The present invention relates to screening methods to identify agents that may be of use for the prevention or treatment of musculoskeletal system disorders, particularly arthritis; the invention also relates to agents for, and methods for, the treatment of musculoskeletal system disorders, particularly arthritis.

The human musculoskeletal system (also known as the locomotor system) is an organ system that gives humans and animals the ability to move using the muscular and skeletal systems. The musculoskeletal system provides form, stability, and movement to the human and animal body.

It is made up of the body's bones (the skeleton), muscles, cartilage, tendons, ligaments, joints, and other connective tissue (the tissue that supports and binds tissues and organs together). The musculoskeletal system's primary functions include supporting the body, allowing motion, and protecting vital organs. The skeletal portion of the system serves as the main storage system for calcium and phosphorus and contains critical components of the hematopoietic system. This system describes how bones are connected to other bones and muscle fibers via connective tissue such as tendons and ligaments. The bones provide the stability to a body in analogy to iron rods in concrete construction. Muscles keep bones in place and also play a role in movement of the bones. To allow motion different bones are connected by joints. Cartilage prevents the bone ends from rubbing directly on to each other. Muscles contract (bunch up) and extend (stretch) to move the bone attached at the joint.

There are, however, diseases and disorders that may adversely affect the function and overall effectiveness of the system. Diseases of the musculoskeletal system mostly encompass functional disorders or motion discrepancies; the level of impairment depends specifically on the problem and its severity. Articular (of or pertaining to the joints) disorders are the most common.

The inventors have investigated the proteolytic disassembly of complex extracellular matrices (ECM), such as cartilage, which occurs during a range of different musculoskeletal system disorders including arthritis. A variety of extracellular matrix degrading proteases have been identified and implicated in neoplastic disease involving tissue invasion, destruction, and/or degradation including urokinase-type plasminogen activator (uPA), various matrix metalloproteinases, and lysosomal cysteine proteases such as cathepsin B. However, none of these enzymes, and specifically matriptase (MTSP-1), an 80-90 kDa trypsin-like, multi-domain type Il trans-membrane serine protease, have been implicated in the aetiology and/or the pathogenesis of other non-neoplastic diseases, and specifically inflammatory and/or autoimmune diseases. The inventors therefore used an in vitro model of arthritis to look at the role of matriptase in the development of that disease, and have shown that matriptase inhibition both prevents collagen destruction and promotes cartilage collagen to resorb in vitro. Hence compounds that can demonstrate that inhibitory effect in vivo could significantly assist in preserving joint function and slowing disease progression. The results therefore indicate that matriptase could be an important therapeutic target for the treatment of both osteo and rheumatoid arthritis, and may also have a broader utility for the treatment of other musculoskeletal system disorders. Moreover, the inventors have devised an assay and which can be used as a screening tool to identify potential inhibitory agents of matriptase activity.

A first aspect of the invention provides a method of screening for an agent for use as a medicament for the prevention or treatment of a musculoskeletal system disorder, comprising determining whether a test agent modulates the amount and/or activity of matriptase.

The methods of the first aspect of the invention provide screening methods for drugs or lead compounds.

An embodiment of screening methods of the invention is wherein the method comprises the steps of: (A) contacting matripase with a quantity of a test agent; and (B) determining the effect of the test agent on the amount and/or activity of matriptase.

Hence in this embodiment of the invention the method may comprise an in vitro/ex vivo screening method in which native and/or recombinant matriptase polypeptide is contacted with the test agent, and the effect of the test agent on amount and/or activity of matriptase polypeptide is determined. Methods for producing recombinant polypeptides are well known in the art. Methods of determining the effect of the test agent on amount and/or activity of matriptase are discussed below.

A further embodiment of the screening method of the invention is wherein the method comprises the steps of: (A) contacting a cell capable of expressing matripase with a quantity of a test agent; and (B) determining the effect of the test agent on the amount and/or activity of matriptase.

Hence in this embodiment of the invention the method may comprise a screening method in which a cell expressing native and/or recombinant matriptase polypeptide is contacted with the test agent, and the effect of the test agent on amount and/or activity of matriptase polypeptide is determined. Examples of cells producing expressing native and/or recombinant polypeptides are well known in the art, as mentioned below. Methods of determining the effect of the test agent on amount and/or activity of matriptase are discussed below.

A further embodiment of the screening method of the invention is wherein the method comprises administering the test agent to a non-human animal and the effect of the test agent on the amount and/or activity of matriptase is determined.

Hence in this embodiment of the invention the method may comprise a screening method in which a non-human animal expressing native and/or recombinant matriptase is contacted with the test agent, and the effect of the test agent on amount and/or activity of matriptase is determined. Examples of non-human animals producing expressing native and/or recombinant polypeptides are well known in the art, as mentioned below. Methods of determining the effect of the test agent on amount and/or activity of matriptase are discussed below.

As mentioned above, the inventors have identified matriptase is significantly elevated in osteoarthritic cartilage. They have also identified that matriptase can locally induce and activate procollagenases, leading to collagenolysis and cartilage ECM degradation. Hence agents that can modulate, preferably decrease, the amount and/or activity of matriptase could be used as medicaments for the prevention or treatment of musculoskeletal system disorders, particularly arthritis.

While matriptase is a well known serine protease, the expression of this protease has been associated with breast, colon, prostate, and ovarian tumors, which implicates its role in cancer invasion, and metastasis, it has also been associated with embryo development Until the present invention, matriptase had not previously been thought to be associated with cartilage ECM degradation As mentioned above, the inventors have determined that matriptase is significantly elevated in osteoarthritic cartilage, and that matriptase locally activates procollagenases leading to collagenolysis They have shown that matripase promotes cartilage to resorb, and that matriptase inhibition prevents collagen release from cartilage Hence it is likely that a reduction in the amount of matriptase would be beneficial for reducing cartilage ECM degradation

Accordingly therefore a preferred embodiment of the screening method of the invention is wherein the method further comprises the step of selecting an agent that decreases the amount and/or activity of matriptase

As mentioned above, musculoskeletal is a general term which is defined as relating to muscles and the skeleton Thus, the musculoskeletal system involves the muscles, bones, joints, bursa, ligaments and tendons Disorders of the musculoskeletal system can be categorised as being caused by inflammation, such as rheumatoid arthritis, psoriatic arthritis, bursitis, tendinitis, and the like, or disorders which are noninflammatory, such as osteoarthritis, muscular sclerosis (an auto-immune disease), Systemic Lupus Erythematosus (also thought to be an-immune disease), and further conditions which are non-inflammatory in aetiology, pathogenesis, and primary patho-physiology

Preferably by "musculoskeletal system disorder" we include arthritis, also preferably by musculoskeletal system disorder" we include non-inflammatory disorders such as those discussed above As mentioned above, matriptase is a small subfamily of type Il transmembrane serine proteases characterised by having related serine protease domains Members of the matriptase polypeptide subfamily include matrιptase-1 , -2 and -3 as well as polyserase-1 As shown in the accompanying example, matriptase can activate proMMP-1 , proMMP-3, and the G-protein-coupled receptor PAR-2, matriptase also enhances cartilage collagenolysis These biological functions of matriptase can be used as a basis for assays to determine whether a test agent modulates the amount and/or activity of matriptase Information concerning the amino acid sequence, and encoding nucleic acid sequence, of each of these polypeptides can be readily obtained from, for example, GenBank or UniProt, and can be easily obtained from those sources by a person skilled in the art. An example of an amino acid sequence of the matriptase polypeptide is provided herein. This also includes a URL for the UniProt entry (obtained by searching the database with the name of the polypeptide).

Hence an example of matriptase that can be used in the screening methods of the invention can be found at UniProt accession number Q9Y5Y6; the amino acid sequence of this representative matriptase is provided at the end of the description as SEQ ID NO:1.

By "matriptase" we include the polypeptide subfamily members mentioned above, as well as fragments or variants of such polypeptides, and further homologues, orthologues or paralogues of matriptase subfamily members which have biological functions including those mentioned above, e.g. proMMP-1 , proMMP-3 and PAR-2 activation; and cartilage collagenolysis. A "fragment" of said polypeptide will preferably comprise less than the total amino acid sequence of the full native polypeptide; preferably the fragment retains its biological activity.

A preferred fragment of matriptase that can be used in the screening methods of the invention comprises the catalytic domain region from amino acid position 596 to 855 of the sequence given in SEQ ID NO:1.

A "variant" of the polypeptide also refers to a polypeptide wherein at one or more positions there have been amino acid insertions, deletions, or substitutions, either conservative or non-conservative, provided that such changes result in a protein whose basic properties, for example protein interaction, thermostability, activity in a certain pH- range (pH-stability) have not significantly been changed. "Significantly" in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein.

By "conservative substitutions" is intended combinations such as GIy, Ala; VaI, lie, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr. Such variants may be made using the methods of protein engineering and site-directed mutagenesis as would be well known to those skilled in the art. By "fragment" or "variant" of the matriptase polypeptide we include a polypeptide that can be used in the screening methods of the invention. Such a variant may be encoded by a gene in which different codons can be substituted which code for the same amino acid(s) as the original codons. Alternatively, the substitute codons may code for a different amino acid that will not affect the function or immunogenicity of the protein or which may improve its function or immunogenicity. For example, site-directed mutagenesis or other techniques can be employed to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions.

We also include "fusions" of the matriptase polypeptide in which said polypeptide is fused to any other polypeptide. For example, the said polypeptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said polypeptide. Examples of such fusions are well known to those skilled in the art. Similarly, the said polypeptide may be fused to an oligo-histidine tag such as His6 or to an epitope recognised by an antibody such as the well known Myc tag epitope.

It will be recognised by those skilled in the art that the amino acid sequence of the matriptase polypeptide can be used to identify homologues to that polypeptide (or nucleic acid encoding the polypeptide).

Methods by which homologues (or orthologues or paralogues) of polypeptides can be identified are well known to those skilled in the art: for example, in silico screening or database mining. Preferably, such polypeptides have at least 40% sequence identity, preferably at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to the matriptase polypeptide.

Methods of determining the percent sequence identity between two polypeptides are well known in the art. For example, the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally. The polypeptide, or fragments, variants or homologues thereof, may originate from any organism. However, a preferred embodiment of the first aspect of the invention is wherein the matriptase polypeptide components, or fragments, variants or homologues thereof, are mammalian; more preferably they are human.

