WO2020000063A1

WO2020000063A1 - Rheumatoid arthritis treatment

Info

Publication number: WO2020000063A1
Application number: PCT/AU2019/050695
Authority: WO
Inventors: Christopher John Jackson; Meilang Xue
Original assignee: Novapep Pty Ltd
Priority date: 2018-06-28
Filing date: 2019-06-28
Publication date: 2020-01-02

Abstract

The invention relates to a method of minimising a symptom of rheumatoid arthritis and other inflammatory joint disease using a TR47 related peptide, wherein the TR47 related peptide generally refers to a peptide having an N-terminal sequence NH₂-NPND or NH₂-NPNDKY, which is derived from the N-terminal sequence of the Met1-Arg46 deleted human PAR1 (protease activated receptor 1) sequence.

Description

Rheumatoid arthritis treatment

Field of the invention

The invention relates to rheumatoid arthritis (RA) and to methods for treatment of RA.

Background of the invention

Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

PAR-1 has been considered as a potential target for development of inhibitors of inflammation. This is because in some inflammatory conditions, PAR-1 is activated resulting in the generation of pro-inflammatory signals.

The mechanism of action is believed to be that thrombin cleaves PAR-1 to generate an S⁴²FLLRN N -terminus which ostensibly acts as a tethered ligand for binding to loop 2 and likely other portions of PAR-1. The binding of the tethered ligand to loop 2 leads to PAR-1 conformation changes, rapid internalisation to the cytosol and concomitant generation of pro-inflammatory signals [Coughlin SR and Camerer E 2003 J. Clin. Investig 111 :25-27; Ramachandran R. et al. 2012 Nature Reviews Drug Discovery 11 : 69:86.

One outcome of the structural changes to PAR-1 arising from protease cleavage of PAR-1 (especially by thrombin) is that PAR-1 activation is irreversible [Trejo, J. 2003 J. Pharmacol and Exper Therap 307:437-4421.

A range of compounds have been proposed to inhibit inflammation by antagonising the development of PAR-1 related pro-inflammatory signals. One approach has been to block signal development by blocking the extracellular domains for PAR-1 , for example, using thrombostatins and modified bradykinin derived blocking peptides [Derian CK et al. 2003 Expert Opin Investig Drugs 12:209-2211. Another approach has been to develop monoclonal antibodies against the cleavage site of PAR 1 to block cleavage and activation of PAR-1 [O’Brien PJ et al. 2001 Oncogene 20:1570-15811.

In another approach, small molecule PAR-1 antagonists have been generated based on the structure of the tethered ligand for PAR-1. These small molecule antagonists function by blocking interaction of the tethered ligand with binding sites on the extracellular face of the receptor but do not inhibit thrombin binding or receptor cleavage [Hollenberg MD and Compton SJ 2002 Pharmacol Rev 54:203-2171.

PAR-1 activation may also lead to the transmission of anti-inflammatory signals. It has been shown that this activation can involve cleavage of PAR-1 by APC to form a PAR-1 receptor having an N-terminus of N⁴⁷PNDKY [Mosnier LO et al. 2012 Blood 120:5237-52461. The substrate for the reaction for the production of N-terminal N⁴⁷PNDKY is non-cleaved PAR-1. In particular, PAR-1 having the N-terminus of N⁴⁷PNDKY is not generated if PAR-1 has been prior cleaved by another enzyme such as thrombin [Mosnier LO et al. 2012 Blood 120:5237-52461.

In studies performed in vitro in artificial systems using endothelial cells, thrombin has a much higher kinetic efficiency for cleavage of PAR-1 than does APC [Ludeman MJ. Et al. 2005 J. Biol.Chem 280:13122-81. The extension of this is that where thrombin is in a higher relative abundance, PAR-1 is irreversibly activated for transmission of pro- inflammatory signals.

It has been reported that a peptide having a PAR-1 sequence with an N-terminus of N⁴⁷PNDKY may have anti-inflammatory effects in neural cells in in vitro conditions [Gorbacheva LR et al. 2017 Biochemistry (Moscow) 82:778-7901. As discussed, these peptides are unlikely to have anti-inflammatory effects on PAR-1 receptors that have been irreversibly activated by thrombin.

To summarise the above, PAR-1 is considered as a potential therapeutic target for treatment of some inflammatory disorders, either to minimise pro-inflammatory signals or to maximise anti-inflammatory signals, and a number of different approaches dependent of the structure/function relationships of the variously activated forms of PAR-1 are under consideration for exploring this potential. Rheumatoid arthritis (RA) is a chronic inflammatory disorder having both immunological and inflammatory components. The synovial milieu seen in RA is complex and distinctive from other inflammatory disease in terms of contributing cell type, cytokine, and genetic and epigenetic factors [Anqelotti F et al. 2017 Clin And Experimen Rheumatol 35:368-3781.

It has been established for some time that thrombin is a major factor in the pathogenesis of RA, for example through the observation that hirudin (a thrombin inhibitor) reduces joint inflammation associated with murine antigen induced arthritis by fibrin and fibrin independent mechanisms, the latter including PAR-1 activation [Varisco PA et al. 2000 Ann Rheum Pis 59:781 -7871. Thrombin is present in abnormally excessive amounts in the synovia of RA patients.

A peptide that was understood to be an agonist for development of PAR-1 pro- inflammatory signals, (because it has the S⁴²FLLRN N -terminus) was shown to result in a decrease in various inflammatory cytokines (TNF, IL-17, IL-6) through increase in anti- inflammatory MMP-2 expression [Xue M et al. 2012 Arthritis & Rheumatism 64:88-981.

APC has been found to minimise the level of pro-inflammatory MMP-9 to upregulate the expression of anti-inflammatory MMP-2 in rheumatoid synovial cells and monocytes, acting through its natural receptor, the EPCR [Xue M et al. 2007 Arthritis & Rheumatism 56: 2864-28741.

There is a need for new methods and compositions for minimisation of joint inflammation, including inflammation associated with RA.

Summary of the invention

In one embodiment there is provided a method for minimising a symptom of rheumatoid arthritis in an individual having joint disease including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising a symptom of rheumatoid arthritis in the individual.

In another embodiment there is provided a method for minimising joint pain, swelling, or stiffness in an individual having joint disease including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising joint pain, swelling or stiffness in the individual.

In another embodiment there is provided a method for minimising the degradation of bone or cartilage in an individual having joint disease including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising the degradation of bone or cartilage in the individual.

In the above described embodiments, it is preferred that the joint disease is inflammatory joint disease, more preferably rheumatoid arthritis.

In the above described embodiments, the joint disease is preferably associated with expression or production of a serine protease, preferably thrombin, preferably over production or over expression of thrombin. Preferably the joint comprises thrombin activity arising from over expression or over production of thrombin at the time of administration of the TR47 related peptide.

In another embodiment there is provided a method for minimising a symptom of rheumatoid arthritis in an individual having joint disease including administering to the individual a therapeutically effective amount of:

- a TR47 related peptide and

- an anti-TNF antibody or anti IL-17 antibody or anti IL-23 antibody for minimising inflammation,

thereby minimising a symptom of rheumatoid arthritis in the individual.

