JP2007291022A - Cartilage differentiation-promoting action - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a medicine suppressing articular destruction or having effective action on diseases of bones or joints such as osteoarthritis. <P>SOLUTION: The present invention provides galectin-9, especially stabilized galectin-9 and galectin-9 inducer, exhibiting cartilage differentiation-promoting action, cartilage destruction-suppressing action or functional maintenance and recovery action of damaged cartilage, in which utilization as therapeutic and prophylactic agent of diseases of bones or joints such as osteoarthritis and utilization to treating and preventing method of the diseases can be expected. It is observed that galectin-9, especially stabilized galectin-9 and galectin-9 inducer act together with TGF-β, etc., having anabolic reaction in intra-articular environment. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a cartilage differentiation promoting agent, a cartilage destruction inhibitor, a cartilage destruction inhibitor, and a function maintenance of damaged cartilage, comprising galectin 9 and a variant thereof (stabilized galectin 9) selected from the group consisting of galectin 9 inducer The present invention relates to a recovery agent and an agent for treating and / or preventing a disease or disorder using the cartilage differentiation promoting action, cartilage destruction inhibiting action, or damaged cartilage function maintaining / restoring action, and a therapeutic and / or preventive method for utilizing its activity.

[Mechanism of cartilage destruction in joint diseases]
Articular cartilage contains an abundant cartilage-specific extracellular matrix such as collagen fibers and is an important tissue that supports motor function, but it is a joint disease represented by rheumatoid arthritis (RA) and osteoarthritis (OA). In arthritis, articular cartilage destruction by synoviocytes and chondrocytes is observed, and bone destruction by osteoclasts progresses in bone tissue under cartilage.

In articular cartilage, the enhanced degradation of cartilage extracellular matrix (eg, aggrecan and collagen) is an essential process for its destruction, and a characteristic destruction image is exhibited by the degradation of cartilage proteoglycan (aggrecan) and subsequent degradation of collagen fibers. Among them, matrix metalloprotease (MMP) has a wide range of substrate specificities and is considered to be the center of cartilage destruction in RA. Recently, the role of aggrecanase, ADAMTS (A Disintegrin And Meta11oproteinase with Thrombospondin Motifs) molecule is also attracting attention. Yes.
These proteases are mainly produced by synovial cells and inflammatory cells in synovial fluid and act on cartilage.In RA synovial fluid, multiple proteases have higher concentrations than osteoarthritis (OA). Detected. On the other hand, protease expression from RA chondrocytes themselves has also been confirmed and plays a role in cartilage destruction in this disease. In addition to matrix destruction by this protease, cell damage by nitric oxide (NO), especially apoptosis, is also considered to be a mechanism of RA cartilage destruction. (Non-Patent Documents 1 and 2).

In osteoarthritis (OA), there is a decrease in extracellular matrix (ECM) due to physical damage of articular cartilage and abnormal metabolism of chondrocytes that produce it, or apoptosis of chondrocytes (see above). Current OA treatment has to rely on temporary symptomatic treatments such as supplements such as cartilage constituents and anti-inflammatory drugs, or surgical treatment in severe cases. On the other hand, in recent years, a large number of basic data related to fundamental treatment has been reported, aiming for the correction of metabolic abnormalities in cartilage tissue.
Usually, TGF beta, which has an anabolic effect, and IL-1, TNF alfa, which has an anabolic effect in the joint environment, are thought to function in a balanced manner. Moreover, the production increase of the inflammatory cytokine which has a catabolic effect is recognized notably (nonpatent literature 3), and high production of iNO which has an apoptosis induction | guidance | derivation action is also recognized in OA joint locality (nonpatent literature 4). It is thought that these factors are suppressed and the progression of the disease state is important for the treatment of OA at an early stage.

[Proteoglycan]
Proteoglycan is a general term for molecules to which glycosaminoglycan (GAG) is covalently bound. It is classified into chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, and hyaluronic acid according to the skeletal structure.
Proteoglycans assemble various molecular groups through sulfated glycosaminoglycan and core protein parts, contribute to tissue construction, and set up a reception environment for various extracellular signal transduction factors. In other words, proteoglycans function not only as an extracellular matrix but also as an important factor in signal transduction.
The most typical proteoglycan is a macromolecule present in articular cartilage called aggrecan containing chondroitin sulfate, and occupies the majority of proteoglycans present in animals. The core protein of aggrecan has a domain (hyaluronan binding site) that specifically binds to hyaluronan (hyaluronic acid), a type of glycosaminoglycan, near the amino terminus, and in the cartilage tissue together with another protein (link protein) It forms a huge aggregate and becomes the source of compressive impact force absorption, an important function of cartilage.
It is also used as a therapeutic application for osteoarthritis (eg, glucosamine hydrochloride, sulfate).

[Galectin 9]
Galectins are a lectin family that has an affinity for β-galactoside and a conserved region on the primary sequence. To date, more than 10 types of mammalian galectins have been discovered, and various biological activities such as cell-cell adhesion or cell-extracellular matrix adhesion, cell activation, cell proliferation, and apoptosis have been reported.
The group of the present inventors succeeded in cloning a human T cell-derived eosinophil migration factor, which is a variant of human galectin 9 (human galectin 9: hGal9, non-patent document 5) reported by Tureci et al. (Non-Patent Document 6). Furthermore, the inventors' group clarified that ekalectin and Gal9 are the same substance, and human Gal9 has three types, short type, medium type, and long type, depending on the length of the link peptide. This was also clarified (Non-Patent Document 7). WO 2004/064857 that Gal9-containing drugs are promising as antitumor agents (anticancer agents), antiallergic agents, immunosuppressive agents, autoimmune disease agents, anti-inflammatory agents and corticosteroid substitutes (2004.08.05) [Patent Document 1] Gal9 has also been reported to induce apoptosis in activated T cells. Furthermore, stabilized Gal9 (Gal9 variant) and its use are disclosed in WO 2005/093064 (2005.10.06) [Patent Document 2].

WO 2004/064857 (2004.08.05) WO 2005/093064 (2005.10.06) CLINICAL CALCIUM 2003 June (Vol.13 No.06) p24 (702) ~ p30 (708) "Special feature of bone diseases with inflammation" Review Mechanism of cartilage destruction in rheumatoid arthritis Harumoto Yamada , Professor of Orthopedic Surgery, Fujita Health University), Yasuo Yoshihara, Lecturer of Department of Orthopedic Surgery, National Defense Medical College Department of Pathology, Keio University School of Medicine, Yasunori Okada, Proteinases in joint destruction, Department of Patho1ogy, School of Medicine, Keio University "Pathophysiological Mechanisms in Osteoarthritis lead to Novel Therapeutic Strategies", Cells Tissues Organs: Vol. 174, No. 1-2, 2003 "Hsp70 prevents Nitric oxide-induced apoptosis in articular chondrocytes", ARTHRITIS &RHEUMATISM; Vol. 48, No. 6, June 2003, pp 1562-1568 Tureci O. et al., J Biol Chem., 1997, 272 (10): 6416-22 Matsumoto R. et al., J Biol Chem., 1998, 273: 16976-84 Matsushita N. et al., J Biol Chem., 2000, 275: 8355-60

In joint diseases, cartilage destruction is observed, and the suppression of the destruction is considered to be important for the suppression and recovery of the progression of the disease state, and the search for an active substance having such a function is required. Also, in the field of osteoarthritis treatment, it is a situation that has to rely on temporary symptomatic treatments such as supplements such as cartilage constituents and anti-inflammatory drugs, or surgical treatment in severe cases. Attempts have been made to search for substances having a cartilage differentiation promoting action based on the results of research aimed at correcting metabolic abnormalities in cartilage tissue.
Many biologically active substances in vivo have been found so far, but many of them act on normal cells as well as, for example, tumor cells. Just because the activity has been elucidated does not make it easy to use it in medicine. While galectin 9 functions differently depending on its localization, galectin 9 is expected to be involved in various biological functions. There is a need to elucidate the detailed biological activity of galectin 9 and to develop related technologies for galectin 9, including the development of pharmaceuticals based on that elucidation.
The present invention relates to an active agent that controls a factor related to a bone or joint disease or exerts a cartilage differentiation promoting action, and a therapeutic and / or prophylactic agent for a bone or joint disease or disorder using the cartilage differentiation promoting action. And therapeutic and / or preventive methods thereof, as well as drugs and therapeutic and / or preventive methods for treating and / or preventing diseases related to bones and joints, such as cartilage destruction, cartilage differentiation, and functional recovery of damaged cartilage The issue is to provide.

  As a result of diligent research to solve the above problems, the present inventors have found that galectin 9, particularly stabilized galectin 9 (for example, G9NC (null)) exhibits a cartilage destruction inhibiting action and a cartilage differentiation promoting action. Succeeded in finding out. That is, galectin 9 was found to be useful as a cartilage differentiation promoting factor, and it was further confirmed that the Gal9 (particularly stabilized galectin 9) also showed a cartilage destruction inhibiting action. Furthermore, they have also been found to be exerted in association with TGF-β such as TGF-β3. Therefore, galectin 9, especially stabilized galectin 9 (for example, G9NC (null)) can suppress cartilage destruction and promote cartilage differentiation, and maintain and restore the function of damaged cartilage. Can also be expected. Thus, galectin 9, especially stabilized galectin 9 (for example, G9NC (null)) shows bone or joint diseases or diseases such as osteoarthritis (OA) by promoting cartilage differentiation promoting action and cartilage destruction inhibiting action. And / or a prophylactic agent and a possibility of providing a therapeutic and / or prophylactic method for its active use.

