JP2009533321A - Treatment of tendinopathy by inhibiting molecules that contribute to cartilage formation - Google Patents

Abstract

  The present invention provides a method for treating tendinopathy. Disorders to be treated or prevented include, for example, tendinitis, tendonitis, tendinosis, paratendinitis, tendon synovitis, tendon overuse injury and trauma, peritonitis, paratenonitis, or other tendon degenerative disorders. The disclosed method of treatment comprises administering to a patient an inhibitor of a molecule involved in cartilage or fibrocartilage formation in a tendinopathy that is effective for the treatment or prevention of tendon degenerative disorders, Delay tendon deterioration, restore tendon healthy structure, stimulate tendon regeneration, and / or maintain tendon mass and / or quality.

Description

This application claims priority to US Provisional Patent Application No. 60 / 779,165, filed March 3, 2006, the contents of which are incorporated herein by reference. To be part of

The technical field of the invention relates to the treatment of tendinopathy by reducing the production of cartilage and / or fibrocartilage tissue in damaged tendons. The invention further relates to the inhibition of molecules involved in the production of cartilage and / or fibrocartilage tissue in damaged tendons.

  Tendinopathy is a general term used to describe various tendon disorders. The term “tendinitis” means “inflammation of the tendon” and is often used to describe problems that have occurred in the tendon, but inflammation rarely causes tendon pain. Most commonly, tendon pain is in fact a symptom of a series of microtears in the tendon or surrounding connective tissue, more formally referred to as tendinosis. Other tendon disorders are tendons caused by collagen degeneration with fiber non-orientation, increased mucoid matrix in the tendon, calcification, tendon overuse, vascularization, aging, and friction of the tendon against the body ridge Includes pain. Tendinopathy is being used by more and more tendon specialists to represent these conditions, often tendinitis, tendinosis, paratendinitis, synovitis, paratenonitis ), Characterized as a collective term for tendon overuse injury and trauma. Khan et al., Sports Med 27: 393-408 (1999).

  Normal tendon tissue consists of connective tissue densely packed with regularly arranged bundles of collagen fibers running in the same direction in primary, secondary and tertiary fiber bundles with high tensile strength. Interspersed among these fibers are flat, tapered tendon cells that synthesize a viscous extracellular matrix (ECM) rich in type I collagen. Type I collagen bundles provide tendon flexibility and structural support. In healthy tendons, there is a dynamic equilibrium between ECM synthesis and degradation. However, tendinopathy has an adverse effect on this equilibrium, resulting in structural rearrangement and disorder of the collagen fibers. The disorder of the collagen bundle causes cartilage to appear in the tendon. The pathologic accumulation of glycosaminoglycans (GAGs) in fibroblasts, myofibroblasts, neovascularization, and ECM is clearly prominent in tissues affected by tendinopathy. Tallon et al., Med Sci Sports Exerc 33 (12): 1983-90 (2001); Khan et al., Sports Med 27 (6): 393-408 (1999). Metabolic and morphological changes in the injured tendon result in pain and are susceptible to tearing and destruction.

  Currently, ECM degradation and remodeling of damaged tendons is caused by the release of metalloproteinases from endogenous connective tissue cells and invading inflammatory cells capable of degrading matrix macromolecules such as aggrecan, decorin and biglycan It is believed that. These metalloproteinases include MMPs (Matrix Metalloproteinases), ADAMs (A Disintegrin And Metalloproteinases) and ADAMs (A Disintegrin And Metalloproteinases with Thrombospondin Motifs, eg, aggrecanases). Riley G., Expert Rev Mol Med 7 (5): 1-23 (2005); Rees et al., Biochem J 350: 180-188 (2000). However, clinical trials on broad spectrum inhibitors of MMPs have been unsuccessful in the treatment of osteoarthritis with pathological similarity to tendinopathy. Clark et al., Expert Opin Ther Targets 7 (1): 19-34 (2003). Thus, metalloproteinase inhibition is unlikely to be effective in treating tendinopathy. Indeed, in high-stress tendons such as the supraspinatus tendon and the Achilles tendon, a minimal level of metalloproteinase activity may be required to provide an optimal level of ECM for the tendon. Riley G., Expert Rev Mol Med 7 (5): 1-23 (2005).

  Currently, there are a variety of standard non-surgical and surgical tendinopathy treatments. Non-surgical methods include rest, cryotherapy, activity modification, physical therapy, non-wateroid anti-inflammatory drugs (NSAIDs) and corticosteroids. While rest and activity modification can help some patients with these conditions, there remains a clinically significant population that cannot be treated with these therapies. Oral anti-inflammatory drug therapy has not proved useful in comparative studies, despite the widespread use, and may have undesirable side effects. In addition, some studies suggest that non-steroidal drug therapy can actually have a negative impact on the healing process, by relieving pain and causing patients to ignore the initial symptoms of tendinopathy.

  Corticosteroids are usually used to reduce tissue inflammation, but the use of such drugs for the treatment of tendinopathy is particularly recommended because corticosteroids inhibit collagen synthesis. Absent. In some studies, short-term improvements were observed in patients treated with cortisone, but following more than one year revealed high symptom recurrence rates and suspicious efficacy rates. Cortisone injection also has the risk of tendon destruction, infection, skin depigmentation, and skin atrophy.

  Surgical methods for tendon repair include debridement and repair of damaged tendons. However, open surgery or arthroscopic surgery has the potential for many complications such as deep infections, damage to neurovascular structures and scar formation. The surgery is also expensive and has additional risks associated with local or general anesthesia.

  Accordingly, there is still a need for a treatment protocol for tendon-related injuries that goes beyond existing therapies. Injuries or other damage to flexible tendon tissue is slow to heal and can take months or years to fully repair. Tendon-related injuries have a significant impact on society. Overuse injuries account for nearly 7 percent of all tendon-related outpatient care in the United States. Woodwell et al., Adv Data 346: 1-44 (2004). According to information from the US Bureau of Labor Statistics, such injuries have led to a significant loss of working hours (see http://www.bls.gov/lif).

Summary of the Invention The present invention provides a novel approach to the treatment of tendinopathy. By performing transcriptional profiling of overused rat tendons, it has now been found that the expression levels of cartilage-specific genes such as aggrecan, versican, Col2a1 (type II collagen) and Sox9 are increased in injured tendons . These results suggest that injured tendon tissue is converted to fibrocartilage as a result of overuse. Unlike tendons consisting primarily of type I collagen, fibrocartilage contains type II collagen and large proteoglycans, such as aggrecan and versican. Since cartilage tissue disrupts the closely packed type I collagen fibers of the tendon, the formation of cartilage and fibrocartilage within the tendon is detrimental to tendon repair. The collapse reduces the tensile strength and elasticity of the tendon.

  Accordingly, the present invention provides a method of treating tendinopathy in a patient by reducing the formation of cartilage-specific proteoglycans in the patient's tendon tissue. The present invention also provides the use of a compound that reduces the formation of cartilage-specific proteoglycans for the manufacture of a medicament for the treatment of tendinopathy.

  In some embodiments, the methods and uses include reducing the activity of enzymes involved in the synthesis of aggrecan and / or versican in a patient's tendon tissue. In certain embodiments, the methods and uses include chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CS-GalNAcT-1) and / or galactosamine polypeptide N-acetylgalactosaminyltransferase 1 (GalNT) in patient tendon tissue. -1) reducing the activity. In other embodiments, the methods and uses include, but are not limited to, UDP-D-xylose: core protein β-D-xylosyltransferase, Gal transferase, GlcA transferase, GlcNAc transferase, sulfotransferase, chondroitin sulfate synthase And reducing the activity of one or more other enzymes involved in cartilage proteoglycan synthesis, including heparan sulfate sulfotransferase.

  The activity of enzymes and other proteins involved in the production of cartilage structural protein can be reduced by directly or indirectly inhibiting the function of the enzyme or other protein, or inhibiting the expression of the enzyme or other protein itself Can be reduced.

  The present invention also provides a method of treating tendinopathy in a patient by reducing the expression of cartilage-specific structural proteins in the patient's tendon tissue. The present invention further provides the use of a compound that reduces the expression of cartilage-specific structural protein for the manufacture of a medicament for treating tendinopathy. In some embodiments, for example, but not limited to, expression of cartilage-specific structural proteins such as aggrecan, syndecan 3 (syn-3), versican, and type II collagen is directly inhibited. In other embodiments, the activity of transcription factors involved in the expression of cartilage specific genes is reduced. These transcription factors and signal transduction proteins include, for example, Sox9.

  In other embodiments, the invention administers a test compound to a subject in need of treatment and then inhibits the activity of the drug involved in the synthesis of glycoglycosaminoglycans (GAGs) in tendon tissue. It includes a method for identifying a compound for treating tendinopathy characterized by measuring ability.

  In certain embodiments, a method of identifying a compound for treating tendinopathy or a method for evaluating treatment comprises: (1) Cs-GalNAcT-1, CS-GalNAcT-2, heparan sulfate (glucosamine) 3-O— Providing at least one sample component selected from the group consisting of sulfotransferase 1 (Hs3st1), GalNT-1 and Sox9, (2) combining the sample with a test compound, and (3) the sample responsive to the test compound Measuring the activity of the component, and then (4) determining whether the test compound inhibits the activity of the sample component.

  Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the following detailed description and the appended claims.

  It is to be understood that both the foregoing general description and the following detailed description are exemplary only and are exemplary and not restrictive of the invention, as claimed. is there.

  The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate some embodiments of the present invention and, together with the description, explain the principles of the invention. It is useful to explain.

BRIEF DESCRIPTION OF THE DRAWINGS In FIGS. 1-7, SST indicates the supraspinatus tendon, PT indicates the patella tendon non-injured internal control, and R indicates the animal subjected to overuse protocol (Soslowsky et al., J. Shoulder Elbow Surg. 9: 79-84 (2000)), C shows control animals not subjected to overuse protocol, time course is 1, 2 and 4 weeks and BM shows bone marrow.

  FIG. 1 shows the gene expression profile of Col2a1 (type II collagen) in tendons overworked for 4 weeks using the Affymetrix RAE230 2.0 gene chip (FIG. 1A). The expression level of the Col2a1 gene was also measured in the indicated normal musculoskeletal tissues (FIG. 1B). The expression profile shows that Col2a1 is highly expressed in both cartilage tissue and overworked tendon tissue when compared to normal tendon tissue.

  FIG. 2 shows the gene expression profile of Agcl (Aggrecan) in tendons overworked for 4 weeks using the Affymetrix RAE230 2.0 gene chip (FIG. 2A). The expression level of the Agcl gene was also measured in the indicated normal musculoskeletal tissues (Fig. 2B). The expression profile shows that Agc1 is highly expressed in both cartilage tissue and overused tendon tissue when compared to normal tendon tissue.

  FIG. 3 shows the gene expression profile of Sox9 (sry-type high mobility group box9) in tendons overuse for 4 weeks using the Affymetrix RAE230 2.0 gene chip (FIG. 3A). The expression level of Sox9 gene was also measured in the indicated normal musculoskeletal tissues (FIG. 3B). The expression profile shows that Sox9 is highly expressed in both cartilage tissue and overworked tendon tissue when compared to normal tendon tissue.

  FIG. 4 shows the gene expression profile of Cspg2 (Versican) in tendon overuse for 4 weeks using the Affymetrix RAE230 2.0 gene chip (FIG. 4A). Versican gene expression levels were also measured in the indicated normal musculoskeletal tissues (FIG. 4B). The expression profile shows that versican is highly expressed in both cartilage tissue and overused tendon tissue when compared to normal tendon tissue.

  FIG. 5 shows the gene expression profile of chondroitin sulfate N-acetylgalactosaminyltransferase (CS-GalNAcT-1) in tendon overuse for 4 weeks using the Affymetrix RAE230 2.0 gene chip (FIG. 5A). The expression level of CS-GalNAcT-1 gene was also measured in the indicated normal musculoskeletal tissues (FIG. 5B). The expression profile shows that CS-GalNAcT-1 is highly expressed in both cartilage tissue and overworked tendon tissue when compared to normal tendon tissue.

  FIG. 6 shows GalNT-1 (GalNT1-UDP-N-acetyl-alpha-D-galactosamine: polypeptide-N-acetylgalactosaminyltransferase 1 in an overused tendon over 4 weeks using the Affymetrix RAE230 2.0 gene chip. ) Shows the gene expression profile (FIG. 6A). The expression level of the GalNT-1 gene was also measured in the normal musculoskeletal tissues shown (FIG. 6B). The expression profile shows that GalNT-1 is highly expressed in overused tendon tissue when compared to normal tendon tissue.

  FIG. 7 shows the gene expression profile of Hs3st1 (heparan sulfate (glucosamine) 3-O-sulfotransferase 1) in tendons overuse for 4 weeks using the Affymetrix RAE230 2.0 gene chip (FIG. 7A). The expression level of the Hs3st1 gene was also measured in the indicated normal musculoskeletal tissues (FIG. 7B). The expression profile shows that Hs3st1 is highly expressed in tendon tissue overworked when compared to normal tendon tissue and in cartilage tissue when compared with normal tendon tissue.

Brief Description of Sequences The nucleotide and amino acid sequences of CS-GalNAcT-1 are available from Genbank under the following accession numbers. Human (NM — 018371); mouse (NM — 172753); and rat (XM — 224757).

  The nucleotide and amino acid sequence of CS-GalNAcT-2 is available from Genbank under the following accession number. Human (NM_018590); mouse (NM_030165); and rat (XM_232316).

  The nucleotide and amino acid sequence of GalNT-1 is available from Genbank under the accession number below. Human (NM — 020474); mouse (NM — 013814); and rat (NM — 124373).

  The nucleotide and amino acid sequences of aggrecan are available from Genbank under the following accession numbers. Human (NM_001135); mouse (NM_007427); and rat (NM_022190).

  The nucleotide and amino acid sequences of Col2a1 are available from Genbank under the following accession numbers. Human (NM — 001844); mouse (NM — 031163) and rat (NM — 012929).

  The nucleotide and amino acid sequence of Sox9 is available from Genbank under the accession number below. Human (NM_000346); mouse (NM_011448); and rat (XM_343981).

  Versican nucleotide and amino acid sequences are available from Genbank under the accession numbers below. Human (NM_004385); mouse (XM_488510) and rat (XM_215451).

  The nucleotide and amino acid sequence of heparan sulfate (glucosamine) 3-O-sulfotransferase 1 is available from Genbank under the following accession number. Human (NM_005114), mouse (NM_010474), and rat (NM_053391).

Detailed Description of the Invention The present invention provides methods of treating tendinopathy by reducing the activity of one or more molecules involved in cartilage and / or fibrocartilage formation in tendon tissue.

  In order that the present invention may be more readily understood, certain specific terms are first defined. Additional definitions are set forth throughout the detailed description of the invention.

  As used herein, the term “tendinopathy” includes, but is not limited to, tendinitis, tendonitis, tendinosis, paratendinitis, tendon synovitis, tendon abuse injury and trauma, Includes all conditions that occur around and around the tendon, including perintendinitis and paratenonitis.

  As used herein, the term “tendon defect” refers to any tendon disorder, including but not limited to tendinopathy.

  As used herein, the term “damaged tendon” refers to tendon tissue suffering from tendinopathy or tendon defects.

  As used herein, the term “overuse” refers to a tendon suffering from a tendon defect or tendinopathy as a result of overuse, as opposed to direct trauma.

  As used herein, the term “patient” or “subject” refers to a tendon defect, a tendon defect, or a recovery process from a tendon defect that requires treatment. Any human or animal is indicated.

  As used herein, the term “tendinitis” refers to tendonitis (another spelling), tendinopathy or tendon inflammation.

  As used herein, the term “tendinosis” refers to tendinopathy or any degenerative condition in which a microtear that weakens the tendon occurs in the tendon, often pain, stiffness, force Cause loss.

  As used herein, the term “tendon injury” refers to tendinopathy or any condition in which tendon tissue becomes defective or degenerated.

  As used herein, the term “small molecule” refers to any chemical or other group other than polypeptides and nucleic acids that can act to affect biological processes. Includes.

  As used herein, the term “treat” or “treatment” refers to any application or application of medicine for a disease in mammals, including humans, and refers to inhibiting the disease. Include. It prevents disease onset and reduces disease, for example, by causing regression, restoring or repairing lost, lost or defective functions, or stimulating ineffective processes To include.

  As used herein, the term “activity” refers to the biological function of an enzyme or protein that can be inhibited, reduced or interfered by various means.

  As used herein, the term “enzymatic activity” refers to one or more physiological, catalytic, regulatory or enzymatic activities associated with an enzyme.

  As used herein, the term "effective amount" is sufficient to indicate a significant patient benefit, i.e., tendinopathy cure or symptom cure and improvement rate. The total amount of each inhibitor is meant.

I. Tendon and Cartilage Components A tendon consists of a correctly aligned layer of type I collagen interspersed with small amounts of type III collagen, fibroblast-like tendon cells and chondrocyte-like fibrochondrocytes. Type I collagen is packed into dense collagen fibers, which provide strength and rigidity to the tendon while it is stretched and pulled by the attached muscle. In contrast, cartilage consists of type II collagen and proteoglycans and is designed to resist compression but not tension. Fibrocartilage is a mixture of tendon and cartilage tissue and is found primarily at the intermediate surface between tendon and bone. The formation of cartilage and fibrocartilage within the tendon is detrimental to tendon function as it breaks closely packed type I collagen fibers and reduces the tensile strength of the tendon. Accordingly, the methods of the invention may be used to prevent or reduce the formation of cartilage and / or fibrocartilage in damaged or injured tendon tissue.

