CN101432026A - Treatment of tendinopathy by inhibition of molecules that contribute to cartilage formation - Google Patents

Abstract

The invention provides methods for treating tendinopathy. The disorders treated or prevented include, for example tendinitis, tendonitis, tendinosis, paratendinitis, tenocynovitis, tendon overuse injury and trauma, peritendinitis, paratenonitis, or other tendon degenerative disorders. The disclosed therapeutic methods include administering to a patient an inhibitor of molecules involved in cartilage or fibrocartilage formation in tendinopathic tendon in an amount effective to treat or prevent a tendon degenerative disorder, slow tendon deterioration, restore tendon healthy structure, stimulate tendon regeneration, and/or maintain tendon mass and/or quality.

Description

Treat the tendon pathological changes by suppressing to be beneficial to chondrogenetic molecule

Invention is described

Previous application

The application requires the priority of the U.S. Provisional Patent Application series number 60/779,165 of submission on March 3rd, 2006, and the content of wherein said temporary patent application is incorporated herein by reference herein.

Invention field

Technical field of the present invention relates to by reducing cartilage in the impaired tendon and/or fibrous cartilage and produces and treat the tendon pathological changes.The invention further relates to and suppress to produce relevant molecule with cartilage and/or fibrous cartilage in impaired tendon.

Background of invention

Tendinopathy becomes the common term that is used for describing polytype tendon disease.Term " tendinitis " (expression " inflammation of tendon ") is commonly used to describe the problem of tendon, but inflammation seldom is the reason of tendon pain.The most normally, tendon pain be actually in the tendon or connective tissue on every side in a series of little symptoms of tearing, more suitably be called tendon degeneration (tendinosis).Other tendon disease comprises and results from the friction to the health projection of the key pain, the mucoid substrate increase in the tendon, calcification, excessively use of tendon, vascularization of the collagenous degeneration of following the fiber alignment obstacle, aging and tendon.The tendon expert of more and more quantity uses the tendon pathological changes to describe these diseases, is characterized by tendinitis, tendon degeneration (tendinosis), peritenonitis (paratendinitis), tenosynovitis (tenosynovitis), paratenonitis, tendon usually jointly and excessively uses damage and wound.People such as Khan, SportsMed27:393-408 (1999).

Normal tendinous tissue comprises the connective tissue of tight filling, and it has regularly arranged collagenous fiber bundle, and described collagen fiber operate in elementary, secondary and three grades, have in the fibre bundle of high-tensile with equidirectional.Tendon cell (tenocyte) flat, taper is dispersed in these fibers, the synthetic stickiness extracellular matrix (ECM) that is rich in type i collagen of these tendon cells.The type i collagen bundle provides elasticity and structural support for tendon.In the tendon of health, between synthesizing and degrade, ECM has dynamic equilibrium.Yet the tendon pathological changes influences this balance negatively, and causes collagen fiber structural rearrangement and disintegration.The disintegration of collagen bundle causes tendon cartilage to occur.Clearly observing the pathology of fibroblast among the ECM, myofibroblast, neovascularization and glycosaminoglycans (GAG) in the tendon pathological tissues assembles.People such as Tallon, Med Sci Sports Exerc 33 (12): 1983-90 (2001); People such as Khan, Sports Med27 (6): 393-408 (1999).Metabolism and modal variation cause pain and to tearing and disruptive susceptible in the impaired tendon.

At present, think impaired tendon ECM degraded and reinvent by causing that from settling down the metalloproteases that connective tissue cell and invasive inflammatory cell discharge wherein said metalloprotein endonuclease capable matrix degradation macromole is aggrecan, decorin gene polysaccharide and disaccharidase catenin polysaccharide for example.These metalloproteases comprise MMP (Matrix Metalloproteinases, matrix metalloproteinase), ADAM (ADisintegrin And Metalloproteinase, de-connect albumen and metalloproteases) and ADAMT (ADisintegrin And Metalloproteinase with Thrombospondin Motifs, what have the thrombospondin motif de-connects albumen and metalloproteases, for example aggrecan enzyme).RileyG., Expert Rev Mol Med 7 (5): 1-23 (2005); People such as Rees, BiochemJ350:180-188 (2000).Yet the wide spectrum inhibitor of MMP has in the clinical trial of osteoarthritis of pathology similarity in treatment and tendon pathological changes be unsuccessful.People such as Clark, ExpertOpinTher Targets 7 (1): 19-34 (2003).Therefore, suppress the unlikely tendon pathological changes for the treatment of effectively of metalloproteases.In fact, for example need in tendon of supraspinatus muscle and the heel string metal proteinase activity of minimum level that the tendon of the ECM with proper level is provided at the tendon of highly stressed.Riley?G.，ExpertRev?Mol?Med?7(5)：1-23(2005)。

At present, the non-operation and the operation property tendon lesions treatment that have multiple standards.Non-operation method comprises rest, frigotherapy, active correction, physiotherapy, nonsteroidal and-inflammatory drug (NSAID) and corticosteroid.Have a rest and patient that active correction can help to suffer from some this diseases, but also existence in a large number can not be with the clinical colony of these therapy for treating.Although be extensive use of, the anti-inflammatory drug therapy of per os does not confirm useful as yet in the research of control, and has undesirable side effect.Some researchs show that further in fact, the non-steroid pharmacotherapy has side effect to rehabilitation course, and this is because it makes the patient ignore the early symptom of tendon pathological changes by easing the pain.

Corticosteroid is generally used for reducing the inflammation in the tissue, but does not advise using this type of Drug therapy tendon pathological changes, particularly because corticosteroid suppresses the synthetic of collagen.The improvement of short-term is observed in some researchs in the patient with cortisone treatment, but follow the tracks of the patient surpass 1 year studies show that high symptoms recurrence rate and uncertain effective percentage.The cortisone injection also has the risk of tendon rupture, infection, skin depigmentation and subcutaneous atrophy.In diabetics, injection may cause hyperglycemia.

The surgical method of tendon reparation comprises the reparation of debridement and impaired tendon.Yet open or arthroscopic surgery operation has many potential complication, for example degree of depth infection, injured nerve blood vessel structure and formation scar.Surgical operation is also very expensive, and has the additional risk of following part or general anesthesia.

Therefore, still exist treatment tendon associated injury, be better than the demand of the method for existing therapy.The damage of pliable and tough tendinous tissue or other infringement recovery from illness is slow, and need the several months or even the several years could recover fully.The tendon associated injury has appreciable impact to society.In the U.S., excessively use that damage accounts for that the relevant mechanism of total loss pays a home visit nearly 7%.People such as Woodwell, Adv Data 346:1-44 (2004).Based on the information of industry employment statistics department of Department of Labor (U.S.Department of Labor, Bureau ofLabor Statistics) data, this type of damage causes the working time significantly to be lost (seeing Http:// www.bls.gov/lif).

Summary of the invention

The invention provides the method for new treatment tendon pathological changes.Transcribe profile analysis by rat tendon to excessive use, have been found that now the special gene of cartilage for example the expression of aggrecan, versican, Col2a1 (II Collagen Type VI) and Sox9 in impaired tendon, increase.These results show that impaired tendinous tissue changes fibrous cartilage into owing to excessively using.Different with the tendon that mainly comprises type i collagen, fibrous cartilage contains II Collagen Type VI and big Dan Baijutang, for example aggrecan and versican.Reparation is deleterious for tendon for cartilage and fibrocartilaginous formation in the tendon because cartilage destruction the tendon type i collagen fiber of tight filling.This destruction has been reduced the tensile strength and the elasticity of tendon.

Therefore, the invention provides the method for coming treatment tendon pathological changes in the patient by the formation that reduces cartilage specificity Dan Baijutang in patient's tendinous tissue.The present invention also provides and reduces the purposes of chemical compound in the medicine of preparation treatment tendon pathological changes that the cartilage specificity Dan Baijutang forms.

In some embodiments, these methods and purposes comprise that reduction relates to the activity of synthesizing the enzyme of aggrecan and/or versican in patient's tendinous tissue.In specific embodiments, these methods and purposes are included in the activity that reduces chondroitin sulfate N-acetylamino galactosamine based transferase 1 (CS-GalNAcT-1) and/or galactosamine polypeptide N-acetylamino galactosamine based transferase 1 (GalNT-1) in patient's tendinous tissue.In other embodiments, these methods and purposes comprise that reducing one or more relates to the activity of synthetic other enzyme of chondroproteoglycan, and wherein said enzyme includes but not limited to the UDP-D-xylose: core protein β-D-xylosyltransferase, Gal transferring enzyme, GlcA transferring enzyme, GlcNAc transferring enzyme, sulfotransferase, chondroitin sulfate synthase and Heparan sulfate sulfotransferase.

Relate to and produce the proteinic enzyme of cartilage structure and other activity of proteins and can reduce by inhibitory enzyme or other proteinic function directly or indirectly, perhaps the expression by inhibitory enzyme or other protein self reduces.

The present invention also provides the method for the treatment of patient's tendon pathological changes by the expression that reduces cartilage specificity structural protein in patient's tendinous tissue.The present invention further provides and reduce the purposes of chemical compound in the medicine of preparation treatment tendon pathological changes that the cartilage specificity structural protein is expressed.In some embodiments, the cartilage specificity structural protein is subjected to direct inhibition such as but not limited to the expression of aggrecan, syndecan 3 (syn-3), versican and II Collagen Type VI.In other embodiments, reduced the activity of the transcription factor that relates to cartilage specificity gene expression.These transcription factor and signal transducer matter comprise for example Sox9.