In the present invention, the following modes are provided.
[1] Function of cartilage differentiation promoting agent, cartilage destruction inhibitor and / or damaged cartilage characterized by containing as an active ingredient galectin 9 and its variants and galectin 9-inducing factor Maintenance / recovery agent.
[2] A cartilage differentiation promoter, a cartilage destruction inhibitor, and / or a nucleic acid encoding a galectin-9 and a variant thereof and a nucleic acid encoding a galectin-9 inducer, and / or An agent that maintains and restores the function of damaged cartilage.
[3] A method for treating chondrocytes, comprising bringing chondrocytes, in particular undifferentiated chondrocytes, into contact with a galectin 9 or a variant thereof and a galectin 9-inducing factor selected to promote differentiation. .
[4] Promotion of chondrocyte differentiation, characterized by introducing into a chondrocyte, particularly an undifferentiated chondrocyte, a nucleic acid encoding a gene selected from the group consisting of galectin 9 and a variant thereof and a galectin 9 inducer Law.
[5] A therapeutic and / or prophylactic agent for bone / joint diseases, comprising as an active ingredient a substance selected from the group consisting of galectin 9 and a variant thereof and a galectin 9 inducer.
[6] A therapeutic and / or prophylactic agent for bone / joint diseases characterized by containing as an active ingredient a nucleic acid encoding galectin 9 and a variant thereof and a galectin 9-inducing factor.
[7] Inhibition of cartilage destruction, wherein cartilage tissue or cells with reduced function or failure are brought into contact with galectin 9 and a variant thereof and a galectin 9 inducer selected from the group consisting of galectin 9 and an inhibitor. Law.
[8] A method for inhibiting cartilage destruction, comprising introducing a nucleic acid encoding a galectin-9 or a variant thereof and a galectin-9-inducing factor into a cartilage tissue or cell having a reduced function or failure. .
[9] The agent or method according to any one of [1] to [8] above, which is in the presence of a TGF-β family such as TGF-β3 or a nucleic acid encoding the same.
[10] A therapeutic and / or prophylactic agent for osteoarthritis comprising an active ingredient selected from the group consisting of galectin 9 and a variant thereof and a galectin 9 inducer.

In the present invention, a technique for controlling cartilage destruction, cartilage differentiation, etc., particularly a technique utilizing a cartilage destruction inhibiting action, a cartilage differentiation promoting action, and / or a function maintaining / recovering action of damaged cartilage, is particularly stable. This was made possible by using a selected galectin-9 (for example, G9NC (null)) and a galectin-9 inducer. Thus, a technique for controlling cartilage differentiation is provided, and a technique for treating and / or preventing a disease caused by cartilage destruction and / or metabolic abnormality of cartilage tissue can be developed. By controlling cartilage differentiation and cartilage destruction with one selected from the group consisting of galectin 9, especially stabilized galectin 9 (eg G9NC (null)), and galectin 9 inducer (eg cartilage differentiation A drug capable of effectively treating and / or preventing the bone and / or joint disease or disorder by promoting or enhancing and / or suppressing or inhibiting cartilage destruction) Development is possible.
Other objects, features, excellence and aspects of the present invention will be apparent to those skilled in the art from the following description. However, it is understood that the description of the present specification, including the following description and the description of specific examples and the like, show preferred embodiments of the present invention and are presented only for explanation. I want. Various changes and / or modifications (or modifications) within the spirit and scope of the present invention disclosed herein will occur to those skilled in the art based on the following description and knowledge from other parts of the present specification. Will be readily apparent. All patent documents and references cited herein are cited for illustrative purposes and are not to be construed as a part of this specification. is there.

  Hereinafter, embodiments of the present invention will be described. Inhibition of cartilage destruction of galectin 9 of the present invention, particularly stabilized galectin 9 (for example, G9NC (null)), and those selected from the group consisting of galectin-9 inducers Cartilage destruction inhibitor, cartilage differentiation promoting agent and / or damaged cartilage function maintaining / restoring agent using function as factor, cartilage differentiation promoting factor and / or damaged cartilage function maintaining / restoring factor (osteoarthritis) A therapeutic agent and an agent for treating and / or preventing a disease or disorder using the cartilage differentiation promoting action, cartilage destruction inhibiting action, and / or damaged cartilage function maintaining / restoring action, and chondrocyte dysfunction Galectin 9 (gallectin 9: Gal9), which is a therapeutic and / or preventive agent for pathologic symptoms and / or diseases (abnormality) caused by the above, and is contained as an active ingredient is, for example, human leukocytes having the ability to produce Gal9 The gene encoding natural galectin 9 (native Gal9 or naturally-occurring Gal9) produced from cultured cell lines and Gal9 derived from the leukocytes or specific cultured cell lines is transformed into animal cells or Escherichia coli by gene recombination technology. Recombinant galectin 9 (recombinant galectin 9: rGal9) obtained by incorporation into a microorganism such as the above, and any of them can be advantageously used. In the present invention, high-purity Gal9 that has been highly purified is not limited, and as long as the intended purpose can be achieved, crude Gal9 containing a pharmaceutically acceptable impurity can also be suitably used. . In addition, the drug of the present invention may be administered parenterally or orally, and not only intramuscular injection and intravenous injection, which are preferably of a relatively high purity, but also need to use the highest purity high purity Gal9. It is also preferable that it is oral which can be used without any disadvantage even if it is relatively low purity Gal9. When such Gal9 is used, the drug of the present invention can be produced at a lower cost. Moreover, it is also possible to use a mixture of two or more types of galectin 9 as the Gal9. From the viewpoint of antigenicity, the Gal9 can be advantageously used as human Gal9 (hGal9).

  In the present specification, “galectin 9: Gal9” typically includes natural Gal9. As natural type Gal9, long type (L type) galectin 9 (galectin 9 long isoform or long type galectin 9: Gal9L), medium type (M type) galectin 9 (galectin 9 medium isoform or medium type galectin 9: Gal9M ) And short type (S type) galectin 9 (galectin 9 short isoform or short type galectin 9: Gal9S) have been reported, but Gal9L is N-terminal by the putative linked peptide region of SEQ ID NO: 4 disclosed in WO 02/37114 A1. The domain (N-terminal carbohydrate recognition domain: NCRD) and the C-terminal domain (C-terminal carbohydrate recognition domain: CCRD) are linked, Gal9M NCRD and CCRD are linked by the putative link peptide region of SEQ ID NO: 5 of WO 02/37114 A1, and Gal9S is linked to NCRD and CCRD by the putative link peptide region of SEQ ID NO: 6 of WO 02/37114 A1. Considered to be concatenated Gal9M differs from Gal9L in that the amino acid residue of SEQ ID NO: 7 of WO 02/37114 A1 is deleted from the linked peptide region of Gal9L, and Gal9S differs from the linked peptide region of Gal9M. It differs from Gal9M in that the amino acid residue of SEQ ID NO: 8 of WO 02/37114 A1 is deleted, that is, Gal9S has an amino acid of SEQ ID NO: 9 of WO 02/37114 A1 from the Gal9L putative linked peptide region It differs from L-type galectin 9 in that the residue is deleted. By the way, the amino acid sequence of Gal9L is SEQ ID NO: 1 disclosed in WO 02/37114 A1, the amino acid sequence of Gal9M is SEQ ID NO: 2 disclosed in WO 02/37114 A1, and the amino acid sequence of Gal9S is WO 02/37 SEQ ID NO: 3 disclosed in 37114 A1 shows the respective typical sequences.

As used herein, galectin 9 includes Gal9L, Gal9M and Gal9S, other naturally occurring variants of these galectin9 families, and further artificial mutations (ie, deletions in one or more amino acid residues). , Addition, modification, insertion, etc.) or those containing a partial domain or partial peptide fragment thereof. All disclosed in WO 2004/064857 (2004.08.05), J. Biol. Chem., 275 (12): pp. 8355-8360 (2000) may be included. For example, GST, FLAG (registered trademark, Sigma-Aldrich), tags such as polyhistidine, luminescent jellyfish-derived green fluorescent protein (GFP) such as Aequorea victorea, and modified variants ( GFP variant), e.g. EGFP (Enhanced-humanized GFP), rsGFP (red-shift GFP), yellow fluorescent protein (YFP), green fluorescent protein (GFP), indigo fluorescent RGal9 fused with protein (cyan fluorescent protein: CFP), blue fluorescent protein (BFP), Renilla reniformis GFP, etc., for example, poly-His-hGal9 Can be mentioned. The galectin 9 includes natural galectin 9 variants, and further, “galectin 9 variant”, “galectin 9 variant polypeptide” disclosed in WO 2005/093064 (2005.10.06), and “galectin 9 variant therapeutic agent” All may be included. Particularly preferred is G9NC (null) manufactured and obtained in Example 1 of WO 2005/093064 (2005.10.06) [encoded by the base sequence represented by SEQ ID NO: 1 of WO 2005/093064 (2005.10.06). And a polypeptide having the amino acid sequence represented by SEQ ID NO: 2]. Among “galectin 9 variant” or “galectin 9 variant polypeptide”, it is recognized that more stable properties can be obtained compared to natural galectin 9, and such Is also referred to herein as “stabilized galectin 9”. Representative examples of the stabilized galectin 9 include G9NC (null) described above, but are not limited thereto, and those showing similar properties may be included therein.
The therapeutic and / or prophylactic agent of the present invention and the therapeutic and / or prophylactic method thereof can be implemented according to the disclosure of WO 2004/064857 (2004.08.05) and WO 2005/093064 (2005.10.06), and The description contained therein is included in the disclosure herein by reference thereto.

