WO2007013050A1 - Heparanase-derived peptide dimers and their uses as inhibitors of heparanase - Google Patents

Abstract

Disclosed are peptide dimers, derived from the N' terminus region of heparanase, and which inhibit heparanase catalytic activity. Pharmaceutical compositions, uses thereof and methods utilizing said peptide dimers, for the inhibition of heparanase activity in conditions related to the same, are also disclosed herein.

Description

HEPARANASE-DERIVED PEPTIDE DIMERS AND THEIR USES AS INHIBITORS OF HEPARANASE

Field of the Invention

The present invention refers to the field of drug development, cancer and inflammation. More specifically, the present invention describes a heparanase- derived dimeric peptide, which can be used in the inhibition of heparanase and in the treatment of conditions related to excess heparanase activity.

Background of the Invention

All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.

Heparanase is an endo-β-D-glucuronidase involved in cleavage of heparan sulfate (HS) chains, and hence participates in extracellular matrix (ECM) degradation and remodeling. Heparanase activity has been traditionally correlated with the metastatic potential of tumor-derived cell types [Nakajima, M. et al. (1998) J. Cell. Biochem. 36, 157-167; Vlodavsky, I. et al. (1999) Nat. Med. 5, 793-802; Parish, C. R. et al. (2001) Biochem. Biophys. Acta 1471, M99- M108; Vlodavsky, I. and Priedmann Y. (2001) J. Clin. Invest. 108, 341-347]. Similarly, heparanase has been shown to facilitate cell invasion associated with autoimmunity, inflammation and angiogenesis [Vlodavsky, I. et al. (1992) Invasion & Metastasis 12, 112-127; Dempsey, L. et al. (2000a) Trends Biol. Sd. 25, 349-351; Parish (2001) ibid.]. More recently, heparanase upregulation was detected in a variety of human primary tumors correlating, in some cases, with increased tumor vascularity and poor postoperative survival [El-Assal, O. N. et al. (2001) CZm. Cancer Res. 7, 1299-1305; Gohji, K. et al. (2001) Int. J. Cancer 95, 295-301; Koliopanos, A. et al. (2001) Cancer Res. 61, 4655-4659; Rohloff, J. et al. (2002) J. Cancer 86, 1270-1275]. In addition, increased heparanase expression has been noted in kidney [Levidiotis, V. et al. (2001) Kidney Int. 60, 1287-1296], liver [Xiao, Y. et al. (2003) Hepatology Res. 26, 192-198] and diabetic [Katz, A. et al. (2002) Isr. Med. Assoc. 4, 996-1002 (2002)] disorders.

The heparanase cDNA encodes a polypeptide of 543 amino acids that appears as a ~65 kDa protein in SDS-PAGE analysis. The protein undergoes proteolytic processing which is likely to occur at two potential cleavage sites, Glu^lo9-Serⁿ° and

yielding an 8 kDa polypeptide at the N- terminus, a 50 kDa polypeptide at the C-terminus and a 6 kDa linker polypeptide that resides in-between [Fairbanks, M. B. et al. (1999) J. Biol. Chem. 274, 29587-29590; Parish (2001) ibid.]. Active heparanase enzyme exists as a heterodimer composed of the 8 kDa polypeptide non-covalently associated with the 50 kDa heparanase subunit. Heterodimer formation is necessary and sufficient for heparanase enzymatic activity [Levy-Adam, F. et al. (2003) Biochem. Biophys. Res. Comm. 308, 885-891; McKenzie, E. et al. (2003) Biochem. J. 373, 423-435].

Seeking for functional domains that would serve as a target for drug development, the inventors recently identified a protein domain that locates at the N-terminus of the 50 kDa heparanase subunit and mediate the interaction of heparanase with its heparan sulfate (HS) substrate [Levy-Adam, F. et al. (2005) J. Biol. Chem. 280, 20457-20466]. A synthetic peptide derived from this region of heparanase was shown to inhibit heparanase catalytic activity. Surprisingly, a dimer of this peptide is a much more potent heparanase inhibitor.

Thus, it is an object of the present invention to provide heparanase peptide dimers and uses thereof as heparanase inhibitors, as well as drugs for the treatment of cancer and inflammation. Other uses and objects of the invention will become apparent as the description proceeds. Summary of the Invention

In a first aspect the present invention provides a peptide dimer consisting of two identical monomer peptides chemically linked to each other, wherein said monomer peptide has an amino acid sequence derived from the 5OkDa subunit of heparanase.