In other embodiments, the present invention includes the method for the chemical compound of identifying treatment tendon pathological changes, use test compounds and detect the active ability that this material suppresses to relate to the synthetic enzyme of glycosaminoglycans in the tendinous tissue (glycoglycosaminoglycan) _ (GAG) comprising experimenter to the needs treatment.

In some embodiments, identify that the chemical compound of treatment tendon pathological changes or the method for assessment treatment comprise: (1) provides the sample composition of at least a CS-GalNAcT-1 of being selected from, CS-GalNAcT-2, Heparan sulfate (glycosamine) 3-O-sulfotransferase 1 (Hs3st1), GalNT-1 and Sox9; (2) with sample and test compounds combination; (3) the measuring samples composition is replied the activity of test compounds; And (4) determine whether test compounds suppresses the activity of sample composition.

Purpose that the present invention is extra and advantage provide part in the following description, and part is apparent in description, perhaps can learn by implementing the present invention.Objects and advantages of the present invention by hereinafter detailed description and appended claims in the composition that particularly points out and combination realize and obtain.

Should be appreciated that above-mentioned general description and detailed description hereinafter only are example and indicative, do not limit described the present invention.

The accompanying drawing that mixes and constitute this description part has shown embodiments more of the present invention, and and describes and work to explain the principle of the invention together.

The accompanying drawing summary

In Fig. 1-7, SST represents tendon of supraspinatus muscle; PT represents the unmarred confidential reference items of patellar tendon; The animal of excessive use step people such as (, J.Shoulder Elbow Surg.9:79-84 (2000)) Soslowsky has been carried out in R representative; The control animal that step is excessively used in the C representative; Time course was 1,2 and 4 weeks; And BM represents bone marrow.

Fig. 1 show use Affymetrix RAE230 2.0 gene chips, the gene expression profile in Col2a1 (II Collagen Type VI) 4 weeks in the tendon (Figure 1A) that excessively uses.Shown in figure (Figure 1B), also measured Col2a1 expression of gene level in the normal flesh skeletal tissue.Express spectra shows, compares with normal tendinous tissue, and Col2a1 highly expresses in the tendinous tissue of cartilaginous tissue and excessively use.

Fig. 2 show use Affymetrix RAE230 2.0 gene chips, the gene expression profile in 4 weeks of Agc1 (aggrecan) in the tendon (Fig. 2 A) that excessively uses.Shown in figure (Fig. 2 B), also measured Agc1 expression of gene level in the normal flesh skeletal tissue.Express spectra shows, compares with normal tendinous tissue, and Agc1 highly expresses in the tendinous tissue of cartilaginous tissue and excessively use.

Fig. 3 show use Affymetrix RAE230 2.0 gene chips, the gene expression profile in Sox9 (sry-type high mobility group frame 9) 4 weeks in the tendon (Fig. 3 A) that excessively uses.Shown in figure (Fig. 3 B), also measured Sox9 expression of gene level in the normal flesh skeletal tissue.Express spectra shows, compares with normal tendinous tissue, and Sox9 highly expresses in the tendinous tissue of cartilaginous tissue and excessively use.

Fig. 4 show use Affymetrix RAE230 2.0 gene chips, the gene expression profile in 4 weeks of Cspg2 (versican) in the tendon (Fig. 4 A) that excessively uses.Shown in figure (Fig. 4 B), also measured versican expression of gene level in the normal flesh skeletal tissue.Express spectra shows, compares with normal tendinous tissue, and versican is all highly expressed in the tendinous tissue of cartilaginous tissue and excessively use.

Fig. 5 show use Affymetrix RAE230 2.0 gene chips, the gene expression profile in 4 weeks of chondroitin sulfate N-acetylamino galactosamine based transferase (CS-GalNAcT-1) in the tendon (Fig. 5 A) that excessively uses.Shown in figure (Fig. 5 B), also measured CS-GalNAcT-1 expression of gene level in the normal flesh skeletal tissue.Express spectra shows, compares with normal tendinous tissue, and CS-GalNAcT-1 highly expresses in the tendinous tissue of cartilaginous tissue and excessively use.

Fig. 6 shows use Affymetrix RAE230 2.0 gene chips, GalNT-1 (GalNT1-UDP-N-acetyl group-α-D-galactosamine: the gene expression profile in 4 weeks polypeptide-N-acetylamino galactosamine based transferase 1) in the tendon (Fig. 6 A) that excessively uses.Shown in figure (Fig. 6 B), also measured GalNT-1 expression of gene level in the normal flesh skeletal tissue.Express spectra shows, compares with normal tendinous tissue, and GalNT-1 expresses at the tendinous tissue camber that excessively uses.

Fig. 7 show use Affymetrix RAE230 2.0 gene chips, the gene expression profile in Hs3st1 (Heparan sulfate (glycosamine) 3-O-sulfotransferase 1) 4 weeks in the tendon (Fig. 7 A) that excessively uses.Shown in figure (Fig. 7 B), also measured Hs3st1 expression of gene level in the normal flesh skeletal tissue.Express spectra shows, compares with normal tendon, and Hs3st1 expresses at the tendon camber that excessively uses, and compares at the cartilaginous tissue camber with normal tendinous tissue and to express.

The sequence summary

The nucleotides of CS-GalNAcT-1 and amino acid sequence can be with following accession number from Genbank Obtain: people (NM_018371); Mouse (NM_172753) and rat (XM_224757).

The nucleotides of CS-GalNAcT-2 and amino acid sequence can be with following accession number from Genbank Obtain: people (NM_018590); Mouse (NM_030165) and rat (XM_232316).

The nucleotides of GalNT-1 and amino acid sequence can obtain from Genbank with following accession number: People (NM_020474); Mouse (NM_013814) and rat (NM_124373).

The nucleotides of aggrecan and amino acid sequence can be with following accession number from Genbank Obtain: people (NM_001135); Mouse (NM_007427) and rat (NM_022190).

The nucleotides of Col2a1 and amino acid sequence can obtain from Genbank with following accession number: People (NM_001844); Mouse (NM_031163) and rat (NM_012929).

The nucleotides of Sox9 and amino acid sequence can obtain from Genbank with following accession number: the people (NM_000346); Mouse (NM_011448) and rat (XM_343981).

The nucleotides of versican and amino acid sequence can be with following accession number from Genbank Obtain: people (NM_004385); Mouse (XM_488510) and rat (XM_215451).

Nucleotides and the amino acid sequence of Heparan sulfate (aminoglucose) 3-O-sulfotransferase 1 can To obtain from Genbank with following accession number: people (NM_005114), mouse (NM_010474) and Rat (NM_053391).

Detailed Description Of The Invention

The invention provides by reducing one or more and relate to cartilage and/or fibrocartilage shape in the tendinous tissue The activity of the molecule that becomes is treated the method for tendon pathology.

For the present invention easier to understand, at first define some terms.Extra being defined in the detailed description of the present invention provides.

As used herein, term " tendon pathological changes " comprises that all occur in the pathology around the tendon neutralization, comprising but be not limited to tendinitis, tendinitis, tendon degeneration, peritenonitis, tenosynovitis (tenosynovitis), tendon and excessively use damage and wound, peritendinitis (perintendinitis) and paratenonitis.

As used herein, term " tendon defective " refers to any tendon disease, comprising but be not limited to the tendon pathological changes.

As used herein, term " impaired tendon " refers to the tendinous tissue of suffering from tendon pathological changes or tendon defective.

As used herein, term " excessively uses " tendon to refer to suffer from because excessively use but not directly the tendon defective that causes of wound or the tendon of tendon pathological changes.

As used herein, term " patient " or " experimenter " refer to any to the damaged sensitivity of tendon, to suffer from tendon damaged or be in the damaged recovery process of tendon and need the human or animal of treatment.

As used herein, term " tendinitis " refers to the inflammation of tendinitis (another kind of saying), tendon pathological changes or tendon.

As used herein, term " tendon degeneration " refers to the tendon pathological changes or little tearing is taken place in tendon and causes the tendon reduction, causes pain, stiff and lose any degeneration disease of intensity usually.

As used herein, term " tendon injury " refers to tendon pathological changes or any tendinous tissue disease of defective or degeneration that becomes.

As used herein, term " micromolecule " comprise except that polypeptide and nucleic acid, can work any chemistry or other component that influence the bioprocess effect.

As used herein, term " treatment (verb) " or " treatment (noun) " refer to and arbitrarily mammal are comprised human disease's administration or application scheme, and comprise the inhibition disease.It comprises and stops disease progression and palliate a disease, for example by restoring or recovering or repair and lose, lose or the function of defective or stimulate invalid process.

As used herein, term " activity " refer to can suppress in several ways, attenuating or interferential enzyme or proteinic biological function.

As used herein, term " activity of enzyme " refers to one or more physiologically actives, catalytic activity, adjusting activity or the enzymatic activity relevant with enzyme.

As used herein, term " effective dose " expression enough shows significant patient's benefit, promptly cures tendon pathological changes or increase healing and improve the total amount of every kind of inhibitor of the present invention of the speed of symptom.

