THE TREATMENT OR PROPHYLAXIS OF ORGAN AND TISSUE FIBROSIS VIA MODULATION OF CELL DIVISION AUTIANTIGEN (CDA1)

FILING DATA

5

This application is associated with and claims priority from Australian Provisional Patent Application No. 2009900265, filed on 23 January, 2009, entitled "Treatment of diabetes and other diseases", the entire contents of which, are incorporated herein by reference.

I O FIELD

The present invention relates generally to the field of tissue fibrosis. More particularly, the present invention contemplates the treatment and prophylaxis of disease- or trauma- mediated organopathy which leads to tissue fibrosis. Even more particularly, the present 15 invention is directed to the treatment and prophylaxis of diabetes, cardiovascular disease. Hepatitis infection and scar prevention following physical injury. The present invention enables clinical management of tissue fibrotic complications such as renal fibrosis. atherosclerosis, hepatic fibrosis and scaring. 0 BACKGROUND

Bibliographic details of references provided in the subject specification are listed at the end of the specification. 5 Reference to any prior art is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

Organ and tissue fibrosis results from the accumulation of tough, fibrous, scar tissue. 0 Generally, fibrosis occurs in tissues and organs in response to disease or injury or following exposure to toxins. This is generally encompassed within the term "organopathy". The immune system is activated following the disease or injury which results in an elevation in cytokines and other growth-factors which in turn induce cells to produce collagen, glycoproteins (such as fibronectin), protoglycans and other substances.

These substances cause a build up of extracellular matrix (ECM) which is a non-functional connective tissue. The equilibrium between fibrinogenesis and fibrinolysis is then disrupted and favors excessive scar tissue build up (fibrinogenesis).

Disease and traumatic conditions which lead to organopathy and tissue fibrosis include viral infection such as infection by Hepatitis C virus or Hepatitis B virus. This can lead to hepatic (liver) fibrosis following hepatic organopathy. Physical trauma can lead to skin or organ fibrosis.

Nephropathy and atherosclerosis are two major lethal consequences of chronic diabetes. With the incidence of Type II diabetes measured at 7.6% in 2000 and expected to increase to 1 1.4% by 2025. with perhaps an equal number of patients undiagnosed, the complications of diabetes are already having a significant effect on public health representing a profound unmet clinical need. Furthermore, with an increasing number oi ^' Type Il diabetic subjects being diagnosed at a younger age and the ongoing increase in the number of Type I diabetic subjects, there is likely to be an ongoing burden of relatively young individuals suffering from the chronic renal and vascular complications, as seen in both Type I and Type II diabetes.

A major feature of diabetic nephropathy and atherosclerosis is tissue fibrosis, the condition of excess accumulation of extracellular matrix (ECM) proteins. Λ range of pathways has been implicated in promoting ECM accumulation in progressive renal and cardiovascular disorders. The most widely studied involves TGF-β, which appears to promote fibrosis and to inhibit cell proliferation via intracellular signalling molecules, known as Smads.

Current treatments for nephropathy and atherosclerosis, which arc dominated by angiotensin converting enzyme (ACE) inhibitors and blockers of Angiotensin signalling (ARB 's) both act to modulate blood pressure, which is an accelerator rather than an underlying cause of the vascular pathology. These agents, at best, can only slow disease progression. Together with statins, anti-hypertensive drugs have had a significant impact in reducing cardiovascular mortality in the non-diabetic population, but this benefit is failing to translate fully to patients with diabetes who continue to have increased morbidity and mortality despite current best clinical practice.

Diabetes (Type I and Type II) can induce diabetic nephropathy leading to fibrosis of the kidneys (renal fibrosis) [Oldfield el al J Clin Invest /0#(72j l 853-1863, 2001 1.

Transforming growth factor-β (TGF-β) is a critical player in the iϊbrotic process by promoting production and accumulation of ECM leading to fibrosis. The latter is the key pathological feature, for example, in kidneys of subjects with diabetes which leads to both glomerulosclerosis and tubulointersital fibrosis. Attempts to target TGF-β have been thwarted by the diverse biological role of this growth factor including its involvement in the body's immune system.

There is a need to identify targets or medicaments in the treatment of disease- or trauma- mediated organopathy which leads to tissue fibrosis.

- A -

SUMMARY

Amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>l (SEQ ID NO: 1 ), <400>2 (SEQ ID NO:2), etc. A summary of the sequence identifiers is provided in Table 1. A sequence listing is provided after the claims.

In accordance with the present invention, cell division autoantigen 1 (CDAl) [Chai et al J Biol Chem 276(36)33665-33674, 2001 ], is identified as a link between TGF-β and ECM. Antagonizing CDA l -mediated signalling is proposed herein to assist in ameliorating the effects of tissue fibrosis resulting from disease- or trauma-induced organopathy.

Accordingly, one aspect of the present invention contemplates a method for the treatment or prophylaxis of disease- or trauma-mediated organopathy leading to tissue fibrosis in a subject, the method comprising administering to the subject an amount of an agent which antagonizes CDAl -mediated signalling for a time and under conditions effective to reduce fibrosis.

Another aspect of the present invention provides a method for the treatment or prophylaxis oϊ disease- or trauma-mediated organopathy leading to tissue fibrosis in a subject, the method comprising administering to the subject an amount of an agent which inhibits CDΛl -mediated signalling by antagonizing interaction between CDA l and CDAl BPl for a time and under conditions effective to reduce fibrosis.

Examples of disease- or trauma-mediated organopathy resulting in tissue fibrosis include diabetic nephropathy which can induce kidney (renal) fibrosis following Type I and I ypc 11 diabetes and hepatic organopathy which results in hepatic (liver) fibrosis following infection with a Hepatitis virus (e.g. Hepatitis B virus or Hepatitis C virus). Other examples include scaring following skin or tissue injury and a variety of fibrotic conditions in the heart, lungs and pancreas. Atherosclerosis is also associated with tissue fibrosis and can lead to or result from cardiovascular disorders. In a particular embodiment, the condition is Type I or Type II diabetic nephropathy leading to renal fibrosis. In another embodiment, the condition is Type I or Type Il diabetic atherosclerosis. In a further embodiment, the condition is hepatic organopathy following Hepatitis virus infection leading to hepatic fibrosis.

The present invention provides a method for treating a subject with diabetes or who is at risk of developing same, the method comprising administering to the subject an amount of an agent which inhibits CDAl -mediated signalling for a time and under conditions effective to ameliorate symptoms of diabetic nephropathy leading to renal fibrosis and/or atherosclerosis.

The present invention is also directed a method for treating a subject with diabetes or who is at risk of developing same, the method comprising administering to the subject an amount of an agent which inhibits CDAl -mediated signalling by antagonizing interaction between CDAl and CDAl BPl for a time and under conditions effective to ameliorate symptoms of diabetic nephropathy leading to renal fibrosis and/or atherosclerosis.

Medicaments also form part of the present invention.

Accordingly, another aspect of the present invention is directed to an agent for use in the treatment or prophylaxis of disease- or trauma-mediated organopathy leading to tissue fibrosis, the agent being an antagonist of CDΛ1 -mediated signalling.

More particularly, the present invention provides an agent for use in the treatment or prophylaxis of disease- or trauma-mediated organopathy leading to tissue fibrosis, the agent being an inhibitor of CDAl -mediated signalling by anatgonizing interaction between DCAl and CDAl BPl .

Another aspect of the present invention is directed to an agent for use in the treatment or prophylaxis of disease- or trauma-mediated organopathy leading to tissue fibrosis, the agent being an antagonist of CDAl -mediated signalling wherein the antagonist competes for binding of the C-terminal end region of CDAl binding protein (CDA l BPl ) which comprises the amino acid sequence (in single letter code) LTISTIIR (SEQ ID NO: 1 ) to CDAl .

In an embodiment, the agent is a peptide comprising the amino acid sequence: Xi-X ₍ JR wherein Xi-X ₆ are each an L- or D-amino acid residue or an unnatural amino acid residue or amino acid analog or a derivative or mimetic of the peptide wherein the agent is an antagonist of CDA l -mediated signalling wherein it competes for binding of the C-tcrminal end region of CDA l BPl which comprises the amino acid sequence L TISTIIR to Cl)A l .

In a related embodiment, the agent is a peptide comprising the amino acid sequence X]- X ₆ IRX _V X _S , wherein Xi-X ₈ are each an L- or D-amino acid residue or an unnatural amino . acid residue or amino acid analog or a derivative or mimetic of the peptide wherein the agent is an antagonist of CDAl -mediated signalling wherein it competes for binding of the C-tcrminal end region of CDAl BPl which comprises the amino acid sequence LTISTIl R to CDAl .

A mimetic may be a homolog peptide or a chemical molecule having similar binding or structural properties to a corresponding native peptide.

Examples of peptide derivatives include amino acid residues having hydrogen atoms substituted for deuterium atoms, cyclized peptides, peptides having inverted and/or reversed amino acid sequences and peptides with an acetylated N-terminus and/or an amidated C-terminus. Derivatives also include chimeric molecules linked to a cell penetrating peptide (CPP). Native peptides may also be used. Generally, if native peptides are employed, the peptides are formulated with stabilizing excipients. Immunoglobulins which bind to CDAl or CDAlBPl or which otherwise inhibit CDA1/CDA 1 BP 1 interaction and CDAl -mediated signalling also form part of the present invention.

The present invention is further directed to the use of CDAl binding protein 1 (CDA l BP l ) or a derivative or homolog thereof in the manufacture of a medicament in the treatment of tissue fibrosis resulting from disease- or trauma-mediated organopathy. In an embodiment. the derivative comprises a peptide fragment comprising the amino acid sequence LTlSTIlR or a derivative, homolog or mimetic thereof.

The present invention enables clinical management of tissue fibrotic complications arising from disease or trauma such as but not limited to diabetes, cardiovascular disease, hepatitis infection and scaring following physical trauma.

Amino acid residues are defined using full names, three letter codes and single letter codes. Table 2 provides a summary of amino acid codes.