In the above described embodiments, the TR47 related peptide may be administered by intra-articular injection.

In one embodiment there is provided a method for minimising the production or expression of a molecule selected from the group consisting of TNFa, IL-1 , IL-6, IL-17 and IL-23, by a cell, preferably a synovial cell including the step of contacting a synovial cell with a TR47-related peptide, thereby minimising the production or expression of TNFa, IL-1 , IL-6, IL-17 or IL-23 by a synovial cell. In another embodiment there is provided a method for increasing the production or expression of MMP-2 by a cell, preferably a synovial cell including the step of contacting a synovial cell with a TR47-related peptide, thereby increasing the production or expression of MMP-2 by a synovial cell.

In another embodiment there is provided a method for inducing phosphorylation of Akt Ser⁴⁷³ in a cell, preferably a synovial cell including the step of contacting a synovial cell with a TR47-related peptide, thereby inducing phosphorylation of Akt (Protein Kinase B) Ser⁴⁷³ by a synovial cell.

A joint disorder or rheumatoid arthritis may have, at the time of administration of the TR47 related peptide, an abnormal amount of thrombin activity, and in particular, an amount of thrombin activity not observed in a joint not having rheumatoid arthritis or related inflammation. Thrombin activity may be determined by the skilled worker. See for example Varisco PA et al. supra. One outcome of the thrombin activity may be the cleavage of PAR-1 receptors on cells located in the region of inflammation by thrombin, thus producing the pro-inflammatory S⁴²FLLRN N terminus and induction of inflammation in, or by these cells. The production of the S⁴²FLLRN N terminus can be determined by methods known to the skilled worker. See Coughlin and Camerer supra; Ramachandran et al. supra. A TR47 related peptide may be administered to a joint in these circumstances and enable generation of anti-inflammatory signals. An anti- inflammatory signal may be identified by assessing for anti-inflammatory signalling through the Akt signalling pathway, on the basis of phosphorylation of Akt, for example at Ser⁴⁷³. An increase in Akt phosphorylation generally indicates the formation of an anti-inflammatory response. Akt phosphorylation may be assessed by methods known to the skilled worker, and as exemplified herein. Thus in another embodiment there is provided a method for treatment of an individual for a joint disorder or rheumatoid athritis, the disorder or RA having an overproduction or overexpression of thrombin, the overproduction or overexpression of thrombin providing for, or enabling cleavage of PAR-1 receptors of cells in the joint having the disorder or rheumatoid arthritis, the method including the step of: administering a TR47 related peptide, preferably a TR47 related peptide consisting of SEQ ID No: 17, preferably by intra-articular injection to provide for, or to enable phosphorylation of Akt, preferably phosphorylation of Akt Ser⁴⁷³ of a cell, or to provide for, or to enable anti-inflammatory signalling through the Akt signalling pathway in a cell, the cell located in the joint, thereby treating the individual for the joint disorder or rheumatoid arthritis.

In another embodiment there is provided a method for minimising the proliferation of a cell preferably a synovial cell including the step of contacting a synovial cell with a TR47-related peptide, thereby minimising the proliferation of a synovial cell.

In the above described embodiments, the synovial cell may be a synovial fibroblast, preferably a hyperplastic synovial fibroblast, or a synovial monocyte or synovial macrophage.

In the above described embodiments, the TR47 related peptide may be contacted with the cell, preferably a synovial cell, in the presence of a serine protease, preferably thrombin.

In a further embodiment there is provided an injectable composition formulated for intra articular injection including a therapeutically effective amount of a TR47 related peptide and a pharmaceutically acceptable diluent, solvent or excipient for enabling intra articular injection of the composition.

Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Brief description of the drawings

Figure 1 : Amino acid sequence of PAR 1 (SEQ ID NO: 1 )

Figure 2: Amino acid sequence of 10 mer TR47 related peptide (SEQ ID NO: 2)

Figure 3: Amino acid sequence of 20 mer TR47 related peptide (SEQ ID NO: 3)

Figure 4: Amino acid sequence of 30 mer TR47 related peptide (SEQ ID NO: 4)

Figure 5: Tetra-branched TR47 related peptide (SEQ ID NO: 5)

Figure 6: Amino acid sequence of TRAP peptide (SEQ ID NO: 6)

Figure 7: Amino acid sequence of Akt (SEQ ID NO: 7) Figure 8: A) Relative p-AKT activity in presence of PAR1 peptide and thrombin. B)

Relative p-AKT activity in the presence PAR1 peptide following thrombin treatment, and in presence of PAR1 peptide prior to thrombin treatment.

Figure 9: The therapeutic effects of PAR1 12mer in antigen-induced arthritis (AIA) model. Animals were treated with different peptides via IP and intra-articular injection three times a week for 3 weeks after intra-articular BSA injection and terminated at day 28. PAR1 =TR 47 12mer; Control= 20mg/kg PAR1 scrambled control (SC). When compared to both Control (scrambled 20mg + 20 mg) anti-TNF treatment, TR47 12mer (PAR1 ) peptide at 10 mg/kg significantly reduced the severity of AIA.

Detailed description of the embodiments

The invention generally relates to improvements in management of joint disease arising from administration of TR-47 related peptide. It is believed that those improvements may be observed at the cellular level including minimisation of production or expression of inflammatory mediators, and at a clinical level in terms of minimisation of key symptoms of joint disease including joint pain, swelling and joint stiffness.

The invention is applicable to joint disease generally including non-inflammatory (such as osteoarthritis) and inflammatory joint disease where there are common clinical manifestations of joint dysfunction. Those symptoms or manifestations are generally joint pain, stiffness or swelling, which may present either separately or in combination.

In preferred embodiments, the invention is applicable to joint disease in the form of inflammatory joint disease, examples of which include RA, ankylosing spondylitis, psoriatic arthritis and disorders arising from conditions such as gout. A common feature of these disorders and diseases is the role of the synovial fibroblast. This is understood to be a key source of inflammatory mediators such as TNF-a, IL-1 and IL-6. It is believed that the anti-inflammatory effects concomitant with the application of TR47 related peptides according to the invention arise from modification of the amount of expression or production of these anti-inflammatory mediators.

While not wanting to be bound by hypothesis, it is believed that an anti- inflammatory signal is provided in inflammatory synovial fibroblast cells by TR47 related peptide through contact of the TR47 related peptide with synovial PAR-1 receptors. It is a surprising finding of the invention that such a signal can be generated by TR47 related peptides where it had been understood that synovial PAR-1 receptors are prior cleaved by thrombin, and therefore irreversibly activated for formation of a pro-inflammatory signal in synovial cells. In contrast it is believed that the invention has shown that synovial fibroblast PAR-1 receptors whether prior activated by thrombin or not, can be activated for formation of an anti-inflammatory signal by TR47 related peptides.

“Rheumatoid arthritis " is a commonly known inflammatory disease of the joint, generally having immune and inflammatory components and involving synovial fibroblasts as a key inflammatory mediator. There are four distinct stages of rheumatoid arthritis progression each with their own treatment courses.

“Stage rheumatoid arthritis or“stage 1 RA " generally refers to the initial stage of disease development and involves initial inflammation in the joint capsule and swelling of synovial tissue. This induces the clear symptoms of joint pain, swelling, and stiffness.