I. the composition of tendon and cartilage

Tendon comprises orderly type i collagen layer, and III Collagen Type VI, fibroblast sample tendon cell and chondrocyte sample fibrocartilage cells in a small amount therebetween is scattered here and there.Type i collagen is filled and enters in the fine and close collagen fiber, and this provides intensity and stiff for it at tendon during by muscle stretch that is attached and tractive.Relatively, cartilage comprises II Collagen Type VI and Dan Baijutang, and design is resisted compression but not tension force.Fibrous cartilage is the mixture of tendon and cartilaginous tissue, and mainly is present between tendon and the bone at the interface.Formation cartilage and fibrous cartilage are deleterious to the function of tendon in tendon, and this is because it destroys tensile strength of the type i collagen fiber and the reduction tendon of closely filling.Therefore, method of the present invention can be used for stoping or reduction cartilage and/or fibrocartilaginous formation in the tendinous tissue of impaired or damage.

A chondrogenetic indication is the Dan Baijutang that occurs the increase level in tissue in the tendon, this usually the existence of glycosaminoglycans (GAG) side chain by these Dan Baijutang detect.Dan Baijutang is the glycoprotein family that is characterized by the core protein with one or more linear GAG chains, and wherein said GAG chain is made up of multiple disaccharide unit.Chondroitin sulfate proteoglycan for example aggrecan and versican is that cartilage is special.Aggrecan is the main constituent of cartilage, and the compressive strength of responsible cartilage and elasticity.The hydration of aggrecan molecule and gathering produce the multiple gel of bearing the compressibility load in cartilaginous tissue.People such as Watanabe, J.Biochem124 (4): 687-93 (1998).Other Dan Baijutang that exists in the cartilaginous tissue comprises heparan sulfate proteoglycan, for example syndecan 3 (syn-3).People such as Kirn-Safran, Birth DefectsRes 72 (C part): 69-88 (2004).

II. promote the molecule that cartilage and fibrous cartilage form

The present invention's part is based on following discovery, and promptly cartilage specificity gene (for example aggrecan, versican and II Collagen Type VI) is expressed increase in impaired tendon.By people such as Soslowsky, this result of the gene expression profile that obtains during the described rat tendon of J Shoulder Elbow Surg.9:79-84 (2000) excessively uses a model show impaired tendon since excessively use change fibrous cartilage into.

The present invention also part promptly relates to the synthetic enzyme of cartilage specificity Dan Baijutang glycosaminoglycans (GAG) side chain and express increase in the tendon that excessively uses based on following discovery.This discovery is that the cartilage specificity enzyme is adjusted to that observed GAG is provided by the mechanism that provides in the tendon that is being badly damaged in impaired tendon.People such as Khan, people such as Sports Med 27:393-408 (1999) and Tallon, Med Sci SportsExerc 33 (12): 1983-90 (2001).The GAG level of these increases shows that the Dan Baijutang for example level of aggrecan, versican and syn-3 increases in impaired tendon.Therefore, can prevent the gathering of cartilage specificity Dan Baijutang by the activity that reduces the enzyme of being responsible for these Dan Baijutang of generation, and then treatment tendon pathological changes.

A. promote in the tendon pathological changes that chondrogenetic molecule is raised

Use Affymetrix

System identifies normal tendon and suffers excessively to use the difference of gene expression between the tendon of step.Gene expression profile produces more than 400 and excessively uses the back gene that difference is regulated in tendon of supraspinatus muscle.By excessively using in 4 weeks, 107 genes are raised, and 27 genes are reduced.Some the gene of high rise be the gene of cartilage specificity, comprising collagen 2 type α-1 chains (Col2a1), Sox9, versican and aggrecan.The gene of other rise comprises the gene that relates to GAG side chain formation on the chondroproteoglycan, comprising CS-GalNAcT-1, GalNT-1 and Hs3st1.Based on these results, these molecules self or other are responsible for the initial of these molecular activities or are kept or the molecule of being responsible for the gene expression of these molecules of coding may relate to cartilage and/or fibrocartilaginous formation in the tendon of excessive use.These molecules can comprise as hereinafter listed enzyme, transcription factor and the signal transduction factor:

Enzyme

A kind ofly suppress method that cartilage in the tendinous tissue or fibrous cartilage produce for suppressing to relate to the expression or the activity of the synthetic enzyme of Dan Baijutang.The full directory that relates to the synthetic enzyme of Dan Baijutang GAG chain is seen people such as Silbert, people such as IUBMB Life 54:177-186 (2002) and Sugahara, IUBMB Life54:163-175 (2002).These enzymes are included in the protein (embodiment 1) of expressing increase in the tendon of excessive use.Therefore, one embodiment of the invention relate to expression or the activity that suppresses following any one enzyme:

CS-GalNAcT-1 and CS-GalNAcT-2 (EC 2.4.1.174 and EC 2.4.1.175) relate to the initial sum of chondroitin sulfate GAG side chain and extend.CS-GalNAcT-1 relates to the initial sum of chrondroitin GAG and extends, and CS-GalNAcT-2 relates to the extension of these identical chrondroitin GAG.About the detailed description of these enzymes, see also people such as Uyama, people such as J.Biol.Chem.277 (11): 8841-8846 (2002), Gotoh, people such as people J.Biol.Chem.278 (5): 3063-3071 (2003), Uyama such as J.Biol.Chem.277 (41): 38189-38196 (2002), Sato, J.Biol.Chem.278 (5): 3072-3078 (2003).

UDP-D-xylose: core protein β-D-xylosyltransferase (EC 2.4.2.269), it relates to initial xylose is partly added on the Dan Baijutang core protein, and this is one of synthetic initial step of GAG side chain;

Gal transferring enzyme (EC 2.4.1.133 and EC 2.4.1.134), it relates to galactose residue is added on the xylose part of new GAG side chain;

GlcA transferring enzyme (EC 2.4.1.135, EC 2..4.1.226 and EC 2.4.1.225), it relates to GlcA is partly added on the galactose residue of new GAG side chain;

GlcNAc transferring enzyme (EC 2.4.1.223 and EC 2.4.1.224), it relates to GlcNAc is partly added on the GalNAc part of the chondroitin sulfate GAG that adds by the CS-GalNAcT enzyme;

Sulfotransferase (EC 2.8.2.5 and EC 2.8.2.1), it relates to transfers to the sulfate radical residue on the chondroitin sulfate GAG;

Chondroitin sulfate synzyme (EC 3.1.6.4 and EC 3.1.6.12), it relates to the sulfate radical residue is added on the chondroitin sulfate GAG for the first time;

GalNT-1 (E.C.2.4.1.41) catalysis forms the first step in the oligosaccharide side chain that O-connects on glycoprotein.People such as White, JBiolChem 270 (41): 24156-65 (1995); And

Hs3st1 (EC 2.8.2.23) catalysis sulfate radical residue adds on the Heparan sulfate GAG.People such as Shworak, JBiolChem 272 (44): 28008-19 (1997)

The activity that directly or indirectly suppresses any one these enzymes will reduce the formation of Dan Baijutang.The method that suppresses these enzymes hereinafter has been discussed.

2. gene expression

Prevent that the alternative approach that cartilage and/or fibrous cartilage form from being to prevent cartilage specificity protein expression in the impaired tendinous tissue directly or indirectly.For example, can reduce versican or aggrecan by many methods, the expression of cartilage specificity Dan Baijutang, wherein said method comprise the gene transcription or the translation of prevent to encode aggrecan or versican or prevent to relate to the expression or the function (this will have the result who prevents these genetic transcriptions) of the transcription factor of aggrecan or versican gene expression.

Implementing importantly to guarantee the inhibitory action of the cartilage specificity factor is confined in the impaired tendon when of the present invention, is that functional articular cartilage and other are organized important composition because many cartilage specificity factors comprise Dan Baijutang and II Collagen Type VI.

It is a kind of that to suppress method that cartilage in the tendinous tissue and/or fibrous cartilage form be to suppress to relate to non-enzymatic protein active of cartilage structure protein expression or express.By suppressing these expression of gene or activity, people can suppress to form the protein expression of cartilage and/or fibrous cartilage tissue indirectly.The protein that relates to the cartilage structure protein expression includes but not limited to Sox9.

The method that cartilage or fibrous cartilage form in other inhibition tendinous tissue relates to and directly suppresses for example expression (and nonactive) of aggrecan, versican and II Collagen Type VI of main cartilage structure protein.Find that following gene is raised in impaired tendinous tissue, and these genes form with cartilage relevant.

a)Sox9

Sox9 (sry-type high mobility group frame 9) is for relating to the transcription factor of cartilage development.Sox9 expresses in chondrocyte, and this expression with collagen α 1 (II) gene (Col2a1) is consistent.Sox9 by activate or strengthen express cartilage structure protein for example the gene transcription of II Collagen Type VI regulate cartilage and take place.People such as Shum, Arthritis Res.4 (2): 94-106 (2002); People such as Bi, Nat.Genet.22 (1): 85-9 (1999).

Col2a1

The main component of Col2a1 coding cartilage, α 1 chain of II Collagen Type VI.People such as Cheah, Proc NatlAcad Sci USA 82 (9): 2555-9 (1985).