TABLE 1

Summary of sequence identifiers

SEQUENCE

DESCRIPTION IDNO:

8mer amino acid sequence in human CDAlBPl required for binding to

CDAl

8mer amino acid sequence in human CDAlBPl showing two amino acids critical for binding to CDAl (X,-X ₆ IR)

19mer amino acid sequence in human CDAl BPl having CDAl binding

8mer

7mer amino acid sequence in human CDAl BPl having CDAl binding

8mer

5 5mer amino acid sequence in human CDAl BPl having CDAl binding

8mer

3mer amino acid sequence in human CDAl BPl having CDAl binding

8mer y 1 lmer amino acid sequence in human CDAl BPl with N-tcrminal truncated CDAl binding 8mer

T 12mer amino acid sequence in human CDAlBPl with CDAl binding

8mer

9 IOmer amino acid sequence in human CDAl BPl with CDAl binding

8mer

7δ ^~ 8mer amino acid sequence in human CDAlBPl with CDAl binding 8mcr

8mer amino acid sequence in human CDAlBPl with CDAl binding

8mer

12 7mer amino acid sequence in human CDAlBPl with CDAl binding

8mer lόmer amino acid sequence in human CDAlBPl with CDAl binding

8mer

15mer amino acid sequence in human CDAl BPl with CDAl binding

8mer

I4mer amino acid sequence in human CDAlBPl with CDAl binding I 8mer j

16 I3mer amino acid sequence in human CDAl BPl with CDAl binding 8mer

I lmer amino acid sequence in human CDAlBPl with CDAl binding

8mer

9mer amino acid sequence in human CDAlBPl with C-terminal truncated CDAl binding 8mcr

Amino acid sequence of human CDAl BPl

20 Amino acid sequence of human CDA 1

21 Amino acid sequence of Tat48-60 SEQUENCE

DESCRIPTION ID NO:

22 Amino acid sequence of IsI-I

23 Amino acid sequence of SAP

24 CDAl BPl siRNA 278

25 6mer amino acid sequence in human CDAl BPl

26 lOmer amino acid sequence in human CDAl BPl

27 12mer amino acid sequence in human CDAl BPl

28 I Omer amino acid sequence in human CDAl BPl showing two amino acids critical for binding to CDAl (Xi-X ₆ IRX ₇ X ₈ )

29 Nucleic acid sequence of CDAl BPl

TABLE 2 Summary of amino acid abbreviations BRIEF DESCRIPTION OF THE FIGURES

Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office.

Figure 1 is a photographic (A) and graphical representation showing that CDA l expression is elevated in diabetic SHR rat kidney. A: Immunohistochcmistry staining for CDA l shows both nuclear and cytoplasm localizations of CDAl in tubule cells (left panel. red arrow head), in podocytes in glomerilus (central panel, green arrow), higher staining intensity in 32 weeks diabetic kidney than control (right panel). B: mRNΛ levels of CDA l in 8 and 32 weeks diabetic rat kidney and controls shown as arbitrary unit. SE (n-6-8) is shown as error bar. Asterisk indicates a statistically significant difference between the diabetic and control groups.

Figure 2 is a graphical representation of expression of genes associated with IiCM production in HK-2 cells transudced with control and CDAl -expressing adenoviruses.

Figure 3 is a graphical representation showing that CDAl was silenced by stable transduction with a retroviral construct expressing a hairpin-structured CDA l si RN A, which resulted in a -30% reduction in CDAl protein expression in a human kidney cell line (A). mRNA levels of CTGF and the HCM genes, fibronectin and various collagens. were dramatically decreased in response to CDAl inhibition (B)

Figure 4 is a graphical representation showing that TGF-β stimulated gene expression of collagen type I and III is enhanced by CDAl overcxpression (CDAl virus). I ^' GF-β action is attenuated by CDAl knockdown (CDAl siRNA).

Figure 5 is a diagrammatic representation of the role of CDAl in relation to TGFβ and the pro-fibrobic effect of diabetic nephropathy.

Figure 6 is a diagrammatic representation of 18 clones derived from the same gene with identical nucleic acid sequences and encoded amino acid sequences aligned to the C- terminus of CDAlBPl .

Figure 7 is a representation of the nucleic acid sequence of the longest clone of CDA l BP l FC78 is shown and numbered from 1 to 476. An open reading frame with corresponding amino acids beginning with Methionine was shown. An in-frame stop codon is shown (*) in the open reading frame.

Figure 8 is a representation of a database search for peptides that are similar to CDA l BP l only finds similarity to a hypothetical protein

Figure 9 is a diagrammatic representation of the interaction of CDAl BPl fragments w ith full length CDA l in plasmid pGBK-T7 was examined by mating the yeasts on SD/- 1 φ- Leu-Λde-His selection medium plates containing Kanamycin and X-α-gal.

Figure 10 is a diagrammatic representation of a yeast 2 hybrid study o^ CDA l Bi ³ I constructs. FC57- 1 and FC57-2 with C -terminal deletion of 6 and 12 amino acid residues, respectively, interact with CDAl ; FC57-3 and FC57-4. with deletion of C-terminal 1 8 and 24 residues, respectively, lose the ability to interact with CDAl .

Figure 11 is diagrammatic representation of point mutagenesis studies of CDA l binding region of CDAl BPl to better define critical determinants of binding motif.

Figure 12 is a diagrammatic representation of constructs containing various fragments of CDA l molecule were examined for their ability to bind to CDA l BPl by the yeast 2-hybrid system.

Figure 13 is a graphical representation that the limited pro-fibrotic effect of CDA l on collagen III gene expression is further enhanced by additional CDA l BP l .

Figure 14 is a graphical representation showing that si RNA knockdown of CDA l BP l resulted in decrease of gene expression of TGF-β by -60%. collagen I by -40% and collagen III by -70%.

Figure 15 is a photographic representation showing that CDAl and the receptor

CDAl BPl co-express in human kidney. A: Western blot showing CDAl and CDA l BP l

5 in human kidney (K), lung (L) and a human kidney proximal tubule cell line HK-2 (HK.2) infected by a Myc-tagged CDAl adenovirus. The blots were reprobed for house keeping proteins α-tubuline or GAPDH to show the loadings of total proteins. B:

Immunohistochemistry staining for CDAl showing CDAl in tubule cells (red arrow head) and in podocyte in glomerulus (green arrow). C: RT-PCR amplification of CDA l and

10 CDAl BPl from total RNA extracted from a human kidney tissue. 1 , RT-PCR; 2. No reverse transcriptase control.

Figure 16 is a photographic representation of Western blotting with anti-CDAl and anti- CDAl BPl . Co-incubation of CDAl BPl with a-fib 19mer, but not the control peptide (oil s fib 19mer mutant), inhibits binding of CDAlBPl to CDAl .

Figure 17 is a graphical representation of GST-CDAl (CDAl ) or GST-CDA l C-tcrminus fusion protein (200 ng per well) coated onto a 96-well ELlSA plate; recombinant GST- CDA l BPl protein or GST control protein expressed and purified from E. coli was added at 0 various concentrations to incubate with and to bind to the coated CDAl proteins. The bound CDAl BPl proteins were then detected by a specific antibody to the N-terminus of CDA l BPl followed by a HRP-conjugated secondary antibody and a colorimetric substrate solution. 5 Figure 18 is a graphical representation of an ELlSA format CDA1/CDABP1 binding inhibition assay: maltose binding protein-CDAl fusion protein (MBP-CDA l ) was coated on a 96-well plate. GST-CDAI BPl fusion protein and inhibiting or control peptides at various concentrations were co-incubated with the coated MBP-CDA l and the bound GS T-CDA I BPl was then detected by the specific CDAl BPl Abs.

30 DETAILED DESCRIPTION

Throughout this specification, unless the context requires otherwise, the word ''comprise ^"" . or variations such as "'comprises' ^" or "'comprising", are understood to imply the inclusion of a stated element or integer or step or group of elements or integers or steps but not the exclusion of any other element or integer or step or group of elements or integers or steps.

Λs used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a formulation" includes a single formulation, as well as two or more formulations: reference to "an agent" includes a single agent, as well as two or more agents; reference to "the invention" includes single or multiple aspects of the invention; and so forth.

The present invention is predicated in part on the identification of a therapeutic target to treat or reduce tissue fibrosis following disease- or trauma-mediated organopathy ^' 1 he target is cell division autoantigen 1 (CDAl -mediated signalling). This molecule is described in Chai el al 2001 supra. It is proposed that inhibiting CDAl -mediated signalling facilitates the clinical management of pro-fibrotic conditions arising from disease or injury.

Accordingly, one aspect of the present invention contemplates a method lor the treatment or prophylaxis of disease- or trauma-mediated organopathy leading to tissue fibrosis in a subject, the method comprising administering to the subject an amount of an agent which antagonizes CDAl -mediated signalling for a time and under conditions effective to reduce fibrosis.

Another aspect of the present invention provides a method for the treatment or prophylaxis of disease- or trauma-mediated organopathy leading to tissue fibrosis in a subject, the method comprising administering to the subject an amount of an agent which inhibits CDA l -mediated signalling by antagonizing interaction between CDAl and CDA l binding protein 1 (CDAl BPl ) for a time and under conditions effective to reduce fibrosis. Refcrence to "organopathy" includes any inflammatory condition arising from disease or trauma of tissue including organs and blood vessels. In one embodiment, the condition is diabetic nephropathy resulting in renal fibrosis. In another embodiment, the condition is hepatic organopathy resulting in fibrosis of the liver. In still another embodiment, the condition is scaring due to injury to the skin or internal organs. In yet a further embodiment, the condition is cardiovascular disease leading to or resulting from atherosclerosis.

Reference to "diabetic nephropathy" includes renal fibrosis and associated or related conditions arising from Type I or Type II diabetes including atherosclerosis. Reference to "hepatic organopathy" includes hepatic fibrosis arising following infection by a Hepatitis virus (e.g. Hepatitis B virus or Hepatitis C virus).

Accordingly, another aspect of the present invention provides a method for treating a subject with diabetes or who is at risk of developing same, the method comprising administering to the subject an amount of an agent which inhibits CDAl -mediated signalling for a time and under conditions effective to ameliorate symptoms of diabetic nephropathy leading to renal fibrosis and/or atherosclerosis.

Yet another aspect of the present invention relates a method for treating a subject with cardiovascular disease or who is at risk of developing same, the method comprising administering to the subject an amount of an agent which inhibits CDAl -mediated signalling for a time and under conditions effective to ameliorate symptoms of atherosclerosis.

Still a further aspect of the present invention is directed a method of reducing scar tissue following physical trauma or injury, the method comprising administering to the subject an amount of an agent which inhibits CDAl -mediated signalling for a time and under conditions effective to ameliorate symptoms of scaring.

In an embodiment, the agent has the binding specificity of or competes with CDA l - binding protein 1 (CDAlBPl) which binds to CDΛ1. The amino acid sequence of CDAl BPl is set forth as SEQ ID NO: 19. The present invention encompasses an isolated protein having the amino acid sequence set forth in SEQ ID NO: 19 or homologs or derivatives thereof having at least about 80% similarity to SEQ ID NO: 19 after optimal alignment. The present invention extends to derivatives and fragments of CDAl BP 1.

Accordingly, another aspect of the present invention provides a method for treating a subject with diabetes or who is at risk of developing same, the method comprising administering to the subject an amount of an agent which inhibits CDAl -mediated signalling by antagonizing interaction between CDAl and CDAl BPl for a time and under conditions effective to ameliorate symptoms of diabetic nephropathy leading to renal fibrosis and/or atherosclerosis.

Another aspect of the present invention relates a method for treating a subject w ith cardiovascular disease or who is at risk of developing same, the method comprising administering to the subject an amount of an agent which inhibits CDAl -mediated signalling by antagonizing interaction between CDAl and CDAl BPl for a time and under conditions effective to ameliorate symptoms of atherosclerosis.