The matriptase polypeptide to be used in the screening methods of the invention may be produced using a number of known techniques. For instance, the matriptase polypeptide may be isolated from naturally occurring sources. Indeed, such naturally occurring sources of the matriptase polypeptide may be induced to express increased levels of the matriptase polypeptide, which may then be purified using well- known conventional techniques. Alternatively cells that do not naturally express the matriptase polypeptide may be induced to express the polypeptide. It is possible to isolate matriptase polypeptide using a molecule which can specifically bind to matriptase polypeptide, such as an antibody. Using such a binding molecule in conditions that preserve the integrity of the matriptase polypeptide, such as non- denaturing conditions, the polypeptide can be isolated substantially pure of any contaminants.

For example, a culture of cells that contain the matriptase polypeptide can be grown in vitro, the polypeptide extracted from the cells, and using an antibody to matriptase, preferably under non-denaturing conditions, the polypeptide can be isolated. A further suitable technique to isolate the matriptase polypeptide involves cellular expression of a fusion between a matriptase and a fusion tag or label, such as a his construct. The expressed polypeptide, and hence matriptase, may subsequently be highly purified by virtue of the his "tag". Cells may be induced to express increased levels of the matriptase polypeptide. This effect may be achieved either by manipulating the expression of endogenous matriptase, or causing the cultured cells to express exogenous matriptase. Expression of exogenous polypeptide may be induced by transformation of cells with well-known vectors into which cDNA encoding matriptase may be inserted. It may be preferred that exogenous matriptase is expressed transiently by the cultured cell (for instance such that expression occurs only during ex vivo culture). Methods of producing recombinant polypeptides are well known in the art. Any suitable cell types can be used to express the polypeptide, for example bacterial or eukaryotic cells including simple yeast such as S. cerevisiae, or Pichia pastoris. Further preferred cells include insect cells, which are commonly used for the production of recombinant polypeptides.

As discussed above, information concerning nucleic acid sequences encoding matriptase polypeptide can be obtained from, for example, GenBank or UniProt, and can be easily obtained from those sources by a person skilled in the art. It will be appreciated that the nucleic acid encoding matriptase polypeptide may be delivered to the biological cell without the nucleic acid being incorporated in a vector. For instance, the nucleic acid encoding matriptase polypeptide may be incorporated within a liposome or virus particle. Alternatively a "naked" DNA molecule may be inserted into the biological cell by a suitable means e.g. direct endocytotic uptake.

The exogenous genes encoding matriptase (contained within a vector or otherwise) may be transferred to the biological cells by transfection, infection, microinjection, cell fusion, protoplast fusion or ballistic bombardment. For example, transfer may be by ballistic transfection with coated gold particles, liposomes containing the exogenous gene, and means of providing direct DNA uptake (e.g. endocytosis).

The matriptase used in the screening methods of the invention may be an isolated polypeptide. That is, a sample of matriptase can be prepared using the methods set out herein. In such circumstances the matriptase will be placed into a biologically suitable environment and then exposed to a quantity of the test agent. The effect of the test agent on the matriptase can then be determined using the experimental approaches set out below.

Alternatively the matriptase is present within a suitable test cell. The cell could be any cell having matriptase, including for example an insect cell. However it is preferred that the cell is a mammalian cell containing a mammalian matriptase; preferably a human cell. Alternatively, it is preferred that the cell is a yeast cell; preferably Saccharomyces cerevisiae. We include cells including nucleic acid sequence encoding the matriptase. Such nucleic acid sequence may be a "native" gene present in the genome of that cell, or it may be an extrachromosomal nucleic acid molecule. Another alternative is where the matriptase is present within non-human animal. The animal could be any animal having the matriptase polypeptide. However it is preferred that the non-human animal is a mammalian organism containing mammalian matriptase. We include non-human animals including nucleic acid sequence encoding the specified polypeptide. Such nucleic acid sequence may be a "native" gene present in the genome of that organism, or it may be an extrachromosomal nucleic acid molecule.

The non-human animal may be any non-human animal, including non-human primates such as baboons, chimpanzees and gorillas, new and old world monkeys as well as other mammals such as cats, dogs, rodents, pigs or sheep, or other animals such as poultry, for example chickens, fish such as zebrafish, or amphibians such as frogs. However, it is preferred that the animal is a rodent such as a mouse, rat, hamster, guinea pig or squirrel. Preferably the animal is mouse. Preferably the non- human animal has a nucleic acid sequence encoding matriptase.

The methods of the invention are "screening methods" to identify agents of use in preventing or treating a musculoskeletal system disorder. For the reasons outlined above, an agent that modulates the amount and/or activity of matriptase is considered an agent that could be of use in preventing or treating a musculoskeletal system disorder; preferably the agent decreases the amount and/or activity of matriptase.

In order to assess whether the test agent modulates the amount and/or activity of matriptase, it is useful to compare matriptase exposed to the test agent to a "reference sample", i.e. a sample of matriptase not exposed to the test agent. By comparing matriptase in a sample exposed to the test agent, to a sample of matriptase not exposed to the test agent, it is possible to determine the effect of the test agent on the amount and/or activity of matriptase.

Hence the test agent may produce an elevation, reduction or no effect on the amount of matriptase or the amount of nucleic acid encoding matriptase; an alteration or no effect on the function of matriptase; a potentiation, inhibition or no effect on the activity of matriptase.

The step of "determining the effect of the test agent on the amount and/or activity of matriptase" may be performed using a number of different experimental techniques. Non-exhaustive examples of methods of determining the amount of matriptase (and nucleic acids encoding this polypeptide) may be performed using a number of different methods, which are discussed below. Further information regarding some of the experimental procedures set out below are described further in Sambrook et al. (2000) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Assaying protein levels in a sample can be performed using any art-known method. Total protein levels within a sample can be measured using Bradford reagent, fluorescamine dye or by using the Lowry method: these techniques are standard laboratory procedures.

It will be appreciated that the amount of a polypeptide may be measured by labelling a compound having affinity for that particular polypeptide. For example, antibodies, aptamers and the like may be labelled and used in an assay. Preferred for assaying protein levels in a biological sample are antibody-based techniques. Examples of immunoassays include immunofluorescence techniques known to the skilled technician, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay analyses.

Antibodies that specifically bind to matriptase are available commercially; for example, Calbiochem (www.calbiochem.com) supply a polyclonal matriptase antibody (catalogue number IM1014).

Levels of mRNA encoding particular polypeptides may be performed using the RT- PCR method. Briefly, this method involves converting mRNA isolated from a sample to cDNA using a reverse transcriptase enzyme. The cDNA products are then subject to PCR according to conventional techniques. After a suitable number of rounds to achieve amplification, the PCR reaction product corresponding to the mRNA encoding the particular polypeptide is quantified. Variations on the RT-PCR method will be apparent to the skilled artisan. Any set of oligonucleotide primers which will amplify reverse transcribed target mRNA can be used and can be designed as will be well known to those skilled in the art.

Levels of mRNA encoding the particular polypeptide can also be assayed using northern blotting, a method well known to those skilled in the art. Further methods which may be of use in measuring mRNA levels include in situ hybridisation, in situ amplification, nuclease protection, probe arrays and amplification based systems. In addition, microarray analysis, a technique well known to those skilled in the art, may also be used to assess the amount of mRNA encoding a particular polypeptide.

Using such techniques common in the art, it would be possible to determine the amount of expression of particular polypeptide.

Also, the expression of a certain gene can be measured using promoter-reporter constructs, a technique well known to the skilled person.

The screening methods of the invention may also include assessing the effect of the test agent on the activity of matriptase. By "activity" we also include the biological function of matriptase. In this respect, assays can be devised that examine the function of matriptase, and the effect of the test agent on that function can be assessed; such as an alteration or no effect. Assays can also be devised that examine the activity of matriptase and the effect of the test agent on that activity can be assessed; such as potentiation, inhibition or no effect.

As shown herein matriptase can activate proMMP-1 , proMMP-3, and the G-protein- coupled receptor PAR-2; matriptase also enhances cartilage collagenolysis. These biological functions of matriptase can be used as a basis for assays to determine whether a test agent modulates the activity of matriptase.

For example, exogenous matriptase can induce significant levels of collagenolysis when human cartilage is exposed to matriptase; hence an assay could be used in which matriptase is contacted with a test agent before or during the incubation of the cartilage with matriptase, and the effect of the test agent on matriptase-mediated collagenolysis determined. Alternatively, a method for ProMMP activation by matriptase is described in the accompanying example: this could be used as a basis for an assay in which a test agent is introduced to the ProMMP activation assay, and the effect of that on matriptase-mediated ProMMP activation measured. Further such assays can be readily devised and are well known in the art, and can be used as a basis for further means of determining the effect of the test agent on the amount and/or activity of matriptase.

Also, the inventors suggest that matriptase activity in promoting cartilage degradation leads to the induction of collagenolytic MMPs, which they suggest is via PAR-2 activation. Hence a further assay can be used to identify agents which prevent PAR- 2 activation by matriptase using the experimental protocols provided herein.

The screening methods of the invention relates to screening methods for drugs or lead compounds. The test agent may be a drug-like compound or lead compound for the development of a drug-like compound.

The term "drug-like compound" is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament. Thus, for example, a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons and which may be water-soluble. A drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes, but it will be appreciated that these features are not essential.

The term "lead compound" is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, nonselective in its action, unstable, poorly soluble, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.

The screening methods of the invention can be used in "library screening" methods, a term well known to those skilled in the art. Thus, for example, the methods of the invention may be used to detect (and optionally identify) a test agent that modulates the the amount and/or activity of matriptase. Aliquots of a library may be tested for the ability to give the required result. Hence by "test compound", we include where matriptase is exposed to more than one compound at the same time, as is commonly performed in high throughput screening assays well known in the art. An embodiment of the screening methods of the invention is wherein the method further comprises the step of selecting an agent that increases the amount and/or activity of matriptase. By "increases" we include where the matriptase has, for example, 110%, 125%, 130%, 140%, 150%, 200%, 250%, 500%, 1000%, or 10000% of the amount and/or activity of matriptase when compared to that of the reference sample.

By "increases" we also include where the matriptase has, for example, 10, 100, 1000, 10,000, 100,000 or 1 ,000,000 times more of the amount and/or activity of matriptase when compared to that of the reference sample.

An embodiment of the screening methods of the invention is wherein the method further comprises the step of selecting a compound that decreases the amount and/or activity of matriptase.

