WO2020000061A1

WO2020000061A1 - Renal treatment

Info

Publication number: WO2020000061A1
Application number: PCT/AU2019/050693
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 for minimizing a symptom of glomerulonephritis in an individual having a kidney or renal disease, comprising the administration of a TR47 related peptide to the individual. The TR47 related peptide generally refers to a peptide that has a N-terminal sequence NH2-NPND or NH2-NPNDKY, which is derived from the N-terminal residues of Met1-Arg46 deleted human PAR1 (protease activated receptor 1) sequence.

Description

Renal treatment

Field of the invention

The invention relates to kidney disease, to glomerulonephritis and to compositions and methods for treatment of same.

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. Glomerulonephritis (GN) is a group of kidney diseases characterised by injury to the glomeruli. One consequence of GN is the passage of protein, blood or other into the urine. Untreated GN may lead to end stage renal disease.

Most forms of GN present within one of two syndromes, namely nephrotic syndrome or nephritic syndrome, the former being characterised by massive proteinuria, hypoalbuminemia, oedema and hyperlipidemia and hyperlipiduria, the latter syndrome characterised by hematuria, oliguria, azotemia and hypertension.

In individuals presenting with nephrotic syndrome, there is often damage to podocytes leading to changes in the filtration slit defined by the podocyte and the capillary that the podocyte overlays. These changes enable the proteins to escape from the blood into the urine. Nephrotic syndrome may generally arise from primary disease (such as minimal change disease, focal segmental glomerulosclerosis, membranous GN) and other diseases may cause nephrotic syndrome (diabetes mellitus, systemic lupus erythrmatosus (SLE), malignancy and viral infection). The primary diseases presenting as nephrotic syndrome are generally recognised as non-proliferative GN.

Nephritic syndrome arises generally from proliferation of cells within the glomeruli, along with inflammation in the glomerus. This allows leakage of blood into the urine and the classical symptom of hematuria. Diseases presenting as nephritic syndrome are sometimes known as proliferative GN diseases and include IgA nephropathy, membranoproliferative GN, and post infectious (strep) GN.

Rapidly progressive (cresentic) GN is another disease which is considered by some to be a form of proliferative GN and to present as nephritic syndrome. It includes type I disease (Goodpasture’s syndrome), type II disease (immune complex mediated eg Henoch-Schonlein purpura) and type III disease (pauci immune GN).

GN diseases, whether presenting as nephrotic or nephritic, are generally immune related. Generally there is a humoral aspect, (for example an antibody directed against a glomerular component, or immune complexes lodge into the glomerulus). Sometimes GN diseases may be associated with inflammation that contributes to the disease aetiology, although this is not always the case. Sharma R et al. 2017 J Am Soc Nephrol 28:2618-2630 showed that thrombin generation is enhanced in nephrotic syndrome and that thrombin is a contributor to podocyte injury which is mediated through podocyte PARs.

Guan Y et al. 2017 J. Pharm Sci 135 81 -88 showed in a model presenting as nephrotic syndrome, that PAR-1 contributes to development of podocyte and glomerular injury and suggested that PAR-1 antagonists have therapeutic potential. The data supported PAR-1 inhibition as a potential therapeutic strategy, and was seen as being consistent with another study in which a mouse model of diabetic nephropathy having increased PAR-1 expression and increased podocyte damage: Sakai T et al. 2009 Biochem Biophys res Commun 384: 173-179. Similarly, Waasdorp M. et al. 2016 Sci Rep 6, 33030: doi:10.1038/srep33030 showed in a model of diabetic nephropathy that despite having similar glucose levels, PAR-1 deficient mice developed less kidney damage after induction of diabetes as compared with wild type mice, suggesting PAR-1 inhibition as a strategy for future therapy.

Both thrombomodulin and APC variants having a reduced anticoagulant function have been proposed for nephron-protection, thereby treating renal ischemia reperfusion injury (IRI) [Dong W. et al. 2015 J Am Soc Nephrol 26:2789-27991, a condition associated with glomerular inflammation. IRI presents as a nephrotic syndrome.