Still a further aspect of the present invention is directed a method of reducing scar tissue following physical trauma or injury, the method comprising administering to the subject an amount of an agent which inhibits CDAl -mediated signalling by antagonizing interaction between CDAl and CDAl BPl for a time and under conditions effective to ameliorate symptoms of scaring

In particular, the agent has the binding specificity (or competes for binding to) of the C- terminal end portion of CDAl BPl which comprises the amino acid sequence LTlSTI IR, using single amino acid code. This sequence defines the portion of CDA l BPl which is the critical binding determinant to CDAl . The 8 amino acids are located in the C-terminus of CDAl BPl . Reference to "at least about 80%" includes 80. 81. 82, 83, 84. 85. 86. 87, 88. 89, 90, 91 , 92, 93, 94. 95, 96, 97, 98, 99 and 100%. Hence. the present invention contemplates a method for treating a subject with disease- or trauma-mediated organopathy leading to tissue fibrosis or who is at risk of developing same, the method comprising administering to the subject an amount of an agent which competes for binding of the C-terminal end portion of CDAl BPl which comprises the amino acid sequence LTISTIIR (SEQ ID NO: 1 ) to CDAl , which agent antagonizes CDA l -mediated signalling, for a time and under conditions effective to ameliorate symptoms of tissue fibrosis.

Another aspect of the present invention provides a method for the treatment or prophylaxis of diabetic nephropathy in a subject, the method comprising administering to the subject an amount of an agent which competes for binding of the C-terminal end portion of CDA l BPl which comprises the amino acid sequence LTISTIIR (SEQ ID NO: 1 ) to CDΛ 1 . which agent antagonizes CDA 1 -mediated signalling, for a time and under conditions to ameliorate symptoms of renal fibrosis and/or atherosclerosis.

Still another aspect of the present invention contemplates a method for the treatment or prophylaxis of hepatic organopathy in a subject, the method comprising administering to the subject an amount of an agent which competes for binding of the C-terminal end portion of CDAl BP l which comprises the amino acid sequence L fISTlIR (SEQ ID NO: 1 ) to CDAl , which agent antagonizes CDAl -mediated signalling, for a time and under conditions to ameliorate symptoms of hepatic fibrosis.

Similar methods are provided to treat or prevent cardiovascular disease an to reduce caring following tissue trauma or injury. The term "tissue trauma" includes surgery.

In an embodiment, the agent is a peptide comprising the amino acid sequence Xi-X ₆ IR or its reverse form Rl X ₆ -Xi wherein X)-X ₆ are each an L- or D-amino acid residue or an unnatural amino acid residue or an amino acid analog or is an amino acid residue having a hydrogen atom substituted for a deuterium atom or a peptide derivative or mimetic thereof. In a related aspect, the sequence comprises Xi-X ₆ RIX ₇ X ₈ wheren Xi-Xs are each an L- or D-amino acid residue or an unnatural amino acid residue or an amino acid analog or is an amino acid residue having a hydrogen atom substituted for a deuterium atom or a peptide derivative or mimetic thereof. A "peptide derivative" includes peptides with an acetylated N-tcrminus and/or an amidated C-terminus or is a cyclic form of the peptide. Λ he derivative may also comprise a chimeric molecule having a peptide portion having the binding specificity of or which competes for binding with the C-terminal end portion of CDAl BPl which comprises the amino acid sequence LTISTIIR (SEQ ID NO: 1 ) and a cell penetrating peptide (CPP) portion. The two portions may also be separated by a cleavablc linker such as a disulfide bond. As indicated above, an inverted sequence such as RIX ₆ -X] is also contemplated herein wherein Xi-X ₆ are as defined above. In a particular embodiment, the peptide inverted sequence comprises the amino acid sequence RI l I Sl I L or is a peptide derivative or mimetic thereof, as defined above.

Hence, the present invention provides an agent for use in the treatment or prophylaxis of tissue fibrosis in a subject, the agent being an antagonist of CDA l -mediated signalling wherein the antagonist competes for binding of the C-terminal portion of CAl BP l which comprises the amino acid sequence LTISTIIR (SEQ ID NO: 1) to CDAl .

More particularly, the agent is for use in the treatment or prophylaxis of diabetic nephropathy, the agent being an antagonist of CDAl -mediated signalling wherein the antagonist competes for binding of the C-terminal portion of CDAlBP l which comprises the amino acid sequence LTISTIIR (SEQ ID NO: 1 ) to CDAl .

As indicated above, diabetic nephropathy includes renal fibrosis.

Yet another aspect provides an agent for use in the treatment of cardiovascular disease, the agent being an antagonist of CDAl -mediated signalling wherein the antagonist competes for binding of the C-terminal portion of CDA l BPl which comprises the amino acid sequence Ll ISTIIR (SEQ ID NO: 1 ) to CDAl .

fhe present invention further provides an agent comprising the amino acid sequence selected from Xi-X ₆ IR and RlX ₆ -Xi, wherein each of X]-X ₆ is an L- or D-amino acid or amino acid analog or an unnatural amino acid wherein the agent competes for binding of the C-terminal portion of CDAlBPl comprising the amino acid sequence LTIS fIlR to CDΛ 1 or a derivative or mimetic of the peptide.

The present invention further provides an agent comprising amino acid sequence selected from X]-X ₆ RIX ₇ X ₈ and X ₈ XvRIX ₆ -Xi, wherein each of Xi-X ₈ is an L- or D-amino acid or amino acid analog or an unnatural amino acid wherein the agent competes for binding of the C-terminal portion of CDAlBPl comprising the amino acid sequence LTIS FIlR to CDΛ 1 or a derivative or mimetic of the peptide.

Hence, the agents contemplated herein are proposed in the treatment, prophylaxis and/or amelioration of symptoms of:

(i) diabetic nephropathy leading to renal fibrosis;

(ii) diabetic nephropathy leading to atherosclerosis;

(iii) cardiovascular disease leading to or resulting from atherosclerosis;

(iv) hepatic organopathy leading to liver fibrosis; and/or (v) scar tissue formation following physical trauma or injury.

Disease conditions contemplated herein include Type I and/or Type II diabetes, Hepatitis infection (B and/or C. chronic or acute) and cardiovascular disease.

In an embodiment, the treatment or prophylaxsis is effected by an agent which antagonizes interaction between CDAl and CDAl BPl thereby inhibiting CDΛl -mediated signalling.

In a particular embodiment, the agent antagonizes interaction between the C-terminal end region of CDAl BP l and CDAl . Even more particularly, the present invention is directed to an isolated peptide comprising the amino acid sequence LTISTlIR (SRQ ID NO. l ) or a derivative or mimetic thereof. The peptide includes a non-full length CDA l BP l protein or a derivative of CDA l BPl protein which binds to CDA l but does not induce Cl)A l signalling. For example, the peptide may be a C-terminal end fragment comprising from about 8 to about 50 amino acids in length such as 8, 9, 10. 1 1. 12, 13, 14, 15, 16. 1 7. 1 8. 19, 20, 21 , 22. 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38. 39, 40. 41 . 42. 43, 44, 45, 46, 47, 48, 49 or 50 embodiments. As indicated above, derivatives of peptides contemplated herein include peptides with an acetylated N-terminus and/or an amidated C-terminus, cyclized peptides and chimeric molecules comprising a CPP portion. Derivatives also include amino acid residues having deuterium atoms substituted for hydrogen atoms.

Other derivatives include side chain modifications of the amino acid residues. Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH ₄ ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH ₄ .

The guanidinc group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedionc. phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride. 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with Ni- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobcnzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by 5 alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids, amino acids and derivatives and amino acid analogs during peptide synthesis include, but are not limited to, use of norlcucine. 4- I O amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohcxanoic acid. ι- butylglycine, norvaline, phenylglycine, ornithine, sarcosine. 4-amino-3-hydroxy-6- mcthylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids, amino acid derivatives and amino acid analogs contemplated herein is shown in Table 3.

TABLE 3 Codes for non-conventional amino acids

Non-conventional Code Non-conventional Code amino acid amino acid α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylargininc Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohcxylalanine Chexa L-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Ninmct

D-cystcine Dcys L-N-methylnorleucine Nmnlc

D-glutamine DgIn L-N-methylnorvalinc Nmnva

D-glutamic acid DgIu L-N-methylornithine N morn

D-histidine Dhis L-N-methylphenylalanine Nmphc

D-isoleucine DiIe L-N-methylproline Nmpro

D-lcucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methioninc Dmet L-N-methyltryptophan Nmlrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmctg

D- serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine NIc

D-tryptophan Dtrp L-norvalinc Nva

D-tyrosine Dtyr α-methyl-aminoisobυtyrate Maib

D-valine Dval α-methyl-γ-aminobutyrate Mgabu

D-α-methylalanine Dmala α-methylcyclohexylalanine Mchcxa

D-α-methylargininc Dmarg α-methylcylcopentylalanine Mcpcn

D-α-mcthylasparaginc Dmasn α-methyl-α-napthylalanine Manap

D-α-mcthylaspartatc Dmasp α-methylpcnicillamine M pen

D-α-methylcysteine Dmcys N-(4-aminobutyl)g]ycine NgIu

D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-α-mcthylhistidine Dmhis N-(3-aminopropyl)glycine Norn

D-α-melhylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu

D-α-methylleucinc Dmleu α-napthylalanine Λnap Non-conventional Code Non-conventional Code amino acid amino acid

D-α-mcthyllysine Dmlys N-benzylglycine Nphe

D-α-mcthylmethionine Dmmet N-(2-carbamylethyl)glycine NgIn

D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-α-methylphenylalanine Dmphe N-(2-carboxyethy])glycine NgIu

D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp

D-α-methylserine Dmscr N-cyclobutylglycine Ncbut

D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep

D-α-methyltryptophan Dmtrp N-cyclohexylglycinc Nchcx

D-α-methyltyrosinc Dmty N-cyclodccylglycine Ncdcc

D-oc-methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-methylalaninc Dnmala N-cyclooctylglycine Ncoct

D-N-methy largininc Dnmarg N-cyclopropylglycine N c pro

D-N-methylasparagine Dnmasn N-cycloundecylglycinc Ncund

D-N-methy [aspartate Dnmasp N-(2,2-diphenylethyl)glycinc Nbhm

D-N-methylcysteinc Dnmcys N-(3,3-diphenylpropyl)glycinc Nbhc

D-N-methylglutaminc Dnmgln N-(3-guanidinopropyl)glycine Narg

D-N-methy Iglutamate Dnmglu N-(I -hydroxyethyl)glycine Nlhr

D-N-methylhistidinc Dnmhis N-(hydroxyethyl))glycine Nscr

D-N-mcthylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis

D-N-methy!leucinc Dnmleu N-(3-indolylyethyl)glycine Nhtrp

D-N-methyllysinc Dnmlys N-methyl-γ-aminobutyrate Nmgabu

N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dn mm ct

D-N-methylornithine Dnmorn N-mcthylcyclopentylalanine Nnicpeπ

N-methylglycine NaIa D-N-methylphenylalanine Dnmphe

N-methylaminoisobutyrate Nmaib D-N-methylproline Dn m pi o

N-( 1 -methylpropyl)glycine Nile D-N-methylserinc Dnmscr

N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtrp N-(l -methylethyl)glycine Nval