By "decreases" we include where the matriptase, has, for example, 90%, 80%, 70%, 60%, 50%, 25%, 10%, 5%, 1%, or less of the amount and/or activity of matriptase when compared to that of the reference sample.

By "decreases" we also include where the matriptase has, for example, 0.1 , 0.01 , 0.001 , 0.0001 , 0.00001 or 0.000001 or less of the amount and/or activity of matriptase when compared to that of the reference sample. An embodiment of the screening methods of the invention is wherein the method has the additional step of mixing the selected agent (or a derivative or analogue thereof) with a pharmaceutically acceptable vehicle.

A further aspect of the invention provides a method of making a pharmaceutical composition comprising the screening method of the invention and the additional step of mixing the selected agent (or a derivative or analogue thereof) with a pharmaceutically acceptable carrier.

A still further aspect of the invention provides the use of matriptase in a method of screening for an agent for use as a medicament for the prevention or treatment of a musculoskeletal system disorder. An embodiment of this aspect of the invention is wherein the matriptase polypeptide comprises the catalytic domain region from amino acid position 596 to 855 of the sequence given in SEQ ID N0:1. The matriptase polypeptide for use in this aspect of the invention can be prepared using the methods described above in relation to the first aspect of the invention.

The various elements required for a technician to perform the methods of aspects of the invention may be incorporated in to a kit.

A further aspect of the invention provides a kit of parts for use in a method of screening for an agent for use as a medicament for the prevention or treatment of a musculoskeletal system disorder comprising matriptase, or a cell capable of expressing matripase.

An embodiment of this aspect of the invention is wherein the kit further comprises one or more materials capable of determining the amount and/or activity of matriptase. Such materials can include, for example, binding agents that specifically bind to matriptase; PCR primers that can be used in methods to measure matriptase mRNA levels; materials used in the ProMMP activation assay described herein; materials used in the matriptase-mediated collagenolysis assay described herein.

The kit of the invention may also comprise relevant buffers and regents for conducting such methods.

The buffers and regents provided with the kit may be in liquid form and preferably provided as pre-measured aliquots. Alternatively, the buffers and reagents may be in concentrated (or even powder form) for dilution. A further aspect of the invention provides an agent capable of modulating the amount and/or activity of matriptase for use as a medicament for the prevention or treatment of a musculoskeletal system disorder.

A further aspect of the invention provides a method of preventing or treating a musculoskeletal system disorder comprising administering to a subject in need thereof a suitable quantity of an agent that modulates the amount and/or activity of matriptase. Agents of use in the above aspects of the invention which modulate the amount and/or activity of matriptase are useful for preventing or treating a musculoskeletal system disorder A preferred embodiment of this aspect of the invention is wherein the disorder is arthritis, including rheumatoid arthritis and osteoarthritis

Examples of agents which may be used according to the invention include where the agent may bind to the matriptase polypeptide and increase or prevent matriptase activity, e g antibodies and fragments and derivatives thereof (e g domain antibodies or Fabs) Alternatively the agent may act as a competitive inhibitor to matriptase by acting as, for example, an antagonist Alternatively the agent may be an activator of matriptase by acting as an agonist Alternatively the agent may inhibit or activate enzymes or other molecules in the matriptase biological pathway Alternatively the agent may bind to mRNA encoding matriptase polypeptide in such a manner as to lead to an increase or reduction in that mRNA and hence a modulation in the amount of matriptase

Alternatively the agent may bind to a nucleic sequence encoding matriptase polypeptide in such a manner that it leads to an increase or reduction in the amount of transcribed mRNA encoding matriptase polypeptide For instance the agent may bind to coding or non-coding regions of the genes or to DNA 5' or 3' of the genes and thereby reduce or increase expression of matriptase protein

The agent may have been identified from the screening methods of the invention as being of use in the prevention or treatment of musculoskeletal system disorder

A preferred embodiment of the invention is wherein said agent decreases the amount and/or activity of matriptase An example of an agent which decreases the amount and/or activity of matriptase is N1-3-methylbutyryl-N4-6-amιnohexanoylpιperazιne (ENMD-1068) Using the assay methods of the invention disclosed herein, the inventors have surprisingly determined that ENMD-1068, a small molecule Proteinase Activated Receptor 2/PAR2 antagonist, decreases the amount and/or activity of matriptase Accordingly therefore a preferred embodiment of this aspect of the invention is wherein the agent is N1-3-methylbutyryl-N4-6-amιnohexanoylpιperazιne (ENMD-1068) N1-3-methylbutyryl-N4-6-aminohexanoylpiperazine (E N M D- 1068) can be obtained commercially from a number of different suppliers; for example BIOMOL International (http://www.biomol.com). Further examples of such agents which may be used in the invention include compounds #21 , #414, #432 and #433 are illustrated below. These compounds are all secondary amides of sulfonylated 3-amidinophenylalanine. As shown in the accompanying examples, compounds #21 , #414, #432 and #433 are matriptase inhibitors and can reduce cartilage breakdown.

Compound #21. K ₁ for matriptase = 3.8 nM

Compound #414. K ₁ for matriptase = 6.1 nM

Compound #432. K ₁ for matriptase = 5.0 nM

Compound #433. K ₁ for matriptase = 24 nM

Accordingly, therefore a preferred embodiment of this aspect of the invention is wherein the agent is compounds #21 , #414, #432 and/or #433, or suitable salts or derivatives of such compounds.

Information concerning the synthesis of compounds #21 , #414, #432 and #433 is provided in the accompanying example. Compounds #21 , #414, #432 and #433 can be readily prepared by the skilled person from information provided herein and from common general knowledge.

Compounds #21 , #414, #432 and #433 as hereinbefore described may be converted into their salts and derivatives by techniques well known in the art. Preferred salts are pharmaceutically acceptable salts. Preferred derivatives are pharmaceutically acceptable derivatives. Preferred salts are those that retain the biological effectiveness and properties of the compounds of the present invention and are formed from suitable non-toxic organic or inorganic acids. Adid addition salts are preferred. Representative examples of salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, trifluoro acetic acid and the like. The modification of a compound into a salt is a technique well known to chemists to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds.

Further agents for use in the aspects of the invention may bind to matriptase polypeptide or to a nucleic acid encoding matriptase polypeptide.

When the agents binds to matriptase polypeptide, it is preferred that the agent binds to an epitope defined by the matriptase polypeptide that has been correctly folded into its native form. It will be appreciated, that there can be some sequence variability between species and also between genotypes. Accordingly other preferred epitopes will comprise equivalent regions from variants of the gene. Equivalent regions from further polypeptides can be identified using sequence similarity and identity tools, and database searching methods, outlined herein. It is most preferred that the agent binds to a conserved region of the polypeptide or a fragment thereof. An embodiment of the invention is wherein the agent is an antibody or fragment thereof. Antibodies that specifically bind to matriptase are available commercially; for example, Calbiochem (www.calbiochem.com) supply a polyclonal matriptase antibody (catalogue number IM1014). The use of antibodies as agents to modulate polypeptide activity is well known. Indeed, therapeutic agents based on antibodies are increasingly being used in medicine. It is therefore apparent that such agents have great utility as medicaments for the improving the prevention or treatment of a musculoskeletal system disorder. Antibodies for use in treating human subjects may be raised against matriptase polypeptide per se or peptides derived from the polypeptide, or peptides comprising amino acid sequences corresponding to those found in the polypeptide. Antibodies may be produced as polyclonal sera by injecting antigen into animals. Preferred polyclonal antibodies may be raised by inoculating an animal (e.g. a rabbit) with antigen (e.g. all or a fragment of the matriptase polypeptide) using techniques known to the art.

Alternatively the antibody may be monoclonal. Conventional hybridoma techniques may be used to raise such antibodies. The antigen used to generate monoclonal antibodies for use in the present invention may be the same as would be used to generate polyclonal sera.

According to another embodiment of the invention, peptides may be used to modulate the amount and/or activity of matriptase. Such peptides represent other preferred agents for use according to the invention. These peptides may be isolated, for example, from libraries of peptides by identifying which members of the library are able to modulate the amount and/or activity of matriptase. Suitable libraries may be generated using phage display techniques.

Aptamers represent another preferred agent of the invention. Aptamers are nucleic acid molecules that assume a specific, sequence-dependent shape and bind to specific target ligands based on a lock-and-key fit between the aptamer and ligand. Typically, aptamers may comprise either single- or double-stranded DNA molecules (ssDNA or dsDNA) or single-stranded RNA molecules (ssRNA). Aptamers may be used to bind both nucleic acid and non-nucleic acid targets. Accordingly aptamers may be generated that recognise and so modulate the activity or amount of matriptase. Suitable aptamers may be selected from random sequence pools, from which specific aptamers may be identified which bind to the selected target molecules with high affinity. Methods for the production and selection of aptamers having desired specificity are well known to those skilled in the art, and include the SELEX (systematic evolution of ligands by exponential enrichment) process. Briefly, large libraries of oligonucleotides are produced, allowing the isolation of large amounts of functional nucleic acids by an iterative process of in vitro selection and subsequent amplification through polymerase chain reaction. Antisense molecules represent another preferred agent for use according to the aspects of the invention. Antisense molecules are typically single-stranded nucleic acids, which can specifically bind to a complementary nucleic acid sequence produced by a gene and inactivate it, effectively turning that gene "off ¹ . The molecule is termed "antisense" as it is complementary to the gene's mRNA, which is called the "sense" sequence, as appreciated by the skilled person. Antisense molecules are typically are 15 to 35 bases in length of DNA, RNA or a chemical analogue. Antisense nucleic acids have been used experimentally to bind to mRNA and prevent the expression of specific genes. This has lead to the development of "antisense therapies" as drugs for the treatment of cancer, diabetes and inflammatory diseases. Antisense drugs have recently been approved by the US FDA for human therapeutic use. Accordingly, by designing an antisense molecule to polynucleotide sequence encoding polypeptide it would be possible to reduce the expression of that polypeptide in a cell and thereby reduce matriptase activity.

