown in FIG. 6, the positive area ratio (%) of type I collagen was 5.16 ± 0.98% in the control group, whereas it was 1.61 ± 0.29% in the Chase ABC enzyme group and in the Chase AC enzyme group. It was 4.30 ± 0.18% and 3.38 ± 0.50% in the Chase B enzyme group. Compared with the control group, the positive area ratio (%) of type I collagen decreased significantly in all enzyme groups (Chase ABC enzyme group: p = 0.0018, Chase AC enzyme group: p = 0.023, Chase B enzyme group: p = 0.0059; all t-test). In particular, in the Chase ABC enzyme group, the positive area ratio (%) of type I collagen was statistically significantly lower than the Chase AC enzyme group and Chase B enzyme group (compared with the Chase AC enzyme group). Significant difference: p = 0.0008, significant difference from Chase B enzyme group: p = 0.003; both are t-tests), indicating that the type I collagen deposition inhibitory effect is extremely high. On the other hand, there was no statistically significant difference between the Chase AC enzyme group and the Chase B enzyme group (p = 0.068, t test). The same analysis was performed on normal liver collected from normal mice not administered with carbon tetrachloride. The positive area ratio of type I collagen was 0%. From this result, it was shown that the deposition of type I collagen in the fibrotic liver can be remarkably suppressed by administration of chondroitinase ABC.

Example 5: Effect of suppressing chondroitin sulfate (CSPG) deposition of chondroitinase ABC (Chase ABC) in a mouse cirrhosis model
In order to confirm that chondroitin sulfate (CSPG) deposited in the fibrotic liver is decomposed by administration of Chase ABC, the control group prepared in Example 3 and the liver tissue samples of the Chase ABC enzyme group were compared with Example 1 Similarly, immunohistological analysis was performed using anti-CSPG antibody, anti-C4SPG antibody, and anti-C6SPG antibody as primary antibodies.
An example of the obtained immunostained image of the liver tissue is shown in FIG. In FIG. 7, the left column is the control group, and the right column is the Chase ABC enzyme group. As shown in FIG. 7, in the control group, a clear positive signal was observed for each of CSPG, C4SPG, and C6SPG in the same manner as the cirrhotic liver of Example 1. On the other hand, in the Chase ABC enzyme group, the signals for CSPG, C4SPG, and C6SPG were clearly attenuated as compared to the control group. In particular, in the stained image of C6SPG (bottom row), in the control group, a clear positive signal was detected along the periphery of the pseudolobule where elongation of collagen was observed in Examples 4 and 5, whereas Chase ABC In the enzyme group, the signal disappeared almost completely.
From the above results, it was demonstrated that CSPG deposited in fibrotic liver was degraded by administration of chondroitinase ABC.

Example 6: Effect of suppressing chondroitinase ABC (Chase ABC) in mouse liver cirrhosis model Fibroblast accumulation suppressive activity Fibroblasts are mainly active fibroblasts. Fibroblast over-accumulation, colonization and activation are said to exacerbate fibrotic lesions. Therefore, an immunohistological study was conducted on the fibroblast dynamics in a cirrhosis model.
First, a rat anti-mouse fibroblast antibody (clone ER-TR7; 1: 500 dilution; manufactured by BMA) was added as a primary antibody to the section sample derived from the control group and Chase ABC enzyme group prepared in Example 3. The reaction was allowed to proceed for 1 hour at room temperature. Subsequently, peroxidase-labeled anti-rat IgG (1: 200 dilution; manufactured by Biosource) was added as a secondary antibody and further reacted at room temperature for 30 minutes, and then DAB substrate (manufactured by Nichirei) was added. The sample was observed under an optical microscope (manufactured by Leica), and antibody binding visualized with a brown signal was confirmed. The same immunohistological analysis was performed on normal liver collected from normal mice not administered with carbon tetrachloride.