D-N-methy ltyrosinc Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-mcthylpenicillaminc Nmpcn γ-aminobutyric acid Gabu N-(/>hydro\yphenyl)glycine Nhtyr

L-/-butylglycinc Tbug N-(thiomcthyl)glycinc Ncys

L-ethylglycine Et ₈ penicillamine Pen

L-homophenylalanine Hphe L-α-methylalanine Mala

L-α-methylargininc Marg L-α-methylasparaginc Masn

L-α-methylaspartate Masp L-α-methyl-/-butylglycine Mtbug

L-α-methylcysteine Mcys L-methylethylglycine Metg

L-α-mcthylglutamine MgIn L-α-methylglutamate MgIu

L-α-methylhistidine Mhis L-α-methylhomophenylalanine M hphe

L-α-mcthylisolcucine Mile N-(2-methylthioethyl)glycine Nmct Non-conventional Code Non-conventional Code amino acid amino acid

L-α-methylleucine Mleu L-α-methyllysine Mlys

L-α-mcthylmethionine Mmet L-α-methylnorleucinc MnIc

L-α-mcthylnorvaline Mnva L-α-methylornithine Morn

L-α-mcthylphenylalanine Mphe L-α-methylproline M pro

L-α-methylscrine Mser L-α-methylthreonine Mthr

L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr

L-α-mcthylvaline Mval L-N-methylhomophenylalanine Nmhphe

N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhc carbamylmethyl)glycine carbamylmethyl)glycine

1 -carboxy- 1 -(2,2-diphenyl- Nmbc ethylamino)cyclopropane

Crosslinkers can be used, for example, to stabilize 3D conformations or to cycii/c peptides, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CH ₂ )n spacer groups with n = 1 to n = 6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N- hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformational Iy constrained by, for example, incorporation of C _n and N _α -methylamino acids, introduction of double bonds between C _n and Cp atoms of amino acids and the formation of cyclic peptides or analogs by introducing covalcnt bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

The present invention extends to mimetics of the anti-fibrotic peptides.

Mimctics contemplated herein modulate CDA l -mediated signalling. The term is intended to refer to a substance which has some chemical similarity to the molecule it mimics but which antagonizes its interaction with a target (i.e. CDAl which has the amino acid sequence set forth in SEQ ID NO:20). A peptide mimetic may be a peptide-containing molecule that mimics elements of protein secondary structure (Johnson el cil. Peptide Turn Mimetics in Biotechnology and Pharmacy, Pezzuto et al. Eds., Chapman and Hall, New York, 1993) corresponding to, for example, a region on CDAlBPl such as but not limited to the LTISTIIR (SFiQ ID NO: 1 ) sequence. The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions such as those of antibody and antigen, enzyme and substrate or scaffolding proteins. A peptide mimetic, therefore, is designed to permit molecular interactions similar to the natural molecule.

The present invention is further directed to the use of an agent which antagonizes CDA l - mediated signalling, the agent competing for binding between CDAl BPl and CDA l in the manufacture of a medicament for the treatment or prophylaxis of tissue fibrosis.

More particularly, the present invention provides for the use of an agent which antagonizes CDA l -mediated signalling, the agent competing for binding between CDA l BP l and CDAl in the manufacture of a medicament for the treatment or prophylaxis of diabetic nephropathy.

In an embodiment, the agent antagonizes interaction between the C-terminal end portion of CDA l BPl and CDAl .

Hence, the present invention is further directed to the use of an agent which antagonizes CDAl -mediated signalling, the agent competing for binding of the C-terminal portion of CDAl BPl which comprises the amino acid sequence LTISTIIR to CDAl in the manufacture of a medicament for the treatment or prophylaxis of tissue fibrosis.

More particularly, the present invention provides for the use of an agent which antagonizes CDA l -mediated signalling, the agent competing for binding of the C-terminal portion of CDA l BPl which comprises the amino acid sequence LTlS TlIR to CDA l in the manufacture of a medicament for the treatment or prophylaxis of diabetic nephropathy.

In an alternative embodiment, the present invention is directed to the use of an agent which antagonizes CDA l-mediated signalling, the agent competing for binding of the C-terminal portion of CDAl BP l which comprises the amino acid sequence LTISTlIR to CDA l in the manufacture of a medicament for the treatment or prophylaxis of a disease condition selected from Type I and/or Type II diabetes, Hepatitis infection, cardiovascular disease and physical trauma or injury to tissue.

In an embodiment, the agent is a peptide comprising the amino acid sequence:

X ₁ -X ₆ IR wherein Xi-X ₆ are each an L- or D-amino acid residue or an unnatural amino acid residue or amino acid analog or a derivative or mimetic of the peptide.

In a related embodiment, the agent is a peptide comprising the amino acid sequence: Xi- X ₆ RIX ₇ Xg, wherein Xi-X ₈ are each an L- or D-amino acid residue or an unnatural amino acid residue or amino acid analog or a derivative or mimetic of the peptide.

Examples of peptide derivatives include amino acid residues having hydrogen atoms substituted for deuterium atoms, cyclized peptides, inverted and reversed peptides and peptides with an acetylated N-terminus and/or an amidated C-terminus. Chimeric peptides having a carrier portion such as a cell penetrating peptide (CPP) are also contemplated herein.

In a particular embodiment, the agent is a peptide comprising the amino acid sequence L fISTIlR (SHQ ID NO: 1) or a derivative or mimetic of the peptide. This embodiment encompasses, therefore, native and modified peptides.

The peptide includes a soluble form of CDAl BPl which binds to CDΛ1 but docs not induce CDAl signalling. Hence, the present invention provides an isolated derivative of CDA l HPl which comprises an amino acid sequence having at least 80% similarity to Sl-Q ID NO: 19 after optimal alignment wherein the derivative competes with CDA l BP l for binding to CDAl but does not induce CDA l -mediated signalling.

Reference to "at least 80%" in this context includes at least 80. 81. 82, 83, 84, 85. 86. 87. 88, 89, 90. 91 , 92. 93. 94, 95, 96, 97, 98 and 99%. CDAl BPl was identified following yeast two hybrid screening and its amino acid sequence as set forth in SEQ ID NO: 19. The 8mer amino acid sequence LiT ₂ I ₃ S ₄ T^I ₆ I ₇ R _X (SEQ ID NO: 1 ), is in its C-terminal end region. The residues I7R. ₈ are critical for binding to CDAl . Agents which have the binding specificity of the C-terminal end comprising LTISTlIR or which compete with such a C-terminal end for binding to CDA l arc contemplated herein. By "compete" means that the agent is able to bind to CDA l in exclusion of CDAl BPl . This therefore reduces CDAlBPl binding and stimulation of CDA l .

The present invention extends to the generation of immunoglobulin molecules which selectively inhibit CDA l -mediated signalling via CDA l BPl

The immunoglobulin molecules may be directed to CDAl or CDAl BPl and inhibits interaction between CDAlBPl and CDAl . More particularly, the immunoglobulin disrupts the binding of the C-terminal end portion of CDAl BPl to CDAl and thereby prevents CDA l -mediated signalling.

Accordingly, one aspect of the present invention is directed to an isolated immunoglobulin molecule which selectively inhibits binding between CDAl BPl and CDAl wherein the inhibition of binding prevents or reduces CDAl -mediated signalling.

The present invention further provides an isolated immunoglobulin or a dcimmuni/ed, humanized or chimeric form thereof or functional equivalent or binding equivalent thereof which inhibits binding of CDAI BP l to CDA l .

More particularly, the present invention is directed to an isolated immunoglobulin molecule which selectively inhibits binding between the C-terminal end portion of CDAl BPl to CDA l wherein the C-terminal end portion comprises the amino acid sequence LTISTIIR and wherein the inhibition of binding prevents or reduces CDA l - mediated signalling.

The present invention further provides an isolated immunoglobulin or a deimmunized. humanized or chimeric form thereof or functional equivalent or binding equivalent thereof which inhibits binding of the C-terminal end portion of CDAlBPl to CDAl .

A "functional equivalent" or "binding equivalent" of the immunoglobulin includes a cartilage vertebrate marine animal-derived immunoglobulin new antigen receptor (IgNAR ) and a catalytic antibody.

An IgNAR is an antibody isotype found only in cartilaginous vertebrate marine animals such as sharks and rays (Greenberg et al. Nature 374: 168-173, 1995; Nuttall el al. MoI Immunol 38:313-326. 2001). IgNAR's are bivalent, but target antigen through a single immunoglobulin variable domain (~14kDa) displaying two complementarity determining region (CDR) loops attached to varying numbers of constant domains (Nuttall el al. Eur J Biochem 270:3543-3554, 2003; Roux et al. Proc Natl Acad Sc i USA 95: 1 1804- 1 1 80'). 1998).

Reference to a "cartilaginous marine animal" includes a member of the families of shark and ray. Reference to a "shark" includes a member ot order Squatini formes. Pristiophoriformes, Squaliformes, Carcharinformes, Laminilbrmes, Orectolobi formes, Heterodonti formes and Hexanchieformes. Whilst not intending to limit the shark to any one genus, immunoglobulins from genus Orectolobus are particularly useful and include the bamboo shark, zebra shark, blind shark, whale shark, nurse shark and Wobbegong. Immunoglobulins from Orectolobus maculates (Wobbegong) are exemplified herein.

With respect to these embodiments, the immunoglobulin may also be referred to as an antibody. The term "immunoglobulin" or "antibody" includes full length molecules as well as fragments thereof and synthetic, recombinant, chimeric and modified forms thereof as well as functional equivalents or binding equivalents.

Accordingly, the term "'immunoglobulin" is used herein to refer to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized immunoglobulin genes include the K. λ, α, γ (IgGi, IgG ₂ , IgG ₃ . IgG ₄ ), δ, ε and μ constant region genes, as well as the myriad of other immunoglobulin variable region genes. In addition to antibodies, immunoglobulins may exist in a variety of other forms including, for example. Fv, scFv, Fab, Fab' and (Fab') ₂ . Immunoglobulins may be polyclonal or monoclonal antibodies. The present invention extends to all forms of immunoglobulins such as catalytic antibodies, synthetic antibodies, chimeric antibodies, dcimmuni/cd antibodies and fragments of antibodies.

The immunoglobulins of the present invention are generally generated following immunization. In relation to monoclonal antibodies, immunization and subsequent production of monoclonal antibodies may be done using any methods known to those of skill in the art. For examples see: Kohler and Milstein Nature 256:495-499, 1975: Kδhler and Milstein. Eur J Immunol (5:51 1 -519, 1976; Coligan et ul Current Protocols in Immunology. John Wiley & Sons, Inc, 1991-1997 or Toyama et al. Monoclonal Antibody, Experiment Manual, published by Kodansha Scientific, 1987.