In one preferred embodiment of the invention, said two monomers are linked via one of covalent bonds and a chemical linker.

In another preferred embodiment of the invention, said sequence comprises a hep arin-bin ding domain, and is preferably derived from the N' terminus region of heparanase.

In a further preferred embodiment of the invention, said sequence comprises amino acid residues selected from the group consisting of: Lys¹⁵⁸ to Asp¹⁷¹, as denoted by SEQ ID NO:1, Lys²6² _to Lys²⁸⁰, as denoted by SEQ ID NO:2, and Lys⁴¹¹ to Arg⁴³², as denoted by SEQ ID NO:3 of human heparanase, or any functionally equivalent fragment, derivative and variant thereof.

In yet another preferred embodiment of the peptide dimer of the invention, said two monomers are linked to each other via one additional cysteine residue at the N'- or C'-terminus. Thus, said monomers are linked to each other by disulfide bonds between their C- and/or N'-termini.

In a second aspect the present invention provides a peptide dimer consisting of two identical monomer peptides chemically linked to each other, wherein said monomer peptides are as denoted by any one of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. In one preferred embodiment of the second aspect of the invention, said monomer peptides are linked to each other by di-sulfi.de bonds between their C-termini.

The peptide dimer of the invention, as described above as a first or second aspect of the invention, is a heparanase inhibitor.

In another aspect the present invention provides a pharmaceutical composition comprising as active ingredient the peptide dimer of the invention, substantially as described above.

In one preferred embodiment of the pharmaceutical composition of the invention, said composition is for the inhibition of heparanase catalytic activity.

In another embodiment, said pharmaceutical composition comprising as active ingredient the peptide dimer of the invention substantially as described above, is for medical use.

In a further embodiment said pharmaceutical composition is for the treatment of a condition associated with heparanase catalytic activity. Said condition is defined as one of angiogenesis, tumor formation, tumor progression, tumor metastasis, malignant proliferative disorder, inflammatory disorder, kidney disorder, and autoimmune disorder. In particular, said malignant proliferative disorder is any one or solid and non-solid tumor selected from the group consisting of carcinoma, sarcoma, melanoma, leukemia, lymphoma and glioma.

In a yet further embodiment, the pharmaceutical composition described herein above further comprises a pharmaceutically acceptable carrier, diluent, excipient and/or additive. In yet another aspect the present invention provides the use of the peptide dimer as defined above in the preparation of a pharmaceutical composition for the treatment of a condition related to heparanase catalytic activity, wherein said condition is one of angiogenesis, tumor formation, tumor progression, tumor metastasis, malignant proliferative disorder, inflammatory disorder, kidney disorder, and autoimmune disorder.

In a further aspect, the present invention provides the use of a peptide dimer as defined above in the preparation of a composition for the inhibition of heparanase catalytic activity.

Further, said dimeric peptide of the invention may be used as an agent for the inhibition of heparanase catalytic activity.

In a yet further aspect the present invention provides a method for the inhibition of heparanase catalytic activity, said method comprising administering a therapeutically effective dosage of a peptide dimer as defined above, or a composition comprising the same.

In a last aspect the present invention provides a method for the treatment of conditions related to heparanase catalytic activity, comprising administering a therapeutically effective dosage of a peptide dimer as defined in the invention, or a composition comprising the same, to a subject in need.

The method of claim 23, wherein said condition is one of angiogenesis, tumor formation, tumor progression, tumor metastasis, malignant proliferative disorder, inflammatory disorder, kidney disorder, and autoimmune disorder. Brief Description of the Figures

Figure 1: KKDC peptide dimerization facilitates the inhibition of heparanase enzymatic activity.

Heparanase was incubated (2 h, 37⁰C) with ³⁵S-labeled ECM in the absence (■) or presence of 50μg/ml of the KKDC peptide (A), KKDC peptide in a dimer form (Δ) or its control, scrambled peptide (□). Heparin (15μg/ml; •) was included as a positive control. Labeled degradation products released into the incubation medium were analyzed by gel filtration, as described in "Experimental Procedures". Note inhibition of heparanase activity by the KKDC peptide, and complete inhibition by the KKDC in its dimmer form. Abbreviations: Sulph., sulphate; lab., labeled; mat., material; cont., control; hep., heparin; pep., peptide; scr., scrambled; dim., dimer.