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, represent further preferred agents for use according to the aspects of the invention. It will be apparent that siRNA molecules that can reduce polypeptide expression may have utility in the preparation of medicaments for the prevention or treatment of musculoskeletal system disorders. siRNA are a class of 20-25 nucleotide-long RNA molecules are involved in the RNA interference pathway (RNAi), by which the siRNA can lead to a reduction in expression of a specific gene, or specifically interfere with the translation of such mRNA thereby inhibiting expression of protein encoded by the mRNA. Essentially any gene of which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA. Given the ability to knockdown essentially any gene of interest, RNAi via siRNAs has generated a great deal of interest in both basic and applied biology. Hence an embodiment of the aspects of the invention is wherein the agent is a siRNA molecule having complementary sequence to polynucleotide encoding matriptase. Using such information it is straightforward and well within the capability of the skilled person to design siRNA molecules having complementary sequence to such polynucleotides. For example, a simple internet search yields many websites that can be used to design siRNA molecules. By "siRNA molecule" we include a double stranded 20 to 25 nucleotide-long RNA molecule, as well as each of the two single RNA strands that make up a siRNA molecule. It is most preferred that the siRNA is used in the form of hair pin RNA (shRNA). Such shRNA may comprise two complementary siRNA molecules that are linked by a spacer sequence (e.g. of about 9 nueclotides). The complementary siRNA molecules may fold such that they bind together.

A ribozyme capable of cleaving RNA or DNA encoding polypeptide components of the protein complex of the invention represent another preferred agent of the aspects of the invention.

It will be appreciated that the amount of an agent needed according to the invention is determined by biological activity and bioavailability which in turn depends on the mode of administration and the physicochemical properties of the agent. The frequency of administration will also be influenced by the abovementioned factors and particularly the half-life of the agent within the target tissue or subject being treated.

Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials etc), may be used to establish specific formulations of the agents and precise therapeutic regimes (such as daily doses and the frequency of administration).

Generally, a daily dose of between 0.01 g/kg of body weight and 0.1g/kg of body weight of an agent may be used in a treatment regimen; more preferably the daily dose is between 0.01mg/kg of body weight and 100mg/kg of body weight.

By way of example a suitable dose of an antibody is 10g/kg of body weight; 1g/kg of body weight; 100mg/kg of body weight, more preferably about 10mg/kg of body weight; and most preferably about 6mg/kg of body weight.

Daily doses may be given as a single administration (e.g. a single daily injection or a single dose from an inhaler). Alternatively the agent (e.g. an antibody or aptamer) may require administration twice or more times during a day. Medicaments should comprise a therapeutically effective amount of the agent and a pharmaceutically acceptable vehicle. A "therapeutically effective amount" is any amount of an agent which, when administered to a subject leads to an improvement in the musculoskeletal system disorder. A "subject" may be a vertebrate, mammal, domestic animal or human being. It is preferred that the subject to be treated is human. When this is the case the agents may be designed such that they are most suited for human therapy (e.g. humanisation of antibodies as discussed above). However it will also be appreciated that the agents may also be used to treat other animals of veterinary interest (e.g. horses, dogs or cats).

A "pharmaceutically acceptable vehicle" as referred to herein is any physiological vehicle known to those skilled in the art as useful in formulating pharmaceutical compositions.

In one embodiment, the medicament may comprise between about 0.01 μg and 0.5 g of the agent. More preferably, the amount of the agent in the composition is between 0.01 mg and 200 mg, and more preferably, between approximately 0.1 mg and 100 mg, and even more preferably, between about 1mg and 10mg. Most preferably, the composition comprises between approximately 2mg and 5mg of the agent.

Preferably, the medicament comprises approximately 0.1 % (w/w) to 90% (w/w) of the agent, and more preferably, 1% (w/w) to 10% (w/w). The rest of the composition may comprise the vehicle.

Nucleic acid agents can be delivered to a subject by incorporation within liposomes, Alternatively the "naked" DNA molecules may be inserted into a subject's cells by a suitable means e.g. direct endocytotic uptake. Nucleic acid molecules may be transferred to the cells of a subject to be treated by transfection, infection, microinjection, cell fusion, protoplast fusion or ballistic bombardment. For example, transfer may be by ballistic transfection with coated gold particles, liposomes containing the DNA molecules, viral vectors (e.g. adenovirus) and means of providing direct DNA uptake (e.g. endocytosis) by application of the DNA molecules directly to the target tissue topically or by injection.

The antibodies, or functional derivatives thereof, may be used in a number of ways. For instance, systemic administration may be required in which case the antibodies or derivatives thereof may be contained within a composition which may, for example, be ingested orally in the form of a tablet, capsule or liquid. It is preferred that the antibodies, or derivatives thereof, are administered by injection into the blood stream. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion). Alternatively the antibodies may be injected directly to the liver.

Nucleic acid or polypeptide therapeutic entities may be combined in pharmaceutical compositions having a number of different forms depending, in particular on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micelle, transdermal patch, liposome or any other suitable form that may be administered to a person or animal. It will be appreciated that the vehicle of the composition should be one which is well tolerated by the subject to whom it is given, and preferably enables delivery of the therapeutic to the target cell, tissue, or organ.

In a preferred embodiment, the pharmaceutical vehicle is a liquid and the pharmaceutical composition is in the form of a solution. In another embodiment, the pharmaceutical vehicle is a gel and the composition is in the form of a cream or the like.

Compositions comprising such therapeutic entities may be used in a number of ways. For instance, systemic administration may be required in which case the entities may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. Alternatively, the composition may be administered by injection into the blood stream. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion). The entities may be administered by inhalation (e.g. intranasally). Therapeutic entities may also be incorporated within a slow or delayed release device. Such devices may, for example, be inserted on or under the skin, and the compound may be released over weeks or even months. Such devices may be particularly advantageous when long term treatment with an entity is required and which would normally require frequent administration (e.g. at least daily injection). In a preferred embodiment of the invention, the agent capable of modulating the amount and/or activity of matriptase is formulated as a medicament for intra articular administration. In a further preferred embodiment of the invention, the method of preventing or treating a musculoskeletal system disorder comprises administering to a subject in need thereof a suitable quantity of an agent that modulates the amount and/or activity of matriptase, the administration being an intra articular administration. As discussed above, the inventors have determined that agents capable of modulating the amount and/or activity of matriptase, preferably reducing matriptase activity, could be used as medicaments for the prevention or treatment of musculoskeletal system disorders, particularly arthritis. Since the site of musculoskeletal system disorders, particularly arthritis and osteoarthritis, is within the joint, then it is preferred that the medicaments are formulated for administration to a joint, i.e. intra articular administration, and that the medicament of administered to intra articular site.

Examples of administration sites include the knee, shoulder, temporo-mandibular and carpo-metacarpal joints, elbow, hip, wrist, ankle, and lumbar zygapophysial (facet) joints in the spine.

When used in this embodiment of the invention, preferably the medicament is formulated as a sterile, injectable aqueous solution, including any necessary pharmaceutically acceptable excipients as discussed above.

Preferably the agent that reduces matriptase activity is N1-3-methylbutyryl-N4-6- aminohexanoylpiperazine (EN M D- 1068), or compounds #21 , #414, #432 and #433 as illustrated above.

All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention will now be further described with reference to the following examples and Figures.

Figure 1 : Matriptase expression is elevated in OA cartilage. Gene expression levels in hip cartilage from patients with OA (closed circles; n = 13) or normal controls (NOF) (open squares; n = 12) of (A) several membrane serine proteinases or (B) HAI- 1 and -2 (performed in a separate set of samples; n = 12 for OA and NOF) were determined as described in Materials and Methods and normalised to the level of 18SrRNA. Since all primer/probe combinations amplify with essentially equal efficiencies, gene expression levels are directly comparable within each separate set of samples. Significant differences between the normal and OA groups were determined using a two-sided Mann-Whitney U test, where * p<0.05, ** p<0.01 , ^* ** p<0.001 and ns = not significant. All detectable data points are shown with the line representing the mean. (C), Human OA cartilages were embedded in OCT and 10 μm sections obtained using a cryostat. Matriptase was detected in three separate cartilage samples using rabbit anti-matriptase polyclonal antibody (panels i - iii), whilst panel iv is SAM-1 1 monoclonal antibody detection of PAR-2 (same sample as in panel iii). The bars represent 100 μm (50 μm for panels iii and iv). Panel i also contains a higher power magnification as an inset.

Figure 2: Cleavage sites in the propeptide regions of MMP-1 and MMP-3 by matriptase. Recombinant human proMMP-1 or proMMP-3ΔC was incubated with recombinant human matriptase (molar ratios 1 :5 and 1 :15, respectively) for various time points. Products generated from (A) proMMP-1 and (B) proMMP-3ΔC were resolved by SDS-PAGE. (C) Mass spectrometry and N-terminal sequence analyses were also performed on the 1 - 4 h incubations. The data presented include several known cleavage sites within the propeptide regions (in italics); residue numbering is for the proform (see (35) and references therein). The 'bait region ¹ is boxed whilst the cysteine switch region is underlined. Cleavage sites marked with upward pointing closed arrows are for matriptase (+GM6001), open arrows are matriptase-generated in the absence of GM6001. Ct = chymotrypsin; He = human neutrophil elastase; Pk = plasma kallikrein; Tp = trypsin.

Figure 3: Cytokine-induced cartilage collagenolysis is enhanced by matriptase. Bovine cartilage explants were incubated with IL-1+OSM ± matriptase (at 100 nM unless indicated otherwise) for 14 days, with fresh medium, cytokines and reagents at Day 7 where appropriate. Increasing concentrations of I L- 1 +OSM were used as follows: High (1 ng/ml + 10 ng/ml), Medium (0.6ng/ml + 6 ng/ml), Low (0.25ng/ml + 2.5 ng/ml). The collagen release by day 7 (open bars) and the cumulative day 14 release (closed bars) were determined by OHPro measurement and expressed as a percentage of the total for each treatment. All data are representative of at least three separate experiments and are presented as mean (± SD, n = 4) where ^* * ^* , p<0.001 , * ^* , p<0.01 for IL-1 +OSM+matriptase-treated compared to IL-1+OSM.