In relation to monoclonal antibodies, the present invention further extends to hybridomas or other cells which produce monoclonal antibodies which selectively inhibit the binding of CDAl BPl to CDA l , such as the C-terminal end portion of CDAl BPl to CDA l . Where single chain antibodies are produced, these may be generated from recombinant yeast, bacterial (e.g. E coli). insect cells or plants.

The subject invention extends, therefore, to antibodies from any source and dcimmuni/cd or mammalianized for use in any host. Examples of animal sources and hosts include, but are not limited to. humans and non-human primates (e.g. guerilla, macaque, marmoset), livestock animals (e.g. sheep, cow, horse, camel, donkey, pig), companion animals (e.g. dog. cat), laboratory test animals (e.g. mouse, rabbit, rat, guinea pig, hamster) and captive wild animals (e.g. fox. deer). The deimmunized antibodies or part thereof may also be generated in non-animal sources, such as but not limited to. plants. In this regard, plants are particularly useful as a source of "'plantibodies" such as single chain antibodies. Other non-animal cells include bacteria, yeast and insect cells.

The agents of the present invention may be formulated with one or more pharmaceutically acceptable carriers, diluents and/or excipients. Furthermore, the formulations may comprisc a preservative and microbial retardants including EDTA. benzyl alcohol, bisυlfites, monoglyceryl ester of lauric acid (Monolaurin), and a member selected from (a) capric acid and/or its soluble alkaline salts or its monoglyceryl ester (Monocaprin). (b) cdetate, and (c) capric acid and/or its soluble alkaline salts or its monoglyceryl ester (Monocaprin) and edetate.

The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral (including intravenous) formulations. In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavouring agents. preservatives, coloring agents and the like in the case of oral liquid preparations, such as. for example, suspensions, elixirs and solutions; or carriers such as starches, sugars. microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations such as, for example, powders, capsules and tablets, with the solid oral preparations being preferred over the liquid preparations. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques.

Pharmaceutical compositions of the present invention used for anti-fibrotic therapy suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil- in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both. and then, if necessary, shaping the product into the desired presentation. For example, a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine. the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.

The components and/or extracts identified of the present invention may be administered orally, parenterally (including subcutaneous injections, intravenous, intramuscular, intrastcrnal injection or infusion techniques), by inhalation spray, or rectally, in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers. adjuvants and vehicles.

When administered by nasal aerosol or inhalation, these compositions arc prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing ben/yl alcohol or other suitable preservatives. absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubili/ing or dispersing agents known in the art.

fhe anti-fibrotic agents identified by the present invention may also be administered m intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinan skill in the pharmaceutical arts. When administered by injection, the injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parentcrally-acceptable diluents or solvents, such as mannitol, 1 ,3-butancdiol. water. Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting antl suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or di- glycerides, and fatty acids, including oleic acid.

When rectally administered in the form of suppositories, these compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter. synthetic glyccridc esters or polyethylene glycols, which are solid at ordinars temperatures, but liquidity and/or dissolve in the rectal cavity to release the drug. The effective dosage of the agents employed in therapy may vary depending on the particular compound employed, the mode of administration, the condition being treated and the severity of the condition being treated. Thus, the dosage regimen utili/ing the compounds of the present invention is selected in accordance with a variety of factors including type, age, weight, sex and medical condition of the patient; the severity oϊ the condition to be treated; the route of administration; the renal and hepatic function of the patient: and the particular compound thereof employed. A physician, clinician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving plasma concentration of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. T his involves a consideration of the distribution, equilibrium, and elimination of a drug. The present invention contemplates effective plasma concentrations from about 0.1 μM to l OOμM including about 0.1. 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8. 0.9, 10, 1 1 , 1 2. 13. 14, 15, 16, 17. 18, 19. 20, 21 , 22, 23. 24, 25, 26, 27, 28, 29, 30. 31, 32. 33, 34, 35. 36. 37. 38, 39, 40. 41 , 42, 43. 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54. 55, 56. 57. 58, 59. 60. 61. 62. 63. 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73. 74. 75, 76, 77, 78. 79, 80. 81. 82. 83. 84. 85. 86. 87. 88, 89, 90, 91. 92, 93, 94, 95, 96, 97, 98, 99 and l OOμM.

The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e.. salts that retain the desired biological activity of the parent compound and do not impart undcsired toxicological effects thereto.

The present invention also includes pharmaceutical compositions and formulations which include genetic molecules which encode peptide or protein agents which inhibit CDA l - mcdiatcd signalling The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration of oligonucleotides may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral Parcnteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2'-O-methoxyethyl modification arc considered to be useful for oral administration Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable

The present invention also extends to other genetic approaches to down-regulate ClM l and/or CDA l BPl . T he gentic approach includes the use of siRNΛ. dsRNA. ssRNΛ, hairpin RNΛ. looped and folded RNA, sense suppression, antisensc suppression as vscll as ribo/ymcs, dicer complexes and RNAi molecules and complexes. The genetic molecules including phosphodiester linkages may be chemically modified to enhance serum half-life or to enhance cell penetration. The genetic molecules may also be native nucleic acid molecules or expressed as DNA-derived RNΛ molecules such as part of a viral vcctoi DNA molecules comprising RNA-encoding sequences operably linked to a promoter (constitutive or inducible) may also be employed. Alternatively, the nucleic acid molecules are chemically modified as described above. This is particularly useful for synthetic RNAi and siRNΛ molecules.

Accordingly, the present invention contemplates a method for the treatment or prophylaxis of tissue fibrosis in a subject, the method comprising the administration of a genetic molecule or chemically modified form thereof which specifically or selectively down- regulates expression of CDAl and/or CDAl BPl .

In a particular embodiment, the genetic molecule is an siRNA or RNAi molecule, DN Λ. derived or synthetic.

Still another aspect of the present invention provides a method for the treatment or prophylaxis of tissue fibrosis in a subject, the method comprising the administration o[ a genetic molecule which encodes a peptide which inhibits CDA l -mediated signalling by antagonizing interaction between CDAl and CDAl BPl for a time and under conditions effective to inhibit tissue fibrosis.

Still another aspect of the present invention provides a method for the treatment or prophylaxis of tissue fibrosis in a subject, the method comprising the administration of a genetic molecule which encodes a peptide which inhibits CDAl -mediated signalling by antagonizing interaction between CDAl and the C-terminal end portion of CDAl BPl for a time and under conditions sufficient to inhibit tissue fibrosis.

Yet even another aspect of present invention relates to a method for the treatment or prophylaxis of tissue fibrosis in a subject, the method comprising the administration of a genetic molecule which encodes a peptide which inhibits CDAl -mediated signalling by antagonizing interaction between CDAl and the C-terminal end portion of CDA l BP l which C-terminal end portion comprising the amino acid sequence: X ₁ -X ₆ IR; or

RlX ₆ -Xi, or X ₁ -X ₆ RlX ₇ X ₈ Or X ₈ X ₇ RIX ₆ -X, wherein Xi-X ₈ are each an L-amino acid residue, for a time and under conditions sufficient to inhibit tissue fibrosis.

As indicated above, condition include diabetic nephropathy, cardiovascular disease. Hepatitis infection, atherosclerosis and tissue scaring. Genetic molecules are as defined above and include siRNA's and RNATs.

The agents of the present invention may be used alone or in combination with one or more other medicaments such as an agent which inhibits TGFβ-mediated signalling or immune function. In addition, the construct includes the agent which inhibits CDAl -mediated signalling and one of insulin or an anti-Hepatitis virus agent (e.g. a nucleoside or nucleotide analog) or a compound to treat scarring. Λccordingly, the administration of the CDAl -mediated signalling antagonists may be part of a broader clinical management protocol for the treatment or prophylaxis of tissue fibrotic conditions such as renal fibrosis involved in diabetic nephropathy, atherosclerosis or hepatic organopathy following infection by a Hepatitis virus or tissue scaring following physical trauma or injury.

Hence, the present invention provides anti-fibrotic formulations comprising: (a) a peptide having the amino acid sequence: RlX ₆ -X,, or X ₈ X ₇ RlX ₆ -X, wherein Xi-Xx are each any L- or D-amino acid residue or an amino acid analog or unnatural amino acid or derivative or mimetic of the peptide; and (b) a pharmaceutically acceptable carrier, excipient or diluent.

The formulation may further comprise additional active agents such as insulin, a cytokine or antagonist thereof, a sedative, an antibiotic or a nucleoside or nucleotide analog.

The anti-fibrotic formulations defined herein arc proposed for use in a range of conditions such as but not limited to Type I or Type II diabetes, cardiovascular disease, infection by pathogenic agents such as Hepatitis viruses and physical trauma of tissue material. Λs indicated above, physical trauma includes accidental injury or surgical or medical intervention.

The formulations here encompass native peptides such as peptides comprising the amino acid sequence LTISTIIR or a derivative, analog or mimetic thereof. Such derivatives, analogs and mimetics include: cyclised peptides, chimeric peptides, deuterated peptides (where an amino acid residue comprises a deuterium atom in place of a hydrogen atom), reverse and/or inverted peptides (RIlTSlTL) and peptides with an acetylated N-terminus and/or amidated C-terminus. The terms "agent", "reagent", "compound", "pharmacologically active agent", "medicament", "therapeutic", "active" and "drug" are used interchangeably herein to refer to a chemical or biological entity which induces or exhibits a desired effect such as inhibiting or antagonizing CDAl -mediated signalling and ameliorating the effects of tissue fibrosis. Terms such as "CDAl signalling antagonist" and "anti-fibrotic agent" may also be used. The terms also encompass pharmaceutically acceptable and pharmacologically active ingredients of those active agents specifically mentioned herein. The terms "agent", "reagent", "compound", "pharmacologically active agent", "medicament", "therapeutic", "active" and "drug" includes active entity per se as well as pharmaceutically acceptable derivatives, such as esters, amides, pro-drugs, metabolites, analogs, etc as well as pharmacologically acceptable salts thereof.

Reference to an "agent", "chemical agent", "compound", "pharmacologically active agent". "medicament", "therapeutic", "active" and "drug" includes combinations of two or more active agents. A "combination" also includes multi-part such as a two-part composition where the agents are provided separately and given or dispensed separately or admixed together prior to dispensation. For example, a multi-part pharmaceutical pack may have two or more agents separately maintained. Hence, this aspect of the present invention includes combination therapy. Combination therapy includes the co-administration ol the CDAl antagonist and another active such as insulin or a CDAl antagonist and a nucleotide or nucleoside analog or interferon.