“Stage 2” rheumatoid arthritis or“stage 2 RA" generally refers to the moderate stage of rheumatoid arthritis, wherein the inflammation of synovial tissue becomes severe enough that it causes cartilage damage. In this stage, symptoms of loss of mobility and range of motion become more frequent.

“Stage 3” rheumatoid arthritis or “stage 3 RA" generally refers to severe rheumatoid arthritis. Inflammation in the synovium is now destroying not only the cartilage of the joint but the bone as well. Potential symptoms of this stage include increased pain and swelling and a further decrease in mobility and even muscle strength. Physical deformities on the joint may start to develop as well.

“Stage 4" rheumatoid arthritis or“stage 4 RA" generally refers to the end stage of rheumatoid arthritis where the inflammatory process ceases and joints stop functioning altogether. Pain, swelling, stiffness and loss of mobility are still the primary symptoms in this stage.

RA is a disease that may display, from stage to stage, all of the classical hallmarks of inflammation including swelling, redness, heat, pain and loss of function. A“symptom of RA” generally refers to swelling, redness, heat, pain or loss of function.

“Joint disease” generally refers to a condition or pathology of a joint, particularly a joint having an articular surface. A joint disease may present with one or more symptoms of inflammation, although the pathogenesis may not be based on inflammation. Osteoarthritis is one example of a non-inflammatory joint disease.

An“inflammatory joint disease” generally refers to a condition or pathology of a joint, where the pathogenesis of the disease arises significantly from inflammation. RA, ankylosing spondylitis and psoriatic arthritis are examples of inflammatory joint disease.

“Minimising a symptom” generally refers to at least reducing symptom severity, for example reducing swelling, stiffness or pain. It does not necessarily mean ablating a symptom.

A“TR47-related peptide " generally refers to a peptide having an N terminal sequence NH₂ - NPND or NH₂ - NPNDKY.

“Akt generally refers to a polypeptide having a sequence shown in SEQ ID No:

7.

A“therapeutically effective amount’ generally refers to an amount of TR 47 related peptide that is effective for minimising one or more symptoms of RA, or for minimising the production or expression of inflammatory mediators from a synovial cell.

" Comprise " and variations of the term, such as " comprising ", " comprises " and " comprised ', are not intended to exclude further additives, components, integers or steps unless the context requires otherwise.

In another embodiment there is provided a method for minimising joint pain in an individual having joint disease including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising joint pain in the individual.

In another embodiment there is provided a method for minimising joint swelling in an individual having joint disease including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising joint swelling in the individual.

In another embodiment there is provided a method for minimising joint stiffness in an individual having joint disease including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising joint swelling in the individual.

In another embodiment there is provided a method for minimising a symptom of inflammatory joint disease, preferably RA, including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising a symptom of inflammatory joint disease or RA in the individual.

Where the individual requiring treatment has RA, the TR47 related peptide may be given before commencement of, or during any one of the clinically recognised stages of RA so as to at least minimise a symptom of the relevant stage. For example, the TR47 related peptide may be administered to at least minimise swelling (i.e. to reduce swelling below that which might obtain where TR47 related peptide is not administered) whether the individual is at stage 1 , 2, 3 or 4 of RA. The TR47 related peptide may be administered to a joint with RA that contains an increased or abnormal amount of serine protease activity, preferably thrombin activity. In a particularly preferred embodiment, the TR47 related peptide is given at an early stage of RA, preferably prior to or during stage 1 to reduce joint swelling, joint stiffness and/or joint pain.

In certain embodiments, a TR47 related peptide may be administered with another therapeutic compound indicated for treatment of joint disease, especially for treatment of inflammatory joint disease. Examples include steroids and inflammatory cytokine inhibitors such as antibodies and other cytokine antagonists. Thus the invention further relates to a method for minimising a symptom of rheumatoid arthritis in an individual having joint disease including administering to the individual a therapeutically effective amount of:

- a TR47 related peptide and

- an anti-TNF antibody for minimising inflammation,

thereby minimising a symptom of rheumatoid arthritis in the individual.

As described further herein, the TR47 related peptide may be administered by intra-articular injection.

The invention further relates to utilising TR47 related peptides to minimise the production or expression of inflammatory mediators by cells of an articulated joint, in particular by synovial cells. These embodiments may also be useful in ex vivo applications for conditioning synovial cells so that they define an anti-inflammatory profile, or in in vitro embodiments for monitoring or determining minimisation of inflammation in synovial cells obtained from an articulated joint.

In accordance with the above, in one embodiment there is provided a method for minimising the production or expression of a molecule selected from the group consisting of TNFa, IL-1 and IL-6, by a synovial cell including the step of contacting a synovial cell with a TR47-related peptide, thereby minimising the production or expression of TNFa, IL-1 and IL-6 by a synovial cell.

In another embodiment there is provided a method for increasing the production or expression of MMP-2 by a synovial cell including the step of contacting a synovial cell with a TR47-related peptide, thereby increasing the production or expression of MMP-2 by a synovial cell. In another embodiment there is provided a method for inducing phosphorylation of Akt Ser⁴⁷³ in a synovial cell including the step of contacting a synovial cell with a TR47-related peptide, thereby inducing phosphorylation of Akt Ser⁴⁷³ by a synovial cell.

In another embodiment there is provided a TR47 related peptide, preferably a TR47 related peptide consisting of SEQ ID NO: 17 or composition comprising same for use in minimising or treating a joint disorder, preferably rheumatoid athritis having an overproduction or overexpression of thrombin providing cleavage of PAR-1 receptors, wherein the TR47 related peptide enables phosphorylation of Akt, preferably phosphorylation of Akt Ser⁴⁷³, by a cell in the region of the joint disorder.

In another embodiment there is provided a method for minimising the proliferation of a synovial cell including the step of contacting a synovial cell with a TR47-related peptide, thereby minimising the proliferation of a synovial cell.

In another embodiment there is provided a TR47 related peptide or composition comprising same for use in minimising a symptom of rheumatoid arthritis in an individual having joint disease, or for treating rheumatoid arthritis, or for minimising the progression of rheumatoid arthritis.

In another embodiment there is provided use of a TR47 related peptide or composition comprising same for the manufacture of a medicament for use in minimising a symptom of rheumatoid arthritis in an individual having joint disease, or for treating rheumatoid arthritis, or for minimising the progression of rheumatoid arthritis.

In another embodiment there is provided a use of a TR47 peptide or composition comprising same for minimising a symptom of rheumatoid arthritis in an individual having joint disease, or for treating rheumatoid arthritis, or for minimising the progression of rheumatoid arthritis.

The invention provides TR47 related peptides which are generally anti inflammatory to the extent that they antagonise the production, expression or action of inflammatory mediators. These peptides generally activate the PI3k-Akt signalling pathway, inhibit secretion of TNF-a by synovial cells, and reduce synovial cell NFKB activation.

Typically, a TR47 related peptide has an N terminal sequence of at least NPND, or an N terminal sequence that is homologous to NPND.