Figure 4. OA cartilage collagenolysis is enhanced by matriptase. Human OA cartilage explants were incubated with control medium ± matriptase (100 nM) ± IL- 1 +OSM (1 ng/ml + 10 ng/ml, respectively) ± GM6001 (or its negative control; both at 10 μM) or L-873724 (at 10 nM) for 7 days. The collagen release by day 7 (open bars) and cumulative day 14 (closed bars) was determined by hydroxyproline measurement and expressed as a percentage of the total for each treatment. For clarity, data are presented separately where the prime stimulus was (A) matriptase or (B) matriptase+IL-1+OSM; for each stimulus, significant collagen release (p<0.001) was observed compared to control. The data are representative of at least three separate experiments and presented as mean (± SD, n = 4) where ^* * ^* , p<0.001 for statistical comparisons against the GM6001-ve control treatment; ANOVA. (C) RNA isolated from the day 7 cartilage was subjected to real-time PCR for MMP gene expression. The data were normalised to 18SrRNA and presented as fold induction compared to basal (mean ± SEM ₁ n = 4), where ^* *, p<0.001 , *, p<0.05 for statistical comparison against basal; ANOVA. Data are representative of three separate experiments. Figure 5. PAR-2 is an in vivo substrate of matriptase and is expressed in an OA model. Sequential laser Doppler images (25 in total) showing vasodilatation within 1 - 2 min after topical administration of 10 μg of recombinant matriptase (denoted by the black arrow) to (A) PAR-2 ^* ' ^* or (B) PAR-Z' ^' mice. Perfusion was measured in arbitrary flux units and color coded (dark blue = lowest, dark red = highest). (C) The percentage change in perfusion is presented as mean ± SEM (n = 3). (D) Consecutive knee joint sections (6 μm) from either DMM or sham-operated C57BL/6J mice (4 weeks post-surgery) were stained for matriptase or PAR-2. Bars represent 20 μm. Data are representative of at least three separate animals. Figure 6. Matriptase-enhanced OA cartilage collagenolysis is PAR-2-mediated.

Human OA cartilage explants were incubated with control medium ± either a neutralizing antibody to PAR-2 (SAM-11) at 400ng/ml final concentration or the same antibody that had been previously heat-denatured (SAM-11 ^d ), or ENMD-1068 at 10 mM final concentration for 72 hours. Subsequently, matriptase (100 nM) was added such that the SAM-11 antibody and ENMD-1068 were at final concentrations of 200 ng/ml and 5 mM, respectively, for 7 days. The collagen release was then determined by hydroxyproline measurement and expressed as a percentage of the total for each treatment. The data are representative of three separate experiments and presented as mean (± SD, n = 4) where ***, p<0.001 vs matriptase, ^m p<0.001 vs control; ANOVA. Figure 7: Serine proteinase gene expression data in OA cartilage compared to normal. Gene expression levels in hip cartilage from patients with OA (closed circles; n = 12) or normal controls (NOF) (open squares; n = 13) of serine proteinases were determined as described in Materials and Methods and normalised to the level of 18SrRNA. Since all primer/probe combinations amplify with essentially equal efficiencies, gene expression levels are directly comparable. Significant differences between the normal and OA groups were determined using a two-sided Mann- Whitney U test, where * p<0.05, ** p<0.01 , * ^* * pθ.001. All detectable data points are shown with the line representing the mean. Genes are only shown where≥20% of samples had detectable mRNA or where all detectable signals were in one sample group only.

Figure 8: Fold change of serine proteinase gene expression in OA cartilage compared to normal. Gene expression levels in hip cartilage from patients with OA (n = 12) or normal controls (NOF) (n = 13) of serine proteinases were determined as described in Materials and Methods and normalised to the level of 18SrRNA. The fold changes between OA and NOF are shown, along with the median threshold cycles for each group. Significant differences between the OA and NOF groups were determined using a two-sided Mann-Whitney U test. Figure 9. Matriptase inhibitors prevent matriptase-mediated breakdown of cartilage from osteoarthritis patients. The effect of compounds #21 , #414, #432 and #433 on cartilage breakdown is presented. The release of (A) sulphated glycosaminoglycans (a measure of proteoglycan) and (B) hydroxyproline (a measure of collagen) were determined for Day 0 - 7 and cumulative (Day 0 - 14). The data are expressed as a percentage of the total (n = 4). Example 1 : Matriptase is a novel initiator of cartilage matrix degradation in osteoarthritis Objective. Increasing evidence implicates serine proteinases in pathological tissue turnover. The inventors have assessed the role of the transmembrane serine proteinase matriptase in cartilage destruction in osteoarthritis (OA).

Methods. Serine proteinase gene expression in femoral head cartilage from either hip OA or fracture to the neck of femur (NOF) patients was assessed using a low density array. The effect of matriptase on collagen breakdown was determined in cartilage degradation models, whilst the effect on matrix metalloproteinase (MMP) expression was analyzed by real-time PCR. ProMMP processing was determined using SDS-

PAGE/N-terminal sequencing, whilst its ability to activate protease-activated receptor-2 (PAR-2) was performed in a synovial perfusion assay in mice.

Results. Matriptase gene expression was significantly elevated in OA cartilage compared to NOF, and matriptase was immunolocalized to OA chondrocytes. The inventors showed matriptase activated proMMP-1 and processed proMMP-3 to its fully active form. Exogenous matriptase significantly enhanced cytokine-stimulated cartilage collagenolysis, whilst matriptase alone caused significant collagenolysis from OA cartilage which was metalloproteinase-dependent. Matriptase also induced MMP-1 , -3 and -13 gene expression. Synovial perfusion data confirmed matriptase to activate PAR-2, and the inventors demonstrated that matriptase-dependent enhancement of collagenolysis from OA cartilage is blocked by PAR-2 inhibition.

Conclusions. Elevated matriptase expression in OA, its ability to activate selective proMMPs as well as induce collagenase expression make this serine proteinase a key initiator and inducer of cartilage destruction in OA. The inventors propose that the indirect effects of matriptase are PAR-2-mediated, and a more detailed understanding of these mechanisms may highlight important new therapeutic targets for OA treatment.

Introduction

Metalloproteinases, especially matrix metalloproteinases (MMPs), are considered to be the most important class of proteinase in terms of cartilage degradation since collectively they can degrade all components of this complex extracellular matrix (ECM). Indeed, type Il collagen is a major structural component of this ECM, and collagenolysis is an essentially irreversible step making this proteolysis a major therapeutic target. Collagens are remarkably resistant to proteolysis, and relatively few enzymes are able to effect their hydrolysis. MMP-1 , -8 and -13 are 'classical collagenases' with MMP-1 and -13 most strongly linked to cartilage degradation in rheumatoid arthritis (RA) and osteoarthritis (OA).

Articular cartilage provides a friction-free articulating joint surface and is an unusual tissue since it is only sparsely populated by a single cell-type, the chondrocyte.

These cells are responsible for ECM development and maintenance and are subject to a variety of stimuli in both normal and patho-physiological settings. During disease states, abnormal stimuli including abnormal loading, as well as pro-inflammatory stimuli such as interleukin-1 (IL-1 ) and tumor necrosis factor α (TNFα), prevail that lead to uncontrolled ECM turnover. These factors are known to be present in both RA and OA, and the inventors have demonstrated that the IL-6-family cytokines IL-6 and oncostatin M (OSM) markedly exacerbate the catabolic potential of these mediators.

Indeed, the inventors have demonstrated synergistic, MMP-dependent cartilage catabolism in vitro and in vivo, and the inventors and others use IL-1 +OSM as a potent stimulus for inducing cartilage destruction.

In the last few years the full repertoire of human proteinases, termed the degradome, has been defined. These studies revealed many new serine proteinases, such that there are almost as many as there are metalloproteinases. It is now emerging that serine proteinases have many diverse roles in both normal and pathological scenarios, and this also applies to destructive joint diseases. The inventors have previously reported that serine proteinase inhibition with inhibitors of urokinase-type plasminogen activator (uPA) and furin-like enzymes can block cytokine-stimulated collagenolysis. The inventors findings have consistently supported the notion that serine proteinase pathways activate latent, inactive proMMPs, evidence that suggests proteolytic networks exist in resorbing cartilage. These findings therefore make serine proteinases a potential therapeutic target although the identity of specific enzymes remains elusive. In OA, collagenolysis initially occurs around the chondrocytes, implicating proteinases associated with the cell surface. Using a serine proteinase active site- specific probe, the inventors previously identified fibroblast activation protein alpha (FAPα), an integral membrane serine proteinase. Of note, FAPα expression is significantly higher in OA cartilage compared to phenotypically normal articular cartilage although the role of this transmembrane dipeptidyl peptidase has not yet been deduced. The inventors have now extended this initial study and performed a screen of OA and normal cartilages for serine proteinase gene expression in order to identify potential candidates important in the proposed serine proteinase pathways. This highlighted matriptase-1 (hereafter referred to as matriptase), one of twenty type Il transmembrane serine proteinases (TTSPs). The biological functions of the matriptase subfamily, which includes matriptase-1 , -2 and -3 as well as polyserase-1 , are poorly defined although matriptase is known to be important in a variety of processes including embryonic development and tumor invasion. This enzyme is not expressed in normal cartilage, whilst its activation and activity are regulated by the endogenous transmembrane Kunitz-type serine proteinase inhibitors hepatocyte growth factor activator inhibitor (HAI)-I and -2.

The inventors propose matriptase is an important mediator of catabolic events in diseased cartilage. Specifically, the inventors demonstrate matriptase and HAI-1 expression are elevated in OA cartilage, addition of matriptase to cartilage enhances collagenolysis as well as inducing the expression of both MMP-1 and MMP-3. The inventors show that matriptase is an activator of proMMP-1 and -3 as well as proteinase-activated receptor-2 (PAR-2). Thus, matriptase has direct and indirect roles that drive cartilage breakdown in OA.

MATERIALS AND METHODS

Materials. All chemicals of the highest purity available were obtained from Sigma Chemical Co (Poole, UK) unless otherwise stated. All cytokines and proteins used were recombinant human. IL-1α was a generous gift from Dr Keith Ray (GlaxoSmithKline, Stevenage, UK). OSM was produced in-house. ProMMP-3(ΔC) was a generous gift from Dr Rob Visse (Kennedy Institute, London, UK) whilst human matriptase (catalytic domain, residues 596-855) was prepared as described as were proMMP-1 and proMMP-13. A matriptase antibody (cat. No. IM1014), GM6001 and its negative control, and the FS-6 substrate were from Calbiochem (Nottingham, UK). A PAR-2 antibody (SAM-11 ; azide-free) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). ENMD-1068 was from Enzo Life Sciences (Exeter, UK). The cathepsin K inhibitor L-873724 was a kind gift from Dr Cameron Black (Merck Frosst, Kirkland, Canada). Animals. Experiments were performed on adult wild-type (PAR-2 ^+/+ ) C57BL/6J mice (body weight 25-30 g) housed in standard cages with food and water available ad libitum (maintained in a thermoneutral environment). PAR-2-deficient (PAR-2 ^" ' ^" ) mice were genetically modified as described previously and maintained under the same conditions. All procedures were performed in accordance with current UK Home Office regulations. OA in C57BL/6J mice was induced following surgical destabilisation of the medial meniscus (DMM) by sectioning of the medial menisco- tibial ligament; this results in medial and posterior rotation of the medial meniscus leading to a mild form of OA. Sham operations were performed in a subset of mice. Animals were left for 4 weeks after which the knee joint was harvested for histology.