As used herein, a galectin-9 inducer is characterized by having an activity that induces the expression of galectin-9. The factor is characterized by significantly inducing the expression of galectin-9 by its presence or its expression, for example, WO 2004/096852 (2004) [WO2004 / 096851, A1 (2004)] And those disclosed in Japanese Patent Application No. 2004-312771 (filing date: October 27, 2004) and PCT / JP2005 / 021942 (filing date: November 22, 2005). Is hereby incorporated by reference into it).
Representative galectin-9 inducers include radiation-treated B cell lymphoma-derived cell lines, such as those obtained from BALL-1 cells, such as fractions that have been separated and purified, and the like. . Those containing the galectin-9 inducing factor include B cell line-derived cell membrane solubilized fractions (membrane fraction: mf, for example, human cell line: BALL-1 established from human acute lymphoblastoid leukemia (ALL)) mf (BALL-1 mf)), mf eluted in its concanavalin A adsorption fraction, mf eluted from Resource Q ^™ ion exchange column, fraction eluted from hydroxyapatite column, and the like. The galectin-9 inducing factor can be confirmed by detecting and measuring its biological activity, for example, galectin-9 inducing activity in vitro or in vivo. For example, in vitro galectin-9-inducing activity can be achieved by stimulating Gal-9 producing / free cells as described above with mf, followed by RT-PCR, Western blotting, flow cytometry, immunohistochemical staining, ELISA Measured by quantitative or qualitative analysis of Gal-9 mRNA and Gal-9 protein by ELISPOT method, RIA method, etc. Using the cell culture solution of the cells, Gal-9 protein can be quantitatively or qualitatively determined by RT-PCR, Western blotting, flow cytometry, immunohistochemical staining, ELISA, ELISPOT, RIA, etc. It can also be analyzed and measured. In vivo galectin-9-inducing activity can be measured using mf to animals such as mice, rats, guinea pigs, rabbits, monkeys, etc., and then using invasion of galectin-9-producing cells and enhancement of Gal-9 release as indicators . It can also be measured using the direct or indirect enhancement of Gal-9 in tumor cells as an indicator. Examples of such animals include experimental animals, and examples of administration methods include intradermal, subcutaneous, intramuscular, intravenous or arterial, intraperitoneal injection, and eating and drinking. The human-derived galectin-9 inducer may include those selected from the group consisting of proteins 80K-H, GRP94, GRP78, GRP58, and S100 calcium-binding protein, and their degradation products.
The therapeutic and / or prophylactic agent and the therapeutic and / or prophylactic method of the present invention are described in WO 2004/064857 (2004.08.05), WO2005 / 093064, A1 (2005), WO2004 / 096851, A1 (2004), Japanese Patent Application 2004. -312771 (filing date: October 27, 2004) and PCT / JP2005 / 021942 (filing date: November 22, 2005) Is included in the disclosure herein.
The invention described in this specification is based on research and patent applications published so far, for example, WO 2004/064857 (2004.08.05), WO2005 / 093064, A1 (2005), WO2004 / 096851, A1 (2004), Reference is made to Japanese Patent Application No. 2004-312771 (filing date: October 27, 2004) and PCT / JP2005 / 021942 (filing date: November 22, 2005), the contents of which are disclosed in this specification. Included in.

  In the present invention, the construction or acquisition of a predetermined nucleic acid (polynucleotide) or a predetermined peptide (polypeptide) using “gene recombination technology”, isolation / sequencing, Can be made. Examples of gene recombination techniques (including recombinant DNA techniques) that can be used in the present specification include those known in the art. For example, J. Sambrook et al., “Molecular Cloning: A Laboratory Manual (2nd edition) (1989) & 3rd edition (2001) ", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; DM Glover et al. Ed.," DNA Cloning ", 2nd ed., Vol. 1 to 4, (The Practical Approach Series), IRL Press, Oxford University Press (1995); edited by the Japanese Biochemical Society, “Sequence Biochemistry Experiment Course 1, Genetic Research Method II”, Tokyo Chemical Doujin (1986); 2. Nucleic acid III (recombinant DNA technology), Tokyo Chemical Doujin (1992); MJ Gait (Ed), Oligonucleotide Synthesis, IRL Press (1984); BD Hames and SJ Higgins (Ed), Nucleic Acid Hybridization, A Practical Approach , IRL Press Ltd., Oxford, UK (1985); BDHames and SJ Higgins (Ed), Transcription and Translation: A Practical Approach (Practical Approach Serie s), IRL Press Ltd., Oxford, UK (1984); B. Perbal, A Practical Guide to Molecular Cloning (2nd Edition), John Wiley & Sons, New York (1988); JH Miller and MP Calos (Ed), Gene Transfer Vectors for Mammalian Cells, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1987); RJ Mayer and JH Walker (Ed), Immunochemical Methods in Cell and Molecular Biology, Academic Press, (1987); RK Scopes et al (Ed), Protein Purification: Principles and Practice (2nd Edition, 1987 & 3rd Edition, 1993), Springer-Verlag, NY; DM Weir and CC Blackwell (Ed), Handbook of Experimental Immunology, Vol.1, 2, 3 and 4, Blackwell Scientific Publications, Oxford, (1986); LA Herzenberg et al. (Ed), Weir's Handbook of Experimental Immunology, Vol. 1, 2, 3 and 4, Blackwell Science Ltd. (1997); RW Ellis (Ed ), Vaccines new approaches to immunological problems, Butterworth-Heinemann, London (1992); R. Wu ed., "Methods in Enzymology", Vol. 68 (Recombinant DNA), Academic Press, New York (1980); R. Wu et al. Ed., "Methods in Enzymology", Vol. 100 (Recombinant DNA, Part B) & 101 (Recombinant DNA, Part C), Academic Press, New York (1983); R. Wu et al. Ed., "Methods in Enzymology", Vol. 153 (Recombinant DNA, Part D), 154 (Recombinant DNA, Part E) & 155 (Recombinant DNA, Part F), Academic Press, New York (1987) JH Miller ed., "Methods in Enzymology", Vol. 204, Academic Press, New York (1991); R. Wu et al. Ed., "Methods in Enzymology", Vol. 218, Academic Press, New York ( 1993); S. Weissman (ed.), "Methods in Enzymology", Vol. 303, Academic Press, New York (1999); JC Glorioso et al. (Ed.), "Methods in Enzymology", Vol. 306, Academic Press, New York (1999); Jeremy Thorner et al. (Ed.), "Methods in Enzymology", Vol. 326 to 328, Academic Press, New York (2000); David R. Engelke et al. (Ed. ), "Methods in Enzymology", Vol. 392, Academic Press, New York (2005), etc. It can be performed by the same method or modified method (the description in them is included in the disclosure of the present specification by referring to it) (hereinafter, all of these are referred to as “gene recombination techniques”).

A wide variety of unicellular and multicellular expression systems (ie, host-expression vector combinations) may be used to produce the galectin-9 polypeptides of the invention, or galectin-9 inducer polypeptides. Possible types of host cells include, but are not limited to bacteria, yeast, insects, mammals, plants, and the like. In the case of E. coli, examples include those derived from E. coli K12 strain or B strain, such as NM533, XL1-Blue, C600, DH1, DH5, DH11S, DH12S, DH5α, DH10B, HB101, MC1061, JM109, Examples of the STBL2 and B834 strains include BL21 (DE3) pLysS, and an expression vector suitable for them can be selected and used. The yeast may include baker's yeast, fission yeast and the like, for example, the genus Saccharomyces, the genus Schizosaccharomyces, and the genus Pichia. More specifically, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, and the like can be selected and used. Representative expression vectors include, for example, pBR322, pUC18, pUC19, pUC118, pUC119, pSP64, pSP65, pTZ-18R / -18U, pTZ-19R / -19U, pGEM-3, pGEM-4, pGEM-3Z, pGEM-4Z, pGEM-5Zf (-), pGEMEX-1 (Promega), pBC KS (Stratagene), pBC SK (Stratagene), pBluescript ^TM SK (Stratagene), pBluescript ^TM II SK (Stratagene), pBluescript ^TM II KS (Stratagene), pBS (Stratagene), pAS, pKK223-3 (Amersham Pharmacia Biotech), pMC1403, pMC931, pKC30, pRSET-B (Invitrogen), pSE280 (Invitrogen), pBTrp2 (Boehringer Mannheim), pBTac1 (Boehringer Mannheim), pBTac2 (Roche), pGEX (Amersham Biosciences), pQE series (QIAGEN), pET Expression System (Novagen), YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15, and invitrogen vectors for Mammalian Expression (e.g. BsdCassette ^TM Vectors, Epitope-Tagged pcDNA ^TM Vectors, Eukaryotic TA Expression Kit, Flp-In ^TM Expression Vectors and Kits, Flp-In ^TM T-REx ^TM Expression Vectors, FreeStyle ^TM 293 Expression System, GeneSwi tch ^TM System, pBC1 Milk Expression Vector Kit, pBudCE4.1, pcDNA ^TM Gateway ^TM Vectors, pcDNA ^TM 3.1 Directional TOPO ^TM Expression Kit, pcDNA ^TM 3.1-E Echo ^TM Expression Vector Kit, pcDNA ^TM 3.1 / V5-His TOPO ^TM Expression Kit, pcDNA ^TM 4 / HisMax and pcDNA ^TM 4 / HisMax TOPO ^TM TA Expression Kit, pcDNA ^TM 4 / TO-E Echo ^TM Vector Expression Kit, pcDNA ^TM 6 BioEase ^TM Gateway ^TM Biotinylation System, pcDNA / V5-GW / D- TOPO ^TM Vectors, pCEP4 and pREP4, pDisplay ^TM Vector, pEF6 / V5-His TOPO ^TM TA Expression Kit, pFRT / lacZeo and pFRT / lacZeo2, pSecTag2 and pSecTag2 / Hygro, pShooter ^TM Vectors, pVAX ^TM 200-DEST Vector System, pVAX1 , pZeoSV2, T-REx ^TM Gateway ^TM Vectors, T-REx ^TM System, Tag-On-Demand ^TM Technology, Untagged pcDNA ^TM Vectors, Vivid Colors ^TM pcDNA ^TM 6.2 Fluorescent Protein Gateway ^TM Destination Vectors, ZeoCassette ^TM Vectors etc.), Clontech vectors (for ^{example, Adenoviral Expression Vectors, Creator TM System} Vectors, IRES Bicistronic Expression Vectors, Living Colors TM Fluorescent P rotein Vectors, MATCHMARJER Vectors, Retroviral Expression Vectors, Signal Transduction Vectors, Tet Expression System Vectors, BacPak Baculovirus Expression Vectors, HAT Protein Expression System Vectors), HaloTag ^TM pHT2 Vector, pACT Vectors, pBIND Vectors, pCAT ^TM 3 Vectors, pCI Vectors , phRG Vectors, phRL Vectors (Promega Corp.), Novagen vectors for protein expression (for example, pTriEX ^TM Multisystem Expression Vectors, pBiEX ^TM Multisystem Expression Vectors, pTandem ^TM -1 Vector, pTK-neo DNA, etc.) In addition, those known in the field or commercially available can be appropriately selected and used.