Agc1

The core protein of Agc1 coding aggrecan Dan Baijutang.People such as Doege, Extracellular Matrix Genes (Sandel, L.J.; Boyd, C.D. writes .) AcademicPress (New York) 137-152 (1990).As used herein, term " aggrecan " refers to the special large protein polysaccharide of cartilaginous tissue.Aggrecan comprises by the protein core of many chondroitin sulfate GAG parts.Aggrecan is the primary structure composition of cartilage, and can bound water and the gel-like substance that forms viscosity.This hydration provides elasticity and compressive strength for cartilaginous tissue.

Cspg2

The core protein of the multifunctional coded Dan Baijutang proteoglycan of Cspg2.Versican is another large protein polysaccharide with many chondroitin sulfate side chains.As aggrecan, the versican bound water, and form increase tissue intensity and elastic viscogel.People such as Knudson, Sem Cell Dev Biol 12:69-78 (2001).

B. by suppressing to promote chondrogenetic molecule to treat the tendon pathological changes

The invention provides by reducing the above expression and/or the active method for the treatment of or preventing the tendon pathological changes of listed molecule.The present invention further provides the method that the tendon pathological changes was treated or prevented to the inhibitor of listed molecule above of using.

Therapeutic strategy

The present invention part is based on following discovery, and the expression of gene level of the aggrecan of promptly encoding, versican Sox9, II Collagen Type VI, CS-GalNAcT-1, GalNT-1 and HS3st1 is compared remarkable increase (Fig. 1-7) with control tissue in the tendinous tissue that excessively uses.This result shows, relates to the enzyme of Dan Baijutang route of synthesis or the expression increase of other molecule and causes impaired tendinous tissue to change cartilage and/or fibrous cartilage into.Therefore, in one embodiment, the present invention includes the method for treatment tendon pathological changes, comprising reducing cartilage and/or fibrocartilaginous formation in the tendinous tissue by activity, expression or the gathering that suppresses to relate to cartilage and/or the synthetic enzyme of fibrous cartilage or other molecule.

Blocking-up relates to enzyme that cartilage in the tendon or fibrous cartilage form or the inhibitor of other molecular activity is useful in the present invention.The inhibitor that is used for the inventive method is by optional glycosylated, adding polymer Polyethylene Glycol or that be connected in another nonprotein.Inhibitor can be modified to the glycosylation pattern (promptly compare with initial or natural glycosylation pattern and change) with change.As used herein, " change " expression is compared with initial inhibitor and to be added or deleted one or more saccharides parts and/or add or deleted one or more glycosylation sites.Adding glycosylation site to inhibitor can contain glycosylation site consensus sequence well known in the art and finish by aminoacid sequence is changed into.The another kind of mode that increases sugar moieties quantity is by glucosides chemistry or enzymatic being coupled to the amino acid residue of inhibitor.These methods are described among the Crit Rev Biochem22:259-306 (1981) people such as WO87/05330 and Aplin.Be present in removing of any sugar moieties on the substrate can chemistry or enzymatic finish, as people such as Sojar, people such as Arch Biocem Biophys 259:52-57 (1987), Edge, people such as Anal Biochem 118:131-137 (1981) and Thotakura are described in the MethEnzymol 138:350-359 (1987).

Comprise that for example the protein and the nonprotein inhibitor of peptide, micromolecule and nucleic acid also can be used for method of the present invention.

A) peptide and micromolecule

The inhibitor that is used for the inventive method comprises little organic peptide and molecule and little inorganic molecule.These peptides and micromolecule comprise the naturally occurring inhibitor of synthetic and purification.Well known evaluation special target is in the peptide and the micromolecular method of destination protein matter.For example, can use the mensuration of target protein enzymatic and/or biological function to screen peptide and/or micromolecule library to for example inhibition of CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1 and/or Sox9 of target protein.In another embodiment, can target protein for example CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1 and/or Sox9 competitive radioligand in conjunction with in measuring with its substrate or ligand screening micromolecule and/or peptide library.

Alternatively, can use to measure and identify inhibitor based on for example time-resolved FRET of the mensuration of FRET (fluorescence resonance energy transfer) (FRET) (TR-FRET).In exemplary, with the core protein of Dan Baijutang with His or GST label, and use coupling the anti-His or the GST antibody of europium (fluorogen) come labelled protein.Alternatively, core protein is carried out non-specific labelling with europium on lysine or cysteine residues.Donor glycan molecule Cy5 (fluorescent dye) labelling.The tabulation that can be used for the donor of this mensuration and acceptor molecule can be people such as Uyama, and J BiolChem 278 (5): find in the table 1 of 3072-78 (2003).

Receptor and donor molecule with different ratios with and do not make up with the target enzyme.TR-FRET measures and to carry out through the following steps, promptly the 340nm activating system, respectively 615 and 665nm measure the emission of europium and Cy5 and calculate the ratio of Cy5 emission and europium emission.

When not having enzyme, donor and acceptor molecule each other can not be closely close, and there is not cancellation in the individual fluorescence of Cy5 and europium molecule, and then produce the ratio of baseline values.When adding the target enzyme in test macro, the donor glycan molecule is transferred to receptor protein, and the fluorescence of cancellation system.The ratio of Cy5 and europium fluorescence reaches maximum along with the glycan molecule quantity that shifts and increases.

In order to identify the inhibitor of target enzyme, in the mensuration system, add peptide or micromolecule.When test compounds can suppress sugar to proteinic transfer, the cancellation of fluorescence will reduce.If the transfer activity that test peptides can inhibitory enzyme 100%, the Cy5 emission is a baseline values just with the ratio of europium emission so.In system, be evaluated at the chemical compound that suppresses 50% enzymatic activity in this mensuration at least then and evaluate the ability that it reduces GAG synthetic (as mentioned below) based on cell.

The present invention also provides and uses extra Screening test for example secondary and three grades of mensuration (secondaryand tertiary assay), further identifies the effect of this quasi-molecule to the tendon form, for example uses the above-detailed test.

B) dominant negative mutant

In some embodiments of the present invention, the CS-GalNAcT-1 of sudden change, CS-GalNAcT-2, GalNT-1, Hs3st1 or Sox9 protein can be used in the method for the invention as inhibitor.For example naturally occurring variant or the congener through transforming that all has a dominant negative regulation effect with external activity to above-mentioned molecule in vivo can be used for method of the present invention.

C) nucleic acid

Can block the active nucleic acid of above listed target molecule is useful in the present invention.This type of inhibitor can be encoded and for example CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1, Hs3st1 or Sox9 interacting proteins.Alternatively, this type of inhibitor can encode can with the substrate of target molecule (for example GalNAc) or the protein of ligand interaction, if and if coded protein blocking-up target molecule and its substrate or part combine or its blocking-up substrate or part binding target molecule after activity, this type of inhibitor is effective in the method for the invention just so.Certainly, the inhibitor protein with target molecule and substrate or ligand interaction of can encoding simultaneously.This type of nucleic acid for example can be used for expressing, and the inhibitor of CS-GalNAcT-1, CS-GalNAcT-2, Hs3st1, GalNT-1 or Sox9 is used for method of the present invention.

Method of the present invention also comprises the expression of using interferential RNA molecule (" RNAi ") to reduce above listed any molecule or its protein bound gametophyte, and wherein said interferential RNA molecule includes but not limited to the hairpin RNA (shRNA) and the short double-stranded RNA (sdsRNA) of short interferential RNA (siRNA), weak point.RNAi can be next initial by introducing for example synthetic short interference siRNA of nucleic acid molecules or rnai agent, and then inhibition or reticent target gene expression.See, for example U.S. Patent Publication No. 2003/0153519 and 2003/01674901 and U.S. Patent number 6,506,559 and 6,573,099.

SiRNA can chemosynthesis, produces by in vitro transcription, perhaps produces in host cell.Generally speaking, siRNA for example grows 20,21,22,23,24,25,26,27,28,29 or 30 nucleotide to a youthful and the elderly 15-50 nucleotide.SiRNA can be about 15 double-stranded RNAs to about 40 length of nucleotides (dsRNA), for example about 15 length to about 28 nucleotide, comprising about 19,20,21 or 22 length of nucleotides, and can on every chain, comprise 3 ' and/or 5 ' jag with about 0,1,2,3,4,5 or 6 length of nucleotides.SiRNA can suppress target gene by Transcriptional Silencing.Preferably, siRNA can by degraded target messenger RNA or specifically PTGS (PTGS) target messenger RNA promote RNA to disturb.

The siRNA that is used for the inventive method also comprises little hairpin RNA (shRNA).ShRNA comprises the antisense strand of short (for example about 19 to about 25 nucleotide), then is about 5 nucleotide ring and similar sense strands (analogous sense strand) to about 9 nucleotide.Alternatively, sense strand can be before the nucleotide ring structure, and antisense strand can after.These shRNA can be included in plasmid and the viral vector.

The target area of siRNA molecule can be selected from the target sequence of being given.For example, nucleotide sequence can be initial from start codon downstream about 25-100 nucleotide.Nucleotide sequence can contain 5 ' or 3 ' untranslated region, and the zone of close start codon.Well known design and prepare the method for siNRA molecule is comprising the rule of multiple choices sequence as the RNAi reactant.See people such as Boese for example, Methods Enzymol 392:73-96 (2005).