Lattenist L et al 2016 Thromb and Haem 116.1 utilised mutant forms of APC lacking either anticoagulant or signalling function in a mouse model of IRI. They found that APC did not influence cytokine or chemokine production or apoptosis and proposed that this was because of a local deficit of EPCR which they considered to be critical for APC protective effect. Kerschen E et al. 2010 J Clin Invest 120: 3167-3178 showed that both EPCR and PAR-1 were required for APC mediated signalling in CD8+ dendritic cells.

In a model of lupus nephritis, it was shown that APC improved nephritis by suppressing the abnormal autoimmunity of SLE [Lichtnekert et al. 2011 J Immunol doi: 10.40491.

There is a need for new methods and compositions for minimising podocyte injury. There is a need for new methods and compositions for minimising a symptom of glomerulonephritis. Summary of the invention

In one embodiment there is provided a method for minimising a symptom of glomerulonephritis (GN) in an individual having a renal disease or condition including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising a symptom of GN in the individual.

In another embodiment, there is provided a method for minimising a symptom of GN selected from the group consisting of proteinuria, hypoalbuminemia, edema, hyperlipidemia, hyperlipiduria, hematuria, oliguria, azotemia or hypertension in an individual having a renal disease or condition including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising the symptom of GN in the individual.

In another embodiment there is provided a method for minimising fibrosis of renal interstitial tissue in an individual having a renal disease or condition including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising fibrosis of renal interstitial tissue in the individual.

In the above described embodiments, the renal disease or condition typically presents as a nephrotic or a nephritic syndrome.

The renal disease or condition may be a primary or secondary disease of the kidney.

The renal disease or condition may be proliferative or non proliferative GN.

The renal disease or condition may be associated with ischemic tissue, with infarcted tissue or with fibrotic tissue.

The renal disease or condition may be a chronic renal disease (CRD).

The renal disease or condition may be acute kidney injury (AKI). AKI may arise from a CRD or from acute injury arising from hypoxia or toxin.

In the above described embodiments, the renal disease or condition is preferably associated with expression or production of a serine protease, preferably thrombin, preferably an over production or over expression of thrombin. Preferably the renal tissue, more preferably, glomerular tissue comprises thrombin activity arising from over express 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 GN in an individual having a renal disease or condition including administering to the individual a therapeutically effective amount of:

- a TR47 related peptide and

- an anti-inflammatory agent,

thereby minimising a symptom of GN in the individual.

In the above described embodiments, the TR47 related peptide may be administered by intra-venous 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 glomerular cell, including the step of contacting a glomerular cell with a TR47-related peptide, thereby minimising the production or expression of TNFa, IL-1 , IL-6, IL-17 and IL-23 by a glomerular cell.

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

Glomerular or renal tissue 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 glomerular or renal tissue not having glomerulonephritis. Thrombin activity may be determined by the skilled worker. See for example Sharma 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 renal or glomerular tissue 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 glomerulonephritis, the disorder 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 glomerular tissue with associated glomerulonephritis, 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-venous 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 inflamed glomerular tissue, thereby treating the individual for glomerulonephritis.

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

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

In the above described embodiments, the glomerular cell may be a podocyte, a proximal convoluted tubule (PCT) cell, a glomerular capillary endothelial cell, or an epidermal cell of Bowmans capsule.

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

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

Detailed description of the embodiments

The invention generally relates to improvements in the management of renal diseases and conditions arising from administration of a 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 glomerulonephritis.

The invention is applicable to a renal disease or condition involving glomerulonephritis generally where there are common clinical manifestations of inflammation of glomeruli. Those symptoms or manifestations are described in more detail below and include abnormal amount of protein, lipid or other components in blood and/or urine.

In preferred embodiments, the invention is applicable to renal disease or conditions where glomerulonephritis arises as condition that is secondary to another disease or condition. For example the renal disease or condition may be diabetic nephropathy, arising from diabetes, or lupus nephritis arising from SLE. In other embodiments the renal disease or condition arises as a primary disease of the kidney, in which case the initial injury is to renal tissue and the disease may more or less localise to the kidney.