  The target product contained in cells (including cultured cells), culture supernatant, or extract can be purified by appropriately combining per se known separation and purification methods, such as ammonium sulfate precipitation. Gel filtration by salting out, Sephadex, etc., for example, ion exchange chromatography using a carrier having a diethylaminoethyl group or carboxymethyl group, for example, a carrier having a hydrophobic group such as butyl group, octyl group, phenyl group, etc. It can be obtained by purifying by a hydrophobic chromatography method, dye gel chromatography method, electrophoresis method, dialysis, ultrafiltration method, affinity chromatography method, high performance liquid chromatography method, etc. Preferably, it can be purified and separated by treatment with polyacrylamide gel electrophoresis, affinity chromatography with a ligand or the like immobilized. Examples of the ligand include an antibody including a monoclonal antibody that specifically recognizes or a fragment thereof, a lectin, a sugar, and one of a binding pair. Examples thereof include immunoaffinity chromatography, gelatin-agarose affinity chromatography, heparin-agarose chromatography and the like. In particular, it can be produced as a fusion protein or polypeptide using genetic recombination technology, using a fusion part (tag), and using a specific binding ligand such as an antibody, affinity chromatography, etc. It can be easily purified.

  Galectin 9 and its variants, as well as galectin 9 inducer, may be chemically synthesized. For example, methods known in the peptide synthesis field, for example, chemical synthesis methods such as a liquid phase synthesis method and a solid phase synthesis method can be used for the synthesis of a protein and a partial peptide thereof. In such a method, for example, a resin for protein or peptide synthesis is used, and appropriately protected amino acids are sequentially bonded onto the desired amino acid sequence on the resin by various known condensation methods. For the condensation reaction, various activating reagents known per se are preferably used. As such reagents, for example, carbodiimides such as dicyclohexylcarbodiimide can be preferably used. When the product has a protecting group, the desired product can be obtained by removing the protecting group as appropriate. Similarly, a nucleic acid encoding a galectin-9 and a variant thereof and a member selected from the group consisting of a galectin-9 inducer may be chemically synthesized. For example, it can be chemically synthesized by a method such as a phosphophotoester method, a phosphodiester method, a phosphite method, a phosphoramidite method, or a phosphonate method. It is known that normal synthesis can be conveniently carried out on a modified solid support, for example using an automated synthesizer, which is commercially available. Within the sequence of the nucleic acid encoding galectin 9, and variants thereof, and those selected from the group consisting of galectin 9 inducers, for example, modified codons suitable for suitable expression in the selected host cell ( For example, codon substitution), restriction enzyme sites may be provided, and target control genes, such as expression control sequences and regulatory sequences for facilitating the expression of target genes. Convenient or useful, including sequences useful for controlling linkers, adapters, etc., antibiotic resistance, etc., controlling auxotrophy / metabolism, sorting / detection, etc. Modification may be given.

When the active ingredient of the present invention is used as a medicine, it can be administered as a pharmaceutical composition or a pharmaceutical preparation, usually alone or mixed with various pharmacologically acceptable adjuvants. Preferably, it is administered in the form of a pharmaceutical preparation suitable for use such as oral administration, topical administration, or parenteral administration, and depending on the purpose, any dosage form (including inhalation or rectal administration) is preferred. Good. Each of the active ingredients of the present invention may be used as a mixture, for example, a mixture of galectin 9 and a galectin 9 inducer, a mixture of native galectin 9 and a galectin-9 variant, and the like.
In addition, the active ingredient of the present invention includes various drugs such as antitumor agents (anticancer agents), antibiotics, tumor transfer inhibitors, thrombus formation inhibitors, joint destruction treatment agents, osteoarthritis treatment agents, analgesics, It can also be used in combination with anti-inflammatory agents, antiallergic agents, immunomodulators and / or immunosuppressants, and they can be used without limitation as long as they have an advantageous function, for example, known in the art You can choose from things.
The parenteral dosage form may include topical, transdermal, intravenous, intramuscular, subcutaneous, intradermal or intraperitoneal administration, but can also be administered directly to the affected area. Is also preferred. Preferably, orally or parenterally to mammals including humans (eg, intracellular, tissue, intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathoracic, intrathecal, instillation, injection Administration to the intestines, rectum, ear drops, eye drops, nose, teeth, skin and mucous membranes, etc.). Specific preparation preparation forms include solution preparations, dispersion preparations, semi-solid preparations, granular preparations, molded preparations, leachable preparations, etc., for example, tablets, coated tablets, sugar-coated preparations, Agents, troches, hard capsules, soft capsules, microcapsules, implants, powders, powders, granules, fine granules, injections, solutions, elixirs, emulsions, irrigation agents, syrups, Water solution, emulsion, suspension, liniment, lotion, aerosol, spray, inhalant, spray, ointment, plaster, patch, pasta, poultice, cream, oil, suppository ( For example, rectal suppositories), tinctures, skin preparations, eye drops, nasal drops, ear drops, coatings, infusions, liquids for injection, freeze-dried preparations, gel preparations, etc. Can be mentioned.

The pharmaceutical composition can be formulated according to a usual method. For example, as necessary, physiologically acceptable carriers, pharmaceutically acceptable carriers, adjuvant agents, excipients, excipients, diluents, flavoring agents, fragrances, sweeteners, vehicles, preservatives, stable Agent, binder, pH adjuster, buffer, surfactant, base, solvent, filler, extender, solubilizer, solubilizer, isotonic agent, emulsifier, suspending agent, dispersant , Thickeners, gelling agents, curing agents, absorbents, adhesives, elastic agents, plasticizers, disintegrating agents, propellants, preservatives, antioxidants, sunscreen agents, moisturizers, relaxation agents, antistatic agents, By using a soothing agent alone or in combination with the protein of the present invention and the like, it can be produced into a unit dosage form required for the practice of a generally accepted formulation.
Formulations suitable for parenteral use include sterile solutions or suspensions of the active ingredient with water or other pharmaceutically acceptable media such as injections. In general, water, saline, dextrose aqueous solution, other related sugar solutions, and glycols such as ethanol, propylene glycol, and polyethylene glycol are preferable liquid carriers for injections. When preparing an injection, the solution is prepared by a method known in the art using a carrier such as distilled water, Ringer's solution, physiological saline, an appropriate dispersing agent or wetting agent and suspending agent. Injectable forms such as suspensions and emulsions.

Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride, etc.), and are pharmacologically acceptable. It may be used in combination with an appropriate solubilizing agent such as alcohol (for example, ethanol), polyalcohol (for example, propylene glycol, polyethylene glycol, etc.), nonionic surfactant (for example, polysorbate 80 ^™ , HCO-50, etc.) . Examples of the oily liquid include sesame oil and soybean oil. As a solubilizing agent, benzyl benzoate, benzyl alcohol and the like may be used in combination. Further, a buffer (for example, phosphate buffer, sodium acetate buffer, etc.) or a reagent for adjusting osmotic pressure, a soothing agent (for example, benzalkonium chloride, procaine, etc.), a stabilizer (for example, human) Serum albumin, polyethylene glycol, etc.), preservatives (eg, benzyl alcohol, phenol, etc.), antioxidants such as ascorbic acid, absorption promoters and the like. The prepared injection solution is usually filled in a suitable ampoule.

For parenteral administration, a solution in a sterile pharmaceutically acceptable liquid such as water, ethanol or oil, with or without the addition of surfactants and other pharmaceutically acceptable auxiliaries, or Formulated in the form of a suspension. The oily vehicle or solvent used in the formulation includes natural, synthetic or semi-synthetic mono-, di- or triglycerides, natural, semi-synthetic or synthetic fats or fatty acids, such as peanut oil, corn oil, large Examples include vegetable oils such as bean oil and sesame oil. For example, this injection can be prepared so that it usually contains about 0.1 to 10% by weight of the compound of the present invention.
Preparations suitable for topical, eg oral, or rectal use include, for example, mouthwash, toothpaste, oral spray, inhalant, ointment, dental filler, dental coating, dental paste, suppository, etc. Is mentioned. The mouthwash and other dental agents are prepared by a conventional method using a pharmacologically acceptable carrier. As an oral spray and an inhalant, the compound of the present invention itself or a pharmacologically acceptable inert carrier can be dissolved in an aerosol or nebulizer solution, or can be administered to teeth as a fine powder for inhalation. The ointment is prepared by a conventional method by adding a commonly used base such as an ointment base (white petrolatum, paraffin, olive oil, Macrogol 400, Macrogol ointment, etc.) and the like.