  One sign of cartilage formation in tendons is the presence of increased proteoglycan levels in the tissue, often detected by the presence of the glycosaminoglycan (GAG) side chain of these proteoglycans. Proteoglycans are a family of glycoproteins characterized by a core protein having one or more linear GAG chains, made up of repeating disaccharide units. Chondroitin sulfate proteoglycans such as aggrecan and versican are cartilage specific. Aggrecan is a major structural component of cartilage and contributes to the compressive strength and elasticity of cartilage. Hydration and aggregation of aggrecan molecules creates a viscous gel that absorbs compressive loads in cartilage tissue. Watanabe et al., J. Biochem 124 (4): 687-93 (1998). Other proteoglycans present in cartilage tissue include heparan sulfate proteoglycans, such as syndecan 3 (syn-3). Kirn-Safran et al., Birth Defects Res 72 (Part C): 69-88 (2004).

II. Molecules that Promote Cartilage and Fibrocartilage Formation The present invention is based, in part, on the finding that the expression of cartilage specific genes (eg, aggrecan, versican and type II collagen) is increased in injured tendons. The results were obtained using the gene expression profile obtained in the rat tendon abuse model described in Soslowsky et al., J Shoulder Elbow Surg. 9: 79-84 (2000). Suggests conversion to fibrocartilage.

  The present invention is also based in part on the finding that the expression of enzymes involved in the synthesis of the glycosaminoglycan (GAG) side chain of the cartilage-specific proteoglycan is increased in overused tendons. The finding that cartilage-specific enzymes are upregulated in damaged tendons provides a mechanism for GAG accumulation observed in severely damaged tendons. Khan et al., Sports Med 27: 393-408 (1999) and Tallon et al., Med Sci Sports Exerc 33 (12): 1983-90 (2001). These increased GAG levels indicate that the levels of proteoglycans such as aggrecan, versican and syn-3 are increased in damaged tendons. Thus, tendinopathy can be treated by preventing the accumulation of these proteoglycans by reducing the activity of the enzymes responsible for the production of cartilage-specific proteoglycans.

A. Molecules that promote cartilage formation are upregulated in tendinopathy.
Affymetrix GeneChip ^R (TM) system was used to identify differences in gene expression between the tendon attached to normal tendon and overuse protocols. The gene expression profile produced over 400 genes that were differentially regulated in the supraspinatus tendon after overuse. Over 4 weeks of abuse, 107 genes were up-regulated and 27 genes were down-regulated. Some of the most highly upregulated genes are cartilage specific genes, including collagen type 2 alpha-1 chain (Col2a1), Sox9, versican and aggrecan. Other upregulated genes include those involved in the formation of GAG side chains on cartilage proteoglycans, including CS-GalNAcT-1, GalNT-1 and Hs3st1. Based on these results, these molecules themselves, or the other molecules involved in the initiation or maintenance of the activity of these molecules or the expression of the genes encoding these molecules, are cartilage and / or in overused tendons. Or it may be involved in the formation of fibrocartilage. These molecules can include enzymes, transcription factors and signal transduction factors as listed below.

1. Enzymes One method of inhibiting the production of cartilage or fibrocartilage in tendon tissue is to inhibit the expression or activity of enzymes involved in proteoglycan synthesis. For a complete list of enzymes involved in proteoglycan GAG chain synthesis, see Silbert et al., IUBMB Life 54: 177-186 (2002) and Sugahara et al., IUBMB Life 54: 163-175 (2002) thing. These enzymes include proteins whose expression increases in overused tendons (Example 1). Accordingly, one embodiment of the invention includes inhibiting the expression or activity of any one of the following enzymes:

  CS-GalNAcT-1 and CS-GalNAcT-2 (EC 2.4.1.174 and EC 2.4.1.175) involved in chondroitin sulfate GAG side chain initiation and elongation. CS-GalNAcT-1 is involved in the initiation and elongation of chondroitin GAGs, while CS-GalNAcT-2 is involved in the elongation of the same chondroitin GAGs. For a detailed description of these enzymes, see Uyama et al., J. Biol. Chem. 277 (11): 8841-8846 (2002); Gotoh et al., J. Biol. Chem. 277 (41): 38189 -38196 (2002); Sato et al. J. Biol. Chem. 278 (5): 3063-3071 (2003); Uyama et al., J. Biol. Chem. 278 (5): 3072-3078 (2003) checking ...

  UDP-D-xylose: core protein β-D-xylosyltransferase (EC 2.4.) Involved in the addition of the first Xyl group onto the proteoglycan core protein, one of the first steps of GAG side chain synthesis. 2.269).

  Gal transferase (EC 2.4.1.133 and EC 2.4.1.134) involved in the addition of galactose residues on the Xyl group of the novel GAG side chain.

  GlcA transferases (EC 2.4.1.135, EC 2.4.4.126, and EC 2.4.1.225, which are involved in the addition of GlcA groups onto galactose residues of the novel GAG side chain ).

  GlcNAc transferase involved in addition of GlcNAc group to GalNAc group of chondroitin sulfate GAGs added by CS-GalNAcT enzyme (EC 2.4.1.223 and EC 2.4.1224).

  Sulfotransferases involved in the transfer of sulfate residues on chondroitin sulfate GAGs (EC 2.8.2.5 and EC 2.8.2.1).

  Chondroitin sulfate synthase (EC 3.1.6.4 and EC 3.1.6.12) involved in the initial addition of sulfate residues to chondroitin sulfate GAGs.

  GalNT-1 (EC 2.4.4.11) catalyzes the first step in the formation of O-linked oligosaccharide side chains on glycoproteins. White et al., J Biol Chem 270 (41): 24156-65 (1995).

  Hs3st1 (EC 2.8.2.23) catalyzes the addition of sulfate residues on heparan sulfate GAGs. Shworak et al., J Biol Chem 272 (44): 28008-19 (1997).

  Inhibiting the activity of any one of these enzymes, either directly or indirectly, will reduce proteoglycan formation. Methods for inhibiting these enzymes are discussed below.

2. Gene Expression Another way to prevent the formation of cartilage and / or fibrocartilage is to prevent, directly or indirectly, the expression of cartilage specific proteins in damaged tendon tissue. For example, expression of versican or aggrecan, cartilage-specific proteoglycan prevents transcription or translation of the gene encoding aggrecan or versican, or prevents the expression or function of transcription factors involved in expression of aggrecan or versican gene As a result, it can be reduced by several methods including preventing transcription of these genes.

  Since many cartilage-specific factors, including proteoglycans and type II collagen, are important elements of functional articular cartilage and other tissues, when practicing the present invention, inhibition of cartilage-specific factors can be applied to damaged tendons. It is important to ensure that it is localized.

  One method for inhibiting the formation of cartilage and / or fibrocartilage in tendon tissue is to inhibit the activity or expression of non-enzymatic proteins involved in the expression of cartilage structural proteins. By inhibiting the expression or activity of these genes, the expression of proteins constituting cartilage and / or fibrocartilage tissue can be indirectly inhibited. Proteins involved in the expression of cartilage structural protein include, but are not limited to, Sox9.

  Other methods for inhibiting the formation of cartilage or fibrocartilage in tendon tissue directly inhibit the expression (as opposed to activity) of major cartilage structural proteins such as aggrecan, versican, and type II collagen Including doing. The following genes have been found to be upregulated in damaged tendon tissue and are associated with cartilage formation.

a) Sox9
Sox9 (sry-type high mobility group box9) is a transcription factor involved in cartilage development. Sox9 is expressed in chondrocytes and is consistent with the expression of the collagen alpha 1 (II) gene (Col2a1). Sox9 regulates cartilage formation by activating or increasing transcription of genes that express cartilage structural proteins such as type II collagen. Shum et al., Arthritis Res. 4 (2): 94-106 (2002); Bi et al., Nat. Genet. 22 (1): 85-9 (1999).

b) Col2a1
Col2a1 encodes the alpha 1 chain of type II collagen, which is a major component of cartilage. Cheah et al., Proc Natl Acad Sci USA 82 (9): 2555-9 (1985).

c) Agc1
Agc1 encodes the core protein of aggrecan proteoglycan. Doege et al., Extracellular Matrix Genes (Sandel, LJ; Boyd, CD, eds.) Academic Press (New York) 137-152 (1990). As used herein, the term “aggrecan” refers to large proteoglycans specific for cartilage tissue. Aggrecan consists of a protein core coated with a number of chondroitin sulfate GAG groups. Aggrecan is a major structural component of cartilage and can combine with water to form a viscous gel-like substance. This hydration provides the cartilage tissue with elasticity and compressive strength.

d) Cspg2
Cspg2 encodes the core protein of versican proteoglycan. Versican is another large proteoglycan with many chondroitin sulfate side chains. Like aggrecan, versican combines with water to form a viscous gel that increases tissue strength and elasticity. Knudson et al., Sem Cell Dev Biol 12: 69-78 (2001).

B. Treatment of tendinopathy by inhibiting molecules that promote cartilage formation The present invention provides methods for treating or preventing tendinopathy by reducing the expression and / or activity of the molecules listed above. Furthermore, the present invention provides methods for administering inhibitors of the molecules listed above to treat or prevent tendinopathy.