The TR47 peptide, including NPND, may contain a total of 4, 5, 6, 7, 8, 9, 10, 15, 20, 50, 100, 200, 300 or more amino acid residues in length. Some of the polypeptides comprise from about 4 amino acid residues to about 100 amino acid residues. Some of the polypeptides comprise from about 8 amino acid residues to about 50 amino acid residues.

In one embodiment, the TR47 related peptide has an amino acid sequence selected from the group consisting of: NPND (SEQ ID No. 11 ), NPNDK (SEQ ID No. 12), NPNDKY (SEQ ID No. 13), NPNDKYE (SEQ ID No. 14), NPNDKYEP (SEQ ID No. 15) and NPNDKYEPF (SEQ ID No. 16). In one embodiment, the TR47 related peptide has an amino acid sequence selected from the group consisting of NPNDX-Y wherein:

X-Y is a sequence from K⁵¹ of SEQ ID NO:1 to L⁶⁶ of SEQ ID NO: 1 ;

X-Y is a sequence from K⁵¹ of SEQ ID NO:1 to L⁹⁶ of SEQ ID NO:1 ;

X-Y is a sequence from K⁵¹ of SEQ ID NO:1 to T¹⁴⁶ of SEQ ID NO:1 ; X-Y is a sequence from K⁵¹ of SEQ ID NO:1 to V²⁴⁶ of SEQ ID NO:1 ; and

X-Y is a sequence from K⁵¹ of SEQ ID NO:1 to T³⁴⁶ of SEQ ID NO:1.

A few specific examples of the PAR 1 -derived cytoprotective polypeptides of the invention are shown in Table 1 :

In a preferred embodiment, the TR47 related peptide has the sequence of SEQ

ID No: 17 NPNDKYEPFWED utilised in Example 15 herein.

In some embodiments, a TR47 related peptide has at least 70%, preferably 80%, preferably 90%, preferably 95%, preferably 98%, 97%, 98%, or 99% identity to a peptide shown in Table 1 , provided that the peptide has the N terminal sequence NPND. Percent sequence identity may be determined by conventional methods, by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 53711 ) as disclosed in Needleman, S. B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453, which is hereby incorporated by reference in its entirety. GAP is used with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.

In some embodiments, the TR47 related peptides can contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids, for example, 4-bydroxyproline, 5-hydroxylysine, 3-methy!histidine, homoserine, ornithine or carboxyglutamate, and can include amino acids that are not linked by polypeptide bonds. Similarly, they can also be cyclic polypeptides and other conformationaily constrained structures. Methods for modifying a polypeptide to generate analogs and derivatives are well known in the art, e.g , Roberts and Vellaccio, The Peptides: Analysis Synthesis , Biology, Eds. Gross and Meinhofer, Vol. 5, p. 341 , Academic Press, Inc , New York, N.Y. (1983); and Burger’s Medicinal Chemistiy and Drug Discovery, Ed. Manfred E. Wolff, Ch. 15, pp. 819-820, John Wiley & Sons Inc., New York, N.Y (1995). Some other derivative compounds of the TR47 related peptides are peptidomimetics. Peptidomimeties based on TR47 related peptides substantially retain the activities of the TR47 related peptide. They include chemically modified polypeptides, polypeptide-!ike molecules containing non-naturally occurring amino acids, peptoids and the like, have a structure substantially the same as the reference polypeptides upon which the peptidomimetic is derived (see, for example, Burger's Medicinal Chemistry and Drug Discoveiy , 1995, supra). For example, the peptidomimetics can have one or more residues chemically derivatized by reaction of a functional side group. In addition to side group derivatizations, a chemical derivative can have one or more backbone modifications including alpha-amino substitutions such as N-methyi, N-ethyl, N-propyi and the like, and alpha-carbonyl substitutions such as thioester, thioamide, guanidino and the like. Typically, a peptidomimetic shows a considerable degree of structural identity when compared to the reference polypeptide and exhibits characteristics which are recognizable or known as being derived from related to the reference polypeptide. Peptidomimetics include, for example, organic structures which exhibit similar properties such as charge and charge spacing characteristics of the reference polypeptide. Peptidomimetics also can include constrained structures so as to maintain optimal spacing and charge interactions of the amino acid functional groups.

In some other embodiments, the TR47 related peptide can be dimerized or muitimerized by covalent attachment to at least one linker moiety. For example, the peptides or polypeptides can be conjugated with a C1-12 linking moiety optionally terminated with one or two— NH— 0 linkages and optionally substituted at one or more available carbon atoms with a lower alkyl substituent. The TR47 related peptide described herein can be joined by other chemical bond linkages, such as linkages by disulfide bonds or by chemical bridges. In some other embodiments, t TR47 related peptide can be linked physically in tandem to form a polymer of TR47 related peptides. The peptides making up such a polymer can be spaced apart from each other by a peptide linker. In some embodiments, molecular biology techniques well known in the art can be used to create a polymer of TR47 related peptides. In some embodiments, polyethylene glycol (PEG) may serve as a linker that dimerizes two peptide monomers. For example, a single PEG moiety containing two reactive functional groups may be simultaneously attached to the N-termini of both peptide chains of a peptide dimer. These peptides are referred to herein as“PEGy!ated peptides.” In some embodiments, the peptide monomers of the invention may be oligomerized using the biotin/streptavidin system.

In one embodiment, the TR47 related peptide comprises at least 2, preferably 3, preferably 4, preferably 5 or more TR47 related peptide sequences.

In one embodiment the TR47 related peptide is defined by the general formula:

A Li B 1.2 ~ C wherein each of A, B and C are C1-C12 alkyl; wherein each of A, B and C are substituted with one or more peptides having an amino acid sequence selected from the group consisting of SEQ ID No: 2 to 4, or 8 to 10; and wherein Li and L2 are each independently linker groups.

Preferably Li and/or L2 comprise an amide group.

In a particularly preferred embodiment, the TR47 related peptide for use in a method described herein has a structure shown below:

NPNDKYE PFWEBEEKN E SGL

WFPEYKDN PN Methods for stabilizing peptides known in the art may be used with the methods and compositions described herein. For example, using D-amino acids, using reduced amide bonds for the peptide backbone, and using non-peptide bonds to link the side chains, including, but not limited to, pyrrolinone and sugar mimetics can each provide stabilization. The design and synthesis of sugar scaffold peptide mimetics are described in the art, e.g., Hirschmann et al., J. Med. Chem. 36, 2441 -2448, 1998. Further, pyrroiinone-based peptide mimetics present the peptide pharmacophore on a stable background that has improved bioavailabiiity characteristics (see, e.g., Smith et al., J. Am. Chem. Soc. 122, 11037-11038, 2000).