SiRNA can use people such as Hannon, people such as Nature418:244-251 (2002), McManus, people such as NatReviews 3:737-747 (2002), Heasman, people such as Dev Biol 243:209-214 (2002), Stein, J Clin Invest, people such as 108:641-644 (2001) and Zamore, the described standard technique of NatStruct Biol8 (9): 746-750 (2001) produces.Preferred siRNA is 5 ' phosphorylation.

SiRNA can be used for targeting for example CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1, Hs3st1, Sox9, Agc1, Cspg1 or Col2a1 or above listed other molecule and substrate or part.

Nucleic acid can be from its natural environment, with form acquisition pure substantially or homogeneity, separation and/or purification.Know in multiple different host cells the system of clone and express polypeptide.Appropriate host cell comprises antibacterial, mammalian cell, yeast and baculovirus (baculovirus) system.The mammal cell line of the available expressing heterologous polypeptide in this area comprises for example Chinese hamster ovary cell, HeLa cell, baby hamster kidney cell, NS0 mouse black-in tumor cell and many other cells.See Gene Expression Systems about being fit to produce proteinic other cell, write people such as .Fernandez, Academic Press (1999) from nucleic acid.The RNAi molecule can chemosynthesis, perhaps expresses from the DNA sequence of coding siRNA or shRNA sequence.

In order to produce above listed molecule dominant negative mutant, can select or make up the suitable carrier that contains suitable adjusting sequence, wherein said adjusting sequence comprises promoter sequence, terminator sequence, polyadenylic acid sequence, enhancer sequence, selection or marker gene and other suitable sequence.As required, carrier can be plasmid or virus, for example phage or phasmid.More details referring to, Molecular Cloning:A Laboratory Manual for example, people such as Sambrook, the 2nd edition, Cold Spring Harbor Laboratory Press (1989).The known technology of many operation nucleic acid and method, for example prepare nucleic acid construct, mutation, order-checking, DNA is introduced in cell and gene expression and the protein analysis, all at Current Protocols in Molecular Biology, people such as Ausubel edit, the 2nd edition, John Wiley ﹠amp; Describe in detail among the Sons (1992).

Nucleic acid can be merged other sequence to the extra peptide sequence of encoding, for example usefulness marks or the sequence of reporter gene.The example of labelling or reporter gene comprises lactamase, chloramphenicol acetyltransferase (CAT), ADA Adenosine deaminase (ADA), aminoglycoside phosphotransferase (being responsible for neomycin (G418) resistance), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (coding tilactase), xanthine-guanine phosphoribosyl transferase (XGPRT), luciferase.Many other genes are also known in this area.

2. inhibitory enzyme activity

In the specific embodiment of the present invention, the method of treatment tendon pathological changes comprises reductions, suppresses or reduces the activity that relates to the biosynthetic enzyme of Dan Baijutang, and wherein said enzyme for example relates to and adds chondroitin sulfate GAG on aggrecan and/or the versican those.These enzymes comprise for example CS-GalNAcT-1, CS-GalNAcT-2, UDP-D-xylose: core protein β-D-xylosyltransferase, Gal transferring enzyme, GlcA transferring enzyme, GlcNAc transferring enzyme, sulfotransferase, chondroitin sulfate synthase, Hs3st1 and GalNT-1.

In exemplary, the present invention includes reduction, suppress or reduce the activity of CS-GalNAcT-1, CS-GalNAcT-2 in the impaired tendon, GalNT-1 and/or Hs3st1 enzyme.In other embodiments, the present invention includes reduction, suppress or reduce the activity of Xyl transferring enzyme in the impaired tendon, Gal transferring enzyme, GlcA transferring enzyme, GalNAc transferring enzyme, GlcNAc transferring enzyme, sulfotransferase or chondroitin sulfate synthase.Above-mentioned peptide or micromolecular inhibitor can be used for carrying out these methods.

A) inhibitor of evaluation enzymatic activity

The present invention further comprises the method for identifying and be evaluated in the impaired tendinous tissue as the effect of the material of the inhibitor of CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1, Hs3st1, Xyl transferring enzyme, Gal transferring enzyme, GlcA transferring enzyme, GalNAc transferring enzyme, GlcNAc transferring enzyme, sulfotransferase or chondroitin sulfate synthase.

This type of inhibitor blocking-up or reduce relates to the activity of the enzyme that cartilage in the tendon or fibrous cartilage form.These inhibitor comprise soluble substrate, protein, micromolecule and the nucleic acid of modification.

The method of identifying the material of treatment tendon pathological changes comprises measures that each independent material interacts to above enzymatic activity, protein-target of listed enzyme or the effect of protein protein interaction.In the multiple body or external mensuration can be used for measuring the effect of inhibitor for treating tendon pathological changes.In an exemplary, by being similar to the method for high flux screening (HTS), by measure the micromolecule influence for example above the enzymatic activity of listed enzyme, target in conjunction with or the ability of protein protein interaction and its potential therapeutic use has been carried out external assessment.

In typical HTS measures, candidate compound to enzymatic activity for example, ligand-receptor in conjunction with etc. the inhibition effect can be by will have the concentration known candidate time measurements determination with measure with reference to comparing, there is not candidate in wherein said reference and/or is carrying out when having known inhibitor compound.Usually, can use conventional dyestuff, fluorescence or radioactive activity molecule to monitor the effect of candidate compound.Therefore, for example, can identify the candidate compound that suppresses part and its receptors bind or inhibitory enzyme activity, reduces the enzyme process turnover.

Exemplary of the present invention is provided at the method for the material of external evaluation treatment tendon pathological changes, and the sample of the sample composition of at least a CS-GalNAcT-1 of being selected from, CS-GalNAcT-2, GalNT-1 and Hs3st1 is provided comprising (1); (2) combined sample composition and test substances; (3) the working sample composition is replied the activity of test substances; And (4) determine whether test substances suppresses the activity of sample composition.

In an exemplary, identify that the in vitro method of the material of treatment tendon pathological changes comprises that handling cartilage explant, elementary chondrocyte granule culture or chondrocyte with BMP-2 protein or the proteinic adenovirus of expressed BMP-2 is.BMP-2 stimulates ECM and GAG to synthesize.Then test compounds is added in the culture, and assess inhibitor to the synthetic effect of GAG by measuring 35S mixing and/or measuring in the GAG side chain by DMMB.The effect of test compounds will compare with the appropriate carriers contrast.

Mensuration can comprise in the body of potential enzyme inhibitor: (1) repeats to use to mammal (for example Sprague-Dawley rat) the test inhibitor of at least 2,4,6 or 8 time-of-weeks; (2) mammal 5 days weekly, 1 hour every day are carried out descending running scheme (embodiment 1) with 17 meters/minute; And (3) pass through DMMB or AB measures or measure the effect of inhibitor to tendon of supraspinatus muscle by the gene expression profile analysis, wherein think the slowing down of tendon degeneration (for example cell and morphologic unusual), and show that inhibitor is effective for treatment tendon degeneration disease owing to inhibitor.

Should be appreciated that the test inhibitor that is used for the inventive method can assess the animal model and/or the people of one or more tendon degeneration diseases.

Known in the art multiple in vivo with the external mensuration of when having inhibitor, measuring enzymatic activity.The example of the mensuration that some are used always comprises that the spectrophotography of spectrophotography, fluorescence spectrophotometer algoscopy, circular dichroism (circular dichroism), automatization and fluorescence spectrophotometer algoscopy, coupling are measured, acid or alkali titration, radiometric method, unmarked optical detection and the EIA of automatization.In exemplary, people such as the enzymatic activity of CS-GalNAcT-1 and/or CSGalNAcT-2 such as Uyama detect described in J Biol Chem 277 (11): the 8841-46 (2002).In another exemplary, people such as the enzymatic activity of GalNT-1 such as White, the described detection of J Biol Chem 270 (41): 24156-65 (1995).

For example, be used for the inventive method enzyme inhibitor can with the enzyme interacting of its inhibition.Alternatively, inhibitor can interact with for example zymolyte (for example GalNAc) or other binding partners.If inhibitor blocking-up enzyme and its substrate combine and/or if inhibitor is blocked activity behind the substrate desmoenzyme, it can reduce or be and effective in the present invention so.Certainly, inhibitor can with enzyme and second kind of factor for example its substrate all interact.Aspect this, the inhibitor of CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1 and/or Hs3st1 comprises soluble substrate, other protein (protein that comprises desmoenzyme and/or zymolyte), the enzyme of modified forms or the analogies of for example modifying of its fragment, propetide, peptide and all these inhibitor.

The inhibitor of enzyme can for example be direct enzyme inhibitor, in conjunction with and in and the activity of enzyme; Reduce the expression of enzyme; Influence the stability of precursor molecule or to transformation active, mature form; Interferases combines with its one or more substrates; The perhaps endocellular function of its interferases.

3. inhibition transcription factor activity

The transcription factor inhibitor that is used for the inventive method can be directly by combination or indirectly by disturbing its protein protein interaction and/or binding characteristic to suppress or reduce the activity of the factor.Aspect this, the inhibitor of Sox9 can comprise modified soluble ligand or target molecule, micromolecule and nucleic acid.