A common feature of glomerulonephritis, particularly glomerulonephritis presenting as a nephrotic syndrome, is the role of the cells of the glomerulus, especially the podocyte, and PCT cells. These cells are 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 glomerular cells by TR47 related peptide through contact of the TR47 related peptide with glomerular cell 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 glomerular PAR-1 receptors are prior cleaved by thrombin, and therefore irreversibly activated for formation of a pro-inflammatory signal in glomerular cells. In contrast, it is believed that the invention has shown that podocyte PAR-1 receptors whether prior activated by thrombin or not, can be activated for formation of an anti-inflammatory signal by TR47 related peptides.

“ Glomerulonephritis” is a commonly known inflammatory condition primarily affecting cells of the glomerulus, especially causing injury to podocytes and PCT cells. “A symptom of glomerulonephritis" generally refers to one or more of the following manifestations: proteinuria, hypoalbuminemia, edema, hyperlipidemia, hyperlipiduria, hematuria, oliguria, azotemia or hypertension.

“ Proteinuria " generally refers to >3 .5 g of protein excreted in the urine over 24 hours

“Hypoalbuminemia" generally refers to serum albumin concentration <25 g/litre

“Edema" generally refers to an excess of watery fluid collecting in the cavities or tissues of the body

“ Hyperlipidemia " generally refers to raised levels of total blood lipids >10 g/dL

“ Hyperlipiduria " generally refers to lipoproteins that leak into the urine resulting in free fat or oval fat bodies in urine

“ Hematuria " generally refers to the presence red blood cells in the urine

“ Oliguria " generally refers to abnormally small production of urine of less than 500 ml_ per day in adults

“ Azotemia " generally refers to elevation of blood urea nitrogen (BUN) exceeds that of creatinine (i.e. , BUN>12*creatinine)

“ Hypertension " generally refers to a blood pressure higher than 130 over 80 mm of mercury (mmHg)

“A renal disease or condition" generally refers to a pathology of the kidney organ itself. The pathology may be primary, i.e. having aetiology in the kidney proper, or it may be secondary i.e. arising from an aetiology in another tissue or organ which injures the kidney. A renal disease or condition may or may not be an inflammatory condition of the kidney.

“An inflammatory renal disease or condition" generally refers to a pathology wherein inflammation is a key contributor to the development of the disease or condition. Generally, in such a condition, treatment of an anti-inflammatory agent, such as a steroid, has effect of minimising or reducing the pathology. An“autoimmune renal disease or condition” generally refers to a pathology of the kidney organ arising from an autoimmune condition or related condition in which glomerular injury arises from deposit of antibody or immune complex in the kidney.

“Minimising a symptom” generally refers to at least reducing symptom severity, for example, reducing proteinuria, hypoalbuminemia, edema, hyperlipidemia, hyperlipiduria, hematuria, oliguria, azotemia or hypertension. 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.

“Akf 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 GN, or for minimising the production or expression of inflammatory mediators from a glomerular 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 one embodiment there is provided a method for minimising a symptom of glomerulonephritis (GN) in an individual having a renal disease or condition including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising a symptom of glomerulonephritis in the individual.

In one embodiment there is provided a method for minimising a proteinuria in an individual having a renal disease or condition including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising proteinuria in the individual.

In one embodiment there is provided a method for minimising hypoalbuminemia in an individual having a renal disease or condition including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising hypoalbuminemia in the individual.

In one embodiment there is provided a method for minimising edema in an individual having a renal disease or condition including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising edema in the individual.

In one embodiment there is provided a method for minimising hyperlipidemia in an individual having a renal disease or condition including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising hyperlipidemia in the individual.

In one embodiment there is provided a method for minimising hyperlipiduria in an individual having a renal disease or condition including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising hyperlipiduria in the individual.

In one embodiment there is provided a method for minimising hematuria in an individual having a renal disease or condition including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising hematuria in the individual.

In one embodiment there is provided a method for minimising oliguria in an individual having a renal disease or condition including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising oliguria in the individual.

In one embodiment there is provided a method for minimising azotemia in an individual having a renal disease or condition including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising azotemia in the individual.