Drugs for topical application to teeth, skin can be formulated into solutions or suspensions in appropriately sterilized water or non-aqueous excipients. Additives include, for example, buffering agents such as sodium bisulfite or disodium edetate; antiseptics including antibacterial and antifungal agents such as acetic acid or phenylmercuric nitrate, benzalkonium chloride or chlorohexidine and hypromelrose A thickener is mentioned.
Suppositories are carriers well known in the art, preferably non-irritating suitable excipients such as polyethylene glycols, lanolin, cocoa butter, fatty acid triglycerides, etc., preferably solid at normal temperature but at the temperature of the intestinal tract. Although it is prepared by a conventional method using a liquid that melts in the rectum and releases a drug, it is usually prepared so as to contain about 0.1 to 95% by weight of the compound of the present invention. Depending on the excipient and concentration used, the drug can be suspended or dissolved in the excipient. Adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle. Examples of preparations suitable for oral use include solid compositions such as tablets, pills, capsules, powders, granules, and troches, and liquid compositions such as liquids, syrups, and suspensions. Can be mentioned. When preparing the formulation, formulation adjuvants known in the art are used. Tablets and pills can also be manufactured with an enteric coating. When the dispensing unit form is a capsule, a liquid carrier such as fats and oils can be further contained in the above-mentioned type of material. In the preparation for oral administration, a stabilizer is preferably blended. The stabilizer refers to a drug capable of stabilizing galectin 9 and its variants and / or galectin 9 inducer, for example, glucose, galactose, xylose, fructose, sucrose, maltose, trehalose, neotrehalose, isotrehalose , Sugars and sugar alcohols such as sorbitol, mannitol, maltitol, lactitol, lactosucrose, maltooligosaccharides and polysaccharides, cyclodextrins, hydroxyethyl starch, dextrin and dextran, sodium glucuronate, phosphates and metal salts One or more selected from serum albumin, gelatin, amino acids, nonionic surfactants, and the like can be used.

In addition, when the active ingredient is a protein or polypeptide, polyethylene glycol (PEG) is particularly useful for binding to it because it has extremely low toxicity in mammals. In addition, when PEG is conjugated, the immunogenicity and antigenicity of the heterologous compound may be effectively reduced. The compound may be provided in a microcapsule device. Polymers such as PEG can be α-amino groups of amino-terminal amino acids, ε-amino groups of lysine side chains, carboxyl groups of aspartic acid or glutamic acid side chains, α-carboxyl groups of amino acids at the carboxy terminal, or certain types It can be conveniently attached to an activated derivative of a glycosyl chain attached to an asparagine, serine or threonine residue.
Many activated forms of PEG suitable for direct reaction with proteins are known. PEG reagents useful for reacting with amino groups of proteins include active esters of carboxylic acids, carbonate derivatives, especially those with leaving groups of N-hydroxysuccinimide, p-nitrophenol, imidazole, or 1-hydroxy-2- Examples thereof include nitrobenzene-4-sulfonate. Similarly, PEG reagents containing aminohydrazine or hydrazide groups are useful for reaction with aldehydes generated by periodate oxidation in proteins.

  A gene therapy vehicle can be conveniently obtained using genetic recombination techniques, and is a coding sequence (eg, galectin-9 code) that is the therapeutic agent of the present invention to be delivered to the mammal for expression in the mammal. For delivery of a construct comprising a sequence, or a galectin 9 inducer coding sequence), or all or part of galectin 9 (or galectin 9 inducer) and for delivery For delivery of the construct also comprising a nucleic acid sequence, which can be administered either locally or systemically. These constructs can utilize viral vector approaches or non-viral vector format approaches, in vivo or ex vivo. Such coding sequences can be expressed by induction using endogenous mammalian promoters or heterologous promoters. Expression of the coding sequence in vivo is either done constitutively or regulated. When galectin 9 (or galectin 9 inducer) is expressed in mammals, it can be expressed as soluble galectin 9 (or soluble galectin 9 inducer) and in some cases membrane-bound galectin 9 ( Alternatively, it can be expressed as a membrane-bound galectin-9 inducer). In both or any of these, they are, for example, all galectin-9 (or galectin-9 inducer), or a biologically active portion, variant, variant of galectin-9 (or galectin-9 inducer) , Derivatives, or fusions. Those intended for co-expression of the required gene may also be included. Accordingly, a TGF-β family such as TGF-β3 that can be introduced into the gene together may be included. Since galectin 9 or a galectin 9 variant (or galectin 9-inducing factor) has a TGF-β signal downstream factor expression / activation action, a technique using it may also be included in the present invention.

  The present invention provides a gene transfer vector capable of expressing the required galectin-9 nucleic acid sequence and / or galectin-9 inducer nucleic acid sequence. The gene transfer vector is preferably a viral vector, more preferably a viral vector such as a retrovirus, adenovirus, adeno-associated virus (AAV), herpes virus, or alphavirus. Examples of viral vectors further include viral vectors such as astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, and togavirus, such as HVJ Envelope Vector (Ishihara). Industry). Regarding the gene transfer vector, generally, D. Jolly, Cancer Gene Therapy, 1 (1): 51-64 (1994); O. Kimura et al., Human Gene Therapy, 5: 845-852 (1994); Reference can be made to S. Connelly et al., Human Gene Therapy, 6: 185-193 (1995); MG Kaplitt et al., Nature Genetics, 8: 148-153 (1994).

In the present invention, the bone / joint disease may be a general term for diseases that cause main lesions in the whole body or joints, and which has an abnormality of bone and cartilage forming the skeleton as one of the symptoms. More specifically, it can also be defined as a disease in which bone differentiation and cartilage differentiation are associated with the onset of lesions. Representative bone and joint diseases can include osteoarthritis, rheumatoid arthritis, and the like. Juvenile rheumatoid arthritis, psoriatic arthritis, Reiter syndrome, systemic lupus erythematosus (SLE), progressive systemic sclerosis, Charcot joint (neuropathic arthropathy), CPPD crystal deposition disease, BCP crystal deposition disease, or gout In some cases, arthritis is accompanied by one of the lesions and may be included in the bone / joint disease in the present invention. In addition, osteoporosis is also included in the bone / joint diseases in the present invention in terms of the relationship between bone differentiation and lesions.
Osteoarthritis is a disease characterized by articular cartilage degeneration, wear and subchondral bone sclerosis, proliferative changes, and secondary synovitis is also observed. Osteoarthritis is considered to be a multifactorial disease based on aging. In addition to aging, gender (female), obesity, trauma (ligament, meniscus injury, etc.) are considered as risk factors. However, there are many unclear points in the etiology. It is known that osteoarthritic articular cartilage has increased expression and mineralization of hypertrophic cartilage markers that are not found in normal permanent cartilage, and cartilage hypertrophy (increased differentiation) is caused by osteoarthritis. It has been implicated in the onset. Therefore, it is possible to improve osteoarthritis symptoms by controlling cartilage differentiation.
In the specification and drawings, the terms are based on the meanings of terms used by the IUPAC-IUB Commission on Biochemical Nomenclature or conventionally used in the art.

The present invention will be described in detail with reference to the following examples, which are provided merely for the purpose of illustrating the present invention and for reference to specific embodiments thereof. These exemplifications are for explaining specific specific embodiments of the present invention, but are not intended to limit or limit the scope of the invention disclosed in the present application. In the present invention, it should be understood that various embodiments based on the idea of the present specification are possible.
All examples were performed or can be performed using standard techniques, except as otherwise described in detail, and are well known and routine to those skilled in the art. .
In the following examples, unless otherwise indicated, specific procedures and processing conditions are described in DNA cloning by J. Sambrook et al., “Molecular Cloning: A Laboratory Manual (2nd edition (1989) & 3rd edition (2001) ", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York and DM Glover et al. Ed.," DNA Cloning ", 2nd ed., Vol. 1 to 4, (The Practical Approach Series), IRL Press, Oxford University Press (1995); When using PCR, HA Erlich ed., PCR Technology, Stockton Press, 1989; DM Glover et al. Ed., "DNA Cloning", 2nd ed., Vol. 1, (The Practical Approach Series), IRL Press, Oxford University Press (1995) and MA Innis et al. Ed., "PCR Protocols", Academic Press, New York (1990). In addition, when commercially available reagents or kits are used, the attached protocols (protocols) and attached chemicals are used.
G9NC (null) is International Publication No. 2005/093064 Pamphlet (2005) (WO2005 / 093064, A1 (2005), Publication Date: October 06, 2005: PCT / JP 2005/006580 (Filing Date 2005.03.29)). And manufactured and obtained in Example 1 of WO2005 / 093064.

1) In mice / collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) which are rheumatoid arthritis models, significant cartilage destruction occurs, but suppression of cartilage destruction was observed by administration of stabilized galectin 9 (FIG. 1). ). In FIG. 1, in both collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA), while significant cartilage destruction is observed, by administration of stabilized galectin 9 (galectin-9 variant, G9NC (null)) Inhibition of cartilage destruction is observed.
〔Method〕
Each sample was fixed with formalin and embedded in paraffin. Slicing is performed at 3 μm, and after deparaffinization and dexylene processes, it is immersed in iron hematoxylin solution for 2 minutes and washed with water. Further, 0.02% fast green staining was allowed to react for 10 minutes, then washed 2 or 3 times with 1% acetic acid solution, and stained in 0.1% safranin-O aqueous solution for 10 minutes. Thereafter, it was dehydrated, sealed and observed.

  It was described in WO2005 / 093064 (publication date: 2005.10.06) that stabilized galectin 9 has an anti-inflammatory effect and a bone destruction-inhibiting action in human rheumatoid arthritis models including mouse CIA and CAIA models. In the present invention, newly stabilized galectin 9 (G9NC (null)) not only suppresses inflammation and bone destruction but also has an effect of inhibiting cartilage destruction by newly staining the joint tissue of the mouse model of the disease with safranin O. I found it. The sample used here was stabilized in a mouse that developed according to “1. Antibody cocktail induction model: galectin variant (iv administration)” in Example 31 of WO2005 / 093064 (publication date: 2005.10.06). A sample in which galectin 9 (galectin-9 variant, 30 μg / mouse) was administered ip and administered once on day 0, and “2-1. CIA (collagen) in Example 31 of WO2005 / 093064 (publication date: 2005.10.06) Sensitization) Model: A sample in an experiment according to “Galectin variant (ip administration)” was used. In both models, stabilized galectin 9 significantly suppressed the onset.