Although the present invention is particularly useful for treating tissue fibrosis such as renal fibrosis arising from diabetic nephropathy, hepatic organopathy. atherosclerosis and/or physical trauma or injury in human subjects, the agents of the present invention also have veterinary applications. A subject regardless of whether a human or non-human animal may be referred to as an individual, patient, animal, host or recipient. The agents and methods of the present invention have applications in human medicine, veterinary medicine as well as in general, domestic or wild animal husbandry. For convenience, an "animal ^" includes an avian species such as a poultry bird (including ducks, chicken. turkeys and geese), an aviary bird or game bird. The condition in a non-human animal may not be a naturally occurring but induced such as in an animal model. Particularly useful subjects include humans, non-human primates such as marmosets, baboons, orangutangs, lower primates such as tupia, livestock animals, laboratory test animals, companion animals or captive wild animals. A human is a particular subject. However, non-human animal models may be used.

Examples of laboratory test animals include mice, rats, rabbits, guinea pigs and hamsters. Rabbits and rodent animals, such as rats and mice, provide a convenient test system or animal model as do primates and lower primates. Livestock animals include sheep, cows. pigs, goats, horses and donkeys. Non-mammalian animals such as avian species, /ebraiϊsh. amphibians (including cane toads) and Drosophila species such as Drosυphila melcmogaster are also contemplated. Instead of a live animal model, a test system may also comprise a tissue culture system.

The present invention is further described by the following non-limiting Examples.

EXAMPLE 1 CDAl expression levels increase at sites of diabetes related end-organ injury

CDAl is present and its gene and protein expression increased at sites of injury in Type I diabetes animal models. Furthermore, CDAl levels correlated with the degree of organ extracellular matrix (ECM) accumulation in the setting of diabetes in two different models. First, in the diabetic spontaneous hypertensive rat (SHR) model which displays functional and structural characteristics with significant similarities to human diabetic ncphropath\ CDA l protein was shown by immunohistochemistry in proximal and distal tubular cells. Strong staining of CDAl is also seen in the podocytes within the glomerulus. The staining intensity of CDA l in diabetic kidney (Diabetic 32 wks) was significantly higher than in control rats (Normal 32 wks). CDAl mRNA levels were significantly increased at least V fold in diabetic SHR kidneys after 32 weeks of diabetes, but not in the animals with shorter-term diabetes (8 weeks). Secondly, in a model of diabetes-associated atherosclerosis it was found that CDAl mRNA levels were approximately 5-fold higher and that CDAl strongly stained in atherosclerotic plaques in the diabetic group in association with increased expression of TGF-β and CTGF as well as an increased collagen deposition (Figure 1 ).

EXAMPLE 2 CDA l overexpression causes increase in collagen production in human renal cells

CDA l expressing adenovirus delivered a ~ 14-fold increase in CDAl mRNA expression levels in a human renal proximal tubule cell line, HK-2, which robustly increased gene expression of collagen type 1 and III by >40- and >70-fold, respectively (Figure 2).

Furthermore, this overexpression also caused significant increase in connective tissue growth factor (CTGF), a profibrotic growth factor in diabetic nephropathy, as well as other extracellular matrix genes including collagen IV and fibronectin. CDAl has no effect on PGF-β. being consistent with CDAl being a downstream molecule of TGF-β. EXAMPLE 3

CDAl knockdown by siRNA results in decrease in CTGF and ECM gene expressions in human renal cells

To demonstrate a physiological role for CDAl in regulating extracellular matrix genes. CDAl was silenced by stable transduction with a retroviral construct expressing a hairpin- structured CDAl siRNA, which resulted in CDAl protein expression reduction to 30% in a human kidney cell line. mRNA levels of CTGF and the ECM genes, fibronectin and various collagens, were dramatically decreased in response to CDAl inhibition (Figure 3 ). The same effect of CDAl knockdown using a different siRNA species was also observed in mouse vascular smooth muscle cells. Thus, these data demonstrate that CDA l is an upstream regulator of genes involved in ECM production.

EXAMPLE 4 CDA J synergistically enhances the proβbrotic effect of TGF-β in human renal cells

Increased CDAl leλ cls by infecting cells with a CDA l expressing adenovirus is associated with enhanced gene expression of collagen I and III in the presence of TGF-β. l GF-β alone caused a ~3-fold increase in collagen 1 and III (Figure 4: HK-2 and Con virus, red bar). CDAl overexpression alone (4-fold) also moderately increased >2 fold collagen expression (CDAl virus, black bar). However, in the cells with increased CDΛ 1 expression, TGF-β stimulated collagen I and III expression by >7-fold. This is particularly relevant in the diabetic context where there is overexpression of both TGF-β and CDΛ 1 .

EXAMPLE 5

Targeting CDA l by siRNA significantly attenuates TG F-β's proβbrotic action in human renal cells

Reducing CDAl levels by 70% via an adenovirus-delivered CDA l siRNA is not only associated with markedly decreased basal levels of collagen I and III. and also significantly attenuated TGF-β ^" s action in stimulating these ECM genes. These findings are critical for the underlying rationale of the present invention on which targeting CDΛ1 is proposed to lead to reduced ECM production and accumulation in disorders such as diabetic nephropathy and hepatic organopathy (Figures 3 and 4).

EXAMPLE 6

Model for CDAl in promoting ECM production and strategy to target the pathological action of CDAl

Based on the data in Examples 1 to 5, a model (Fig 5) is proposed in which TGl' -β activates TβRII via receptor binding, which allows dimerization and activation of TβRI leading to rapid increases in CDAl protein levels, which is attenuated by the TβRI inhibitor, SB431542. CDAl at increased levels further modulates TβRI to amplify the TGF-β signal leading to activation of Smad and ERK MAPK pathways, which can be attenuated by CDAl siRNA knockdown. The existence of a CDAl - TβRI-Smad&I RK MAPK axis is demonstrated by CDA l overexpression-induced phosphorylation of Smad 3 and ERK MAPK. This was attenuated by the TβRI inhibitor. Activated Smad3 and ERK MAPK then translocate to the nucleus to regulate target genes, such as CTGF and various ECM genes (Fig 5). Both TGF-β and CDAl are overexpressed in the sites of injury in diabetes, where CDA l plays a pivotal role in amplifying the TGF-β signal leading to increased gene expression of CTGF and ECM genes, and tissue fibrosis, l argeting CDA 1 by siRNA in a human renal cell line was shown to be effective to remove the CDA l - dcpendent signaling amplification mechanism for TGF-β, hence to block the profibrotic effect of TGF-β treatment in the human renal cells, ITK-2. It is proposed that targeting CDA l would have minimal adverse effect on the other physiological and beneficial roles of TGF-β, since this targeting strategy avoids direct blockage of TGF-β and its receptors, and only targets the enhanced profibrotic effect of TGF-β specifically seen in the pathological conditions such as diabetic nephropathy. EXAMPLE 7 Approach to target CDAl, identification of CDAl interacting proteins

Method: Approximately 5 x 10 ⁶ independent clones of pre-transformed human testis cDlMΛ expression library constructed in pACT2 vector hosted in yeast strain Yl 87 (HD Bioscience) were screened by mating with yeast strain AH 109 harbouring full length CDA l constructed in pGBK-T7 plasmid (BD Bioscience). The interacting clones are selected on SD/-Trp-I.eu-Ade-His /Kan plates containing X-α-gal under stringent selection pressure. Positive colonies were streaked onto new SD/- frp-Leu-Ade-His plates containing Kanamycin and X-α-gal and grew 2-4 days for further identification of putative positive colonies. PCR was used to amplify the insert of positive clones and PCR products were purified and scquenced. The predicted protein sequences were employed to blast searching GcneBank to obtain the interacting genes of CDA 1 . The interaction was confirmed by re-cloning the candidate cDNA in the pACT2 vector plasmid, and yeast 2- Hybrid system was used to confirm the interaction.

Result: Approximately 100 candidate clones were obtained and up to 50 clones were sequcnced. Among the 50 clones sequenced, 18 (Figure 6) clones are derived from the same gene with identical nucleic acid sequences and encoded amino acid sequences aligned to the C-terminus (Figure 7). This gene or protein was named as CDA l binding protein 1 (CDAl BPl ) (SEQ ID NO: 19, with corresponding nucleic acid sequence disclosed in SEQ ID NO:29).

Conclusion: The proteins encoded by all the cDNA clones contain a putative binding site responsible for binding to CDAl protein. The binding site is located at the C-terminal region.

The nucleic acid sequence of the longest clone of CDA l BPl FC78 is shown and numbered from 1 to 476 (Figure 7) (SEQ ID NO:29). An EST sequence (accession number: BX450762) was found to contain identical sequence of entire FC78. The additional nucleic acid sequences obtained from this EST sequence were added to FC78 at both the 5 ^' and 3 ^' ends. An open reading frame with corresponding amino acids beginning with Methionine was shown. Λn in-frame stop codon is shown (*) in the open reading frame. An in- frame stop codon at the 5 ^' end is highlighted in red and underlined. The open reading frame begins with the first in-frame Methione downstream of the 5 ^" end in-frame stop codon. indicating this is the mostly possible start point of the open reading frame. I wo polyadenylation signals (AATAAA) are highlighted, italics and underlined at the 3 ^" untranslated region.

Database search for peptides that are similar to CDAl BPl only finds similarity to a hypothetical protein (Figure 8).

EXAMPLE 8 Identification of CDAl binding motif of CDA IBPl

Method: Several cDNA fragments of CDAl BPl were cloned into pAC Ϊ2 plasmid. and transformed into yeast Yl 87. The interaction of these fragments with full length CDA l m plasmid pGBK- 17 was examined by mating the yeasts on SD/- frp-Leu-Adc-His selection medium plates containing Kanamycin and X-α-gal. Interaction was noted when yeast colonies grew after 2-5 days.

Result: The longest clone FC78 and the shortest clone FC57 were interactive with CDA l (Sec Figure 9). The N-terminal half of the FC78 (FC78 (-FC57)). which is missing in the FC57 clone, was not interactive. The fragment of FC78 with deletion of the C-tcrminal one quarter, which was equivalent to the FC78 clone with deletion of C-tcrminal hall of the FC57 clone (FC78( 1 /2FC57), failed to interact with CDAl .

Conclusion: These results indicated that the binding site is located to the C-terminal quarter. EXAMPLE 9 Determination of the binding site with 6 amino acid residues

Method: Shortening constructs derived from the shortest CDA l BPl clone FC57 were generated by progressive deletion of 6 amino acid residues from the C-tcrminus (Figure 10). These new constructs were then transformed into yeast and then determined for their ability to interact with full length CDAl .

Result: Constructs FC57-1 and FC57-2 with C-terminal deletion of 6 and 12 amino acid residues, respectively, were still able to interact with CDAl . Flowever. FC57-3 and FC57- 4, with deletion of C-terminal 18 and 24 residues, respectively, lost the ability to interact with CDAl .

Conclusion: These results attribute the binding site to the region containing amino acids: L ITSTIIR, which was present in all the interactive constructs including FC57, FC57-1 and FC57-2. but was absent in the non-interactive constructs including FC57-3 and FC57-4

EXAMPLE 10a Point mutagenesis to further characterize the binding site

Method: Point mutagenesis was carried out to substitute the residues in the binding site of CDA l BPl in the shortened construct FC57-2 (Figure 1 1). In Mutant 1 , the first 3 residues (TIl) were substituted with 3 Alanine residues (AAA) and in Mutant 2, the last 3 residues (RFS) were substituted by AAA. In the Mutant 3, 4 and 5, three pairs oi ^~ residues are substituted by Alanines (AA). A construct containing GST protein as a negative control was also constructed. These constructs were transformed into yeast, and determined for their ability to interact with CDAl using the yeast 2-hybrid system.