1. Therapeutic methods of the present invention include, in part, over-used tendon tissue, aggrecan, versican, Sox9, type II collagen, CS-GalNAcT-1, GalNT-1 and HS3st1 Based on the finding that expression levels are significantly increased (FIGS. 1-7). The results indicate that increased expression of enzymes or other molecules involved in the proteoglycan synthesis pathway leads to the conversion of damaged tendon tissue to cartilage and / or fibrocartilage. Thus, in one embodiment, the present invention relates to the cartilage and / or fibrocartilage in tendon tissue by inhibiting the activity, expression or accumulation of enzymes or other molecules involved in cartilage and / or fibrocartilage synthesis. It includes a method for treating tendinopathy characterized by reducing formation.

  Inhibitors that can block the activity of enzymes or other molecules involved in cartilage or fibrocartilage formation in tendons are useful in the present invention. Inhibitors useful in the methods of the invention may be glycosylated, pegylated, or linked to another non-proteinaceous polymer. Inhibitors may be modified to have a glycosylation pattern that is altered (ie, altered from the original or native glycosylation pattern). As used herein, the term “modified” refers to the addition or deletion of one or more carbohydrate moieties and / or one or more glycosylation relative to the original inhibitor. Means that the site has been added or deleted. Addition of glycosylation sites to inhibitors can be accomplished by altering the amino acid sequence to contain glycosylation site consensus sequences well known in the art. Another approach to increase the number of carbohydrate moieties is by chemically or enzymatically coupling glycosides to the amino acid residues of the inhibitor. These methods are described in WO 87/05330 and in Aplin et al., Crit Rev Biochem 22: 259 306 (1981). Removal of any carbohydrate moieties present on the substrate is described by Sojar et al., Arch Biochem Biophys 259: 52-57 (1987); Edge et al., Anal Biochem 118: 131-137 (1981); and by Thotakura It can be accomplished chemically or enzymatically as described by et al., Meth Enzymol 138: 350-359 (1987).

  Also, for example, proteinaceous and non-proteinaceous inhibitors, including peptides, small molecules and nucleic acids, may be used in the methods of the invention.

a) Peptides and small molecules Inhibitors useful in the methods of the invention include small organic peptides and molecules and small inorganic molecules. These peptides and small molecules include synthetic and purified natural inhibitors. Methods for identifying peptides and small molecules that specifically target a protein of interest are well known in the art. For example, using enzymatic and / or biological functional assays of target proteins, peptides and / or small molecule libraries can be converted to target proteins such as CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1 and / Or Sox9 inhibition may be screened. In another embodiment, small molecules and / or peptide libraries are used in competitive radioligand binding assays of target proteins such as CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1 and / or Sox9. You may screen using a substrate or a ligand.

  Alternatively, a fluorescence resonance energy transfer (FRET) based assay, such as a time resolved FRET (TR-FRET) assay, may be used to identify inhibitors. In an exemplary embodiment, the proteoglycan core protein is labeled with a His or GST tag, and the protein is labeled with an anti-His or GST antibody conjugated to europium, a fluorophore. Alternatively, the core protein is non-specifically labeled with europium on lysine or cysteine residues. Donor sugar molecules are labeled with Cy5, a fluorescent dye. A list of donor and acceptor molecules useful in the assay can be found in Table I of Uyama et al., J Biol Chem 278 (5): 3072-78 (2003).

  The acceptor and donor molecules are combined in various ratios with and without the target enzyme. The TR-FRET assay is performed by exciting the system at 340 nm, measuring the emission of europium and Cy5 at 615 and 665 nm, respectively, and then calculating the ratio of Cy5 emission to europium emission.

  In the absence of enzyme, the donor and acceptor molecules are not in close proximity to each other and there is no quenching of the individual fluorescence of the Cy5 and Europium molecules, resulting in a baseline level ratio. When the target enzyme is added to the assay system, the donor sugar molecule moves to the acceptor protein and the fluorescence of the system is quenched. The ratio of Cy5 to europium fluorescence increases to a maximum with the amount of sugar molecules transferred.

  Peptides or small molecules are added to the assay system to identify inhibitors of the target enzyme. If the test compound can inhibit the transfer of sugar to the protein, fluorescence quenching is reduced. If the test compound can inhibit 100% of the transfer activity of the enzyme, the ratio of Cy5 luminescence to europium luminescence is at the baseline level. Compounds that inhibit at least 50% of enzyme activity in the assay are then evaluated in a cell-based system to assess for the ability to reduce GAG synthesis (below).

  The invention also provides for the use of additional screening assays, such as secondary and tertiary assays, to further identify the effects of such molecules on tendon morphology, for example using the assays detailed above. .

b) Dominant negative mutants In certain embodiments of the invention, mutant CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1, Hs3st1 or Sox9 proteins may be used as inhibitors in the methods of the invention. Good. For example, natural variants or artificially modified homologs that have a dominant negative effect on the activity of the molecule both in vivo and in vitro can be used in the methods of the invention.

c) Nucleic acids Nucleic acids that can block the activity of the target molecules listed above are useful in the present invention. Such an inhibitor may, for example, encode a protein that interacts with CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1, Hs3st1 or Sox9. Alternatively, such an inhibitor may encode a protein capable of interfering with a substrate or ligand of a target molecule (eg, GalNAc), and the encoded protein is directed to that substrate or ligand of the target molecule. It may be useful in the methods of the invention when blocking binding or blocking the activity of a substrate or ligand after binding of a target molecule. Of course, the inhibitor may encode a protein that interacts simultaneously with the target molecule and its substrate or ligand. Such nucleic acids can be used, for example, to express CS-GalNAcT-1, CS-GalNAcT-2, Hs3st1, GalNT-1 or Sox9 inhibitors useful in the methods of the invention.

  The methods of the invention also include, but are not limited to, short interfering RNA (siRNA), short hairpin RNA (shRNA) and the like to reduce expression of any of the above molecules or their protein binding partners. Includes the use of interfering RNA (RNAi), including short double stranded RNA (sdsRNA). RNAi can be initiated by introducing a nucleic acid molecule, such as a synthetic short interfering siRNA or RNA interfering agent, to inhibit or silence the expression of the target gene. See, for example, US Patent Publications 2003/0153519 and 2003/01674901 and US Pat. Nos. 6,506,559 and 6,573,099.

  siRNA may be synthesized chemically, produced by in vitro transcription, or produced in a host cell. Typically, siRNA is at least 15-50 nucleotides in length, for example 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. The siRNA may be a double-stranded RNA (dsRNA) that is about 15 to about 40 nucleotides long, including about 19, 20, 21 or 22 nucleotides long, eg, about 15 to about 28 nucleotides long, in each strand. It may contain 3 'and / or 5' overhands about 0, 1, 2, 3, 4, 5, or 6 nucleotides long. siRNA can inhibit target genes by transcriptional silencing. Preferably, the siRNA can promote RNA interference by degradation of the target messenger RNA or specific post-transcriptional gene silencing (PTGS).

  The siRNA used in the methods of the present invention also includes small hairpin RNAs (shRNAs). The shRNA consists of a short (eg, about 19-25 nucleotides) antisense strand followed by a nucleotide loop of about 5 to about 9 nucleotides, and similar sense strands. Alternatively, the sense strand may precede the nucleotide loop structure and may follow the antisense strand. These shRNAs may be contained in plasmids and viral vectors.

  The target region of the siRNA molecule can be selected from a predetermined target sequence. For example, the nucleotide sequence can begin about 25-100 nucleotides downstream of the start codon. The nucleotide sequence can contain a 5 'or 3' untranslated region, as well as a region near the start codon. Methods for designing and preparing siRNA molecules are well known in the art and include various rules for selecting sequences as RNAi reagents. See, for example, Boese et al., Methods Enzymol 392: 73-96 (2005).

  siRNA has been found in Hannon et al., Nature 418: 244 251 (2002); McManus et al., Nat Reviews 3: 737 747 (2002); Heasman et al., Dev Biol 243: 209 214 (2002); Stein et al , J Clin Invest, 108: 641 644 (2001); and Zamore et al., Nat Struct Biol 8 (9): 746 750 (2001). . Preferred siRNAs are 5 'phosphorylated.

  siRNA inhibitors, for example, to target CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1, Hs3st1, Sox9, Agc1, Cspg1 or Col2a1, or other molecules as described above, and their substrates or ligands Can be used for

  Nucleic acids can be obtained, isolated and / or purified from their natural environment in substantially pure or homogeneous form. Systems for cloning and expression of polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include, for example, Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells, and the like. For other cells suitable for producing proteins from nucleic acids, see Gene Expression Systems, Eds. Fernandez et al., Academic Press (1999). RNAi molecules may be chemically synthesized or expressed from a DNA sequence that encodes an siRNA or shRNA sequence.

  For the production of dominant negative mutants of the molecules described above, suitable regulatory sequences are included, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, selection or marker genes and other sequences as appropriate. Vectors can be selected or constructed. The vector may be plasmid or viral, for example, phage or phagemid, as appropriate. For further details, see, eg, Molecular Cloning: A Laboratory Manual, Sambrook et al., 2nd ed., Cold Spring Harbor Laboratory Press (1989). For example, many known techniques and protocols for working with nucleic acids in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and protein analysis include Current Protocols in Molecular Biology, Eds. Ausubel et al., 2nd ed., John Wiley & Sons (1992).