2) Expression of galectin 9 for staining of human lesion tissue Safranin-O (Safranin-O) staining of the extracellular matrix of cartilage lesions of osteoarthritis patients was performed and compared with normal regions. FIG. 2 shows a comparison result of human cartilage part: normal part and OA lesion part. As a result of safranin O staining, the number of sites with positive safranin 0 staining was reduced in OA lesions compared to normal areas. In the lesion, the hyaline cartilage area is reduced. The causes are thought to be chondrocyte organization and decreased production of substrate due to apoptosis.
〔Method〕
Samples from osteoarthritis patients were used. The method was performed as in FIG.

FIG. 3 shows the results of examining the expression of galectin 9 and HSP-70 in the OA lesion cartilage. In normal cartilage tissues, no expression / localization of galectin 9 was observed. On the other hand, in the OA lesion cartilage, localization in the surviving chondrocytes was confirmed. HSP70 shows the same localization as galectin 9 in OA lesions.
〔Method〕
Articular cartilage samples from osteoarthritis patients were fixed with formalin and embedded in paraffin. Slicing was performed at 3 μm, followed by deparaffinization and dexylene steps, and then fixed with 4% PFA / PBS (−) for 30 minutes. The plate was washed with PBS (−) and blocked with 3% BSA / PBS (−) at 37 ° C. for 1 hour. Subsequently, anti-human galectin 9 polyclonal antibody (Galpharma) diluted 100% (1.2 μg / mL) with 3% BSA / PBS (−), anti-HSP70 antibody (abcam; Cat. No, ab5439-50) diluted 100 times ) And antigen-antibody reaction was performed at 4 ° C overnight. After the primary antibody reaction, it was washed with PBS (−). For secondary antibodies, anti-rabbit antibody (ENVISION System HRP Rabbit, Dako, Carpinteria, California) and HRP-labeled anti-mouse antibody (ENVISION System HRP mouse, Dako, Carpinteria, California) were applied to the samples stained with galectin 9 and HSP70, respectively. Used and processed at 37 ° C. for 1 hour. Histofine DAB substrate kit (Nichirei Bioscience) was used for color development. After coloring, it was sealed and observed.

Our group has revealed that HSP70 is a factor that binds to galectin 9 (WO2006 / 009309: publication date 2006.1.26). Many survival activities of chondrocytes expressing HSP70 have already been reported, and gene therapy applications using viruses, vectors, etc. using the gene as a transgene are expected.
A variety of functions of the heat shock protein (HSP) family of stress response proteins have been reported so far. Among them, HSP70 has been reported to inhibit caspase3, which is involved in apoptosis in articular chondrocytes, and is treated for OA. There have been many reports on the development of gene transfer methods with an eye toward application.
1) Pathophysiological Mechanisms in Osteoarthritis lead to Novel Therapeutic Strategies.Cells Tissues Organs: Vol. 174, No. 1-2, 2003
2) Hsp70 prevents Nitric oxide-induced apoptosis in articular chondrocytes ARTHRITIS &RHEUMATISM; Vol. 48, No. 6, June 2003, pp 1562-1568
3) Induction of heat shock protein 70 (Hsp70) by proteasome inhibitor MG 132 protects articular chondrocytes from cellular death in vitro and in vivo. Biorheology. 2004; 41 (3-4): 521-34.
4) Genes transfer with HSP70 in rat chondrocytes confers cytoprotection in vitro and during experimental osteoarthritis The FASEB Journal Research communication 66 Vol.20 January 2006
5) Kubo, T., Arai, Y., Takahashi, K., Ikeda, T., Ohashi, S., Kitajima, I., Mazda, O. Takigawa, M., Imanishi, J. and Hirasawa, Y. : Expression of transduced HSP70 gene protects chondrocytes from stress.Journal of Rheumatology (2001) 28, 330-335.

3) Stabilized galectin 9 promotes the differentiation-inducing action of human mesenchymal stem cells (hMSCs) into chondrocytes by TGF-β.
(1) Construction of evaluation system (hMSCs in vitro cartilage differentiation system)
a) Three-dimensional culture method: The Milomass pellet culture method was used.
・ Outline of the method: As shown in FIG.
-Confirmation of construction system: as shown in Fig. 5: Morphological observation In Fig. 5, abundant cartilage-specific extracellular matrix (ECM) was detected 21-28 days after addition of TGF-β3, and Chondroid cells surrounded by ECM were observed (Day 28). It was confirmed that differentiation into cartilage was induced.
[References]:
1) Multi lineage potential of adult human mesenchymal stem cells Science; 284 (1999) 143-147
2) Cartilage-derived morphogenetic protein-1 promotes the differentiation of mesenchymal stem cells into chondrocytes Biochemical and Biophysical Research Communications 325 (2004) 453-460
-Expression of galectin 9: The results shown in Fig. 6 were obtained.
From FIG. 6, in human mesenchymal stem cells miromass pellet culture (under cartilage differentiation conditions), endogenous galectin-9 expression was observed at the early stage of differentiation (GAG protein detection time). It was found to decrease with cartilage differentiation. It was suggested that galectin 9 is involved in cartilage differentiation.
-In addition, as a typical three-dimensional culture method, there is an alginate bead method.

(2) Action of stabilized galectin 9
a) Pellet size: as shown in FIG.
From the graph shown in FIG. 7, the cell aggregation action is essential in cartilage differentiation and is an important phenomenon. Compared to the case of stimulation with TGF-β3 alone, a significant increase in pellets was observed in the system to which stabilized galectin 9 was added. This phenomenon is not observed with stabilized galectin 9 alone.

b) Effect on glucosaminoglucan (GAG) production: shown in FIGS. 8 and 9 (concentration dependence).
From the graph shown in FIG. 8, the production of glucosaminoglucan (GAG) was induced with the differentiation into chondrocytes, and the addition of the stabilized galectin 9 increased the production of GAG. Only stabilized galectin 9 does not induce GAG production. From the graph shown in FIG. 9, a cartilage-specific extracellular matrix production promoting action was observed depending on the concentration of the stabilized galectin 9 (stabilized galectin 9 density-dependent cartilage-specific extracellular matrix production promoting action. ).
Also in the RT-PCR method, it was confirmed that stabilized galectin 9 has a cartilage-specific extracellular matrix production promoting effect in a concentration-dependent manner.

c) Influence on collagen production: as shown in FIGS. 10 and 11 (concentration dependence).
FIG. 10 shows the results of examining the effect of stabilized galectin 9 on the production of Type II Collagen and Type I Collagen. Type I collagen is produced in immature cell masses of cartilage differentiation, It declines as it progresses, and is no longer recognized in later stages of differentiation. On the other hand, Type II collagen is observed in the middle to late differentiation (day 21-). In the system in which stabilized galectin 9 was added to TGF-β3 after 21 days of culture, more type II collagen was produced than in the case where TGF-β3 alone was added, and promotion of cartilage differentiation was confirmed.
FIG. 11 shows the results of analyzing the influence of stabilized galectin 9 using the degree of differentiation as the cell morphology and the production of proteoglycan and collagen as indicators. Abundant production of ECM and cells surrounded by it were observed under the condition of adding 100 nM of stabilized galectin-9 (Alcian Blue staining). When 10 nM of stabilized galectin 9 was added, a region in which ECM production was not observed was observed, but when 100 nM was added, ECM production was uniformly observed in the pellet. Type II collagen was locally observed under the condition of 10 nM stabilized galectin-9 added. Under the condition of adding 100 nM, it was observed at the edge of the pellet.

d) Expression and activation of TGF-β3 downstream factor: as shown in FIG.
FIG. 12 shows the influence on the signal of stabilized galectin 9 in the induction of cartilage differentiation. TGF-β signal downstream factor expression / activation effect of stabilized galectin 9 was examined. Stabilized galectin 9 promotes activation of TGF beta downstream factor, which is important for cartilage differentiation. In particular, p38 and smad2 showed this effect even in the absence of TGF-β3. In addition, for ERK1 / 2 expression, in the presence or absence of TGF-β3, the expression was promoted depending on the stabilized galectin 9 concentration.

〔Method〕
[Evaluation of action of stabilized Gal-9 on mesenchymal stem cell (hMSC) cartilage differentiation]
* Culture methods are common items
1) Culture method
hMSC (Cat. No. CCS-PT-2501), basal medium for mesenchymal stem cells (Cat. No. CCM-PT-3001), and differentiation medium kit (Cat. No. CCM-PT-3003) are Takara Bio. The product was purchased from the company and cultured according to the attached protocol. The pre-culture was performed after calculating the required number of cells and then culturing in a basal medium for mesenchymal stem cells until the cells were 90% confluent. Moreover, all the hMSC cells used in this study were those within 5 passages.

1-1) Dish culture
hMSC cells were seeded at 1.0 × 10 ⁴ cells and 3.0 × 10 ⁵ cells in an 8 wells chamber slide (Nalge Nunc) or 10 cm culture dish, respectively, and cultured to 90% confluence. Thereafter, the cells were cultured and fixed under the conditions where TGF-β3 was added / not added at 10 ng / mL, and stabilized Gal-9 was added / not added at 0, 1, 10, 100 nM.