Result: When the residue Arginine (R) was mutated (Mutant 2 and 4), the interaction between CDA l BPl and CDAl was disabled. The negative control GST did not bind as expected. Conclusion: These results indicate that the Arginine residue in the binding site of CDAl BPl is critical to determine the functional binding.

EXAMPLE 10b Substitution of the Arginine residue in the CDAlBPl binding site with various other amino acid residues to determine its critical role for binding

Method: Point mutagenesis was carried out to generate substitutive amino acid residue to replace the Arginine residue in the CDAlBPl binding site (Figure 1 1 ). The constructs were then transformed into yeast and their ability to interact with CDAl was determined by yeast 2-hybrid system.

Result: All the mutants including those with the Arginine substituted by Alanine. Lysine or Glutamine were still interactive with CDAl .

Conclusion: These results indicate that change of the Arginine residue alone in the binding site of CDAl BPl is insufficient to abolish the function of CDA I BPl /CDAl binding.

However, the Arginine residue and its surrounding context residues, including IR or RhS

(Figure 1 1 ) are critical for binding.

EXAMPLE I l Approach to identify CDAlBPl binding motif of CDA l

Method: Using the same approach as described in Example 7, constructs containing various fragments of CDAl molecule as shown in Figure 12 were examined for their ability to bind to CDA l BPl by the yeast 2-hybrid system.

Result: CDAl BPl bound to the full length CDAl and its C-terminal acidic fragments including the acidic domain 1 (ADl) and acidic domain 2 (AD2). while CDAl BP l failed to bind to the N-tcrminal fragment of CDAl with the C-terminal acidic tail deleted. This indicates that CDAl BPl binds to the C-terminal acidic tail of CDAl . CDAl BPl also bound to the fragments containing each half of the ADl and AD2 domains including ΛD 1 - 1, AD 1-2, AD2-1 and AD2-2.

Conclusion: These results indicate that CDAl BPl bind to multiple sites in the C-terminal acidic regions of CDA 1.

EXAMPLE 12

The pro-fibrotic effect of CDAJ on collagen III gene expression is enhanced by additional CDAlBPl

Method: Three adenoviruses were used in this experiment: a vector control adenovirus (Ad-control), adenovirus expressing CDAl (Ad-CDAl ) and adenovirus expressing CDA l BPl (Ad-CDA l BPl ). These viruses were diluted to have the same concentration (~6 x 10 ⁷ pfu/μL). A total 45 μL virus or mixtures of viruses (2.7 x 10 ⁴ pfu) as specified below were used to infect cells (2 x 10 ⁶ cells) in 10 mL medium per dish. After overnight incubation with the adenoviruses, the medium containing viruses was aspirated and cells were washed and incubated with complete medium for a further 24 hrs. Total RNA was extracted from the cells and real-time RT-PCR was carried out to measure mRNΛ levels of CDAl , collagen 1, III and other genes.

Result: HK-2 cells were infected with no virus (Figure 16). CDAl mRNA levels increased >200-fold in the HK-2 cells infected with adenoviruses containing 25 μL Ad-CDA l . 1 he 200-fold increase in CDA l mRNΛ levels increased collagen 111 by ~70-fold (1- igurc 16: 3rd column). Addition of Ad-CDAl BPl further increased collagen 111 levels in a dose- dependent manner (Figure 13).

Conclusion: The higher levels of CDAl overexpression had a similar effect in stimulating collagen, suggesting that effect of CDAl was saturated, probably due to the limited availability of endogenous CDAl BPl . Indeed, provision of increasing CDA l Bl ³ I by adenovirus infection was shown to further increase collagen III gene expression, in a dose- dependent manner. These results clearly demonstrate the profibrotic effect of CDA l BP l . in association with CDAl , consistent with CDAl BPl being a key functional interacting protein for CDΛ1 in promoting gene expression of various collagens. This further shows that the CDA1/CDΛ1 BP1 interaction is a target for inhibiting the profibrotic effects of CDAl and related factors such as TGF-β.

EXAMPLE 13 si RNA knockdown of CDAlBPJ inhibits CDAl mediated signalling

Method: Λ human kidney cell line (HK2) was stably transduced with a retrovirus construct expressing a CDABPl siRNΛ 278 (target sequence 5 ^" - GGCTTACCA fCTC fACCATCAT -3') (Sl-Q ID NO:24) or irrelevant siRNΛs as controls. The total cell lysate was extracted for WB and total RNA was extracted from these cells for real-time PCR (Figure 14).

Result: CDAl BPl retroviral-delivered siRNA 278 (target sequence: 5 ^" - GGCTTACCA fCTCTACCATCAT -3') (SEQ ID NO:24) (red bar) reduced CDΛ 1 BP 1 mRNA levels to 40% when compared to the no virus (blue bar) and vector control retrovirus (green bar) transduced HK-2 cells. 1 his knockdown of CDA l BP l resulted m decrease of gene expression of TGF-β by -60%, collagen I by -40% and collagen I I I by

-70%. The knockdown in mRNA expression of these genes further demonstrates the importance of CDA l BPl in tranduction of CDAl signalling.

EXAMPLE 14 CDA lBPl co-expresses with CDA l in relevant renal cells

Since this protein was initially pulled out from a testis library, it was necessary to determine if this binding protein is present in the kidney and specifically in cell types which considered relevant to CDATs biological actions and to the pathogenesis of diabetic nephropathy.

The co-expression of CDAl and CDAl BPl is shown in human kidney tissue (Hg 15 ) Western blotting showed CDAl and CDAl BPl detected in human kidney and lung tissues by respective specific antibodies, with human renal proximal tubule cell line. HK-2 (1 11C2) infected with Myc-tagged CDAl adenovirus as a positive control for CDAl (Fig 15A). A double band of CDAl , as expected, was shown in HK-2 cells representing the transfcctcd Myc-tagged CDAl (upper band) and the endogenous CDAl (lower band) (Fig 15Λ) lmmunohistochemistry staining of a human kidney paraffin section showed CDAl in proximal tubules (Fig 15B, red arrow head) and in podocytes in glomerulus (Fig 15 Ii, green arrow). RT-PCR detected mRNA of both CDAl and CDAl BPl in human kidney (Fig 15C).

These results indicate co-localization of this "'receptor' ^" with CDAl in the relevant cell types of the human kidney, and therefore is consistent with an important biological function as a result of the interaction between these two proteins. Indeed, interruption of such a protein-protein interaction would block the potentially deleterious effects of CDA l . thus preventing, retarding or reversing diabetic nephropathy.

EXAMPLE 15a

Development of short mimetic peptides to inhibit CDA1/CDΛ1BPI interaction

Peptides were synthesized using standard Fmoc-solid-phase peptide synthesis (Fmoc- SPPS) protocols using a Symphony synthesiser (Protein Technologies), with standard coupling chemistry and deprotection. All amino acids used arc appropriately protected at the side-chain if required, and pseudoproline residues may be used where appropriate to increase the crude purity of the peptides. The peptides are purified by reverse-phase I IPLC' using I FA containing buffers unless otherwise specified, and characterised using IvSl-MS and LC-MS.

A 19mer peptide (SLQ ID NO:3) was designed and synthesized based on the sequence of CDA l BPl and a control peptide with the binding site mutated (αfib 19mer mutant), and have shown the activity of α-fib 19mer in interrupting binding between CDA l and CDAl BPl in vitro (Figure 16) Western Blotting showed undetectable levels of CDA l in no virus and control-virus samples, and expression of CDAl delivered by CDA l adenovirus (lanes 3 and 4). Recombinant CDAl BPl protein was added to a duplicate blot allowing it to bind to the CDAl bands, and then detected by a specific antibody to CDAl BPl . The peptide α-fib 19mer blocked the binding of CDA l BPl to CDAl , whereas the control compound was not able to block the binding.

EXAMPLE 15b A novel I9mer synthetic peptide inhibits CDA1/CDA1BP1 binding in vitro

Method: An adenovirus construct expressing CDAl (Ad-CDAl) was used to infect a human renal proximal tubule cell line HK-2 to overexpress CDAl (Figure 16) (lanes 3 and 4). HK-2 cells with no virus (lane 1) or infected with a control adenovirus (lane 2) were used as controls. Cell lysates were electrophoresized by SDS-PAGE and transferred to a PVDF membrane. Western blotting was carried out to visualize the overexpressed CDA l bands (Figure 16; middle row, lanes 3 and 4) with GAPDH shown to indicate equal protein loading per lane (Figure 16; bottom row). A duplicate blot was then incubated with a purified recombinant CDAl BPl protein (50 μg/mL) in the presence of a synthetic 19mer peptide containing the binding site of CDA l BPl (α-fib 19mer) or a similar peptide with the binding site mutated (α-fib 19mer mutant). The bound CDA l BPl was then detected by an affinity purified antibody to the N-terminus of CDA l BPl (Figure 16; top row).

Results: Western Blotting shows (Figure 16) undetectable levels of CDAl in no virus (lane 1 ) and control-virus (lane 2) samples, and expression of CDAl delivered by a CDA l adenovirus (lanes 3, 4). The peptide α-fib 19mer blocked the binding of CDAl BP l to CDAl (red arrow), whereas the control peptide was not able to block the binding (blue arrow head).

Conclusion: The activity of the α-fib 19mer in interrupting binding between CDA l and CDAl BPl in vilro has been demonstrated. EXAMPLE 16a

Development of ELISA to measure inhibition ofCDAl/CDAlBPl interaction

Method: Based on the assay of Western blot format of Examples 15a and 15b an IiLISA- like binding assay was developed. This is a rapid, specific and easy to perform assay which is suitable for testing large number of samples. GST-CDAl (CDAl ) or GST-CDA l C- terminus fusion protein (200 ng per well) were coated onto a 96-wcll ELlSA plate. Recombinant GST-CDAI BPl protein or GST control protein expressed and purified from E. coli was added at various concentrations to incubate with and to bind to the coated CDAl proteins. The bound CDAl BPl proteins were then detected by a specific antibody to the N-terminus of CDAl BPl followed by a HRP-conjugated secondary antibody and a colorimetric substrate solution. The OD reading of the substrate solution represents the amount of bound CDA l BPl per well, which was found to be quantitatively dependent on the concentrations of the input CDAl BPl protein at the specified range of concentrations (Figure 17).

These results demonstrated the ability of the recombinant CDAl BPl protein to bind to the GS T-CDA l fusion proteins coated on the ELISA plate, whereas the GST control protein did not produce a significant signal.