In some embodiment, derivative compounds of the TR47 related peptides include modifications within the sequence, such as, modification by terminal-Nhh acylation, e.g., acetylation, or thioglycolic acid amidation, by terminal-carboxylamidation, e.g., with ammonia, methyiamine, and the like terminal modifications. One can also modify the amino and/or carboxy termini of the polypeptides described herein. Terminal modifications are useful to reduce susceptibility by proteinase digestion, and therefore can serve to prolong half-life of the polypeptides in solution, particularly in biological fluids where proteases may be present. Amino terminus modifications include methyiation (e.g., — HCHs or — N(CHh)2), acetylation (e.g., with acetic acid or a halogenated derivative thereof such as a-chloroacetic acid, a-bromoacetic acid, or a- iodoacetic acid), adding a benzyioxycarbonyl (Cbz) group, or blocking the amino terminus with any blocking group containing a carboxyiate functionality defined by RCOO— or sulfonyi functionality defined by R— SO2— , where R is selected from the group consisting of alkyl, aryl, heteroaryl, alkyl aryl, and the like, and similar groups. One can also incorporate a desamino acid at the N-terminus (so that there is no N- terminal amino group) to decrease susceptibility to proteases or to restrict the conformation of the peptide compound. In some embodiments, the N-terminus is acetylated with acetic acid or acetic anhydride.

Carboxy terminus modifications include replacing the free acid with a carboxamide group or forming a cyclic lactam at the carboxy terminus to introduce structural constraints. One can also cyclize the peptides described herein, or incorporate a desamino or descarboxy residue at the termini of the peptide, so that there is no terminal amino or carboxyl group, to decrease susceptibility to proteases or to restrict the conformation of the peptide. Methods of circuiar peptide synthesis are known in the art, for example, in U S. Patent Application No. 20090035814; and Muralidharan and Muir, Nat. Methods, 3:429-38, 2008. C-terminai functional groups of the peptides described herein include amide, amide lower alkyl, amide diflower alkyl), lower a!koxy, hydroxy, and carboxy, and the lower ester derivatives thereof, and the pharmaceutically acceptable salts thereof.

The TR47 related polypeptides described herein, including variants and derivatives thereof, can be chemically synthesized and purified by standard chemical or biochemical methods that are well known in the art. Some of the methods for generating analog or derivative compounds of the TR47 related polypeptides are described above. Other methods that may be employed for producing the TR47 related polypeptides and their derivative compounds, e.g., solid phase peptide synthesis, are discussed below. For example, the peptides can be synthesized using t-Boc (tert-butyloxycarbonyl) or FMOC (9-flourenylmethloxycarbonyl) protection group described in the art. See, e.g., “Peptide synthesis and applications” in Methods in molecular biology Vol. 298, Ed. by John Howl;“Chemistry of Peptide Synthesis” by N. Leo Benoiton, 2005, CRC Press, (ISBN-13: 978-1574444544); and“Chemical Approaches to the Synthesis of Peptides and Proteins” by P. Lioyd-Wiiliams, et. al., 1997, CRC~Press, (ISBN-13: 978- 0849391422), Methods in Enzymo!ogy, Volume 289: Solid-Phase Peptide Synthesis, J. N. Abelson, M. I. Simon, G. B. Fields (Editors), Academic Press; 1 st edition (1997) (ISBN-13: 978-0121821906); U S. Pat. Nos. 4,965,343, and 5,849,954.

Solid phase peptide synthesis, developed by R. B. Merrifield, 1983, J. Am. Chem. Soc. 85 (14): 2149-2154, was a major breakthrough allowing for the chemical synthesis of peptides and small proteins. An insoluble polymer support (resin) is used to anchor the peptide chain as each additional alpha-amino acid is attached. This polymer support is constructed of 20-50 pm diameter particles which are chemically inert to the reagents and solvents used in solid phase peptide synthesis. These particles swell extensively in solvents, which makes the linker arms more accessible. Organic linkers attached to the polymer support activate the resin sites and strengthen the bond between the alpha- amino acid and the polymer support. Chioromethyi linkers, which were developed first, have been found to be unsatisfactory for longer peptides due to a decrease in step yields. The PAM (phenylacetamidomethyl) resin, because of the electron withdrawing power of the acid amide group on the phenylene ring, provides a much more stable bond than the classical resin. Another alternative resin for peptides under typical peptide synthesis conditions is the Wang resin. This resin is generally used with the FMGC labile protecting group.

A labile group protects the alpha-amino group of the amino acid. This group is easily removed after each coupling reaction so that the next alpha-amino protected amino acid may be added. Typical labile protecting groups include t-Boc (tert- buty!oxycarbonyl) and FIV!OC. t-Boc is a very satisfactory labile group which is stable at room temperature and easily removed with dilute solutions of trifluoroacetic acid (TFA) and dich!oromethane. FMOC is a base labile protecting group which is easily removed by concentrated solutions of amines (usually 20-55% piperidine in N-methylpyrrolidone). When using FMOC alpha-amino acids, an acid labile (or base stable) resin, such as an ether resin, is desired.

The stable blocking group protects the reactive functional group of an amino acid and prevents formation of complicated secondary chains. This blocking group must remain attached throughout the synthesis and may be removed after completion of synthesis. When choosing a stable blocking group, the labile protecting group and the cleavage procedure to be used should be considered. After generation of the resin bound synthetic peptide, the stable blocking groups are removed and the peptide is cleaved from the resin to produce a “free” peptide. In general, the stable blocking groups and organic linkers are labile to strong acids such as TFA. After the peptide is cleaved from the resin, the resin is washed away and the peptide is extracted with ether to remove unwanted materials such as the scavengers used in the cleavage reaction. The peptide is then frozen and lyophilized to produce the solid peptide. This is generally then characterized by HPLC and MALDI before being used. In addition, the peptide should be purified by HPLC to higher purity before use.

Commercial peptide synthesizing machines are available for solid phase peptide synthesis. For example, the Advanced Chemtech Model 398 Multiple Peptide Synthesizer and an Applied Biosystems Model 432A Peptide synthesizer are suitable. There are commercial companies that make custom synthetic peptides to order, e.g., Abbiotec, Abgent, AnaSpec Global Peptide Services, LLC., Invitrogen, and rPeptide, LLC.

The TR47 related polypeptides and derivatives thereof can also be synthesized and purified by molecular methods that are well known in the art. Recombinant polypeptides may be expressed in bacteria, mammal, insect, yeast, or plant cells.

Cell-free expression systems can also be used for producing TR47 related polypeptides of the invention. Cell-free expression systems offer several advantages over traditional cell-based expression methods, including the easy modification of reaction conditions to favor protein folding, decreased sensitivity to product toxicity and suitability for high-throughput strategies such as rapid expression screening or large amount protein production because of reduced reaction volumes and process time. The cell-free expression system can use plasmid or linear DMA. Moreover, improvements in translation efficiency have resulted in yields that exceed a milligram of protein per milliliter of reaction mix. An example of a cell-free translation system capable of producing proteins in high yield is described by Spirin et. al. , Science 242:1162, 1988. The method uses a continuous flow design of the feeding buffer which contains amino acids, adenosine triphosphate (ATP), and guanosine triphosphate (GTP) throughout the reaction mixture and a continuous removal of the translated polypeptide product. The system uses E coll lysate to provide the cell-free continuous feeding buffer. This continuous flow system is compatible with both prokaryotic and eukaryotic expression vectors. An example of large scale cell-free protein production is described in Chang et. al., Science 310:1950-3, 2005.

Other commercially available cell-free expression systems include the Expressway™ Cell-Free Expression Systems (invitrogen) which utilize an E. coil- based in-vitro system for efficient, coupled transcription and translation reactions to produce up to milligram quantities of active recombinant protein in a tube reaction format; the Rapid Translation System (RTS) (Roche Applied Science) which also uses an E coli- based in- vitro system; and the TNT Coupled Reticulocyte Lysate Systems (Promega) which uses a rabbit reticulocyte-based in-vitro system.