  The nucleic acid can be fused to other sequences encoding additional polypeptide sequences, for example, sequences that function as markers or reporters. Examples of marker or reporter genes include lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (causing neomycin (G418) resistance), dihydrofolate reductase (DHFR), hygromycin- Includes B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding galactosidase), xanthine guanine phosphoribosyltransferase (XGPRT), luciferase. Many others are known in the art.

2. Inhibition of Enzymatic Activity In certain embodiments of the invention, methods of treating tendinopathy include enzymes involved in proteoglycan biosynthesis, eg, enzymes involved in the addition of chondroitin sulfate GAGs to aggrecan and / or versican Reducing, inhibiting or down-regulating the activity of These enzymes include, for example, CS-GalNAcT-1, CS-GalNAcT-2, UDP-D-xylose: core protein β-D-xylosyltransferase, Galtransferase, GlcA transferase, GlcNAc transferase, sulfotransferase, chondroitin sulfate synthase , Hs3st1 and GalNT-1.

  In an exemplary embodiment, the present invention provides a method for reducing, inhibiting or downregulating the activity of CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1, and / or Hs3st1 enzyme in a damaged tendon. Include. In other embodiments, the present invention provides a method of reducing, inhibiting or downregulating Xyl transferase, Gal transferase, GlcA transferase, GalNAc transferase, GlcNAc transferase, sulfotransferase, or chondroitin sulfate synthase activity in damaged tendons. Include. The above peptides or small molecule inhibitors may be used to carry out these methods.

a) Identification of Inhibitors of Enzymatic Activity The present invention further relates to CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1, Hs3st1, Xyltransferase, Galtransferase, GlcA transferase, Methods of identifying and evaluating the efficacy of agents that can act as inhibitors of GalNAc transferase, GlcNAc transferase, sulfotransferase, and / or chondroitin sulfate synthase are included.

  Such inhibitors block or reduce the activity of enzymes involved in cartilage or fibrocartilage formation in tendons. These inhibitors include modified soluble substrates, proteins, small molecules and nucleic acids.

  A method of identifying an agent for treating tendinopathy includes assaying the effect of an individual agent on the enzyme, enzyme activity, protein-target interaction, or protein-protein interaction of the enzyme. . Various in vivo or in vitro assays may be used to determine the efficacy of an inhibitor in the treatment of tendinopathy. In one exemplary embodiment, the small molecule is capable of high-throughput screening (HTS) for its effects on the enzyme, eg, its enzymatic activity, target binding, or protein-protein interaction. By assaying by such methods, the potential for its therapeutic utility is assessed in vitro.

  In a typical HTS assay, the inhibitory effect of a candidate compound, eg, on enzyme activity, ligand-receptor binding, etc., may be determined by determining the endpoint of the assay in the presence of a known concentration of the candidate substance in the absence and / or known inhibition of the candidate substance. It can be measured by comparison with a control carried out in the presence of the compound. In general, conventional dyes, fluorescent or radioactive molecules are used to monitor the effects of candidate compounds. Thus, for example, candidate compounds can be identified that inhibit binding of a ligand to its receptor, or inhibit enzymatic activity, thereby reducing turnover of the enzymatic process.

  Exemplary embodiments of the present invention provide (1) at least one sample selected from the group consisting of CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1, and Hs3st1, and (2) a test agent And (3) measuring the activity of the sample component in response to the test agent and then (4) determining whether the test agent inhibits the activity of the sample component In vitro methods of identifying agents for treating tendinopathy are provided.

  In one exemplary embodiment, an in vitro method for identifying an agent for treating tendinopathy comprises: cartilage explants, primary chondrocyte pellet cultures, or chondrocyte lineage BMP-2 protein or BMP -2 treatment with an adenovirus expressing the protein. BMP-2 stimulates ECM and GAG synthesis. Test compounds are then added to the culture and the effect of the inhibitor on GAG synthesis is assessed by measuring 35S incorporation into the GAG side chain and / or by DMMB assay. The effect of the test compound is compared to the appropriate vehicle control.

  In vivo assays of potential enzyme inhibitors include: (1) repeated administration of a test inhibitor to a mammal (eg, Sprague-Dawley rat) for at least 2, 4, 6 or 8 weeks; Animals were subjected to a downhill running protocol at 17 m / min for 1 hour / day, 5 days / week (Example 1), and then (3) the effect of the inhibitor on the supraspinatus tendon in DMMB or AB assay Or by gene expression profiling, wherein the slowing of tendon degeneration (eg, cellular and morphological abnormalities) is believed to be due to the inhibitor, and the inhibitor is It is effective in treating degenerative disorders.

  It will be appreciated that test inhibitors useful in the methods of the invention can be evaluated in one or more animal models of tendon degenerative disorders and / or in humans.

  Various assays are known in the art for measuring enzyme activity in the presence of inhibitors in vivo and in vitro. Some examples of more frequently used assays are spectrophotometry, fluorescence spectroscopy, circular dichroism, autospectrophotometry and fluorescence spectroscopy, conjugate assays, acid or base autotitration, radioactivity, Includes label-free optical detection and enzyme inhibition assays. In an exemplary embodiment, the enzyme activity of CS-GalNAcT-1 and / or CSGalNAcT-2 is measured as described in Uyama et al., J Biol Chem 277 (11): 8841-46 (2002). The In another exemplary embodiment, the enzyme activity of GalNT-1 is measured as described in White et al., J Biol Chem 270 (41): 24156-65 (1995).

  Enzyme inhibitors useful in the methods of the invention can interact, for example, with the enzyme they inhibit. Alternatively, the inhibitor may interact with, for example, an enzyme substrate (eg, GalNAc) or other binding partner. Inhibitors may be useful in the present invention if they block the binding of the enzyme to the substrate and / or block the activity of the substrate after binding of the enzyme. Of course, the inhibitor can interact with both the enzyme and the second factor, eg, a substrate. In this regard, CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1, and / or Hs3st1 inhibitors may be modified, eg, modified soluble substrates, other proteins (those that bind to enzymes, and / or enzymes). Substrate), modified forms of enzymes or fragments thereof, propeptides, peptides, and mimetics of all these inhibitors.

  The enzyme inhibitor may be, for example, a direct enzyme inhibitor, may bind to the enzyme to neutralize enzyme activity, may decrease the level of enzyme expression, and active maturation of the precursor molecule. It may affect the stability or conversion to form, may interfere with the binding of the enzyme to one or more substrates, or may interfere with the intracellular function of the enzyme.

3. Inhibition of Transcription Factor Activity Transcription factor inhibitors useful in the methods of the present invention are either directly by binding or indirectly by interfering with protein-protein interactions and / or binding. Can be inhibited or reduced. In this regard, inhibitors of Sox9 can include modified soluble ligands or target molecules, small molecules, and nucleic acids.

  Assays for measuring Sox9 activity in the presence of inhibitors in vivo and in vitro are known in the art. Some examples of more frequently used assays for Sox9 inhibitors include expression levels of chondrocyte specific genes (eg, Col2a1, Agc1) in the presence of the yeast 2 hybrid system, luciferase reporter gene, Sox9 inhibitor. Includes PCR assays to determine, Western blot analysis of Sox9 or chondrocyte specific genes, transient transfection, chloramphenicol acetyltransferase (CAT) assay, and Northern blot analysis.

Various protein-protein or protein-nucleic acid interaction assays based on fluorescence resonance energy transfer (FRET) may be utilized to assay the effect of inhibitors on the interaction of Sox9 with its binding partner. For example, can be used AlphaScreen ^TM (TM) amplification luminescence proximity homogeneous assay (PerkinElmer ^R), or by using a bioluminescence resonance energy transfer (BRET ^TM of PerkinElmer ^R), interfere with inhibition Sox9 binding interactions The agent may be assayed in vitro.

  In one embodiment, the present invention provides (1) a target molecule as described above fused to a donor fluorogenic molecule, (2) the target molecule is combined with a test agent, and (3) fused to an acceptor fluorescent molecule. Adding the binding partner of the target molecule; (4) measuring the level of fluorescence energy transfer between the target molecule and its binding partner; and (5) allowing the test agent to interact with the target molecule and its binding partner. An in vitro assay method is provided for identifying an agent for treating tendinopathy comprising determining whether an effect has been inhibited.

  Inhibitors can interfere with the binding of the target molecule to its binding partner and can then affect the FRET level between the two substances. Thus, FRET measurements can be used to identify inhibitors of varying potency. Once an inhibitor of the target molecule has been identified in vitro, further in vivo testing is performed to determine the efficacy and availability of the inhibitor in preventing cartilage and fibrocartilage formation in the tendon Can do.

4). Inhibition of protein expression In another embodiment, methods for treating tendinopathy include CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1, Hs3st1, Col2a1, Cspg1, Agc1, and / or Sox9. Reducing, inhibiting or down-regulating the expression and / or accumulation of the gene and its encoded polypeptide or protein. Compositions useful for inhibiting the expression of these genes include, for example, interfering RNA molecules described in Section II (B) (1) (c) above.