Cartilage degradation assays. Bovine nasal septum cartilage (from a local abattoir) was dissected into ~2x2x2 mm discs, plated into 24-well tissue culture plates (3 discs/well, n = 4) in serum-free medium and incubated for 14 days in the presence of IL-1±OSM (±matriptase), changing media after 7 days as described. Cartilage remaining at day 14 was digested with papain, and all samples stored at -20 ⁰ C until assayed. Viability of cartilage explants was assessed by screening for adenylate kinase (AK) production using the Toxilight bioassay kit (Lonza, Slough, UK). No increase in AK levels with any of the treatments including inhibitors were found (data not shown).

Macroscopically normal articular cartilage was obtained from patients (either hip OA patients or patients with neck of femur fracture (NOF) with no history of OA) undergoing total joint replacement surgery in hospitals in either Newcastle or Norwich, and was prepared and treated as above for bovine cartilage. Cartilage samples were pre-treated for 72 hours with PAR-2 inhibitors to allow efficient tissue penetration prior to stimulation. All subjects gave informed consent and the study performed with Ethical Committee approval.

Collagen and collagenolytic activity assays. Hydroxyproline measurements were used as an estimate of cartilage collagen, and the cumulative release calculated and expressed as a percentage of the total for each well. Collagenolytic activity present in the culture media from cartilage explants was determined using a diffuse fibril assay with ³ H-acetylated collagen as described where one unit of collagenase activity degrades 1 μg of collagen per min at 37°C. RNA extraction from cartilage. Total RNA from hip cartilage samples was prepared as described; NOF cartilages were phenotypically normal and lesion-free. For experiments in which human OA cartilage was cultured prior to RNA isolation, RNA was prepared as previously reported.

Real-time PCR of relative mRNA levels. For TaqMan and SYBR Green PCR, mRNA levels for each gene were obtained from standard curves and corrected using 18S ribosomal RNA levels. Cycling conditions (7900HT system, Applied Biosystems, Foster City, CA) for SYBR Green PCR (using Takara SYBR ExTaq premix; Lonza Biologies, Cambridge, UK) were 95 ⁰ C 10 s, then 40 cycles of 95°C 5 s then 60 ⁰ C 30 s, followed by a standard dissociation curve analysis. Cycling conditions for Taqman PCR (Jumpstart Taq Readymix; Sigma, Poole, UK) were 2 min at 50°C, 10 min at 95°C, then 40 cycles of 15 s at 95°C and 1 min at 60 ⁰ C. Human primer/probe sequences for Taqman PCR were as described.

ProMMP activation assays. Human recombinant and titered matriptase (catalytic domain) was incubated with equal volumes of either proMMP-1 (1 :5 molar ratio) or proMMP-3ΔC (1 :15 molar ratio) at 37°C in a 50 μl final volume. Working dilutions (25 μl) of each enzyme were made using 25 mM sodium cacodylate, 10 mM CaCI ₂ , 0.05% (v/v) Brij35, 0.02% (w/v) sodium azide, pH8.0). Enzyme stocks were stored in: matriptase: 50 mM Tris-HCI, pH 9.0, 10% (v/v) glycerol, 1 mM β-mercaptoethanol, 0.4 M NaCI; proMMP-1 : 20 mM Tris-HCI, pH7.2, 5 mM CaCI ₂ , 0.05% (w/v) sodium azide, 0.01% (v/v) Brij35, 0.5 M NaCI; proMMP-3: 50 mM Tris-HCI, pH 7.5, 0.15 M NaCI, 10 mM CaCI ₂ , 0.02% (w/v) sodium azide, 0.05% (v/v) Brij35, 10% (v/v) glycerol. Equal aliquots (8 μl) were removed at time points up to 24 hours and snap- frozen until being resolved on 10% sodium dodecyl sulphate-polyacrylamide gels. For visualisation, proteins were silver stained. For sequence determination, incubations were performed ± GM6001 (50 μM final concentration), proteins transferred to polyvinylidene fluoride membrane, bands of interest excised after briefly staining with 0.1% (w/v) Coomassie Blue R250 and subjected to amino- terminal sequencing and/or mass spectrometry.

Substrate Assays. Enzymatic assays of MMP activity were performed in 0.1 M Tris- HCI, pH 7.5, 0.1 M NaCI, 10 mM CaCI ₂ , 0.05% Brij35, 0.1% PEG-6000. Enzyme activity was monitored by measurement of the increase in fluorescence (excitation

324 nm; emission 400 nm) from 5 μM Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH ₂ (FS-6) at 37°C in a LS50B fluorometer with microplate accessory reader (Perkin Elmer, Massachusetts, USA). Matriptase activity was confirmed using 25 μM Boc- Gln-Ala-Arg-AMC (excitation 360 nm; emission 460 nm) in 0.1 M Tris-HCI, pH 9.0, 500 μg/ml BSA, 0.01% Brij-35.

Immunohistochemistry. For human cartilage, tissue was frozen in ice-cold iso- pentane. Serial cryostat sections (10 μm) on APES-coated (2%) slides were prepared and immunostained as described (23). Normal rabbit serum (1.5%) was used for blocking (10 minutes) and sections incubated with anti-matriptase antibody (1 :1000 dilution) for 90 minutes at room temperature. Washed sections were then incubated with biotinylated secondary antibody (rabbit anti-sheep IgG ₁ diluted 50-fold in PBS according to the Vectastain kit instructions) in 1.5% rabbit serum in PBS for 30 minutes, followed by incubation with avidin-biotin complex for 30 minutes using Vectastain kit PK-6105 (Vector) according to the manufacturer's instructions. Decalcified murine knee joints were embedded in paraffin wax. and. Subsequently, sections (6 μm) were deparaffinised, rehydrated and stained with SAM-11 antibody at 1 μg/ml using the Animal Research Kit Peroxidase (Dako, Ely, UK) according to the manufacturer's instructions. Signal development was with diaminobenzidine tetrahydrochloride (Dako) following the manufacturer's protocol. Images were captured using a 3-CCD color video camera (JVC, Tokyo, Japan).

PAR-2 activation assay. Synovial perfusion was measured from the exposed medial aspect of knee joint capsules of parallel groups of wild-type and PAR-2 ^' ' ^" mice using high-resolution laser Doppler imaging (Moor Instruments Ltd, Axminster, UK) as described in the art. A series of scans were taken immediately following topical application of saline vehicle, and once a stable baseline was achieved, a further series of scans was taken immediately after topical administration of matriptase. Images were later analyzed by dedicated software to obtain median flux values over the knee joint region. Vascular perfusion responses (in arbitrary perfusion units) are presented as the percentage change from baseline. The carotid artery was cannulated to allow blood pressure monitoring.

Statistical analyses. Significant differences between patient groups were determined using a two-sided Mann-Whitney U test. Standard TaqMan experiments were performed in at least triplicate for a minimum of three separate samples, with data analysed using a two-tailed Student t-test. Cartilage experiments were performed in quadruplicate for three different cartilages, and significance assessed using analysis of variance (ANOVA) with a post-hoc Bonferroni multiple comparison test using commercial software (SPSS, v15.0, SPSS Inc, Chicago, IL). Blood flow measurement data were similarly assessed by ANOVA. For clarity, only selected comparisons are presented in some figures, where ***p<0.001 , ^* *p<0.01, *p<0.05.

RESULTS

Matriptase gene expression is elevated in OA tissues. Analyses of total RNA from OA and normal (NOF) joint tissues revealed a statistically significant increase in matriptase (ST14) gene expression in OA cartilage (p = 0.0039; Fig. 1A). In a separate experiment, HAI-1 gene expression, but not HAI-2 (not shown), was also significantly elevated (p = 0.0039) in OA cartilage (Fig. 1 B). The increase in matriptase gene expression was reflected in detectable matriptase protein in several OA cartilages (Fig. 1 C, panels i - iii), and the inventors also detected PAR-2 in OA cartilage (Fig. 1 C, panel iv).

Of the TTSP genes assessed, none other than matriptase was up-regulated in OA cartilage although both HPN (hepsin; p = 0.00006) and TMPRSS4 (MT-SP2; p = 0.00002) were markedly suppressed compared to normal (Fig. 1A). These TLDA data for cartilage were validated by the significantly elevated FAPα expression (p = 0.0004) as the inventors have previously shown. Furthermore, significant elevation of several other serine proteinase genes was detected including HtrA1 (high- temperature requirement A1 peptidase), PRSS23 (SPUVE), CFD and CFI (complement factors D and I) and PCSK6 (proprotein convertase 6), whilst PCSK1 (proprotein convertase 1) and CFB (complement factor B) were significantly down- regulated in OA cartilage (see Figures 7 and 8).

Matriptase is an activator of proMMP-1. Since matriptase is a known activator of proMMP-3, the inventors assessed its ability to activate proforms of the two major collagenolytic MMPs relevant to cartilage collagenolysis. SDS-PAGE revealed matriptase failed to process proMMP-13 (not shown), but did process proMMP-1 albeit at a slower rate than for proMMP-3 (Figs. 2A and B). Mass spectrometry and amino-terminal sequence analysis of the processed proMMPs revealed cleavages at the Arg74-Cys75 (cysteine switch region), and at Arg35-Arg36 (bait region) for MMP- 3 (MMP-3 numbering; (35)), in the presence of GM6001 (Fig. 2C). For proMMP-1 , cleavage at Thr64-Leu65 occurred in the absence of GM6001 , reflecting the presence of some active MMP-1 in the preparation. Matriptase generated the [Val82]MMP-1 mature form irrespective of GM6001 presence. Full-length mature MMP-3 ([Phe83]MMP-3) was generated in the absence of GM6001 , whilst inclusion of this MMP inhibitor generated [Thr85]MMP-3 (Fig. 2C). IL-1+OSM-mediated cartilage collagenolysis is enhanced by matriptase activity. Having demonstrated matriptase is an activator of both proMMP-1 and proMMP-3, the inventors hypothesized that exogenous addition of active matriptase to stimulated cartilage would enhance collagenolysis. An ex vivo model of cartilage degradation confirmed that at day 7, a time point when little or no collagen release is typically observed, significant levels of hydroxyproline (a measure of collagen) were detected in the culture supematants. Furthermore, even with a low dose of IL-1+OSM, matriptase (100 nM) still significantly enhanced the release by day 14 (Fig. 3). Where collagenolysis was evident at day 7, active collagenase was also detected (data not shown).