Pharmaceutical compositions of the invention can be prepared in accordance with methods well known and routinely practiced in the art. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20^th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions are preferably manufactured under GMP conditions.

A pharmaceutical composition of the invention may be formulated to enable administration by any known route. By way of example, the composition may be administered to enable administration by a mucosal, pulmonary, optical or other localized or systemic route (e.g., enteral and parenteral).

An injectable formulation of a TR47 related peptide may be supplied as a sterile, lyophilized powder for intra articular injection including TR47 related peptide, sucrose, NaCI and sodium citrate. The vials may be reconstituted with sterile water for injection, USP, to give a concentration of about 100 mg/ml TR47 related peptide and this diluted TR47 related peptide may then be added to 0.9% Sodium Chloride Injection to give a concentration of from about 0.1 to about 50000 pg/ml, preferably about 1 to about 1000 pg/ml, preferably about 1 to about 100 pg/ml, TR-47 for administration to a patient. This is a particular preferred formulation for administration of TR47 by intra articular injection.

In certain embodiments, a pharmaceutical composition may include, in addition to a TR47 related peptide, one or more other agents for treatment of a joint disease, especially an anti-inflammatory agent such as a steroid, or anti-inflammatory cytokine or anti-inflammatory antibody, such as an anti TNF antibody. In some embodiments, the one or more other agents for treatment of a joint disease may be provided separately to the composition including the TR47 related peptide.

A therapeutically effective amount of TR47 - related peptide for bolus administration, especially for intra-articular injection can typically be 2 mg/kg or less, 1 mg/kg or less, 0.5 mg/kg or less, 0.04 mg/kg or less, 0.03 mg/kg or less, 0.02 mg/kg or less, 0.01 mg/kg or less, or 0.005 mg/kg or less. Typically, the therapeutic amount may be based on titering to a synovial fluid level amount of about 0.01 pg/ml to about 1.6 pg/ml, preferably from about 0.01 pg/ml to about 0.5 pg/ml. It is also within the skill of the art to start doses at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. It is likewise within the skill of the art to determine optimal concentrations of variants to achieve the desired effects in the in vitro and ex vivo preparations of the invention. Depending on initial assay results, optimal concentrations can be in the range of, e.g., about 1 -1 ,000 nM or about 1 -200 mM depending on the general nature of the compound. In one embodiment, a therapeutically effective amount of TR47 related peptide may be 0.1 -100 mg/kg.

The present disclosure includes methods of administering in vivo or ex vivo a TR47-related peptide described above (or compositions comprising a pharmaceutically acceptable excipient and one or more such peptides) to a subject, including, e.g. , a mammal, including a human.

According to the invention, in in vivo methods, one or more cells or a population of cells of interest of the subject are contacted directly or indirectly with an amount of a TR47 related peptide effective in prophylactically or therapeutically treating the disease, disorder, or other condition. In direct contact/administration formats, the TR47 related peptide is typically administered or transferred directly to the cells to be treated or to the tissue site of interest by any of a variety of formats, including topical administration, injection (e.g., by using a needle or syringe). The TR47 related peptide can be delivered, for example, via intra- articular, sub-cutaneous, parenteral, or intravenous delivery, or placed within a cavity of the body (including, e.g., during surgery).

In in vivo indirect contact/administration formats, the selected TR47 related peptide is typically administered or transferred indirectly to the cells to be treated or to the tissue site of interest, including those described above, by contacting or administering the peptide directly to one or more cells or population of cells from which treatment can be facilitated. This may involve for example administration to a site that is distant from the site where treatment is required. In these embodiments the treatment may involve systemic delivery.

Preferably administration is given by bolus or by continuous infusion. Bolus refers to administration of a drug (e.g., by injection) in a defined quantity (called a bolus) over a period of time. Continuous infusion refers to continuing substantially uninterrupted the introduction of a solution into a blood vessel for a specified period of time. The pharmaceutical compositions may be added to the culture medium. In addition to active compound, such compositions may contain pharmaceutically acceptable carriers and other ingredients known to facilitate administration and/or enhance uptake.

In some aspects, in ex vivo methods, one or more cells or a population of cells of interest of the subject (e.g. , a chondrocyte) are obtained or removed from the subject and contacted with an amount of a TR47 related peptide that is effective in prophylactically or therapeutically treating the disease, disorder, or other condition. The contacted cells are then returned or delivered to the subject to the site from which they were obtained or to another site of interest in the subject to be treated. If desired, the contacted cells can be grafted onto a tissue, organ, or system site of interest in the subject using standard and well-known grafting techniques or, e.g. delivered to the blood or lymph system using standard delivery or transfusion techniques.

In each of the in vivo and ex vivo treatment methods above, a composition comprising an excipient and the TR47 related peptide can be administered or delivered. In one aspect, a composition comprising a pharmaceutically acceptable excipient and a polypeptide is administered or delivered to the subject as described above in an amount effective to treat a joint disorder or disease.

Disease activity can be assessed overall by clinical judgement of a rheumatologist, and by patient questionnaires (a simple version is the Visual Analog Scale (VAS); by joint counts (eg DAS28, which generates a“disease activity score” based on an examination of 28 joints as well as other factors); by laboratory testing including erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) and by imaging techniques including X-ray, MRI, CT or bone scan. Plasma or serum biomarkers of inflammation, such as IL-1 beta, IL-6, may also be measured.

It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention. Examples

Example 1 - Peptides

Peptides used in this study are synthesised according to standard techniques to >95% purity.

Example 2 - SF culture

Human RA synovial tissues are obtained during joint replacement surgery from patients with RA according to the American College of Rheumatology criteria from male and female patients of 60 to 70 years age. Ethical approval and the written consent of every patient is obtained. RSFs are compared with low metabolic dermal fibroblasts, as previously described.

RSFs, human dermal fibroblasts (HDFs) and mouse dermal fibroblasts (MDFs) are obtained from RA synovium, dermis of neonatal foreskin and C57BL/6J mouse skin, respectively, by enzymatic digestions. Briefly, tissues are minced into small pieces and digested with 2 mg/mL collagenase (Sigma-Aldrich, St. Louis, MO, USA) in Dulbecco’s modified Eagle medium (DMEM) containing 100 U/mL penicillin, 100 pg/mL streptomycin, 10 mmol/L HEPES (all from Gibco BRL/Life Technologies, Carlsbad, CA, USA) and 3.7 g/L NaHC03 at 37°C for 3 h, followed by digestion with 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) at 37°C for 30 min. The cells are cultured in DMEM containing 10% fetal bovine serum (FBS) (ICN, Aurora, OH, USA) in 75-cm2 flasks at 37°C in a humidified 5% C02 atmosphere. RSFs are used at passages 1-4 (one passage per experiment) (26). Cultured RSFs abundantly express the fibroblast specific marker cadherin-11 (27) and express barely detectable levels of the myeloid cell marker CD11 b between passages 1 and 4. Cells are plated onto 24- well plates (Corning, Corning, NY, USA) in DMEM containing 10% FBS. To avoid serum effects (24), confluent cells are pre-incubated in DMEM without FBS (basal medium) for 24 h and then used for experiments with or without recombinant TNFa (Pepro Tech, London, UK), using fresh basal media. Example 3 - Proliferation assay