Figure 2: KKDC peptide physically interacts with heparin- Sepharose beads, but not when treated with EDT.

Control, scrambled (Scr), and KKDC peptide in its dimer (KKDC) and monomer (KKDC-EDT) forms were incubated (2h, 4⁰C) with heparin- Sepharose beads and washed with PBS+0.35 M NaCl to reduce nonspecific binding. Sample buffer was then added and following boiling for 5 min, samples were loaded on Tris-Tricine gel and bound peptides were detected with Commassie Blue staining. Note that high affinity and physical interaction of the KKDC peptide with heparin requires peptide dimmer. Abbreviations: Scr., scrambled peptide; KKDC, dimeric peptide; KKDC-EDT, KKDC peptide treated with ethandithiol.

Figure 3A-3D: Binding of the KKDC peptide to cell surface heparan sulfate. CHO Kl (Fig.3A-3C) and HS-deficient CHO 745 (Fig. 3D) were incubated (2h, 4⁰C) with control scrambled (Fig. 3A, Scr) or KKDC-biotin conjugate in the absence (Fig. 3B, 3D) or presence of .heparin (50μg/ml; Fig. 3C; KKDC+Hep). Cells were then washed three times with PBS, fixed with 4% paraformaldehyde, and incubated (60 min, room temperature) with Avidin- FITC conjugate. Following three more washes with PBS slides were mounted and images were taken by confocal microscope. Note the interaction of the

KKDC peptide with cell membrane HS, which is prevented by heparin or the lack of HS (CHO 745 cells).

Fig. 3 A: Scrambled peptide (Scr.).

Fig. 3B: KKDC dimeric peptide localizes to the cell surface of CHO cells.

Fig. 3C: Presence of heparin (Hep.) reduced dimeric peptide binding to the cell surface.

Fig. 3D: No binding of the KKDC dimer in the cell surface of CHO cells devoid of heparan sulfate (KKDC/745).

Detailed Description of the Invention

The inventors have previously described that heparanase-derived peptides, corresponding to the heparin-binding domain, inhibited heparanase catalytic activity [co-pending PCT Application No. IL2005/000068, co-pending US Application Serial No. 10/901,943; Levy- Adam et al. (2005) ibid].

In the present invention, the inventors show that a dimeric peptide is significantly more potent than the monomer peptide in inhibiting heparanase catalytic activity.

As described herein in the Examples below, the inventors prepared homodimeric peptides, and evaluated their capacity to inhibit heparanase catalytic activity. Essentially, the dimers were composed of two monomeric units of one of the following peptides: Lys¹⁵⁸ to Asp¹⁷¹, as denoted by SEQ ID NO:1, Lys²⁶² to Lys²8o, as denoted by SEQ ID NO:2, and Lys⁴¹¹ to Arg«², as denoted by SEQ ID NO: 3.

The sequence of each monomeric peptide is denoted as follows: - SEQ ID NO:1 (also referred to as KKD): KKFKNSTYSRSSVD; - SEQ ID NO:2: KLYGPDVGQPRRKTAKMLK; and

- SEQ ID NO:3: KKLVGTKVLMASVQGSKERKLR.

As detailed below, when a cysteine was introduced in the C-termini of the peptides, the sequence is denoted as follows:

- SEQ ID NO:4 (also referred to as KKDC): KKFKNSTYSRSSVDC;

- SEQ ID NO:5: KLYGPDVGQPRRKTAKMLKC; and

- SEQ ID NO:6: KKLVGTKVLMASVQGSKRRKLRC.

It should be appreciated that as used herein in the specification and in the claim section below, all the amino acid locations (Lys¹⁵⁸ to Asp¹⁷¹, Lys²⁶² to Ly s²⁸⁰ and Lys⁴¹¹ to Arg⁴³²) refer to the amino acid sequence of human heparanase as denoted by GenBank Accession No. AF144325.

Thus, the present invention provides dimeric peptides, derived from the N'- terminus of the 5OkDa subunit of heparanase, which are heparanase inhibitors.

The dimeric peptides were synthesized in a step-wise fashion. Firstly the monomer peptides were synthesized, and then the dimers were formed, through one of dimerization means as detailed below.

Synthesis of the monomer peptides may be through in vivo or in vitro expression systems, as previously described [Baneyx, F. (1999) Curr. Opin. Biotechnol. 10(5): 411-21; Wurm, F. and Bernard, A. (1999) Curr. Opin. Biotechnol. 10(2): 156-9; Hernandez et al. (1997) Biochimie. 79(8): 527-31].