In one embodiment there is provided a method for minimising hypertension in an individual having a renal disease or condition including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising hypertension in the individual.

In one embodiment there is provided a method of minimising an autoimmune renal condition or disease, preferably diabetic nephropathy or lupus nephritis including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising an autoimmune renal condition or disease in the individual.

In one embodiment there is provided a method of minimising an inflammatory renal condition or disease, including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising an inflammatory renal condition or disease in the individual. The inflammatory renal condition or disease may be minimal change disease, focal segmental glomerulosclerosis, membranous GN, IgA nephropathy, membranoproliferative GN, and post infectious (strep) GN. The TR47 related peptide may be administered to glomerular tissue that contains an increased amount of 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 development of renal disease or condition, preferably prior to development of fibrotic, or ischemic or infarct lesions.

In certain embodiments, a TR47 related peptide may be administered with another therapeutic compound indicated for treatment of a renal disease or condition, especially for treatment of inflammatory renal disease or condition. 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 glomerulonephritis in an individual having a renal condition or disease including administering to the individual a therapeutically effective amount of:

- a TR47 related peptide and

- an anti-TNF, or anti-IL-17 or anti-IL-23 antibody for minimising inflammation, thereby minimising a symptom of GN in the individual.

As described further herein, the TR47 related peptide may be administered by intra- venous injection. The invention further relates to utilising TR47 related peptides to minimise the production or expression of inflammatory mediators by glomerular cells, in particular by podocyte, PCT cells and glomerular capillary endothelial cells. These embodiments may also be useful in ex vivo applications for conditioning cells so that they define an anti- inflammatory profile, or in in vitro embodiments for monitoring or determining minimisation of inflammation in cells obtained from a diseased or inflamed kidney.

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 glomerular cell including the step of contacting a glomerular cell with a TR47-related peptide, thereby minimising the production or expression of TNFa, IL-1 and IL-6 by a glomerular cell.

In another embodiment there is provided a method for inducing phosphorylation of Akt Ser⁴⁷³ in a glomerular cell, particularly a podocyte, including the step of contacting a glomerular cell with a TR47-related peptide, thereby inducing phosphorylation of Akt Ser⁴⁷³ by a glomerular cell.

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

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

In the above described embodiments, the glomerular cell may be a podocyte, a PCT cell, a glomerular capillary endothelial cell or an epidermal cell of Bowman’s capsule.

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

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 GN in an individual having a renal disease or condition, or for treating GN, or for minimising the progression of GN.

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

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 glomerular cells, and reduce glomerular 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 6 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 (8EG ID No. 11 ), NPNDK (8EG ID No. 12), NPNDKY (SEG 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 SEG ID NO:1 to L⁶⁶ of SEG ID NO: 1 ;

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

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

X-Y is a sequence from K⁵¹ of SEG ID NO:1 to V²⁴⁶ of SEG 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 NPNDKYEPFWEDE utilised in Example 14 herein.

In some embodiments, a TR47 related peptide has at least 70%, preferably 80%, preferably 90%, preferably 95%, preferably 96%, 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-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, 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 conformationa!ly 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, Bioiogy, Eds. Gross and Meinhofer, Vol. 5, p. 341 , Academic Press, Inc., New York, N.Y. (1983); and Burger's Medicinal Chemistry and Drug Discovery , Ed. Manfred E. Wolff, Ch. 15, pp. 619-820, John Wiley & Sons Inc., New York, N.Y. (1995).

Some other derivative compounds of the TR47 related peptides are peptidomimetics Peptidomimetics based on TR47 related peptides substantially retain the activities of the TR47 related peptide. They include chemically modified polypeptides, polypeptide-like 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 Discovery, 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-methyl, N-etby!, N-propyl 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 - La - 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:

NPNDKYEPFWEDEEKNESGL

WFPEY DN PN

NPNDKYEPFWED

LGSENKEEDEWFPEY DNPN

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 carboxylate functionality defined by RCOO— or sulfonyl 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 cyciize 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 circular peptide synthesis are known in the art, for example, in U.S. Patent Application No. 20030035814; and Mura!idharan and Muir, Nat. Methods, 3:429-38, 2006. C-terminai functional groups of the peptides described herein include amide, amide lower alkyl, amide di(lower alkyl), lower aikoxy, 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. Lloyd-Williams, et. al. , 1997, CRC-Press, (ISBN-13: 978- 0843391422), Methods in Enzymology, 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, 1963, 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. Ch!oromethyi 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 FMOC 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!oxycarbonyi) and FMOC. 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 dichloromethane. 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 ceils.