Cell proliferation is performed as described previously (17) with modification. Briefly, (1 ^c 10⁴ cells/well) confluent cells from 75-cm² flasks are seeded into a 96-well microplate (Corning) to a final volume of 200 pL and incubated for 4 h to allow cells to attach and then pre-incubated in basal media for a further 12 h. Cells are then treated with test agents in serum-free conditions by using fresh basal media. To avoid fibroblast contact inhibition (26), after incubation for 24 h (29,30), culture medium is removed and cells are stained with 1 pg/mL crystal violet (Sigma-Aldrich) dissolved in phosphate- buffered saline (PBS). The unbound dye is removed by washing with tap water, and cells are left to completely dry overnight. Bound crystal violet is solubilized with 0.1 % sodium dodecyl sulfate in PBS. The optical density of each well is determined at a wavelength of 550 nm. Results are expressed as relative to control. We expect the peptides to dose-dependently inhibit synovial fibroblast proliferation.

Example 4 - Cell morphology and death assay

Passage 4 RSFs are seeded into a 24- well plate (Corning) at 1 ^c 10⁵ cells/well to a final volume of 250 pL and incubated for 24 h to allow cells to attach and are then preincubated in basal media for a further 12 h. At 90% confluency, cells are treated with or without peptides at varying concentrations of 10-200 uM in fresh basal media for 24 h. Micrographs are taken by an inverted phase contrast microscope (Nikon Eclipse TE2000-U) fitted with a DS-Fi1 digital camera and DS-U2 camera control unit (Nikon, Tokyo, Japan). RSFs in culture with or without peptide treatment appear elongated by phase light microscopy, sometimes oval or polygonal, with a few branched cytoplasmic processes. Cell death is measured by adding 10 pL 0.5% trypan blue (Sigma-Aldrich) in PBS to 100 pL cell supernatant, and cell viability is assessed by the trypan blue exclusion method on a hemocytometer and may be corrected for the total volume of media in the well. We expect the peptides to dose-dependently stimulate synovial fibroblast death.

Example 5 - Quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR)

Total RNA is isolated from RSFs of passage 1 and passage 4 by using RNAzol (Molecular Research Centre, Cincinnati, OH, USA) according to the manufacturer’s instructions (http:// www.mrcgene.com/rnazol.htm). RNA concentration is determined by NanoDrop spectrophotometry (Thermo Scientific; Scoresby, Australia) and reverse- transcribed into complementary DNA (cDNA) using the cDNA synthesis kit (Bioline; Taunton, MA, USA). Subsequently, qRT-PCR is performed with the Rotor-Gene 6000 Real-Time PCR machine (Corbett Life Science, Mortlake, Australia) by using ImmoMix (Bioline; Taunton) and SYBR Green dye (Qiagen, Hilden, Germany). The reaction mixture consists of 1 pg cDNA template, 0.75 pL each of forward and reverse primer, 12.5 pL ImmoMix and 2.5 pL SYBR Green. Cycling conditions comprise an initial activation step at 95°C for 10 min followed by 45 amplification cycles of 95°C for 15 s (denaturation), 58°C for 20 s (primer annealing) and 20°C for 45 s (extension). Specificity of the amplification reactions is verified by melting curve analysis.

Primers used in the assay are designed and checked for specificity by using the National Center for Biotechnology Information BLAST search tool (http://www.ncbi.nlm.nih.gov/tools/ primer-blast). Data are analysed using the standard curve for absolute quantification method.

Example 6 - Western blotting

Western Blotting is performed as described previously. The primary antibodies used are as follows against pan or phosphospecific forms of Akt, GSK3 , ERK1/2. Immunoreactivity is detected by using the ECL detection system (Amersham Biosciences, Buckinghamshire, UK). Anti-human b-actin (Sigma-Aldrich) antibody is included to normalize for unequal loading. Protein band intensity is evaluated by densitometry by using image analysis.

Example 7 - ELISA

Levels of IL-6, IL-1 and TNFa are measured using ELISA kits in accordance with manufacturer’s instructions.

Example 8 - Rac 1 activation

Briefly, SF cells (5 x 10⁶ per plate) are grown in 100-mm dishes for 48 hours and serum starved overnight before addition of peptides (50mM) for 30 or 180 minutes. Lysates (2 mg) are mixed with GST-PAK1 glutathione-agarose (150 pg) and after washing active GTP-Rad is eluted from GST-PAK1 glutathione-agarose by boiling in reducing SDS sample buffer. Active GTP-Rad is resolved on 12% SDSPAGE, transferred to PVDF membrane, and immunoblotted with a mouse anti-Rad antibody and anti-mouse secondary antibodies. Immunoblots are scanned and integrated fluorescence intensity units are quantified. We expect the peptides to dose-dependently stimulate RAC activation.

Example 11 - Statistical Analysis

The data are expressed as the mean ± SD. Statistical analyses were performed by using the Student t test or analysis of variance (ANOVA) followed by the Bonferroni post hoc test (where appropriate). Statistical significance was accepted at the p < 0.05 level.

Example 12 - Induction of Akt activation in SF

The TR47 peptide is to induce robust and sustained activation of Akt in SF cells as determined by phosphorylation of Ser473. To confirm activation of Akt by TR47, Akt- mediated inactivation of GSK3 via phosphorylation at Ser9 is determined. GSK3 is a well-known downstream substrate for Akt. TR47 is to induce significant Ser9- GSK3 phosphorylation with a time course that falls within the time course of TR47-mediated Akt activation. A scrambled control peptide is used to demonstrate no phosphorylation of Akt at Ser 473. A PAR-1 inhibitor SCH79797 is used to show that activation of Akt is dependent on PAR-1.

Example 13 - Collagen induced arthritis (CIA) model

A CIA model is induced in DBA/J1 mice by immunisation with chicken type II collagen following the methods discussed in [Brand DB et al. 2007 Nature Protocols 2: 1269-12751. Briefly, Male DBA/J1 mice (8-10 weeks old) are immunized with chicken type II collagen (CM) in complete Freund's adjuvant (CFA) at day 0 and day 21 (100 pg Cll/mouse/each time) by intradermally injection into the base of the mouse tail. After second immunization, mice are examined for clinical signs of arthritis. The presence of arthritis is determined by the appearance of the front and hind paws. Severity is graded for each paw and an arthritis score assigned to each mouse. Clinical signs of arthritis are assessed visually in the wrist and ankle joints 3 times weekly, using a semiquantitative scoring system, where 0 = no erythema or swelling, 1 = erythema and mild swelling confined to the tarsal or ankle joint, 2 = erythema and mild swelling extending from the ankle to the tarsal, 3 = erythema and moderate swelling extending from the ankle to the metatarsal joints, and 4 = erythema and severe swelling encompass the ankle, foot and digits, or ankylosis of the limb. All four limbs of a mouse are assessed, and the maximum score for each animal is 16. To test the preventative effects, test compounds were administrated by intraperitoneal injection (IP) after the first immunization twice a week for 3 weeks. To investigate the therapeutical effects, test compounds were administered by IP after second immunization for 3 weeks twice a week. After second immunization, animals are euthanized at day 28 for testing the preventive effect and at day 42 for the therapeutic effect of test compounds. The dosages of TR47 are 1 , 5, 10 and 20 mg/kg, with scrambled control peptide used at 20 mg/kg.