Matriptase enhances OA cartilage collagenolysis and collagenase gene expression in the absence of an inflammatory stimulus. Although the inventors data confirmed a potential role for matriptase in cytokine-stimulated cartilage, previous experiments with matriptase-treated bovine explants failed to demonstrate any collagenolysis (see Fig. 3). However, assessment of the effect of exogenous matriptase on human OA cartilage revealed the striking observation that matriptase alone induced significant collagenolysis which was sensitive to the metalloproteinase inhibitor GM6001 but neither its negative control nor a cathepsin K inhibitor (Fig. 4A). Similar results were found when IL-1+OSM was also included except slightly more collagenolysis was seen (Fig. 4B). The inventors also found significant glycosaminoglycan release following matriptase treatment compared to control (not shown).

The finding that matriptase alone caused significant collagenolysis, and that this was most likely MMP-mediated, led the inventors to hypothesize that matriptase action on OA cartilage led to new collagenase gene expression. Culture of human OA cartilage explants with matriptase for 7 days revealed a significant increase in the expression of both MMP-1 and MMP-3 mRNA, whilst MMP-13 was also elevated although not statistically significant (Fig. 4C). All three MMP genes were detectable (Ct value ranges: untreated human cartilages, 37.9 - 21.2; treated cartilages, 33.5 - 20.2). No increases in MMP-14 were seen whilst di-isopropyl phosphorofluoridate-treated matriptase failed to induce any MMP expression (not shown). PAR-2 is an in vivo substrate of matriptase and is expressed in OA cartilage. To confirm the ability of recombinant matriptase to act as an in vivo activator of PAR-2, the inventors assessed blood perfusion in murine joints following topical administration of matriptase. This demonstrated a rapid and significantly (P<0.0001 ; 2-way ANOVA) greater increase in synovial perfusion comparing PAR-2 ^+/+ (wild-type) animals to their PAR-2-deficient (PAR-2 ^" ' ^" ) littermates (Figs. 5A-C). Arterial blood pressure was not significantly affected by matriptase administration (1.1 ± 0.3 % increase), lmmunohistochemistry of murine knee joints confirmed the presence of chondrocyte PAR-2, and also revealed matriptase expression in chondrocytes from the OA model (DMM), but not sham-operated mice (Fig. 5D). Indeed, matriptase was only detectable at the medial aspect of the joint, corresponding to where OA pathology associated with this model occurs.

Matriptase enhances OA cartilage collagenolysis via PAR-2 Since the inventors had demonstrated an indirect role for matriptase in driving cartilage degradation, they next hypothesised that matriptase mediated this effect by activating PAR-2. They and others have shown PAR-2 is a target of matriptase, and two different PAR-2 inhibitors (SAM-11 or ENMD-1068) both significantly prevented collagenolysis (Fig 6).

DISCUSSION

The burgeoning field of degradomics has enabled research into aberrant proteolysis to make important new discoveries. Cartilage disassembly during disease involves multiple proteinase cascades that are likely to be inter-dependent. The inventors, and others, have reported that human OA cartilage is highly resistant to proinflammatory stimuli despite increased expression of collagenolytic MMPs; the reasons for this resistance are unclear although a failure to activate latent procollagenases would explain this. The inventors previous data strongly implicate serine proteinases with roles in pathological cartilage turnover, especially procollagenase activation, but it has been difficult to identify specific enzymes.

The inventors' current screen of ~100 serine proteinases in OA and phenotypically normal (NOF) cartilages corroborated previous findings of significantly elevated expression in OA for several serine proteinases albeit with differing degrees of significance as recently outlined in the art. The elevated expression of FAPα in the present study corroborates previous independent report known in the art; these data

Ob suggest that this post-prolyl peptidase, known to modify chemokines and bioactive peptides, may have a protective role in OA. Another up-regulated enzyme to be confirmed was HtrA1 which further implicates this proteinase in catabolic ECM turnover since treatment of synovial fibroblasts with HtrA1 or HtrA1 -generated fibronectin fragments induces MMP-1 and -3 expression.

Of the TTSPs screened, most were unaltered and expressed at very low levels. However, one TTSP gene was significantly elevated in OA cartilage compared to normal, namely matriptase which is widely expressed in various cancers. A failure to suppress matriptase expression during disease may therefore have catabolic implications for cartilage. Several putative substrates have been identified including pro-hepatocyte growth factor, and the serine proteinases prourokinase-type plasminogen activator (pro-uPA) and prostasin, with a major role as an initiator of serine proteinase cascades. The inventors have proposed such cascades are important in cartilage degradation, and this initiator hypothesis is further supported in that matriptase activation is autocatalytic, an uncommon occurrence for a latent serine proteinase. In fact, matriptase activation is a complex process requiring its cognate inhibitor, HAI-1, which enables appropriate trafficking to the cell surface. This process therefore directs matriptase activity to the cell surface whilst providing a means of regulating this activity. In turn, this suggests that increased matriptase expression and subsequent activity would only result with a concomitant increase in HAI-1 expression; the inventors found this situation to exist in OA cartilage, data that strongly imply elevated matriptase activity in OA. Some of the potential consequences of matriptase activity in cartilage are well aligned with the inventors' previous data known in the art. MMP-3 is a key activator of procollagenases and the inventors confirm matriptase processes proMMP-3 to a fully active enzyme following an intermediate cleavage within the bait region (Arg-Arg bond) in line with its P1 preference. This intermediate species generates a fully active MMP-3 with an N-terminal Phe. Interestingly, inclusion of a MMP inhibitor revealed cleavage at the Arg-Cys bond within the cysteine switch regions for MMP-1 and MMP-3 proforms; this same bond is cleaved in MMP-7 and -8 by trypsin, and MMP- 13 by plasmin. This presumably results in loss of MMP latency, since in the absence of MMP activity, matriptase ultimately processed proMMP-1 and -3 to less active forms similarly to chymase and trypsin, respectively. However, MMP-3 is typically co-ordinately expressed with MMP-1 and MMP-13 (45) such that in vivo, matriptase action on proMMP-3 will generate MMP-3 enzyme capable of processing collagenolytic MMPs to their most active forms. This is evidenced by the enhanced collagenolysis at Day 7 when cytokine stimulation alone fails to promote such collagenolysis; this therefore provides a mechanism that maximises the degradative potential of the procollagenase pool, and one that preferentially localises this activity at the cell surface to effect pericellular collagenolysis.

Matriptase also activates pro-uPA which generates active plasmin from plasminogen. Although the inventors confirmed the absence of plasminogen expression in OA cartilage (data not shown), this enzyme is expressed in synovial tissues. Both MMP-3 and plasmin activate procollagenases to expedite collagenolysis in resorbing cartilage, and plasmin has several other roles that contribute to ECM remodelling. The inventors have proposed procollagenase activation represents a key and rate- limiting step in cartilage collagenolysis, and confirmation that matriptase also activates proMMP-1 , as well as expedites collagenolysis from pro-inflammatory cytokine-stimulated cartilage (with concomitant increase in detectable active collagenase) further supports the notion that matriptase could be a major player in such processes. The inventors have also shown reduced collagenolysis from stimulated cartilage following addition of anti-thrombin III or plasminogen activator inhibitor-1 (unpublished results); these serpins inhibit matriptase, albeit not specifically.

Historically, human OA cartilage explants are highly resistant to proteolysis even following stimulation with highly catabolic cytokine combinations such as IL-1+OSM. Indeed, only about 25% of cartilages respond with typically very low collagen release. The current study also failed to promote cytokine-induced collagenolysis despite collagenase(s) induction (not shown). However, the most striking observation was that even in the absence of a pro-inflammatory stimulus, matriptase induced significant collagenolysis from OA cartilage which was entirely metalloproteinase- dependent. Inclusion of matriptase with a pro-inflammatory stimulus (IL-1 +0SM) promoted slightly more collagenolysis presumably via the activation of cytokine- induced proMMPs. This new MMP expression occurred as a consequence of matriptase action on OA cartilage which was significant for MMP-1 and -3. Matriptase activity would subsequently activate secreted proMMPs although they cannot exclude the concomitant initiation of another activation cascade.

These highly novel observations suggest matriptase may mediate its affect via a cell surface receptor specifically expressed in OA cartilage since treatment of healthy bovine or non-diseased human cartilages with matriptase fails to promote collagenolysis (data not shown). Matriptase is a known activator of PAR-2, a mechanism involving the proteolytic release of a tethered ligand which can be mimicked by use of a PAR-2-activating peptide). The inventors confirmed this in vivo using wildtype and PAR-2-deficient mice in synovial perfusion assays as previously for β-tryptase. The small vasodilatation in the PAR-2 deficient mice may indicate that matriptase has a minor non-PAR-2-mediated effect. PAR-2 expression is significantly elevated in OA compared to normal cartilage, and synthetic activation of PAR-2 in OA cartilage induces both MMP-1 and MMP-13 in line with their observations using matriptase. The inventors have previously shown that the PAR-2 antagonist, ENMD-1068, is effective at inhibiting PAR-2 when activated proteolytically (trypsin) or via a PAR-2 activating peptide. In the current study, PAR-2 inhibition with a neutralizing antibody (SAM-11) or ENMD-1068 effectively blocked matriptase-induced collagenolysis, thus confirming PAR-2 as a target for matriptase in OA cartilage. The inventors have previously implicated PAR-2 with a role in RA since it is highly expressed in rheumatoid synovium and the spontaneous synovial expression of both IL-1 β and TNFα is markedly reduced following PAR-2 antagonism. Furthermore, chondrocyte PAR-2 expression has been shown to be increased following IL-1β or TNFα stimulation, pro-inflammatory cytokines implicated in OA. The expression of various cell surface receptors is altered in OA (eg. some toll-like receptors (TLRs)) which may well be a consequence of inflammation (IL-1 β, TNFα) and/or aberrant mechanical load. This altered phenotype may then increase susceptibility to further stimuli for ECM degradation. Interestingly, whilst PAR-2 was detectable in chondrocytes from both sham-operated and OA joints, matriptase was consistently only detectable in the latter. Moreover, matriptase was only detected in locations where PAR-2 was also present. This suggests PAR-2 may act as a sentinel for proteinase-mediated injury signals, such as matriptase expression due to joint destabilisation, leading to PAR-2 activation and signalling resulting in cartilage degradation.