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 DNA. 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. a!., Science 242:1182, 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. coil 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 co//- 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 intravenous 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 10 to about 50000 mg/ml TR-47 for administration to a patient. This is a particular preferred formulation for administration of TR47 by intra venous 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 renal disease or condition, especially an anti-inflammatory agent such as a steroid, or anti-inflammatory cytokine or anti-inflammatory antibody. In some embodiments, the one or more other agents for treatment of a renal disease or condition 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-venous 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 plasma 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 pM depending on the general nature of the compound.

Continuous infusion refers to continuing substantially uninterrupted the introduction of a solution into a blood vessel for a specified period of time. Continuous infusion may be particular applicable in the case of treatment or management of acute kidney injury. Treatment may involve a continuous infusion (e.g., for 3 hr after ischemia or reperfusion) or a slow infusion (e.g., for 24 hr to 72 hr when given within 6 hr of acute kidney injury). The therapeutic amount may be about 0.01 mg/kg/hr to about 1 .1 mg/kg/hr if administered by continuous infusion over 4 hour to 96 hour, to as little as about 0.01 mg/kg/hr to about 0.10 mg/kg/hr for about 24 hours. Preferably, the therapeutic dose would be administered by continuous infusion for about 4 to about 72 hours. More preferably, by continuous infusion for about 4 to about 48 hours. More preferably, by continuous infusion for about 12 to about 48 hours. More preferably, by continuous infusion for about 12 to about 36 hours. More preferably, by continuous infusion for about 4 to about 36 hours.

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 glomerular cell, especially a podocyte or proximal convoluted tubule cell) 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 renal disease or condition.

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 - in vitro podocyte culture

Conditionally immortalized mouse podocytes were cultured as previously described. Under growth permissive conditions, cells were seeded on type I collagen coated dishes at 33 ^°C. The growth medium was RPMI 1640 (Sigma-Aldrich) with 10% fetal bovine serum and 20 U/mL mouse interferon-g (Sigma-Aldrich) to drive the expression of a thermosensitive T-antigen. To induce differentiation, cells were maintained at 37 ^°C without interferon- g for 10 to 14 d. Example 3 - Ex vivo podocyte culture

Glomeruli were isolated by differential sieving from mouse renal cortex and disrupted by sonication.

Example 4 - Proliferation assay

Cell proliferation is performed 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. 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. The peptides dose-dependently stimulated proliferation of podocytes.

Example 5 - Cell apoptosis

To induce apoptosis, cells are incubated with glucose at concentrations of 5.6- 30 mmol/l (HG) for 0-48 h. Apoptosis of podocytes is quantified with an Annexin V-FITC apoptosis detection kit. Briefly, podocytes are trypsinized and resuspended in

1 x binding buffer and collected by centrifugation at 1000 ^c g for 5 min. The cell suspension is incubated with Annexin V-FITC and propidium iodide for 10-20 min at room temperature in the dark. Stained cells are immediately detected using a flow cytometer (FACSARIA II; BD Biosciences, San Jose, CA, USA). The peptides dose- dependently inhibit apoptosis in podocytes.

Example 6 - 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 7 - Histology

Kidneys were fixed with 15% formalin (pH 7.4), embedded in paraffin, cut into 2 mmsections and mounted on slides. The sections were then stained with periodic acideSchiff (PAS) reagent and sirius red. Images were evaluated using light microscopy (BX-51/ DP-72; Olympus, Tokyo, Japan). Glomerular sclerotic and sirius red positive areas were determined using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Example 8 - ELISA

Levels of IL-6, IL-1 and TNFa are measured using ELISA kits in accordance with manufacturer’s instructions. All three cytokines are dose-dependently reduced by the peptides.