  An example of an immunostained image of the obtained liver tissue is shown in FIG. In FIG. 8, the leftmost column shows the normal group (normal liver), the middle column shows the control group, and the rightmost column shows the Chase ABC enzyme group. The upper row is an image with a magnification of 100 times, and the lower row is an image with a magnification of 200 times. In untreated normal mouse liver, the positive signal for fibroblasts was strongly distributed around the portal vein. In the control group (cirrhotic liver), fibroblasts were clearly accumulated along the pseudolobule, but in the Chase ABC group, the accumulation was very mild. From this, it was shown that the excessive accumulation or establishment of fibroblasts in the fibrotic liver was significantly suppressed by Chase ABC administration.

  Furthermore, fibroblasts accumulated along the pseudolobule (fibrosis layer) in the cirrhotic liver in this example, and clear CSPG along the pseudolobule circumference in the cirrhotic liver of the control group also in the above example. A positive signal was detected, suggesting that CSPG provides a fibroblast scaffold in liver fibrosis.

Example 7: Macrophage accumulation inhibitory effect of chondroitinase ABC (Chase ABC) in mouse cirrhosis model Cells that produce factors that activate fibroblasts are considered to be mainly macrophages. Therefore, immunohistological studies were also conducted on the dynamics of macrophages in a cirrhosis model.
First, a rat anti-mouse macrophage antibody (clone F4 / 80; 1: 500 dilution; manufactured by BMA) was added as a primary antibody to the section samples derived from the control group and Chase ABC enzyme group prepared in Example 3, and at room temperature. Reacted for 1 hour. Subsequently, peroxidase-labeled anti-rat IgG (1: 200 dilution; manufactured by Biosource) was added as a secondary antibody and further reacted at room temperature for 30 minutes, and then DAB substrate (manufactured by Nichirei) was added. The sample was observed under an optical microscope (manufactured by Leica), and antibody binding visualized with a brown signal was confirmed. The same immunohistological analysis was performed on normal liver collected from normal mice not administered with carbon tetrachloride.

  An example of an immunostained image of the obtained liver tissue is shown in FIG. In FIG. 9, the leftmost column shows the normal group (normal liver), the middle column shows the control group, and the rightmost column shows the Chase ABC enzyme group. As shown in FIG. 9, in normal liver, positive signals were widely distributed in sinusoids, which were considered Kupffer cells. On the other hand, in the cirrhotic liver of the control group, the number of macrophages was clearly increased, and a large number of macrophages were observed beside the collagen observed in Examples 4 and 5 so as to surround the pseudolobule. On the other hand, in the Chase ABC enzyme group, the number of macrophages was clearly reduced compared to the control group. That is, the Chase ABC enzyme group was shown to suppress the accumulation of macrophages in cirrhotic liver. These results suggest that the fibrosis progression is suppressed in the Chase ABC enzyme group, while the control group (cirrhotic liver) is still in the process of progressive fibrotic lesion formation.

Example 8: Treatment effect of mouse cirrhosis model by ADAMTS-4 peptide administration (suppression of fibroblast infiltration):
The mouse carbon tetrachloride-induced cirrhosis model was induced in the same manner as in Example 1. The ADAMTS-4 peptide sequence used for treatment was NH2-DRARSCAIVEDDGLQSAFTA-COOH (SEQ ID NO: 67) (synthesized peptide 336-355, which is a domain having the metalloproteinase activity of mouse ADAMTS-4. Manufactured by Gene World, 1 μg / animal), and vehicle (PBS) was used for the control group. Similar to the additional administration of carbon tetrachloride (4 times in total from the 9th to the 12th time), the peptide or vehicle was intraperitoneally administered 4 times in total. After completion of the additional administration, mice in both groups were sacrificed, liver tissue sections were prepared, and immunohistological examination was performed.
Activated fibroblasts are central to collagen producing cells that promote fibrosis. Fibroblast over-accumulation, colonization and activation are said to exacerbate fibrotic lesions. Therefore, an immunohistological study was conducted on the fibroblast dynamics in a cirrhosis model.