  These inhibitors may be administered by any method, but in certain embodiments, the DNA expressing the RNA inhibitor is incorporated into an adenoviral vector which then requires tendon repair. Inject the patient. In an exemplary embodiment, the nucleotide sequence encoding RNAi is placed downstream of the pol II promoter as described in Wahdwa et al., Curr Opin Mol Ther 6 (4): 367-72 (2004). . Methods for inserting siRNA and shRNA into adenoviral vectors are well known in the art and are described, for example, in Krom et al., BMC Biotech 6 (11): [electronically published before printing]. Yes. Alternatively, as described in Schiffelers et al., Arthritis & Rheumatism 52 (4): 1314-18 (2005), RNAi inhibitors are injected directly into the injured tendon tissue and electroporated to cells Transported in.

5. Tendinopathic Disease The method of the present invention can be used in any mammal in need of treatment, including humans, primates, monkeys, rodents, sheep, rabbits, dogs, guinea pigs, horses, cows, and cats. It can be used to treat or prevent dynopathies. Disorders that can be treated or prevented include, for example, Tendinitis, Tendonitis, Tendinosis, Paratendinitis, Tenosynovitis, Tendon overuse injury, Tendon trauma, Peritonitis, Paratenonitis, and the family of “Tendinopathic” disease Any other state in is included.

  Tendinopathy can be caused by overuse and repetitive movement, sudden injury (mild to severe), gradual degeneration, or aging. Most tendon injuries consist of a series of slow recovering microruptures that weaken the tendon and thereby often cause pain, stiffness, and loss of power. Tendinopathy usually requires several weeks of treatment, reduced or modified activity, and rest. If a damaged tendon is used again too soon, it causes more tendon damage, which makes the damaged tendon susceptible to tearing or breaking. Thus, disorders treated or prevented by the present invention include both acute and chronic tendon degenerative disorders associated with overuse, sports or accident-related injuries and trauma, undernourishment, and older age.

  One example of tendinopathy is lateral epicondylitis, also known as “tennis elbow”, a well-known sports medicine and orthopedic disorder. The underlying pathology of the disorder is related to overuse and microrupture of the elbow short carpal extensor muscles. The body tries to repair these micro-ruptures, but often the healing process is incomplete. Histopathological specimens of patients undergoing surgery for chronic lateral epicondylitis show disordered angiofibroblast dysplasia in the damaged tendon. This incomplete attempt at repair results in degenerated, immature and vascularized tissue. Incompletely repaired tissue is weaker than normal tendon tissue and lacks the power to function properly. This weakened tissue also limits patients by causing pain and negatively impacting quality of life. Similar incomplete repairs include other types of tendon-related injuries or injuries such as patella tendonitis (jumper's knee), Achilles tendonitis (common in runners), and rotator cuff tendonitis (such as baseball pitchers) Found in “overhead” athletes).

  Tendinopathy can also be caused by, for example, older age, dietary deficiencies, sports activities, trauma, injury, or drugs such as pefloxacin. For example, prostaglandin E1 (PEG1)-, prostaglandin E2 (PGE2)-or pefloxacin-induced tendinopathy is a well-established animal model of human tendinopathy. In one embodiment, the present invention provides a method of treating or preventing drug-induced tendinopathy, eg, pefloxacin-induced tendinopathy, in an individual. In other exemplary embodiments, the present invention provides methods for treating or preventing tendinopathy induced by overuse, sports or accident-related injuries and trauma, undernourishment, and aging.

  The present invention further provides CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1, Xyl transferase, Gal transferase, GlcA transferase, GalNAc transferase, GlcNAc transferase, sulfotransferase, chondroitin sulfate synthase, Col2a1, Inhibitors of activity and / or expression of molecules such as aggrecan, versican, Hs3st1, and / or Sox9 in an amount effective to prevent or reduce the activity and / or expression of such molecules in tissues affected by tendinopathy To treat or prevent tendon degeneration disorders, slow tendon deterioration, restore tendon quality, maintain tendon health, treat tendon tissue hypoxia degeneration or Prevent, treat or prevent hyaline degeneration in tendon tissue, treat or prevent mucoid or myxoid degeneration in tendon tissue, treat or prevent fibrinoid degeneration in tendon tissue, treat or prevent lipoid degeneration in tendon tissue To treat or prevent calcification of tendon tissue, to treat or prevent fibrocartilage and ossification of tendon tissue, maintain tendon microstructure, maintain tendon fiber structure, tendon fiber array Maintain normal tendon vascularity, maintain normal cellularity in tendon tissue, restore normal ECM content in tendon tissue, reduce pathological accumulation of GAG in tendon tissue, and / or Alternatively, a method for maintaining tendon mass, tensile strength and / or flexibility is provided.

6). Treatment and Evaluation of Animal Models The methods of the present invention can be used to treat tendinopathy or microdefects in tendon tissue. Tendon quality after or during treatment, for example, by determining tendon microstructure integrity, tendon collagen staining, changes in tendon tissue cellularity, tendon vascularization, and / or GAG content Can do. Methods for assessing tendon health after or during treatment are known in the art and include, but are not limited to, magnetic resonance imaging (MRI), ultrasonography, and functional and / or pain assessment. Is included. Alternatively, tendon health can be measured by assessing the level of GAG in tendon tissue.

  One assay for determining tendon GAG content is the 1,9-dimethylmethylene blue (DMMB) assay for the measurement of sulfated glycosaminoglycan (GAG) concentrations in tendons. Another assay for determining tendon GAG content is histological Alcian blue (AB) staining that can be used to detect an increase in GAG content in tendon tissue.

III. Methods of treatment and pharmaceutical compositions Inhibitors useful in the methods of the invention are administered locally or systemically by topical, oral, intravenous, intraperitoneal, intramuscular, intracavitary, subcutaneous, implantation, or transdermal means. sell.

  In exemplary embodiments, administration of an inhibitor to an individual can also be accomplished by means of gene therapy in which a nucleic acid sequence encoding the inhibitor is administered to a patient in vivo. In an exemplary embodiment, a nucleic acid comprising a promoter sequence and a sequence encoding a nucleic acid inhibitor useful in the methods of the invention is inserted into an adenovirus expression vector. The adenovirus is then injected directly into the patient's tendon, where the nucleic acid inhibitor is expressed. Examples of adenovirus-mediated delivery of nucleic acids to tendon tissue are described, for example, in Mehta et al., J Hand Surg 30A (1): 136-41 (2005); Lou et al., J Orthoped Res 19: 1199-1202 (2001) and Lou, Clin Orthopaed Rel Res 379S: S252-55 (2000).

  In other exemplary embodiments, the nucleic acid inhibitor is incorporated into other viral expression vectors, such as lentiviruses, herpesviruses, and adeno-associated viruses. Appropriate vectors can be selected by assessing the in vitro infectivity of each type of virus in primary tendon cells.

  Alternatively, as described in Schiffelers et al., Arthritis & Rheumatism 52 (4): 1314-18 (2005), nucleic acid inhibitors are injected directly into tendon tissue and migrated into cells by electroporation. Let

  In other exemplary embodiments, peptides or small molecule inhibitors useful in the methods of the invention are as described in Paoloni et al., J Bone Joint Surg 86A (5): 916-22 (2004). It may be coated on a transdermal patch and applied directly to the skin covering the tendon tissue.

  Methods of administering molecules for the treatment of tendinopathy are known in the art. “Administration” is not limited to any particular delivery system, but includes, but is not limited to, parenteral (subcutaneous, intravenous, intramedullary, intra-articular, intramuscular, intracavitary, or intraperitoneal injection. ), Rectal, topical, transdermal, or oral (eg, in capsules, suspensions or tablets). Administration to an individual can be in a single dose or in continuous or intermittent repeated doses, in any of various physiologically acceptable salt forms, and / or as an acceptable pharmaceutical as part of a pharmaceutical composition (above). It may be performed locally or systemically with carriers and / or additives. Physiologically acceptable salt forms and standard pharmaceutical formulation techniques and excipients are well known to those skilled in the art. For example, Physicians' Desk Reference (PDR) 2003, 57th ed., Medical Economics Company, 2002; and Remington: The Science and Practice of Pharmacy, eds.Gennado et al., 20th ed, Lippincott, Williams & Wilkins, 2000) See

Inhibitors useful in the methods of the invention may be administered at a dosage of about 1 μg / kg to about 20 mg / kg, depending on the severity of the symptoms and the degree of disease progression. Suitable effective dosages can be determined by the treating clinician, for example, in the following ranges: from about 1 μg / kg to about 20 mg / kg, from about 1 μg / kg to about 10 mg / kg, from about 1 μg / kg to about 1 mg / kg. , About 10 μg / kg to about 1 mg / kg, about 10 μg / kg to about 100 μg / kg, about 100 μg / kg to about 1 mg / kg, and about 500 μg / kg to about 1 mg / kg.

  In another exemplary embodiment, the inhibitor is for a period of at least 2, 4, 6, 8, 10, 12, 20, or 40 weeks, or at least 1,1,. It is administered repeatedly for 5 or 2 years or until the subject's lifetime. In another embodiment of the invention, the inhibitor can be administered in a single dose, for example to stimulate restoration of the normal structure and composition of the tendon.