Alternatively, monomer peptides may be synthesized by chemical procedures, like solid phase peptide synthesis (SPPS), for example. In this procedure, peptides are produced in a stepwise fashion from C- to N'-terminus in a series of steps whose conditions allow for the chemical formation of peptide bonds between two amino acid residues [Merrifield, B. (1997) Methods Enzymol. 289:3-13].

The monomeric units synthesized by one of the procedures described above are then chemically linked, forming the dimer. The link may be between the monomers N'-termini (head-to-head), between N'-terminus of one and C- terminus of the other (head- to-tail), or between the C'-termini (tail- to-tail).

Moreover, the link may be through covalent bonds or through a linker.

Without being bound by theory, one preferred method of linking monomers is through the addition of a cysteine residue, during synthesis, to the C- terminus of the monomers, and thus forming the dimer. One example of such dimer is the KKDC dimer, consisting of two C-C linked monomers, each as denoted by SEQ ID NO:7, and as presented in the Examples. Other examples of such are the dimer composed of two Lys²⁶²-Lys²⁸⁰-Cys-linked monomers, each as denoted by SEQ ID NO:8, or the dimer composed of two Lys⁴¹¹-Arg⁴³²- Cys linked monomers, each as denoted by SEQ ID NO: 9.

Thus, preferably, the additional cysteine residue is at the C-terminus of the monomer peptide, and the monomers are linked to each other by di-sulfi.de bonds between their C-termini, i.e, tail-to-tail.

Alternatively, the extra cysteine residue may be added to the N'-terminus of the monomers during synthesis.

Monomer linkage (i.e., dimer formation) occurs through the formation of disulfide bonds between the cysteine residues of two monomers. Dimerization may also occur through the formation of dityrosine crosslinks between two monomers [Amado, R. et al. (1987) Meth. Enzymol. :377-383].

Alternatively, dimerization may be effected through an esterification reaction between two monomers, wherein a dialcohol is the linker.

The dimeric peptide of the invention comprises preferably a homodimer of any one of SEQ ID NO: 1 to 6, most preferably SEQ ID NO:1 and 4. However, it is to be understood that the invention pertains to any dimeric peptide comprising sequence structurally similar to the N-terminus of the heparanase sequence with substantially equal or greater activity, and particularly the heparin-binding domains. Changes in the structure of the monomer peptide comprise one or more deletions, additions, or substitutions. The number of deletions or additions, which may occur at any point in the sequence, including within the acetylcholinesterase-derived sequence, will generally be less than 25%, preferably less than 10% of the total amino acid number. This figure does not include additions as described above, e.g., addition of sequences coding for growth factor sequences.

Preferred substitutions in the monomer are changes that would not be expected to alter the secondary structure of the dimeric peptide, i.e., conservative changes. The following list shows amino acids that may be exchanged for the original amino acids.

Original Residue Exemplary Substitution

Ala GlyjSer

Arg Lys

Asn Gln;His

Asp GIu

Cys Ser

GIn Asn GIu Asp

GIy Ala; Pro

His Asn; GIn

He Leu; VaI

Leu lie; VaI

Lys Arg; GIn; GIu

Met Leu; Tyr; lie

Phe Met; Leu; Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp; Phe

VaI He; Leu

Amino acids can also be grouped according to their essential features, such as charge, size of the side chain, and the like. Preferred substitutions would exchange an amino acid present in one group with an amino acid from the same group. The following list shows groups of similar amino acids:

1. Small aliphatic, nonpolar: Ala, Ser, Thr Pro, GIy;

2. Polar negatively charged residues and their amides: Asp, Asn, GIu, GIn;

3. Polar positively charged residues: His, Arg, Lys;

4. Large aliphatic nonpolar residues: Met, Leu, He, VaI, Cys;

5. Large aromatic residues: Phe, Tyr, Trp.

Further comments on amino acid substitutions and protein structure may be found in the literature [Creighton, T.E. (1983) Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, CA].

The preferred conservative amino acid substitutions as detailed above are expected to substantially maintain or increase the function or activity of the dimeric peptide of the invention, as detailed herein below. Of course, any amino acid substitutions, additions, or deletions are considered to be within the scope of the invention where the resulting monomeric peptide forms a homodimeric peptide of the invention, i.e., a peptide which is substantially equal or superior in terms of function to the preferred peptide dimer of the invention.