Rat anti-mouse fibroblast antibody (clone ER-TR7; 1: 500 dilution; manufactured by BMA) was added as a primary antibody to the section samples from the control group and ADAMTS-4 treatment group, and reacted at room temperature for 1 hour. . Subsequently, peroxidase-labeled anti-rat IgG (1: 200 dilution; manufactured by Biosource) was added as a secondary antibody and further reacted at room temperature for 30 minutes, and then DAB substrate (manufactured by Nichirei) was added. The sample was observed under an optical microscope (manufactured by Leica), and antibody binding visualized with a brown signal was confirmed.
An example of an immunostained image of the obtained liver tissue is shown in FIG. In the control group (cirrhotic liver), fibroblasts were clearly accumulated along the pseudolobule, but in the ADAMTS-4 treatment group the accumulation was very mild. From this, it was shown that the excessive accumulation or establishment of fibroblasts in the fibrotic liver was remarkably suppressed by ADAMTS-4 administration.

Example 9: Treatment effect of mouse cirrhosis model by ADAMTS-4 peptide administration (suppression of type III collagen):
A sample of a frozen section of the liver was prepared in the same manner as in Example 8, and immunohistological analysis was performed. Rabbit anti-mouse type III collagen antibody (1: 250 dilution; manufactured by Santa Cruz) was added to the sample as a primary antibody and allowed to react at room temperature for 1 hour. Subsequently, alkaline phosphatase-labeled anti-rabbit IgG (1: 200 dilution; manufactured by Jackson ImmunoResearch) was added as a secondary antibody, and further reacted at room temperature for 30 minutes, followed by alkaline phosphatase chromogenic substrate (Vector Red; Vector Laboratory) Made) was added. The sample was observed under an optical microscope (manufactured by Leica), and antibody binding visualized with a red signal was confirmed.
An example of the obtained immunostained image of liver tissue is shown in FIG. In the control group, type III collagen fibers detected by rabbit anti-mouse type III collagen antibody stretched not only from sinusoids but also from the central vein to the portal vein, and partly formed pseudolobules It was shown that. This was a typical image of cirrhosis. In contrast, almost no elongation of type III collagen was observed in the ADAMTS-4 group.

Example 10: Treatment effect of mouse cirrhosis model by ADAMTS-4 peptide administration (suppression of fibrosis score):
Based on the immunohistological staining performed in Example 9, the degree of liver fibrosis in each sample has been reported (Dai K, et al. World J Gactroenterol. 31: 4822-4826, 2005, Hillebrandt S, et al. Nature Genetics 37: 835-843, 2005). The evaluation criteria for the fibrosis frequency are as follows. 0 degree: normal, 1 degree: a small amount of collagen elongation from the central vein is observed, 2 degrees: obvious collagen elongation from the central vein is observed, but not to surround the entire liver, 3 degrees: central vein 4 degrees: diffuse collagen elongation of the entire liver is observed, forming a pseudolobule.
The results are shown graphically in FIG. Each bar shows the mean value ± standard deviation of the fibrosis frequency in each group. The control group was 3.9 ± 0.4 (n = 6), whereas the ADAMTS-4 treatment group was 2.3 ± 0.4 (n = 6). Compared with the control group, the ADAMTS-4 treatment group (p <0.001, t-test) showed a statistically significant reduction in fibrosis. From the above, it was shown that administration of ADAMTS-4 peptide exerts a clinically excellent liver fibrosis inhibitory effect.