  In general, inhibitors useful in the methods of the invention include dosages of 10-8 and 10-7; 10-7 and 10-6; 10-6 and 10-5; or 10-5 and 10-4 g / kg. Can be administered. The therapeutically effective dose achieved in one animal model can be converted using conversion factors known in the art for use in other animals, including humans. See, for example, Freireich et al., Cancer Chemother Reports 50 (4): 219 244 (1966).

  The exact dosage of the inhibitor to be used in the method of the present invention is empirically determined based on the desired outcome. Exemplary outcomes are: (1) tendon degeneration disorder is treated or prevented, (2) tendon deterioration is delayed, (3) tendon quality is restored, and (4) tendon health is maintained. (5) treatment or prevention of hypoxic degeneration of tendons, (6) treatment or prevention of hyaline degeneration of tendons, (7) treatment or prevention of mucoid or mixoid degeneration of tendons, (8) Treatment or prevention of fibrinoid degeneration of tendons, (9) Treatment or prevention of lipoid degeneration of tendons, (10) Treatment or prevention of tendon calcification, (11) Tendons Treatment or prevention of fibrocartilage and ossification, (12) maintenance of tendon fiber structure, (13) maintenance of tendon fiber arrangement, (14) normal vascularity of tendon Maintained (15) normal cellularity of the tendon It encompasses be equity, (16) the normal ECM contained in the tendon is restored, and / or (17) tendon mass, the tensile strength and / or that the flexibility is maintained. For example, the inhibitor causes at least 20, 30, 40, 50, 100, 200, 300, 400 or 500 tendon deterioration (eg, tendon mass, loss of structural configuration and / or prone to micro-rupture and pain). Administered in an amount effective to delay.

  In some embodiments, the composition used in the methods of the invention further comprises a pharmaceutically acceptable excipient. As used herein, the term “pharmaceutically acceptable excipient” refers to any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like that are compatible with pharmaceutical administration. Examples of tonicity agents and absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition may also contain other active compounds that provide complementary, additional, or enhanced therapeutic functions. The pharmaceutical composition may also be included in a container, pack, or dence dispenser along with instructions for administration.

  A pharmaceutical composition to be administered in connection with the methods of the present invention should be formulated to be compatible with its intended route of administration. Examples of such formulations include crystalline protein formulations that are provided as is or in combination with biodegradable polymers (eg, PEG, PLGA).

Inhibitors useful in the methods of the invention may be administered as a pharmaceutical composition together with a carrier gel and matrix or other compositions used for induced bone regeneration and / or bone replacement. Examples of such matrices include synthetic polyethylene glycol (PEG), hydroxyapatite, collagen and fibutin based matrices, Tisseel ^R fibrin glue and the like.

  In certain embodiments, the inhibitor is in combination with other therapeutic compounds such as NSAIDs, corticosteroids, nitric oxide, testosterone, estrogens, growth factors (eg, BMP-12, BMP-13 and MP52) or You may administer simultaneously.

  Inhibitors useful in the methods of the present invention may be coated on or incorporated into tendon implants, matrices and depot systems to facilitate treatment or prevention of tendon injury.

Example 1: Rat supraspinatus tendon displays overused cartilage markers.
The response of rat supraspinatus tendon to overuse was studied at the molecular level using transcriptional profiling. A rat model of tendon overuse has been previously described. Soslowsky et al., J Shoulder Elbow Surg 9: 79-84 (2000). In this model, inflammation and angiogenesis markers are known to change (Perry et al., J Shoulder Elbow Surg 14: 79S-83S (2005)), but to understand the events surrounding overuse injury, A broader approach was taken.

  Twenty-four male Sprague-Dawley rats (400-450 g) were subjected to the supraspinatus tendon (SST) overuse protocol for 1 week (n = 8), 2 weeks (n = 8), and 4 weeks (n = 8). . The protocol consists of downhill running (10% gradient) at 17 m / min for 1 hour / day, 5 days / week. An additional 6 rats were used as cage activity controls (time 0). At each time point, two additional non-running rats were used as age-matched group controls.

  At necropsy, the supraspinatus tendon from each shoulder and the patella tendon (PT) from each knee were removed. The patella tendon was an internal control of the effects of exercise as it showed no significant signs of overuse using the protocol. The collected tendons were weighed and immediately frozen in liquid nitrogen. The tissue was frozen and cut with TRIzol reagent (Invitrogen). RNA was isolated from the aqueous layer of the extract using the RNeasy kit (QIAGEN). RNA concentration was determined using a spectrophotometer. Transcription profiling to monitor the expression level of over 30,000 transcripts was performed using the Affymetrix rat genome 230 2.0 array. All array images were examined visually for defects and quality. Arrays with high background, low signal intensity or serious defects were excluded from further analysis. The signal value was determined using Gene Chip Operating System 1.0 (GCOS, Affymetrix). For each array, statistics were normalized to an average signal intensity value of 100. Default GCOS statistics were used for all analyses. A gene was considered detectable if the average expression in any tissue exceeded 100 signal units and the percentage of samples with present (P) calls determined by GCOS default settings was 66% or higher. Normalized signal values were converted to log base 10. A gene was considered differentially expressed if the p-value from the ANOVA test was <0.01 and the difference between running and control was at least twice at any time point.

  Over 400 genes were differentially regulated in the supraspinatus tendon after overuse. By running for 4 weeks, 107 genes were up-regulated and 27 genes were down-regulated. Many of the most up-regulated genes are highly expressed in cartilage tissues including collagen type II alpha 1, versican and aggrecan. These results suggested that overused tendons have been converted to a fibrocartilage phenotype. These same genes were not upregulated in the patella tendon of the run animals (see FIGS. 1-7).

Example 2: Preparation of siRNA Inhibitors siRNAs are derived from CS-GalNAcT-1, GalNT-1 or Hs3st1 nucleotide sequences on commercial websites for designing specific siRNAs (eg sirnawizard.com or Ambion ^R siRNA). It is selected by typing in Target Finder. Sequence selection guidelines are embedded in these tools.

  The efficacy of siRNA is to transfect it into C-20 / A4 and / or C-28 / I2 chondrocytes and monitor the expression of CS-GalNAcT-1 and / or GalNT-1 by real-time RT-PCR. Measured by. A suitable scrambled siRNA having the same nucleotide composition is used as an experimental control.

Example 3: In vitro assay system for inhibitors of proteoglycan synthesis siRNA reduces expression of CS-GalNAcT-1, GalNT-1 or Hs3st1 in C-20 / A4 and / or C-28 / I2 chondrocytes If so, it is then tested for the ability to reduce the production of proteoglycan GAG side chains. C-20 / A4 and / or C-28 / I2 chondrocytes are treated with adenovirus expressing BMP-2 protein or BMP-2 to stimulate extracellular matrix and GAG synthesis. siRNA, or alternatively, a small molecule, an inhibitor of GAG synthesis, is added to the culture and the effect of the inhibitor is assessed by comparing 35S incorporation into proteoglycans in the presence or absence of the inhibitor. . Alternatively, inhibitors can be obtained by comparing GAG levels in the presence or absence of inhibitors by DMMB assay as described in Arai et al., Osteoarthritis and Cartilage 12: 599-613 (2004). Evaluate the effect.

Example 4: Protocol for treatment of tendinopathy by siRNA inhibition of CS-GalNAcT-1, GalNT-1 or Hs3st1 Treadmill overuse protocol (Example 1) or week of PGE1, PGE2 or pefloxacin (300 mg / ml) Tendinopathies are induced in rats using any one of 5 weekly, 1 week local injections.

  Once siRNA was found to reduce the in vitro activity of CS-GalNAcT-1, GalNT-1 or Hs3st1, for example, Krom et al., BMC Biotech 6 (11): [electronic publication] It is converted to shRNA as described in, and the shRNA sequence is cloned and inserted into adenovirus. Adenoviruses are used for in vivo expression of shRNA and subsequent inhibition of CS-GalNAcT-1 and / or GalNT-1 expression. The infection method is optimized in primary tendon cells.

  Functional adenovirus expressing shRNA is injected directly into the injured tendon (Mehta et al., J Hand Surg 30A (1): 136-41 (2005)), CS-GalNAcT-1 and / or Alternatively, the expression of the GalNT-1 gene is locally reduced. A scrambled sequence is included as an experimental control. The amount of cartilage tissue formation in the tendon is determined by evaluating the GAG level. Histological Alcian blue staining is used to detect increased amounts of glycosaminoglycan (GAG) in tendon tissue. The amount of sulfated GAG extracted from tendon is quantified using DMMB reagent.

  In vivo assessment of the effect of CS-GalNAcT-1 and / or GalNT-1 inhibition on tendinopathy is determined by histology and by mechanical or functional testing of tendon strength. An inhibitor is considered effective if the GAG level is significantly reduced compared to an untreated control and / or if it is reduced to a level similar to a normal undamaged tendon. Alternatively, an inhibitor is considered effective if a functional or mechanical test reveals improved function and / or reduced pain compared to an untreated control.