Known in the art when having inhibitor in vivo with the active mensuration of in-vitro measurements Sox9.The example of the mensuration of the Sox9 inhibitor that some are used always comprises Western engram analysis, transient transfection, chloramphenicol acetyltransferase (CAT) mensuration and the Northern engram analysis of PCR mensuration, Sox9 or the cartilage specificity gene of chondrocyte specific gene when there are the Sox9 inhibitor in yeast two-hybrid system, luciferase reporter gene, mensuration (for example Col2a1, Agc1) expression.

Can use and multiplely measure inhibitor to Sox9 and the interactional vast usefulness of its binding partners based on the protein-protein of FRET (fluorescence resonance energy transfer) (FRET) or the mensuration of protein-nucleic acid interaction.For example, can use AlphaScreen ^TMThe luminous approximation homogeneity of amplification measure (available from Pu Jinaiermo company (

)), perhaps can adopt the bioluminescence resonance energy shift (Bioluminesence Resonance Energy Transfer) (available from Pu Jinaiermo company (

) BRET ^TM) disturb the external test of the interactional inhibitor of Sox9 associativity.

In one embodiment, the invention provides the method for the external test of the material that is used to identify treatment tendon pathological changes, comprising: (1) provides the above listed target molecule that is blended in the donor fluorescence molecule; Combination target molecule and test substances; (3) adding is blended in the binding partners of the target molecule of acceptor fluorescence molecule; (4) the fluorescence energy transfer level between detection target molecule and its binding partners; And (5) measure the interaction whether test substances suppresses target molecule and its binding partners.

Inhibitor can disturb combining of target molecule and its associativity gametophyte, and and then influence FRET level between the two.Therefore, can use the FRET measurement to identify the inhibitor of multiple effectiveness.In case, just can carry out effect and the suitability of further body build-in test in measuring inhibitor cartilage and fibrous cartilage forming in the prevention tendon at the external inhibitor of identifying target molecule.

4. Profilin matter is expressed

In another embodiment, the method for treatment tendon pathological changes comprises reduction, inhibition or down-regulated gene for example CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1, Hs3st1, Col2a1, Cspg1, Agc1 and/or Sox9 and encoded polypeptides or protein expression and/or gathering.The compositions that is used to suppress these gene expressions for example comprises above (c) the interferential RNA molecule described in the part of II (B) (1).

These inhibitor can be used in any way, but in specific embodiments, and the DNA of expressed rna i inhibitor is mixed adenovirus vector, then described vector injection are entered the patient who needs tendon to repair.In exemplary, the nucleotide sequence of coding RNA i is positioned at the downstream of polII promoter, as people such as Wahdwa, and Curr Opin Mol Ther 6 (4): described in the 367-72 (2004).With the method for siRNA and shRNA insertion adenovirus vector is well known, and people such as Krom, BMCBiotech6 (11): be described in [prior to the electronic distribution of printing].Alternatively, directly the injection of RNAi inhibitor is entered impaired tendinous tissue and by electroporation its transhipment entered cell, as people such as Schiffelers, Arthritis; Rheumatism 52 (4): described in the 1314-18 (2005).

5. tendon pathological changes disease

Method of the present invention can be used in office what is the need for will the mammal of this treatment in treatment or prevention tendon pathological changes, wherein said mammal comprises people, primates, monkey, rodent, sheep, rabbit, Canis familiaris L., Cavia porcellus, horse, cattle and cat.The disease that can treat or prevent comprises that for example tendinitis, tendinitis, tendon degeneration, peritenonitis, tenosynovitis (tenocynovitis), tendon excessively use any other disease in damage, tendon wound, peritendinitis, paratenonitis and " tendon pathological changes " disease family.

The tendon pathological changes can be owing to excessively use and repeating motion, damage (from slightly to severe) suddenly, degeneration or aging causing gradually.Most of tendon injurys are made up of the slowly little laceration of a series of recoveries from illness (tendon degeneration), and described little laceration weakens tendon, causes pain, stiff and lose intensity usually.The treatment that the tendinopathy accommodation often needs several weeks, minimizing or change activity and rest.The too fast impaired tendon of use that recovers will cause more tendon injury, make impaired tendon be more prone to tear or break.Therefore, the disease by the present invention's treatment or prevention comprises the tendon degeneration disease (acute and chronic two kinds) of following excessive use, physical culture or accident associated injury and wound, malnutrition and age growth.

An example of tendon pathological changes is a lateral epicondylitis, also is known as " tennis elbow (tennis elbow) ", and this is sports medicine and the orthopedics's disease of knowing.The potential pathology of this disease relates to excessive use and little extensor carpi radialis brevis tendon of tearing the ancon place.Health is attempted repairing this and is torn slightly, but in many cases, agglutination is incomplete.The pathology sample that carries out the patient of chronic lateral epicondylitis operation shows blood vessel fibroblast abnormal development disorderly in the impaired tendon.This incomplete reparation trial causes the tissue of degeneration, jejune and vascularization.Not exclusively the tissue of repairing than normal tendinous tissue a little less than, and lack the intensity of exercising normal function.Organizing also by causing pain of this reduction, and negatively influence quality of life and limit the patient.Similarly not exclusively repair in the tendon associated injury or infringement that is present in other type, for example patellar tendon inflammation (leaper's knee joint), Achilles Tendonitis (being common in running athlete) and shoulder sleeve tendinitis (being common in the athlete of " crossing the crown ", for example the pitcher).

The tendon pathological changes also can owing to the age that for example increases, bad diet, sports, wound, damage or medicine for example pefloxacin (pefloxacin) cause.For example, PGE1 (PGE1) is inductive, PGE2 (PGE2) is inductive or the inductive tendon pathological changes of pefloxacin is the good animal model that people's tendon pathological changes is set up.In one embodiment, the invention provides in individuality for example method of the inductive tendon pathological changes of pefloxacin of treatment or the inductive tendon pathological changes of prophylactic agent.In other exemplary, the invention provides treatment or prevention because the damage that excessively use, physical culture or accident are correlated with and the method for the inductive tendon pathological changes of wound, malnutrition and age growth.

The present invention further provides by treat or prevent tendon degeneration disease to the inhibitor of administration molecular activity and/or expression, delaying tendon degenerates, recover the tendon quality, keep tendon health, the hypoxia degeneration of treatment or prevention tendinous tissue, the hyaline degeneration of treatment or prevention tendinous tissue, the mucoid or the myxoid degeneration of treatment or prevention tendinous tissue, the fibrinoid degeneration of treatment or prevention tendinous tissue, the lipid degeneration of treatment or prevention tendinous tissue, the calcification of treatment or prevention tendinous tissue, the fibrous cartilage of treatment or prevention tendinous tissue and the metaplasia of bone, keep the microstructure integrity of tendon, keep the fibre structure in the tendon, keep the fiber alignment in the tendon, keep normal tendon vascularity, keep normal cellularity in the tendinous tissue, recover normal ECM content in the tendinous tissue, reducing the pathology of GAG in the tendinous tissue assembles, and/or keep the quality of tendon, the method of tensile strength and/or flexible quality, wherein said molecule is CS-GalNAcT-1 for example, CS-GalNAcT-2, GalNT-1, the Xyl transferring enzyme, the Gal transferring enzyme, the GlcA transferring enzyme, the GalNAc transferring enzyme, the GlcNAc transferring enzyme, sulfotransferase, the chondroitin sulfate synthase, Col2a1, aggrecan, versican, Hs3st1 and/or Sox9, and the amount of using above-mentioned inhibitor is for prevention in the tendon pathological tissues or reduce the effective dose of this type of molecular activity and/or expression.

6. assessment is treated and animal model

Method of the present invention can be used for treating tendon pathological changes or microdefect in tendinous tissue.Tendon quality during treatment back or the treatment can be for example measured by variation, tendon vascularization and/or the GAG content of the microstructure integrity of assessment tendon, tendon collagen stability, tendinous tissue cellularity.Be known in the art in the method for assessment tendon health after the treatment or during the treatment, and include but not limited to nuclear magnetic resonance (MRI), ultrasonography and functional and/or pain assessment.Alternatively, the health of tendon can be measured by the level of GAG in the assessment tendinous tissue.

A kind of mensuration tendon GAG Determination on content is that 9-dimethylated methylene indigo plant (DMMB) is measured for 1 of sulfated glycosaminoglycans (GAG) concentration in the measurement tendon.The another kind of tendon GAG Determination on content of measuring is histological love alizarin blue (AB) dyeing, and it can be used for detecting the GAG that accelerates in the tendinous tissue.

III. Zhi Liao method and pharmaceutical composition

The inhibitor that is used for the inventive method can be by partial, per os, intravenous, endoperitoneal, intramuscular, intracavity, subcutaneous, mode part or systemic administration that implant or percutaneous.

In exemplary, also can use inhibitor to individuality by the mode of gene therapy, wherein use the nucleotide sequence of coding inhibitor in vivo to the patient.In exemplary, will comprise the nucleic acid of sequence that promoter sequence and coding be used for the nucleic acid inhibitor of the inventive method and insert adenovirus expression carrier.Directly the adenovirus injection is entered patient's tendon, express nucleic acid inhibitor in described tendon then.For example, people such as Mehta, people such as J Hand Surg 30A (1): 136-41 (2005), Lou, J Orthoped Res 19:1199-1202 (2001) and Lou have described the example that adenovirus mediated nucleic acid is sent to tendinous tissue among the Clin Orthopaed Rel Res379S:S252-55 (2000).