Example 9 - Rac 1 activation

Briefly, podocytes are grown in a 6 well plate till confluent and incubated in serum free medium without supplements 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.

Example 10 - 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 11 - Western blotting

Briefly, cells are washed three times with PBS and lysed with lysis buffer (0.15 M NaCI, 0.01 mM PMSF, 1 % NP-40, 0.02 M Tris, 6 M urea/H20). Cell lysates are centrifuged at 10,000g for 15 minutes and supernatants are separated by 10% sodium- dodecyl-sulphate polyacrylamide-gel electrophoresis (SDS-PAGE) and transferred to a PDVF membrane. The primary antibodies used are as follows against pan or phospho specific forms of Akt, GSK3 . 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 ImageQuant™ LAS 4000 (GE Healthcare Life Sciences).

TR 47 related peptides are expected to dose-dependently increase phosphorylation of Akt and reduce GSK3 .

Example 12 - Induction of Akt activation in podocytes

The TR47 peptide is to induce robust and sustained activation of Akt in podocytes 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 - Folic acid model

Eight-week-old, male, CD1 mice (Charles Rivers Mouse Farms, Margate, Kent, UK) are administered folic acid (240 mg/kg) in vehicle (0.2 ml of 0.3 M NaHC03) or vehicle only by intraperitoneal injection. This folic acid dose reliably induces severe nephrotoxicity, as assessed by histologic examination, and is associated with a morbidity rate of <5% for the experimental period. Six control kidneys are analyzed before folic acid or vehicle administration. Kidneys are collected at 1 , 2, 3, 7, and 14 d, with 6 folic acid-treated and three vehicle-treated animals at each time point. Left kidneys are used for immunohistochemical analyses and right kidneys for Western blotting. Kidney damage is assessed by histology and creatine levels.

Example 14 - 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. Results:

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. Giindiiz D, Thom J, Hussain I, Lopez D, Hartel FV, Erdoqan 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 glomerular cells and podocytes [Tejada T, Catanuto P, liaz A, Santos JV, Xia X, Sanchez P, et al. Failure to phosphorylate AKT in podocytes from mice with early diabetic nephropathy promotes cell death. Kidney Int 2008;73(12): 1385-93; Bussolati B, Deregibus MC, Fonsato V,

Doublier S, Spatola T, Procida S, et al. Statins prevent oxidized LDL-induced injury of glomerular podocytes by activating the phosphatidylinositol 3-kinase/AKT-signaling pathway. J Am Soc Nephrol 2005; 16(7): 1936-47] 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 glomerulonephritis (GN) in an individual having a renal disease or condition including administering a therapeutically effective amount of a TR47 related peptide to the individual, thereby minimising a symptom of glomerulonephritis in the individual.

2. The method of claim 1 wherein the renal disease or condition presents as a nephrotic or a nephritic syndrome.

3. The method of claim 1 or claim 2 wherein the renal disease or condition is a primary disease of the kidney.

4. The method of any one of the preceding claims wherein the renal disease or condition is chronic kidney disease (CKD).

5. The method of any one of the preceding claims wherein the renal disease or condition is acute kidney injury (AKI).

6. The method of any one of the preceding claims wherein the symptom of GN is selected from the group consisting of proteinuria, hypoalbuminemia, edema, hyperlipidemia, hyperlipiduria, hematuria, oliguria, azotemia and hypertension.

7. The method of any one of the preceding claims wherein the renal disease or condition includes fibrosis of renal interstitial tissue.

8. The method of any one of the preceding claims wherein the renal disease or condition includes deposition of antibody or immune complexes in glomeruli.

9. The method of any one of the preceding claims wherein the renal disease or condition includes ischemic tissue or infarcted tissue.

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-inflammatory compound to the individual.

14. The method of any one of the preceding claims wherein the TR47 related peptide is administered by I.V. or oral route.

15. The method of any one of the preceding claims wherein the individual having the glomerulonephritis 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 glomerular tissue.

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