FIG. 1 shows the gene expression profile of Col2a1 (type II collagen) in tendons overworked for 4 weeks using the Affymetrix RAE230 2.0 gene chip (FIG. 1A). The expression level of the Col2a1 gene was also measured in the indicated normal musculoskeletal tissues (FIG. 1B). The expression profile shows that Col2a1 is highly expressed in both cartilage tissue and overworked tendon tissue when compared to normal tendon tissue. FIG. 2 shows the gene expression profile of Agcl (Aggrecan) in tendons overworked for 4 weeks using the Affymetrix RAE230 2.0 gene chip (FIG. 2A). The expression level of the Agcl gene was also measured in the indicated normal musculoskeletal tissues (Fig. 2B). The expression profile shows that Agc1 is highly expressed in both cartilage tissue and overused tendon tissue when compared to normal tendon tissue. FIG. 3 shows the gene expression profile of Sox9 (sry-type high mobility group box9) in tendons overuse for 4 weeks using the Affymetrix RAE230 2.0 gene chip (FIG. 3A). The expression level of Sox9 gene was also measured in the indicated normal musculoskeletal tissues (FIG. 3B). The expression profile shows that Sox9 is highly expressed in both cartilage tissue and overworked tendon tissue when compared to normal tendon tissue. FIG. 4 shows the gene expression profile of Cspg2 (Versican) in tendon overuse for 4 weeks using the Affymetrix RAE230 2.0 gene chip (FIG. 4A). Versican gene expression levels were also measured in the indicated normal musculoskeletal tissues (FIG. 4B). The expression profile shows that versican is highly expressed in both cartilage tissue and overused tendon tissue when compared to normal tendon tissue. FIG. 5 shows the gene expression profile of chondroitin sulfate N-acetylgalactosaminyltransferase (CS-GalNAcT-1) in tendon overuse for 4 weeks using the Affymetrix RAE230 2.0 gene chip (FIG. 5A). The expression level of CS-GalNAcT-1 gene was also measured in the indicated normal musculoskeletal tissues (FIG. 5B). The expression profile shows that CS-GalNAcT-1 is highly expressed in both cartilage tissue and overworked tendon tissue when compared to normal tendon tissue. FIG. 6 shows GalNT-1 (GalNT1-UDP-N-acetyl-alpha-D-galactosamine: polypeptide-N-acetylgalactosaminyltransferase 1 in an over-worked tendon over 4 weeks using an Affymetrix RAE230 2.0 gene chip. ) Shows the gene expression profile (FIG. 6A). The expression level of the GalNT-1 gene was also measured in the normal musculoskeletal tissues shown (FIG. 6B). The expression profile shows that GalNT-1 is highly expressed in overused tendon tissue when compared to normal tendon tissue. FIG. 7 shows the gene expression profile of Hs3st1 (heparan sulfate (glucosamine) 3-O-sulfotransferase 1) in tendons overuse for 4 weeks using the Affymetrix RAE230 2.0 gene chip (FIG. 7A). The expression level of the Hs3st1 gene was also measured in the indicated normal musculoskeletal tissues (FIG. 7B). The expression profile shows that Hs3st1 is highly expressed in tendon tissue overworked when compared to normal tendon tissue and in cartilage tissue when compared with normal tendon tissue.

Claims (42)

  A method of treating tendinopathy in a patient, characterized by reducing the formation of cartilage-specific proteoglycans in the tendon tissue of the patient.   2. The method of claim 1, wherein the patient is a human.   2. The method of claim 1, wherein the patient is selected from the group consisting of primates, monkeys, rodents, sheep, rabbits, dogs, guinea pigs, horses, cows and cats.   2. The method according to claim 1, wherein the activity of an enzyme involved in the synthesis of proteoglycan selected from the group consisting of aggrecan, versican, and syndecan-3 is reduced in the tendon tissue of the patient.   Reducing the activity of chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CS-GalNAcT-1) and / or galactosamine polypeptide N-acetylgalactosaminyltransferase 1 (GalNT-1) in the tendon tissue of patients The method according to claim 1.   The method of claim 1, wherein the activity is decreased by inhibiting the enzyme activity of CS-GalNAcT-1, CS-GalNAcT-2, Hs3st1, and / or GalNT-1.   The method according to claim 1, wherein the activity is decreased by inhibiting the expression of CS-GalNAcT-1, CS-GalNAcT-2, Hs3st1, and / or GalNT-1 gene.   The method according to claim 1, wherein the activity of a transcription factor involved in cartilage production is decreased.   9. The method of claim 8, wherein the activity of Sox9 is decreased.   10. The method of claim 9, wherein the activity is decreased by inhibiting the ability of Sox9 to increase the expression of a cartilage specific gene.   The method according to claim 8, wherein the activity is decreased by inhibiting expression of the Sox9 gene.   2. The method of claim 1, wherein the expression of cartilage specific protein is reduced.   13. The method of claim 12, wherein the expression of proteoglycan selected from the group consisting of aggrecan, versican, and syndecan-3 is decreased.   13. The method of claim 12, wherein the expression of Col2a1 is decreased.   The method of claim 1, wherein the tendinopathy is tendinitis.   The method of claim 1, wherein the tendinopathy is tendinosis.   The method of claim 1, wherein the tendinopathy is tendon injury.   A method of treating tendinopathy in a patient, comprising administering an inhibitor of CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1, Hs3st1, and / or Sox9 to tendon tissue in the patient .   19. The method of claim 18, wherein the inhibitor reduces CS-GalNAcT-1 and / or GalNT-1 enzyme activity.   19. The method of claim 18, wherein the inhibitor reduces CS-GalNAcT-1 and / or GalNT-1 expression.   19. The method of claim 18, wherein the inhibitor is administered locally.   19. The method of claim 18, wherein the inhibitor comprises an interfering RNA molecule.   The method of claim 18, wherein the inhibitor comprises a small molecule.   A method of treating tendinopathy in a patient, comprising reducing the expression of cartilage-specific protein in the patient's tendon tissue.   25. The method of claim 24, wherein the method inhibits expression of aggrecan, versican, and / or type II collagen.   25. The method of claim 24, wherein the method inhibits Sox9 expression.   25. The method according to claim 24, wherein the method inhibits Sox9 activity.   Administering a test agent to a subject in need of treatment and then measuring the ability of the agent to inhibit the activity of enzymes involved in the biosynthesis of glycoglycosaminoglycans (GAGs) in tendon tissue, A method of identifying a therapeutic agent for tendinopathy.   A test compound comprising administering a test compound to a subject in need of treatment and then measuring the ability of the compound to inhibit the activity of an enzyme involved in the synthesis of glycoglycosaminoglycan in tendon tissue. A method of identifying compounds useful in therapy. further,
(A) providing at least one sample component selected from the group consisting of CS-GalNAcT-1, CS-GalNAcT-2, heparin sulfate (glucosamine) 3-O-sulfotransferase 1, GalNT-1 and Sox9;
(B) combining the sample with a test compound;
(C) measuring the activity of the sample component in response to the test compound;
30. The method of claim 29, comprising determining whether (d) the test compound inhibits the activity of the sample component.   Use of a compound that reduces the formation of cartilage-specific proteoglycans in the manufacture of a medicament for treating tendinopathy.   32. Use according to claim 31, wherein the compound reduces the activity of an enzyme or other protein involved in cartilage synthesis.   33. Use according to claim 32, wherein the compound directly or indirectly inhibits the function of the enzyme or protein.   34. Use according to claim 32 or 33, wherein the compound inhibits the expression of an enzyme or protein.   33. The enzyme selected from the group consisting of UDP-D-xylose: core protein β-D-xylosyltransferase, Gal transferase, GlcA transferase, GlcNAc transferase, sulfotransferase, chondroitin sulfate synthase, and heparin sulfate sulfotransferase. 34. Use according to any one of 34.   The enzymes are chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CS-GalNAcT-1), chondroitin sulfate N-acetylgalactosaminyltransferase 2 (CS-GalNAcT-2), galactosamine polypeptide N-acetylgalactosaminyltransferase 1 ( 36. Use according to claim 35, selected from the group consisting of GalNAcT-1) and heparin sulfate (glucosamine) 3-O-sulfotransferase 1 (Hs3st1).   32. Use according to claim 31, wherein the compound reduces the expression of cartilage specific structural proteins.   32. Use according to claim 31, wherein the compound decreases the activity of transcription factors involved in the expression of cartilage-specific structural proteins.   39. Use according to claim 38, wherein the transcription factor is Sox9.   40. Use according to any one of claims 31 to 39, wherein the proteoglycan is selected from the group consisting of aggrecan, versican, syndecan-3 and type II collagen.   41. Any one of claims 31-40, wherein the tendinopathy is selected from the group consisting of tendonitis, tendinitis, tendinosis, paratendinitis, tendon synovitis, tendon injury, tendon trauma, peri-tendonitis and paratenonitis. Use of description.   The use according to any one of claims 31 to 41, wherein the medicament is administered to a patient selected from the group consisting of primates, monkeys, rodents, sheep, rabbits, dogs, guinea pigs, horses, cows and cats. .

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