In other exemplary, the nucleic acid inhibitor is mixed other virus expression carrier, for example slow virus, herpesvirus and adeno associated virus.Appropriate carriers can be selected by assessing the external infectiousness of every type of virus in elementary tendon cell.

Alternatively, nucleic acid inhibitor direct injection is entered tendinous tissue, and enter cell by the electroporation transfer, as people such as Schiffelers, Arthritis ﹠amp; Rheumatism 52 (4): described in the 1314-18 (2005).

In other exemplary, the peptide or the micromolecular inhibitor that are used for the inventive method can be coated on endermic patch, and directly apply to the skin that covers tendinous tissue, as people such as Paoloni, described in J Bone Joint Surg 86A (5): the 916-22 (2004).

The application process of the molecule of treatment tendon pathological changes known in the art." use " the particular delivery system arbitrarily that is not limited to, and can restrictedly not comprise (for example in capsule, suspensoid or tablet) parenteral (comprising in subcutaneous, intravenous, the marrow, IA, intramuscular, intracavity or endoperitoneal injection), rectum, partial, endermic or per os.Can be local or take place capapie to use to individuality, with single dose, perhaps repetitive administration continuously or discontinuously, and go up acceptable salt form with any multiple physiology, and/or with using as the pharmaceutically suitable carrier and/or the additive of pharmaceutical composition (mentioned above) part.Those skilled in the art know drug preparation technique and the excipient that the physiology goes up acceptable salt form, standard.See Physicians ' Desk Reference (PDR) 2003, the 57 editions, Medical Economics Company, 2002 and Remington:The Science and Practice of Pharmacy, write.People such as Gennado, the 20th edition, Lippincott, Williams ﹠amp; Wilkins, 2000).

The inhibitor that is used for the inventive method can be used from the dosage of the extremely about 20mg/kg of about 1 μ g/kg, and this depends on the severity of symptom and the progress of disease.Suitable effective dose is selected from following scope by the doctor in charge: extremely about 1mg/kg, about 10 μ g/kg of extremely about 10mg/kg, about 1 μ g/kg of for example about 1 μ g/kg extremely about 20mg/kg, about 1 μ g/kg extremely about 1mg/kg, about 10 μ g/kg are extremely about 1mg/kg of about 100 μ g/kg, about 100 μ g and the extremely about 1mg/kg of about 500 μ g/kg extremely.

In another exemplary, the period in repetitive administration inhibitor at least 2,4,6,8,10,12,20 or 40 weeks, perhaps at least 1,1.5 or 2 year or lifelong up to the experimenter, thus for example treat the tendon pathological changes.In another embodiment of the invention, using inhibitor with single dose for example stimulates normal configuration and the composition that recovers tendon.

Generally speaking, the inhibitor that is used for the inventive method can be applied in the dosage between 10-8 and 10-7,10-7 and 10-6,10-6 and 10-5 or 10-5 and the 10-4g/kg.The treatment effective dose of finishing in a kind of animal model can be converted to and be used in another animal and comprise among the people, uses conversion coefficient known in the art.See people such as Freireich for example, Cancer Chemother Reports 50 (4): 219-244 (1966).

The definite dosage of the inhibitor of Shi Yonging is determined by rule of thumb based on the result who wants in the methods of the invention.Example results comprises: (1) tendon degeneration disease obtains medical treatment or prevents; (2) tendon is degenerated and is eased; (3) quality of tendon is restored; (4) health of tendon is kept; (5) the hypoxia degeneration of tendon obtains medical treatment or prevents; (6) the hyaline degeneration of tendon obtains medical treatment or prevents; (7) mucoid of tendon or myxioid degeneration obtain medical treatment or prevent; (8) the fibrinoid degeneration of tendon obtains medical treatment or prevents; (9) the lipid degeneration of tendon obtains medical treatment or prevents; (10) calcification of tendon obtains medical treatment or prevents; (11) metaplasia of the fibrous cartilage of tendon and bone obtains medical treatment or prevents; (12) fibre structure of tendon is kept; (13) fiber alignment in the tendon is kept; (14) the normal vascularity of tendon is kept; (15) normal cellularity is kept in the tendon; (16) normal ECM content is restored in the tendon; And/or the quality of (17) tendon, tensile strength and/or flexible quality are kept.For example, the inhibitor of using effective dose come at least 20,30,40,50,100,200,300,400 or 500% ground slow down tendon degeneration (for example the tendon quality, structure is formed and/or to little loss of tearing with pain sensitivity).

In some embodiments, the compositions that is used for the inventive method also comprises pharmaceutically useful excipient.As used herein, word " pharmaceutically useful excipient " refers to arbitrarily and solvent, dispersant, coating, antibacterial and antifungal, isotonic agent and the delay absorbent etc. of all and medicament administration compatibility.Well known this type of medium and material are as the purposes of pharmaceutically active substance.Compositions also can contain the reactive compound that other provides additional, extra or enhanced treatment function.Pharmaceutical composition also can be included in container, packing or the riffle sampler (dispenser) with using explanation.

Should be formulated into route of administration compatibility with its expection with the co-administered pharmaceutical composition of the inventive method.This type of examples of formulations comprises the crystallization of protein preparation, and it is with exposed or provide with biodegradable polymer (for example PEG, PLGA) combination.

The inhibitor that is used for the inventive method can be used as pharmaceutical composition and uses with carrier gel and substrate or other compositions that is used to guide osteanagenesis and/or bone to replace.The example of this type of substrate comprises synthetic Polyethylene Glycol (PEG), hydroxyapatite, the substrate based on collagen and fibrin, Tisseel

Fibrin adhesive ﹠ etc.

In some embodiments, inhibitor can for example NSAID, corticosteroid, nitric oxide, testosterone, estrogen, somatomedin (for example BMP-12, BMP-13 and MP52) make up or use concomitantly with other therapeutic compound.

The inhibitor that is used for the inventive method can be coated on tendon implant, substrate and storage system or mix and wherein promote treatment or prevention tendon injury.

Embodiment

Embodiment 1: the rat tendon of supraspinatus muscle is owing to excessively use expression cartilage labelling

Use is transcribed profile analysis and at molecular level the rat tendon of supraspinatus muscle is studied the reaction of excessive use.The previous excessively rat model of use of tendon of having described.People such as Soslowsky, JShoulder Elbow Surg 9:79-84 (2000).Show that inflammatory and angiogenic are marked in this model and are changed people such as (, J Shoulder Elbow Surg 14:79S-83S (2005)) Perry, but take widely approach to understand around the incident of excessively using damage.

24 male Sprague-Dawley rats (400-450g) are carried out excessive 1 week of operational version (n=8) of tendon of supraspinatus muscle (SST), 2 weeks (n=8) and 4 weeks (n=8).Scheme was by 5 days weekly, the running (10% gradient) with 17 meters/minute descending and form of 1 hour every day.6 extra rats are as active contrast (time 0) in the cage.At each time point, two extra non-rats of running are used as the cohort contrast of age-matched.

When necropsy, take out tendon of supraspinatus muscle from each shoulder, and take out patellar tendon (PT) from each knee.Patellar tendon is as the confidential reference items of movement effects, because it does not show the obvious symptom of excessive use with this scheme.The tendon that weighing is gathered, and quick-freezing is in liquid nitrogen.To organize freezing crushing, and with TRIzol reagent (hero Life Technologies Corporation (Invitrogen)) extracting.With the aqueous phase isolation of RNA of RNeasy test kit (strong basis is because of company (QIAGEN)) from extract.Concentration with spectrophotometric determination RNA.Monitoring is carried out with Affymetrix rat gene group 230 2.0 arrays greater than the profile analysis of transcribing of 30,000 transcript expressions.All array images are all visually checked defective and quality.The array of in further analyzing, getting rid of high background, low signal intensity or major defect.(GCOS Affymetrix) measures signal value with Gene Chip Operating System 1.0.For every group pattern, statistical value is standardized as 100 average signal strength value.The GCOS statistical value of acquiescence is used for all analyses.Be greater than or equal to 66% as the average expression in the fruit gene meaning tissue in office greater than 100 signal units and sample percent with Present (P) call that measures by the GCOS default setting, so just think that gene is detectable.Changing standardized signal value into the truth of a matter is 10 logarithm.If p value＜0.01 that ANOVA detects, and run at any time and contrast between difference all be at least 2 times, so just think that gene is a differential expression.

After excessively using, in tendon of supraspinatus muscle, surpass 400 genes and be subjected to the difference adjusting.By running for 4 weeks, 107 genes are raised, and 27 genes are reduced.In the gene of the highest rise, manyly express, comprising II Collagen Type VI α 1, versican and aggrecan at the cartilaginous tissue camber.These results show that excessively the tendon that uses changes fibrocartilaginous phenotype into.These homologous geneses are not subjected to raising (seeing Fig. 1-7) in the patellar tendon of animal of running.

Embodiment 2: preparation siRNA inhibitor

The following selection of siRNA is promptly by with the nucleotide sequence of CS-GalNAcT-1, GalNT-1 or Hs3st1 input business website (for example sirnawizard.com or Ambion

SiRNA TargetFinder) designs special siRNA.Sequence selection instructs and is included in these instruments.

The effect of siRNA is by entering its transfection C-20/A4 and/or C-28/I2 chondrocyte, and measures by the expression of real-time RT-PCR monitoring CS-GalNAcT-1 and/or GalNT-1.Suitably out of order (scramble) siRNA that the identical nucleotide of tool is formed is used as experiment contrast.