1-2) .Pellet culture
1. Prepare a cartilage differentiation inducing medium (hereinafter referred to as cartilage differentiation inducing medium (-)).
An additive factor set (1 μM dexamethasone, 1 mM sodium pyruvate, 0.17 mM ascorbic acid-2-phosphate, 0.35 mM proline, 6.25 μg / mL bovine insulin) was added to the basic medium in the differentiation medium kit, and cartilage differentiation induction medium ( -) Was produced. A cartilage differentiation induction medium (+) was prepared by adding TGF-β3 to final 10 ng / mL to this cartilage differentiation induction medium (−) and used for the cartilage differentiation induction experiment.
2. Remove the pre-cultured cells from the dish and transfer to a 50 mL polypropylene tube.
3. Wash hMSCs with an appropriate amount of cartilage differentiation-inducing medium (-).
4. Centrifuge the cells at 150 x G for 5 minutes at room temperature and discard the supernatant.
5. Suspend MSCs in cartilage differentiation induction medium (-) to a concentration of 5.5.0 × 10 ⁵ cells / mL.
6. Apply 0.5 ml (2.5 × 10 ⁵ cells / pellet) of cell suspension in a 15 ml polypropylene culture tube.
7. Centrifuge the cells at 150 x G for 5 minutes at room temperature.
Cultivate under conditions of 8.37 ° C and 5% CO ₂ * Loosen the cap of the tube about 1/2 turn to allow gas exchange
9. After leaving for 24 hours (exhibiting a round shape), add the first cartilage differentiation-inducing medium (+).
* This timing is set to Day 0, and the stabilization galectin 9 addition process is performed simultaneously. The added concentrations were 0, 1, 10, 100 nM (or only 100 nM), and each stabilized galectin 9 was also added when the medium was changed.
10. After changing the medium, tap the bottom of each tube to float the cells. Loosen the cap as above and return to the 37 ° C incubator. The medium was changed every 3 to 4 days.
11. Proteoglycan production is histologically observed about 12-14 days after the start of culture, and abundant type 2 collagen is confirmed after about 21-28 days. Samples were collected at an appropriate timing (see each method item) according to the purpose.

2) Immunohistochemical staining with anti-galectin 9 antibody
hMSC cells were seeded on 8 wells chamber slide (Nalge Nunc) 1.0 × 10 ⁴ and cultured until aggregation was confirmed. The cells were fixed with 4% PFA / PBS (−), washed with PBS (−), and blocked with 3% BSA / PBS (−) at 37 ° C. for 1 hour. After that, anti-human galectin 9 polyclonal antibody (Galpharma) diluted 100% (1.2 μg / mL) with 3% BSA / PBS (-) was applied, and antigen-antibody reaction was performed at 4 ° C overnight, then PBS (-) And then blocked with 3% BSA / PBS (−). As the secondary antibody, an HRP-labeled anti-rabbit antibody (ENVISION System HRP Rabbit, Dako, Carpinteria, California) was used and treated at 37 ° C. for 1 hour. Histofine DAB substrate kit (Nichirei Bioscience) was used for color development. After coloring, it was sealed and observed.

The data shown in FIG. 5 was obtained by the following method.
Using 3D culture system, hMSC culture in the presence of TGF-β310ng / mL, collect pellets in early differentiation (day14), middle phase (day21), late phase (day28), 4% PFA / PBS (-) After fixing with paraffin, it was sliced at 3 μm after embedding in paraffin, and then attached to a slide.
[HE staining]
The sample subjected to deparaffinization and dexylene treatment was stained with hematoxylin for 5 seconds, washed with water, then immersed in 1% hydrochloric acid alcohol and washed with water. Further, the sample stained with eosin was dehydrated and transparent, sealed and observed.
[Alcian Blue staining]
The sample subjected to deparaffinization and dexylene treatment was stained with Alcian blue (pH 2.6) under acidic conditions for 30 minutes, washed with running water, and then nuclear stained with Cologne ethirate. After the dehydration and clearing steps, it was sealed and observed.

The data shown in FIG. 6 was obtained by the following method.
[Expression of galectin 9 associated with cartilage differentiation in hMSC three-dimensional culture (pellet culture)]
The pellets collected on days 14, 21, and 28 were fixed with 4% PFA / PBS (−) under the presence / absence of TGF-β3 stimulation, embedded in paraffin, sliced into 3 μm, and attached to a slide. After performing the deparaffinization and dexylene process (xylene 4 minutes, 3 times → 100% ethanol → 90% ethanol → 80% ethanol → 70% ethanol → washed with running water), slides for galectin 9 staining were autoclaved (121 ° C, The antigen activation treatment was performed for 15 minutes.
Immunostaining was performed according to the above method. For the primary antibody reaction, an anti-human galectin 9 polyclonal antibody (Galpharma) diluted 100 times (final concentration 1.2 μg / mL) was used. After staining, dehydration and clearing were performed, and observation was performed after encapsulation.

The data shown in FIG. 7 was obtained by the following method.
[Comparison of TGF-β3, cartilage pellet size under stabilized / unstimulated galectin-9]
After culturing in a 3D culture system with or without TGF-β3 and with or without stabilized galectin 9 (TGF-β 10 ng / mL, stabilized galectin 9 concentration 100 nM) After dehydration with Kimwipe, the major axis and minor axis were measured under a microscope. The product of the major axis and the minor axis was taken as the volume and compared under each condition.

The data shown in FIG. 8 was obtained by the following method.
[TGF-β3, glycosaminoglycan (GAG) quantification in cartilage pellets under stimulated / unstimulated stabilized galectin 9]
After culturing in a 3D culture system with or without TGF-β3 and with or without stabilized galectin 9 (TGF-β 10 ng / mL, stabilized galectin 9 concentration 100 nM) Digestion was carried out at 60 ° C. for 1 hour in 200 μL of 20 mM sodium phosphate buffer supplemented with papain. After adding iodoacetic acid, 800 μL of Tris buffer (50 mM Tris-Cl pH 8.0) is added ((1)). Add an equal volume of (1) to 500 μL of DMB solution and stir. Measured at an OD of 525 nm, quantified using a predetermined amount of chondroitin sulfate solution as a standard, or compared relative values (graphs are relative values).

The data shown in FIG. 9 was obtained by the following method.
[Glycosaminoglycan protein assay]
After culturing in a 3D culture system with or without TGF-β3 and with or without stabilized galectin 9 (TGF-β 10 ng / mL, stabilized galectin 9 concentration 1,10,100 nM), collect the pellet on day 14 Digest in 200 μL of 20 mM sodium phosphate buffer supplemented with papain at 60 ° C. for 1 hour. After adding iodoacetic acid, 800 μL of Tris buffer (50 mM Tris-Cl pH 8.0) is added ((1)). Add an equal volume of (1) to 500 μL of DMB solution and stir. Measured at an OD of 525 nm, a fixed amount of chondroitin sulfate solution was used as a standard, and the relative value under each condition was compared.

The data shown in FIG. 10 was obtained by the following method.
[Detection of Type II Collagen and Type I Collagen by immunohistological staining]
After culturing in a 3D culture system with or without TGF-β3 and with or without stabilized galectin 9 (TGF-β 10 ng / mL, stabilized galectin 9 concentration 100 nM) The sample was fixed with 4% PFA / PBS (−), embedded in paraffin, sliced at 3 μm, and attached to a slide. The slides subjected to the deparaffinization and dexylene steps were immunostained. Immunostaining was performed according to the above method. Anti-Col.II antibody (chemicon) and anti-Col.I antibody (Santa-Cruz) diluted 20 times (final concentration 10 μg / mL) and 40 times (final concentration 25 μg / mL) were used for the primary antibody reaction, respectively. After dyeing, dehydration and clearing were performed and then sealed and observed.

The data shown in FIG. 11 was obtained by the following method.
[Stabilized Gal-9 cartilage differentiation promoting effect in the presence of TGF-β3 (histological analysis)]
After culturing in a 3D culture system with or without TGF-β3 and with or without stabilized galectin 9 (TGF-β 10 ng / mL, stabilized galectin 9 concentration 10,100 nM), the pellet recovered on day 14 is 4% PFA After fixing with / PBS (-) and embedding in paraffin, it was sliced at 3 μm and then attached to a slide. Deparaffinization and xylene removal processes were performed. HE staining and Alcian Blue staining were performed in the same manner as the data shown in FIG.
Type II Collagen immunohistochemical staining was performed as follows.
After deparaffinization and dexylene treatment, immunostaining was performed. Immunostaining followed the method described above. For the primary antibody reaction, 20-fold diluted anti-Col.II antibody (final concentration 10 μg / mL; chemicon) was used. After staining, dehydration and clearing were performed, and observation was performed after encapsulation.

The data shown in FIG. 12 was obtained by the following method.
hMSC cells were cultured in a 10 cm culture dish to 3.0 × 10 ⁵ cells and 90% confluent. Thereafter, the cells were cultured for 24 hours under conditions where TGF-β3 was added or not added at 10 ng / mL, and stabilized Gal-9 was added or not added at 1, 10, 100 nM, or were collected in SDS sample buffer. Thereafter, the mixture was sonicated and stored at -80 ° C. For SDS-PAGE, 7.5% gel (PAGEL NPU-7.5L Cat. No. 2331153) was used. Mercaptoethanol was added just before the application and boiled for 5 minutes. After electrophoresis, the gel was recovered, and the electrophoretic protein was transferred to a membrane (Immobilon-P SQ transfer membrane Cat. No. ISEQ10100: Millipore). After blocking with 3% skim milk / PBS (−) for 1 hour, the following antibody was used for immunodetection of the target protein. Each primary antibody was reacted at room temperature for 2 hours, washed 3 times with PBS (-), and then each HRP-labeled secondary antibody against the donor animal was reacted at room temperature for 1 hour. For color development, ECL Western blotting detection reagents (Cat. No. RPN2106: Amersham Biosciences) were used, and were exposed and detected with autolafilm.