[Possibility of industrial use]
The hepatic fibrosis inhibitor containing chondroitinase ABC according to the present invention as an active ingredient suppresses the fibrosis of liver tissue, thereby preventing a disease associated with excessive fibrosis of the liver tissue (liver fibrosis disease). Effective for treatment or prevention. The hepatic fibrosis inhibitor according to the present invention has the effect of suppressing the extension of type III collagen, the effect of suppressing the deposition of type I collagen, the effect of suppressing the accumulation of fibroblasts in the fibrotic liver tissue, Since it has the effect of suppressing various signs related to fibrosis such as the effect of suppressing accumulation, it is very useful for suppressing liver fibrosis. The method for treating or preventing a liver fibrosis disease by administering the hepatic fibrosis inhibitor of the present invention can effectively improve the fibrotic lesion by drug therapy, and thus can be an excellent therapy useful for improving the QOL of the patient.
All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

It is a photograph which shows the detection result (brown signal) of CSPG, C4SPG, C6SPG, and HSPG in a cirrhosis model mouse induced by carbon tetrachloride. Magnification 100 times. The name of the antibody clone used for detection is shown in parentheses. It is a photograph which shows the detection result (brown signal) of CSPG after the treatment with PBS (chondroitinase (-)), Chase AC, Chase B, or Chase ABC in cirrhotic liver induced by carbon tetrachloride. 200x magnification. It is a photograph which shows the reduction effect of type III collagen (red signal) after Chase ABC administration in the cirrhosis model mouse | mouth induced | guided | derived with carbon tetrachloride. Magnification 100 times. It is a graph which shows the fibrosis frequency after Chase ABC, Chase AC, or Chase B administration in a cirrhosis model mouse induced by carbon tetrachloride. * P <0.05, ** p <0.01 (t-test). It is a photograph which shows the reduction effect of type I collagen (red signal) after Chase ABC administration in a cirrhosis model mouse induced by carbon tetrachloride. Magnification 100 times. It is a graph which shows the type III collagen positive area rate (%) after Chase ABC, Chase AC, or Chase B administration in the cirrhosis model mouse | mouth induced | guided | derived with carbon tetrachloride. * P <0.05, ** p <0.01 (t-test). It is a photograph which shows the detection result (brown signal) of CSPG, C4SPG, and C6SPG after Chase ABC administration in a cirrhosis model mouse induced by carbon tetrachloride. 200x magnification. It is a photograph which shows the change of the distribution after fibroblast distribution (brown signal) and the Chase ABC administration in the cirrhosis model mouse | mouth induced | guided | derived with carbon tetrachloride. Magnification 100 times (upper), 200 times (lower). It is a photograph which shows the distribution of the macrophage in a cirrhosis model mouse induced by carbon tetrachloride (brown signal) and the change in the distribution after Chase ABC administration. 200x magnification. It is a photograph which shows suppression of the fibroblast infiltration by ADAMTS-4 functional peptide administration. ER-TR7 stained image (brown) in liver cirrhosis model liver tissue. Upper row, 100 times. Bottom, 400 times. It is an image in which formation of pseudolobule is suppressed in addition to suppression of cell infiltration by administration of ADAMTS-4 functional peptide. It is a photograph showing suppression of type III collagen growth by administration of ADAMTS-4 functional peptide. Type III collagen staining image (brown) in liver cirrhosis model liver tissue. Hundredfold. This is an image in which formation of pseudolobule is suppressed by administration of ADAMTS-4 functional peptide. It is a figure which shows suppression of the fibrosis by ADAMTS-4 functional peptide administration. It is the graph which digitized the type III collagen staining findings as a fibrosis score. ADAMTS-4 functional peptide administration significantly suppressed liver fibrosis.

Claims (12)

A liver fibrosis inhibitor comprising a substance that inhibits the production or accumulation of chondroitin sulfate proteoglycan as an active ingredient. 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 drug according to any one of claims 1 to 5, wherein production or accumulation of chondroitin sulfate proteoglycan is inhibited in the liver. The drug according to any one of claims 1 to 6, which is used for treatment or prevention of a liver fibrosis disease. The drug according to claim 7, wherein the liver fibrosis disease is chronic liver disease. The drug according to claim 7, wherein the liver fibrosis disease is chronic hepatitis, liver fibrosis, cirrhosis, liver failure, or liver cancer. A method for screening a liver fibrosis inhibitor, comprising 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 liver fibrosis inhibitor is for treatment or prevention of liver fibrosis disease.

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