The ability of matriptase to activate key MMPs makes it an important effector of cartilage degradation. The inventors recently highlighted the importance of serine proteinases in cartilage breakdown, and propose matriptase as a key initiator of proteinase cascades, including MMPs, that collectively orchestrate ECM turnover. Furthermore, matriptase activity in OA cartilage leads to the induction of collagenolytic MMPs, a process the inventors suggest is via PAR-2 activation. These properties of matriptase offer a paradigm for OA where an initial focal injury or inflammatory event leads to localised, altered chondrocyte receptor expression which combined with matriptase action leads to PAR-2 activation and subsequent MMP activation (eg. MMP-1) at the cell surface; this then facilitates pericellular collagenolysis within this focal region of the cartilage. This could have direct implications since MMP-1 is a known activator of PAR-1 , shown to be expressed in cartilage, leading to new MMP expression. In the context of altered receptor expression in OA cartilage, MMP-mediated proteolysis of the ECM may also generate ligands for TLRs not normally abundantly expressed (eg. TLR2). All, or a combination, of these mechanisms could thus perpetuate cartilage degradation in the absence of classical inflammatory cues, especially in OA. Periodic recurrences of such events over time may also explain the slower progression of OA compared to RA, a disease characterised by more persistent inflammatory stimuli and synovial involvement. In summary, the inventors have shown that the TTSP matriptase is expressed in OA cartilage and that this enzyme is both an inducer and activator of procollagenases. This therefore makes matriptase a highly attractive new target for preventing pathological cartilage breakdown. Example 2: Matriptase inhibitors prevent matriptase-mediated breakdown of cartilage from osteoarthritis patients

It is recognized that potent and selective matriptase inhibitors are disclosed in the literature. For example, Steinmetzer et a/ (2006) Journal of Medicinal Chemistry 49:41 16-4126; Steinmetzer et a/ (2009) Bioorganic & Medicinal Chemistry Letters 2009; 19:67-73; and Schweinitz et al Bioorganic & Medicinal Chemistry Letters 2009; 19: 1960-1965. Such inhibitors have previously been disclosed for the treatment of cancer. Following the inventors finding that matriptase inhibitors may be of use as medicaments for the prevention or treatment of musculoskeletal system disorders, particularly arthritis, the inventors decided to assess the potential utility of some of these compounds. Compounds used in assay Compounds #21 , #414, #432 and #433 as set out below are all secondary amides of sulfonylated 3-amidinophenylalanine.

Compound #21. /<, for matriptase = 3.8 nM

Compound #414. K ₁ for matriptase = 6.1 nM

Compound #432. K ₁ for matriptase = 5.0 nM

Compound #433. K ₁ for matriptase = 24 nM

Detailed information concerning the synthesis of Compounds #21 , #414, #432 and #433 can be found in, for example, Steinmetzer et a/ Bioorganic & Medicinal Chemistry Letters 2009; 19:67-73; and Steinmetzer et al (2006) Journal of Medicinal Chemistry 49:4116-4126. Also, further information concerning the synthesis of these compounds is provided at the end of this example. Compound #21 is referenced as compound 1 in Steinmetzer et al Bioorganic & Medicinal Chemistry Letters 2009;19:67-73, and as compound 59 in Steinmetzer et a/ (2006) Journal of Medicinal Chemistry 49:4116-4126.

Compound #414 is referenced as compound 13 in Steinmetzer et al Bioorganic & Medicinal Chemistry Letters 2009; 19:67-73.

Inhibitor #21 was further converted into a 3 x acetate salt by cation exchange chromatography using Fractogel EMD COO- (Merck, Darmstadt, Germany) and an ammonium acetate gradient. The excess of ammonium acetate was removed by repeated lyophilization from water.

Experiments and results.

Cartilage chips (approx 2 x 2 x 2mm; 1 per well of a 96-well plate) were prepared from human OA obtained at the time of joint replacement surgery. Cartilage was left overnight in 200 μl serum-free culture medium. The following day, cartilage was then treated with serum-free culture medium with or without the addition of matriptase

(10OnM) for 7 days in culture. After this, media were removed and replaced with identical test reagents for a further 7 days. In some instances, compounds that inhibit matriptase were also included (19 μM). Residual cartilage is then papain digested.

The release of (A) sulphated glycosaminoglycans (a measure of proteoglycan) and

(B) hydroxyproline (a measure of collagen) were determined for Day 0 - 7 and cumulative (Day 0 - 14) (see Cawston et al. Arthritis Rheum 1998:41 :1760-1771 ).

The data are expressed as a percentage of the total (n = 4). Statistical significance was assessed by ANOVA with a post-hoc Bonferroni test, where * ^** p< 0.001 compared to matriptase only. This data is presented in Figure 9.

From the data presented in Figure 9, it can be seen that compounds #21 , #414, #432 and #433 inhibit the release of (A) sulphated glycosaminoglycans (a measure of proteoglycan) and (B) hydroxyproline (a measure of collagen) from the cartilage samples. Hence such compounds are considered to prevent matriptase-mediated breakdown of cartilage from osteoarthritis patients. Hence the inventors conclude that compounds #21 , #414, #432 and #433 may be of use as medicaments for the prevention or treatment of musculoskeletal system disorders, particularly arthritis.

Synthesis of compounds. #432 and #433

The synthesis of inhibitors MI-432 and MI-433 was performed by methods described previously (Steinmetzer et al., J. Med. Chem. 49, 4116-4126, 2006 and Steinmetzer et al. Bioorg. Med. Chem. Lett. 19, 67-73, 2009) and is shown below for MI-432. For the synthesis of the analogues inhibitor MI-433 2-fluoro-phenylboronic acid (Aldrich) was used for step c.

Synthesis of Inhibitor MI-432:

Reagents and conditions: (a) dioxane/1 N NaOH, stirring 1 h O ⁰ C, overnight at room temperature; (b) PyBop, 2.0 equiv. DIPEA in DMF, 15 min O ⁰ C and 3 h room temperature; (c) 2 equiv. boronic acid, 1 mol% Pd(OAc) ₂ and 2 mol% S-Phos in toluene containing 3 equiv. 2 M Cs ₂ CO ₃ solution, 4 h reflux; (d) i: 2.5 equiv. hydroxylamine x HCI and 2.5 equiv. DIPEA, reflux in ethanol, 4 h, stirring overnight at room temperature, followed by evaporation of ethanol, ii: 3 equiv. Ac ₂ O in acetic acid, 30 min room temp., evaporation of the solvent; iii: zinc powder in 90% acetic acid, stirring overnight at 3O ⁰ C; iv: 90% trifluoroacetic acid, room temp., 1 h, evaporation, and purification by preparative reversed phase HPLC.

Analytical data for MI-432: MS: calc. 601.2, found: 602.2 (M+H) ⁺ , HPLC: retention time 39.4 min

Analytical data for MI-433: MS: calc. 551.2, found: 552.3 (M+H) ⁺ , HPLC: retention time 23.8 min

Analytical Methods:

Analytical HPLC experiments were performed on a Shimadzu LC-10A system (column: Nucleodur Ci ₈ , 5 μm, 100 A, 4.6 x 250 mm, Machery-Nagel, Dϋren, Germany) with a linear gradient of acetonitrile (10-70 % in 60 min, detection at 220 nm) containing 0.1 % TFA at a flow rate of 1 mL/min. The final inhibitors were purified to more than 95 % purity (detection at 220 nm) by preparative HPLC (pumps: Varian PrepStar Model 218 gradient system, detector: ProStar Model 320, fraction collector: Varian Model 701) using a C ₈ column (Nucleodur, 5 μm, 100 A ₁ 32 x 250 mm, Macherey-Nagel, Dϋren, Germany) and a linear gradient of acetonitrile containing 0.1 % TFA at a flow rate of 20 mL/min. All inhibitors were finally obtained as TFA-salts after lyophilization. The molecular mass of the synthesized compounds was determined using a QTrap 2000 ESI spectrometer (Applied Biosystems).

SEQ ID NO:1 Matriptase amino acid sequence (UniProt accession Q9Y5Y6):

MGSDRARKGGGGPKDFGAGLKYNSRHEKWGLEEGVEFLPVNNVKKVEKHGPGRWWLA A VLIGLLLVLLGIGFLVWHLQYRDVRVQKVFNGYMRITNENFVDAYENSNSTEFVSLASKV KDALKLLYSGVPFLGPYHKESAVTAFSEGSVIAYYWSEFSIPQHLVEEAERVMAEERVVM LPPRARSLKSFWTSWAFPTDSKTVQRTQDNSCSFGLHARGVELMRFTTPGFPDSPYPA HARCQWALRGDADSVLSLTFRSFDLASCDERGSDLVTVYNTLSPMEPHALVQLCGTYPPS YNLTFHSSQNVLLITLITNTERRHPGFEATFFQLPRMSSCGGRLRKAQGTFNSPYYPGHY PPNIDCTWNIEVPNNQHVKVRFKFFYLLEPGVPAGTCPKDYVEINGEKYCGERSQFWTS NSNKITVRFHSDQSYTDTGFLAEYLSYDSSDPCPGQFTCRTGRCIRKELRCDGWADCTDH SDELNCSCDAGHQFTCKNKFCKPLFWVCDSVNDCGDNSDEQGCSCPAQTFRCSNGKCLSK SQQCNGKDDCGDGSDEASCPKVNVVTCTKHTYRCLNGLCLSKGNPECDGKEDCSDGSDEK DCDCGLRSFTRQARWGGTDADEGEWPWQVSLHALGQGHICGASLISPNWLVSAAHCYID DRGFRYSDPTQWTAFLGLHDQSQRSAPGVQERRLKRIISHPFFNDFTFDYDIALLELEKP AEYSSMVRPICLPDASHVFPAGKAIWVTGWGHTQYGGTGALILQKGEIRVINQTTCENLL PQQITPRMMCVGFLSGGVDSCQGDSGGPLSSVEADGRIFQAGWSWGDGCAQRNKPGVYT RLPLFRDWIKENTGV