[Used antibody]
anti total ERK1 / 2 antibody (Cat.No.V1411: Promega)
anti phospholylated ERK1 / 2 antibody (Cat.No.V8031: Promega)
anti total-Smad2 antibody (Cat.No.ab11784: abcam)
anti phospholylated Smad2 antibody (Cat.No.ab5487: abcam)
anti total p38 antibody (Cat.No.ab7952: abcam)
anti phospholylated p38 antibody (Cat.No.V1211: Promega)

Glycosaminoglycan (Aggrecan) mRNA was detected by the following RT-PCR method.
Sample preparation for RT-PCR was performed using the QUIAGEN RNeasy mini kit and following the product protocol. Mercaptoethanol-added buffer RNT (350 μL) is added to the collected sample) and homogenized. Add 70% ethanol equal to Buffer RNT, pipet, and apply to column. After centrifugation at 8000g for 15 seconds, the column is washed with 700 μL of buffer RW. Transfer the column to a new tube, add buffer RPE, and centrifuge under the above conditions. Once the column is dried, add 500 μL of buffer RPE and centrifuge. Transfer column to RNA collection tube and elute with 30 μL RNase free water. After measuring OD260nm and confirming the RNA amount, the sample was mixed with RT-PCR reaction mixture solution (5x buffer, dNTP, DNA synthase, reverse transcriptase, antisense primer, sense primer) and RT-PCR reaction was performed. . Gal-9, Col.II, and aggrecan were all detected using the following cycle configuration.
(1) 50 ℃, 30 minutes → (2) 95 ℃, 15 minutes → (3) 96 ℃, 1 minute → (4) 57 ℃, 4 minutes → (5) 94 ℃, 1 minute → (6) 57 ℃ , 2.5 minutes → (7) 70 ° C, 10 minutes → (8) 4 ° C, ∞

From the above, it is suggested that galectin 9 [galectin-9 (Gal9)] and galectin 9 modified (stabilized galectin 9), particularly G9NC (null), have activity on cartilage. Gal9 and galectin 9 variants (including G9NC (null)) can be used to inhibit cartilage destruction, promote cartilage differentiation, maintain and restore the function of damaged cartilage, and use them as pharmaceuticals or bioactive agents it can. Gal9 and galectin-9 variants (including G9NC (null)) have a cartilage destruction inhibiting and regenerating action in joint disorders, and are promising as drugs utilizing such action.
Thus, those selected from the group consisting of galectin 9 and its variants (stabilized galectin 9) and galectin 9 inducer are cartilage differentiation promoter, cartilage destruction inhibitor, damaged cartilage function maintaining / restoring agent and cartilage It is useful for the treatment and / or prevention agent of the disease or disorder using the differentiation promoting action, the cartilage destruction inhibiting action, the function maintenance / restoration action of the damaged cartilage, and the treatment and / or prevention method of its activity utilization.

[Reference Example 1]
Galectin 9 used in Example 1 is a stable galectin 9, G9NC (null) [WO2005 / 093064 (published), which is manufactured and obtained in Example 1 of WO2005 / 093064 (published date: October 6, 2005). A polypeptide having the amino acid sequence represented by SEQ ID NO: 2 and encoded by the nucleotide sequence represented by SEQ ID NO: 1) was used.
The stable galectin 9 (galectin 9 variant), that is, G9NC (null) can also be prepared as follows.
The expression of stable galectin 9 (G9NC (null)) was induced by using 2% (w / v) glucose and 100 μg / ml of E. coli (BL21 (DE3)) transformed by electroporation with pET-G9NC (null). The cells were cultured in 2 × YT medium containing ml ampicillin, and isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 0.15 mM when the absorbance at 600 nm reached 0.7. After culturing at 20 ° C. for 22.5 hours, the cells were collected by centrifugation and stored at −80 ° C. until purification. Frozen cells were suspended in 10 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 1 mM DTT, 1 mM PMSF, 10 mM MgCl ₂ , 25 μg / ml DNase, 0.2 mg / ml egg white lysozyme. After turbidity, Triton X-100 was added to a final concentration of 1% and sonicated for 18 minutes. After centrifugation at 18,800 xg for 75 minutes, the recombinant protein in the obtained supernatant was purified by affinity chromatography using lactose agarose, and then the buffer was replaced by PBS by dialysis. After removing the precipitate through a 0.22 μm sterilizing filter, endotoxin was removed by Cellulofine ET-Clean L treatment, and then the bacteria and precipitates were removed through a 0.22 μm sterilizing filter to obtain a final G9NC (null) standard. It was a product.

G9NC (null) prepared in this manner does not show a contaminating band by protein polyacrylamide electrophoresis, and the endotoxin amount is 0.5 EU / ml or less. Stable to freeze and thaw, and retains biological activity stably for more than half a year even at 4 ℃.
As the galectin 9, a recombinant body obtained by expressing native L-type galectin 9, M-type galectin 9 and S-type galectin 9 in a host cell using “gene recombination technology” may be used.

The present invention provides a cartilage destruction inhibitory factor, a cartilage differentiation promoting factor, and / or a function maintenance of damaged cartilage, comprising galectin 9 and a variant thereof and a galectin 9-inducing factor 9 as an active ingredient. -Therapeutic agent and / or prevention for diseases or diseases caused by cartilage destruction using the biological activity against cartilage exhibited by those selected from the group consisting of recovery factor, galectin 9 and its variants and galectin 9 inducer By using an agent or a therapeutic and / or prophylactic agent for a disease associated with cartilage differentiation, an excellent activity is effectively obtained for a subject that is difficult to treat. For example, bone and joint diseases including osteoarthritis, etc., and chondrocytes, or diseases and / or prevention techniques that are considered to be involved in cartilage function involving TGF-β such as TGF-β3 Provided. Galectin 9 and its variants (human stable galectin 9, such as G9NC (null)) and / or galectin 9 inducer can be applied to maintain and restore the function of damaged cartilage. Galectin 9 and its variants (human stable galectin 9, such as G9NC (null)) and / or galectin 9 inducer have the potential to be used as osteoarthritis agents. In the present invention, a disease or disease treatment and / or prevention technique for obtaining the above-mentioned effect by introducing a gene of a galectin 9 and a variant thereof and a gene selected from the group consisting of a galectin 9 inducer, and a deformable joint The present invention also provides a technique for treating and / or preventing bone / joint diseases including infectious diseases.
It will be apparent that the invention may be practiced otherwise than as particularly described in the foregoing description and examples. Many modifications and variations of the present invention are possible in light of the above teachings, and thus are within the scope of the claims appended hereto.

The results of assaying the effect of galectin 9 (G9NC (null)) in mouse CIA and CAIA arthritis models by safranin-O staining are shown. It is a microscope picture of the form of a biological tissue. Human articular cartilage: Comparison between normal and diseased areas. Safranin-O staining of the extracellular matrix of cartilage lesions of osteoarthritis patients was performed and compared with normal areas. It is a microscope picture of the form of a biological tissue. The result of having investigated about the expression of galectin 9 and HSP-70 in the OA lesion cartilage part is shown. It is a microscope picture of the form of a biological tissue. Three-dimensional culture method Miromass pellet culture method is shown. On day 0, TGF-β3 was added. On day 14, proteoglycan production occurred in the early stage of differentiation. On day 21, type II collagen was produced in the middle stage of differentiation. On day 28, stages of chondrocyte-like vesicle formation in the late stage of differentiation. The results of morphological observation of the differentiation of human mesenchymal stem cells into chondrocytes by the three-dimensional culture method Miromass pellet culture method are shown. It is a microscope picture of the form of a biological tissue. The result of having investigated the expression of galectin 9 in the cartilage differentiation system in the presence of TGF-β3 is shown. It is a microscope picture of the form of a biological tissue. The result of investigating the influence of galectin 9 in the TGF-β3-induced cartilage differentiation system is shown. The pellet size was examined. The result of investigating the influence of galectin 9 in the TGF-β3-induced cartilage differentiation system is shown. Glycosaminoglycan production was examined. The result of investigating the influence of galectin 9 in the TGF-β3-induced cartilage differentiation system is shown. The dependence on galectin 9 concentration was examined for glycosaminoglycan production. The result of investigating the influence of galectin 9 in the TGF-β3-induced cartilage differentiation system is shown. Collagen production was examined. It is a microscope picture of the form of a biological tissue. The result of investigating the influence of galectin 9 in the TGF-β3-induced cartilage differentiation system is shown. The dependence of collagen production on galectin 9 concentration was examined. It is a microscope picture of the form of a biological tissue. The result of having investigated the influence on the signal of galectin 9 in cartilage differentiation induction is shown. Investigated by Western blotting. It is a photograph of electrophoresis.

Claims (9)

Maintaining / recovering the function of cartilage differentiation promoting agent, cartilage destruction inhibiting agent, and / or damaged cartilage characterized by containing as an active ingredient a substance selected from the group consisting of galectin 9 and its variants and galectin 9 inducer Agent. A cartilage differentiation promoter, a cartilage destruction inhibitor, and / or a damaged cartilage characterized by containing as an active ingredient a nucleic acid encoding a galectin 9 and a variant thereof and a galectin 9 inducer selected from the group consisting of Function maintenance / recovery agent. A method for treating chondrocytes, comprising bringing chondrocytes, particularly undifferentiated chondrocytes, into contact with a galectin 9 and a variant thereof and a galectin 9-inducing factor selected to promote differentiation. A method for promoting chondrocyte differentiation, comprising introducing a nucleic acid encoding a galectin-9, a variant thereof and a galectin-9 inducer into a chondrocyte, particularly an undifferentiated chondrocyte. A therapeutic and / or prophylactic agent for bone and joint diseases, comprising as an active ingredient a substance selected from the group consisting of galectin 9 and a variant thereof and a galectin 9 inducer. A therapeutic and / or prophylactic agent for bone and joint diseases, comprising as an active ingredient a nucleic acid encoding galectin 9 and a variant thereof and a galectin 9-inducing factor. A method for inhibiting cartilage destruction, which comprises contacting a cartilage tissue or cell having a reduced function or failure with a member selected from the group consisting of galectin 9 and a variant thereof and a galectin 9-inducing factor to inhibit cartilage destruction. A method for inhibiting cartilage destruction, comprising introducing a nucleic acid encoding a galectin-9 or a variant thereof and a galectin-9-inducing factor into a cartilage tissue or cell with reduced function or failure. The agent or method according to any one of claims 1 to 8, which is in the presence of a TGF-β family such as TGF-β3 or a nucleic acid encoding the same.