Example 14 - Antigen induced arthritis (AIA) model

An AIA model is induced in C57BI6 mice by intradermal immunisation with methylated bovine serum albumin (mBSA) and intra articular injection of mBSA as described previously (Brackertz, et al. Arthritis Rheum. 1977; 20:841 -850. Male C57BL6 mice (8-10 weeks old) are immunized with mBSA in CFA at the base of tail under general anaesthesia at day 0 and 14. At day 21 , the knee joint is intra-articularly injected with mBSA (10 pi of 20 mg/ml mBSA in phosphate-buffered saline (PBS)) to induce arthritis. Mice will develop arthritis with typical symptom of joint swelling on day 1 post intra-articular mBSA injection, and joint swelling will start to decrease after day 7, falling back to minimum swelling on days 14 and 28. Mice are monitored for joint swelling at day 1 , 4, 7, 14, 21 , 28 after intra articular mBSA injection using a caliper. Joint swelling is expressed as the difference in diameter (mm) between the right (arthritic) and left (control) knee joint.

Mice are also sacrificed 2 mice/group and prepared for histological and immunological evaluation at day 4 (acute phase of arthritis) and 14 (chronic phase of arthritis). At the end of experiment, all the mice will be euthanized under C02.The development of arthritis is assessed histologically at day 4 (acute inflammation) and 14, 28 (chronic inflammation) after intra articular mBSA injection.

To test the preventative effect, test compounds or PBS are administrated by intraperitoneal injection (IP) after the first immunization twice a week for 3 weeks. To investigate the therapeutical effects, test compounds or PBS are administered by IP after second immunization for 3 weeks twice a week 1 hr prior to mBSA intra-articularly injection. The dosages of IP-administered TR47 are 1 , 5, 10 and 20 mg/kg, with scrambled control peptide used at 20 mg/kg.

Example 15 - PAR-1 peptide treatment in presence of and prior to and following thrombin treatment.

Cells and treatment:

AE.Hy926 cells (ATCC^® CRL-2922^™), the human umbilical vein cell line, established by fusing primary human umbilical vein cells with a thioguanine-resistant clone of A549 by exposure to polyethylene glycol (PEG), were cultured in Dulbecco's Modified Eagle's Medium (DMEM, high glucose) containing 10% fetal bovine serum (FBS) in 75 sq cm flasks. When cells reached confluence, they were trypsinzed and seeded into 48 well plates at 1x 10⁴ viable cells/well for 2 days to reach complete confluency. Before treatment, cells were preincubated with DMEM medium (no FBS) for 2 hrs, then switched to fresh DMEM and treated with:

• PAR1 12mer peptide (shown on graphs as PAR1 ) (or scrambled control (SC) of

PAR1 peptide)

• thrombin

either alone or at different combinations for 1 hr. After treatment, media were discarded and 100 pi lysis buffer was added to each wells and cell lysates were collected and stored in -40°C for ELISA.

ELISA:

Human p-GSK and p-AKT were measured using ELISA Dueset kits (R&D Systems) according to manufacturers’ instructions.

The PAR1 peptide induced robust activation of Akt as determined by phosphorylation of Ser⁴⁷³, after 1 hour. The AKT signaling pathway is synonymous with cytoprotection in endothelial cells [Akt mediates cytoprotection of endothelial cells by vascular endothelial growth factor in an anchorage dependent manner. Fujio Y, Walsh

K. J Biol Chem. 1999 Jun 4;274(23): 16349-54; Insulin stabilizes microvascular endothelial barrier function via phosphatidylinositol 3-kinase/Akt-mediated Rad activation. GCindCiz D, Thom J, Hussain I, Lopez D, Hartel FV, Erdogan A, Grebe M,

Sedding D, Piper HM, Tillmanns H, Noll T, Aslam M. Arterioscler Thromb Vase Biol.

2010 Jun;30(6): 1237-45] and other cells including synovial cells as it mediates barrier stabilization, and enhances an anti-inflammatory as opposed to an inflammatory phenotype. In contrast, the latter is induced by thrombin. Fig 8A shows that the addition of thrombin alone at 0.1 and 0.25 nM inhibited phosphorylation of AKT. When lower doses (0.1. or 0.25 uM) of thrombin and PAR1 peptide were added together simultaneously there was increased pAKT compared to adding thrombin alone, indicating that the PAR1 peptide exerts cytoprotective AKT activity even in the presence of the potent inflammatory enzyme, thrombin. At some higher concentrations, eg 10 nM, thrombin in the presence of peptide also significantly enhanced pAKT. Figure 8B shows the effect of adding the PAR1 peptide 15 mins before or after thrombin, and its ability to enhance the Akt activity.

Claims

1. A method for minimising a symptom of rheumatoid arthritis (RA) in an individual having joint disease including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising a symptom of rheumatoid arthritis in the individual.

2. The method of claim 1 wherein the joint disease is inflammatory joint disease

3. The method of claim 2 wherein the inflammatory joint disease is RA.

4. The method of claim 3 wherein the RA is stage 1 RA, or stage 2 RA, or stage 3 RA, or stage 4 RA.

5. The method of any one of the preceding claims wherein the symptom is joint pain, joint swelling, or joint stiffness.

6. The method of any one of the preceding claims wherein the joint disease includes inflammation of synovial tissue.

7. The method of any one of the preceding claims wherein the joint disease includes cartilage degradation.

8. The method of any one of the preceding claims wherein the joint disease includes bone degradation.

9. The method of any one of the preceding claims wherein the joint disease includes a deformed joint.

10. The method of any one of the preceding claims wherein the administration of the TR47 related peptide minimises the production or expression of a molecule selected from the group consisting of TNFa, IL-1 and IL-6 in the individual.

11. The method of any one of the preceding claims wherein the TR47 related peptide is selected from the group consisting of SEQ ID No: 2 to 4 or 8 to 17 or Figure 5.

12. The method of any one of the preceding claims wherein the TR47 related peptide is administered in the form of a pharmaceutical composition.

13. The method of any one of the preceding claims including the step of administering an anti-TNF antibody to the individual.

14. The method of any one of the preceding claims wherein the TR47 related peptide is administered by intra-articular injection.

15. The method of any one of the preceding claims wherein the joint disorder is rheumatoid arthritis and the joint of the individual having the disorder has, at the time of the administration of the TR47 related peptide, an overexpression or overproduction of thrombin, said overexpression or overproduction of thrombin providing for, or resulting in, the cleavage of PAR-1 receptors on cells contained in the joint.

16. The method of any one of the preceding claims wherein the administration of a TR47 related peptide to a joint of the individual having the disorder enables, or causes, the phosphorylation of Akt, preferably phosphorylation of Akt Ser⁴⁷³, by a cell in the joint.