Embodiment 3: the testing in vitro system of the synthetic inhibitor of Dan Baijutang

If siRNA can reduce the expression of CS-GalNAcT-1, GalNT-1 in C-20/A4 and/or the C-28/I2 chondrocyte or Hs3st1, so just test it and reduce the ability that Dan Baijutang GAG side chain produces.Adenovirus processing C-20/A4 and/or C-28/I2 chondrocyte with BMP-2 protein or expressed BMP-2 come synthesizing of irritation cell epimatrix and GAG.With siRNA or alternatively, micromolecule, the synthetic inhibitor of GAG join in the culture, and by relatively when existing or not having inhibitor 35S mix the effect of assessing inhibitor in the Dan Baijutang.Alternatively, by measuring the effect that the level that compares GAG when existing or not having inhibitor is assessed inhibitor, as people such as Arai, described in the Osteoarthritis and Cartilage 12:599-613 (2004) with DMMB.

Embodiment 4: the method that suppresses to treat the tendon pathological changes by the siRNA of CS-GalNAcT-1, GalNT-1 or Hs3st1

With the excessive operational version of treadmill (embodiment 1) or local injection PGE1, PGE2 or 1 week of pefloxacin (300mg/ml) (5 times weekly) in rat, inducing the tendon pathological changes.

In case reduced the activity of CS-GalNAcT-1, GalNT-1 or Hs3st1 at external discovery siRNA, just be converted into shRNA, and with the shRNA sequence clone with insert adenovirus, people such as Krom for example, BMC Biotech 6 (11): described in [electronic distribution].Use adenovirus to express shRNA in vivo, and suppress the expression of CS-GalNAcT-1 and/or GalNT-1 subsequently.Injecting method is optimized in elementary tendon cell.

The functional adenovirus direct injection of expressing shRNA is entered impaired tendon people such as (, JHand Surg 30A (1): 136-41 (2005)) Mehta come local CS-GalNAcT-1 of reduction and/or GalNT-1 expression of gene.Comprise that out of order sequence (Scramble sequence) is as experiment contrast.Assess the amount that cartilaginous tissue forms in the tendon by assessment GAG level.The alizarin blue dyeing of the love that using-system is learned detects the amount of the increase of glycosaminoglycans in the tendinous tissue (GAG).Use the quantitatively amount of extractive sulphation GAG from tendon of DMMB reagent.

Suppressing CS-GalNAcT-1 and/or GalNT-1 measures by the mechanicalness or the functional detection of histology and tendon intensity assessment in the body of the effect of tendon pathological changes.If compare with untreated contrast, the GAG level significantly descends and/or is lowered to and the similar level of normal, intac tendon, just thinks that inhibitor is effective.Alternatively, when comparing, if the function that function or mechanical test proof are improved and/or the pain of reduction just think that inhibitor is effective with untreated contrast.

Claims (42)

1. treat the method for tendon pathological changes in the patient, it comprises the formation that reduces cartilage specificity Dan Baijutang in patient's tendinous tissue.

2. the process of claim 1 wherein that described patient behaves.

3. the process of claim 1 wherein that described patient is selected from primates, monkey, rodent, sheep, rabbit, Canis familiaris L., Cavia porcellus, horse, cattle and cat.

4. the method for claim 1, it comprises and reduces the activity that relates to the synthetic enzyme of Dan Baijutang in patient's tendinous tissue that described Dan Baijutang is selected from aggrecan, versican and syndecan-3.

5. the method for claim 1, it comprises the activity that reduces chondroitin sulfate N-acetylamino galactosamine based transferase 1 (CS-GalNAcT-1) in patient's tendinous tissue and/or galactosamine polypeptide N-acetylamino galactosamine based transferase 1 (GalNT-1).

6. the process of claim 1 wherein that described activity reduces by the enzymatic activity that suppresses CS-GalNAcT-1, CS-GalNAcT-2, Hs3st1 and/or GalNT-1.

7. the process of claim 1 wherein that described activity reduces by suppressing CS-GalNAcT-1, CS-GalNAcT-2, Hs3st1 and/or GalNT-1 expression of gene.

8. the method for claim 1, it comprises and reduces the activity that relates to the transcription factor that cartilage produces.

9. the method for claim 8, it comprises the activity that reduces Sox9.

10. the method for claim 9, wherein said activity reduces by the ability that suppresses Sox9 and strengthen cartilage specificity gene expression.

11. the method for claim 8, wherein said activity reduces by suppressing the Sox9 expression of gene.

12. the method for claim 1, it comprises reduction cartilage specificity protein expression.

13. the method for claim 12, it comprises the expression that reduces the Dan Baijutang that is selected from aggrecan, versican and syndecan-3.

14. the method for claim 12, it comprises the expression that reduces Col2a1.

15. the process of claim 1 wherein that described tendinopathy becomes tendinitis.

16. the process of claim 1 wherein that described tendinopathy becomes the tendon degeneration.

17. the process of claim 1 wherein that described tendinopathy becomes tendon injury.

18. the method for treatment tendon pathological changes in the patient, it comprises the inhibitor of using CS-GalNAcT-1, CS-GalNAcT-2, GalNT-1, Hs3st1 and/or Sox9 in patient's tendinous tissue.

19. the method for claim 18, wherein said inhibitor reduces the enzymatic activity of CS-GalNAcT-1 and/or GalNT-1.

20. the method for claim 18, wherein said inhibitor reduces the expression of CS-GalNAcT-1 and/or GalNT-1.

21. the method for claim 18, wherein said inhibitor local application.

22. the method for claim 18, wherein said inhibitor comprises the interferential RNA molecule.

23. the method for claim 18, wherein said inhibitor comprises micromolecule.

24. the method for treatment tendon pathological changes in the patient, it comprises cartilage specificity protein expression in the reduction patient tendinous tissue.

25. the method for claim 24, it comprises the expression that suppresses aggrecan, versican and/or II Collagen Type VI.

26. the method for claim 24, it comprises the expression that suppresses Sox9.

27. the method for claim 24, it comprises the activity that suppresses Sox9.

28. identify the method for the material of treatment tendon pathological changes, it comprises that the experimenter to the needs treatment uses test substances, and measures the active ability that this material suppresses to relate in the tendinous tissue the biosynthetic enzyme of glycosaminoglycans (GAG).

29. identify the method for chemical compound useful in treatment tendon pathological changes, it comprises that the experimenter to the needs treatment uses test compounds, and measures the active ability that chemical compound suppresses to relate in the tendinous tissue the synthetic enzyme of glycosaminoglycans.

30. according to the method for claim 29, it further comprises step:

(a) provide the sample composition of at least a CS-GalNAcT-1 of being selected from, CS-GalNAcT-2, Heparan sulfate (glycosamine) 3-O-sulfotransferase 1, GalNT-1 and Sox9;

(b) with sample and test compounds combination;

(c) the measuring samples composition is replied the activity of test compounds; And

(d) determine whether test compounds suppresses the activity of sample composition.

31. the purposes of chemical compound in the medicine of preparation treatment tendon pathological changes, described chemical compound reduces the formation of cartilage specificity Dan Baijutang.

32. according to the purposes of claim 31, wherein said chemical compound reduction relates to the synthetic enzyme of cartilage or other activity of proteins.

33. according to the purposes of claim 32, direct or indirect inhibitory enzyme of wherein said chemical compound or proteinic function.

34. according to the purposes of claim 32 or 33, wherein said chemical compound inhibitory enzyme or protein expression.

35. according to purposes any in the claim 32 to 34, wherein said enzyme is selected from the UDP-D-xylose: core protein β-D-xylosyltransferase, Gal transferring enzyme, GlcA transferring enzyme, GlcNAc transferring enzyme, sulfotransferase, chondroitin sulfate synthase and Heparan sulfate sulfotransferase.

36. according to the purposes of claim 35, wherein said enzyme is selected from chondroitin sulfate N-acetylamino galactosamine based transferase 1 (CS-GalNAcT-1), chondroitin sulfate N-acetylamino galactosamine based transferase 2 (CS-GalNAcT-2), galactosamine polypeptide N-acetylamino galactosamine based transferase 1 (GalNAcT-1) and Heparan sulfate (glycosamine) 3-O-sulfotransferase 1 (Hs3st1).

37. according to the purposes of claim 31, wherein said chemical compound reduces the expression of cartilage specificity structural protein.

38. according to the purposes of claim 31, wherein said chemical compound reduces the activity of the transcription factor that relates to the expression of cartilage specificity structural protein.

39. according to the purposes of claim 38, wherein said transcription factor is Sox9.

40. according to purposes any in the claim 31 to 39, wherein said Dan Baijutang is selected from aggrecan, versican, syndecan-3 and II Collagen Type VI.

41. according to purposes any in the claim 31 to 40, wherein said tendon pathological changes is selected from tendinitis, tendinitis, tendon degeneration, peritenonitis, tenosynovitis, tendon injury, tendon wound, peritendinitis and paratenonitis.

42. according to purposes any in the claim 31 to 41, wherein to the patient's drug administration that is selected from primates, monkey, rodent, sheep, rabbit, Canis familiaris L., Cavia porcellus, horse, cattle and cat.

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