EXAMPLE 16b Method for assay of peptide inhibitors

Based on the ELISA format binding assay, a CDA1/CDABP1 binding inhibition assay has been developed. Approximately 2 μg per well maltose binding protein-CDΛl fusion protein (MBP-CDA l ) was coated on a 96-well plate. GST-CDA I BPl fusion protein (60 μg per well) and inhibiting or control peptides at various concentrations were co-incubated with the coated MBP-CDAl and the bound GST-CDA I BPl was then detected by the specific CDA l BPl Abs.

Result: The 19mer CDAl BPl wild type peptide (SEQ ID NO:3) containing the 8-aa binding site (L TlS fIIR) inhibited the binding of GST-CDAl BPl to MBP-CDAl in a dose-dependent manner (Figure 18) with the highest inhibiting activity at 10 μM (red line), whereas a scramble peptide (dark blue) and a peptide with the binding site mutated (light blue) failed to inhibit the binding. Peptide sequences derived from the 19mer also inhibited binding to a similar extent (Table 4).

EXAMPLE 17 Generation of a CDAl knockout mouse

Mice with a conditional gene knockout of CDAl arc generated. Female heterozygous mice with CDAl gene targeted (wt/flox) are obtained and confirmed by Southern blot genotyping. Genomic DNA samples are prepared and digested with EcoRV, from two female offspring (AOOl F and A002F) generated by mating a male coat color mouse v\ ith a wild type C57BL/6 female. Southern hybridization showed an expected wild type band of about 14.6 kb and a 6.8 kb for a targeted genotype. Additional breeding is used to delete the targeted exons 2-5 of CDAl gene and to remove the selection marker gene, nco. These are CDAl gene floxed mice and therefore it will enable generation of tissue specific CDA 1 K. O mice, initially focusing on podocyte specific CDAl KO mice.

EXAMPLE 18

Optimization of the efficacy of the peptide to inhibit CDAl signaling in a cell-based assay

Once the optimum peptide size and sequence is determined, the peptide is tested for efficacy in the cell-based assay of CDA l function. Bioactive peptides are acknowledged Io have poor cell penetration capabilities, and it is anticipated that the peptides from hxample 1 will be modified in order to achieve cell penetration.

One method is the use of cell penetrating peptides (CPP) that are covalently attached to a bio-active peptide or protein. The development of CPPs is based on the observation that a cationic peptide sequence (TaU _8-60 ) which was derived from the human immunodeficicnc} virus (I II V) Tat protein, when conjugated to β-galactosidase and administered b> intraperitoneal injection to mice, resulted in the distribution of the β-galactosidasc actiuty into virtually every organ in the animal, and included crossing the blood brain barrier (Schwarz e/ α/ Science 285 1569-1572, 1999).

The optimi/ed peptide is conjugated to different CPP sequences ( 1 able 5).

The CPP-Peptide constructs are tested for efficacy, first in the cell free CDAl - CDA l BP l binding assay and then in the cell-based assay of CDA l function Whilst a direct measure of CPP-peptide penetration into the cell that is distinct from CDAl function is a useful adjunct to this invention, such assays are complex to establish and validate (see for example Palm el al Peptides 27 1710-1717, 2006). Since the objective is to validate CDAl as a target, and not the validation of CPPs as a technique, assays which focus on the function of CDΛ1 arc used as the primary measures of effectiveness.

TABLE 5 CPP Sequences that used in CPP-cargo constructs and reported to be effective

Name Sequence Reference

I at. 4K-60 GRKKRRQRRRPPQQ Schwaiy Science 285 1569- 1572, 1999

Oligo-R R _n (n = 5-9) uses L and D ammo acids Wender PNAS 97 13003- 13008 2000

IsI- I RVIRVWFQNKRCKDKK KiIk el al Bioconjugate Chemistry /2 91 1 -9 1 6 2001

SAP LGTYTQDFNKFHTFPQTAIGVGAP Machova ChemBioChem 3 672-677, 2002

Cleavable Linker: In addition to exploring different CPP sequences to optimi/.e the activity of the CPP-peptide construct; this example extends to the introduction of a cleavable linkage, in the form of a disulfide bond, between the CPP peptide and the l ϋ bioactivc peptide, as described in Brooks el al Advanced Drug Discovery Reviews 5 ^~ 559- 577. 2005, and Hallbrink el al Biochim Biophys Ada 1515 101 -109, 2001 The disulfide linkage allows for the intracellular release of the bioactive peptide and may improve the peptide's intracellular distribution.

15 Peptide Stability: Stability of therapeutic peptides to degradation by plasma and tissue proteases is a key feature in their in vivo use. In this example, the stability of the peptide constructs to plasma proteolysis is determined using a protocol comprising a known concentration of peptide being exposed to human and mouse plasma under standard conditions and the time-dependant concentration of the peptide determined using 0 LC/MS/MS analysis. Those compounds which show a half-life of greater than 6 hours are selected for further study.

fhere are several methods that have been used to increase the proteolytic stability of peptide therapeutics. These include capping the termini of the peptides: N-terminal 5 acctylation and C-terminal amidation and stabilizing the peptide by cycli/ation, or through the introduction of one or more "unnatural" amino acids, typically replacing L-amino acids with the D-equivalcnts (see Nolan and Walsh ChemBioChem 10 34-53. 2009) 1 his principle, named "retro-inverso" (Fischer Current Protein and Peptide Science 4 339-356, 2003), can also be applied "in total ^" to the bioactive peptide. The N to C sequence is reversed when the full D for L substitution is carried out. The biological activity of the native sequence is maintained in the Retro Inverso sequence; this is confirmed using the appropriate CDΛ 1 assays for efficacy.

EXAMPLE 19 Cell penetration

Another approach to increasing the cellular penetration of the bioactive peptide makes use of passive membrane permeability which is dependant on the hydrophobicity and size of the peπneant molecule. To the extent that solubility in water is not excessively compromised, small, relatively hydrophobic molecules are more likely to passively translocate across biological membranes (Lipinski Journal of Pharmacological and Toxicologic^/ Methods 44:235-249, 2000).

With this in mind, the second cell penetration approach focuses on selecting the smallest. bioactive peptide from Example 18, and introducing an iterative manner the changes to the chemical structure of the compound in order to increase bioactivity and membrane permeability simultaneously. Hydrophobicity often makes a significant contribution to the binding affinity between a drug and its target (Lipinski 2000 supra). 1 he modifications described below arc used. The membrane permeability of the modified peptides is determined using the parallel artificial membrane permeability (PAMPA; Sugano el a/ Journal of Biomolecular Screening 6: 189-196, 2001 ).

Strategy to increase bioactivity and hydrophobicity: End cap peptide. fhis uses with N-acctylation and carboxylate amidation, guided by existing sidcchains of terminal amino acids has been demonstrated to improve membrane permeability; Cyclise peptides. Using known methodology particularly the all hydrocarbon methodology described by Schafmeister et al Journal of the American Chemical Society /22 5891 -5892. 2000. peptides are cyclized. Once a peptide with the target efficacy in the cell-based assay of CDAl function has been identified, the compound is tested to determine if it has the appropriate properties for use in an animal model of diabetic renal fibrosis.

EXAMPLE 20

Optimization of the efficacy of the peptide to inhibit CDAl signaling in an animal model of diabetic complications

For a bioactive peptide to be therapeutically useful, it is tested for ability to undergo proteolysis and rapid renal clearance.

This example described how the bioactive peptide is optimized to be stable towards proteolysis, and it is therefore anticipated that the bioactive peptide will be protcolytically stable.

Pharmacokinetic analysis (PK). fhe plasma half life of the bioactive peptides is tested in a standard model o\ ^' intraperitoneal bioavailability. In this model, separate indwelling catheters are used to administer compound (for IV only) and to sample plasma; a single dose of peptide is administered cither IV or IP and aliquots of blood are collected at defined time points over 24hours The plasma is separated and the concentration of the bioactive peptide is determined using standard techniques on a tandem HPLC-MS-MS instrument. The rate of clearance of the peptide from plasma following an IV dose in this assay provides an indicator of plasma half-life (t|/ ₂ ); the relative area under the curve (AUC) of the plasma concentration of peptide following the IP vs IV dose provides a measure of (he bioavailability of the peptide via the IP route.

fhe clearance of peptides is governed by two factors, the hydrophilicity of peptides which minimizes the non-specific binding to serum proteins (and hence uptake by the reticular endothelial system) and the small size of the peptides which leads to rapid removal by the kidneys. A method to improve the plasma half-life of bioactive peptides has been to increasc the molecular weight by conjugation to large polyethylene glycol (PEG) polymers, however peptides modified in this manner are unable to cross biological membranes (Harris el al. Nature Reviews Drug Discovery 2:214-221 , 2003).

Extending Half life.

To extend peptide half life in plasma the peptide is modified so that it binds, in a reversible or non-reversible manner, to serum albumin (see Knudsen et al Journal of Medicinal Chemistry 43 1664- 1669, 2000). This binding is mediated through hydrophobic modifications to the peptide (conjugation of a fatty acid). The bioactive peptide is modified according to the methods described by Knudsen 2000 supra if it proves necessary to increase the circulating half life. Peptides modified in this way arc tested for efficacy in the cell based assay of CDΛ 1 function.

Tolerability. Whilst one of the benefits of using peptides as therapeutics is an expectation for low toxicity, each bioactive peptide is tested for tolerability in mice using a single dose, escalating dose protocol to establish the acute tolerated dose (A l D). This dose is used to guide dose selection in a low-mid-high dose multiple dose (14 day) study in mice to establish the multiple tolerated dose (MTD).

Efficacy.

The bioactive peptide is selected for administration in the animal model of diabetic renal fibrosis when it can be demonstrated that a trough plasma concentration of drug that is 10- fold greater than the IC ₅ 0 as determined in the cell-based assay of CDA l function, can be safely achieved.

fhose skilled in the art will appreciate that the invention described herein is susceptible Io variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to. or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. BIBLIOGRAPHY

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Doublier et al. Diabetes 52(4): 1023-1030, 2003

Fischer Current Protein and Peptide Science -/.339-356, 2003

Greenberg et al Nature 374: 168-173, 1995

Hallbrink et al. Biochim Biophys Acta 1515: 101 -109, 2001

Harris et al. Nature Reviews Drug Discovery 2.214-221 , 2003

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KiIk et al Bioconjugale Chemistry /2.91 1 -916, 2001

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Kδhlcr and Milstein, Eur J Immunol 6:51 1 -519, 1976

Lipinski Journal of Pharmacological and Toxicological Methods 44 -235-249. 2000

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Nolan and Walsh ChemBioChem IQ- 34-53. 2009

Nuttall el al. Eur J Biochem 270:3543-3554, 2003

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PAM PΛ

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Roux et al. Prυc Natl Acad Sci USA 95: 1 1804- 1 1 809, 1998

Schal ^' meistcr et al. Journal of the American Chemical Society 122 5891 -5892, 2000

Schwarz et al Science 285: 1569- 1572. 1999

Sugano et al Journal of Biomolecular Screening 6: 189- 196, 2001

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Wender PNAS 97: 13003- 13008, 2000