The peptide of the invention may be further modified to improve its function, affinity, or stability. For instance, it may be desirable to link polyethyleneglycol (PEG) groups to the peptide dimer. Such groups may impart enhanced stability upon the peptide dimer. Another effect of these groups may be lowered immunogenicity. This feature of PEG-linked peptides may be particularly desirable when the peptide of the invention is to be used in υiυo. Preparation of PEG-linked peptides has been described previously [Guerra et al. (1998) Pharm. Res. 15:1822-7].

It should be appreciated that as used herein in the specification and in the claim section below, all the amino acid locations (Lys¹⁵⁸ to Asp¹⁷¹, Lys²⁶² to Ly_S28o _an(j Lys⁴¹¹ to Arg⁴³²) refer to the amino acid sequence of human heparanase as denoted by GenBank Accession No. AF144325.

As shown in the examples below, the dimeric peptide described herein is a potent inhibitor of heparanase catalytic activity.

As used herein in the specification and in the claims section below, the phrase "heparanase catalytic activity" or its equivalent "heparanase activity" refers to an animal endoglycosidase hydrolyzing activity which is specific for heparin or heparan sulfate proteoglycan substrates, as opposed to the activity of bacterial enzymes (heparinase I, II and III) which degrade heparin or heparan sulfate by means of β-elimination. Heparanase activity which is inhibited or neutralized according to the present invention can be of either recombinant or natural heparanase. Such activity is disclosed, for example, in US 6,177,545 and US 6,190,875.

As used herein in the specification and in the claims section below, the term "inhibit" and its derivatives refer to suppress or restrain from free expression of activity. According to a preferred embodiment of the present invention at least about 60-70%, preferably, at least about, 70-80%, more preferably, at least about 80-90% of the heparanase activity is abolished by the dimeric peptide of the invention. Without being bound by theory, the dimeric peptide of the invention may compete with the corresponding sequence within the native 50 kDa subunit of heparanase for binding to a substrate.

The expression "monomer peptide" also refers to any functionally equivalent fragment, derivative, analog or variant of the peptides denoted by SEQ ID NO: 1-6.

By "functional fragments" is meant "fragments", "variants", "analogs" or "derivatives" of the molecule. A "fragment" of a molecule is meant to refer to any amino acid subset of the peptides defined by residues Lys¹⁵⁸ to Asp¹⁷¹, Ly_S ²⁶2 to Lys²⁸⁰ and Lys⁴¹¹ to Arg⁴³² of heparanase. A "variant" of such molecule is meant to refer to a naturally occurring molecule substantially similar to either the entire molecule or a fragment thereof. An "analog" of a molecule is a homologous molecule from the same species or from different species. By "functional" is meant having same biological function, for example, being capable of inhibiting heparanase catalytic activity.

Thus, the terms derivatives and functional derivatives as used herein mean homodimeric peptides consisting of two identical monomer peptides, as denoted by any one of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, with any insertions, deletions, substitutions and modifications to the monomeric peptide that do not interfere with the ability of said dimeric peptide to inhibit heparanase catalytic activity (hereafter referred to as "derivative/s"). A derivative should maintain a minimal homology to said amino acid sequence, e.g. even less than 30%. It should be appreciated that the term "insertions" as used herein means any addition of amino acid residues to the peptides of the invention, between 1 to 50 amino acid residues, preferably, between 20 to 1 amino acid residues and most preferably, between 1 to 10 amino acid residues. Another example may be the incorporation of an N-terminal lysyl-palmitoyl tail, the lysine serving as linker and the palmitic acid as a hydrophobic anchor. In addition, the peptides may be extended by aromatic amino acid residue/s, which may be naturally occurring or synthetic amino acid residue/s. A preferred aromatic amino acid residue may be tryptophan. Alternatively, the peptides can be extended at the N-terminus and/or C-terminus thereof with amino acids present in corresponding positions of the amino acid sequence of the naturally occurring N' region of the 50 kDa subunit of heparanase. Nonetheless, according to the invention, the dimeric peptides of the invention may be extended at the N-terminus and/or C-terminus thereof with various identical or different organic moieties which are not naturally occurring or synthetic amino acids. As an example for such extension, the peptide may be extended at the N-terminus and/or C-terminus thereof with an N- acetyl group.

The lack of structure of linear peptides renders them vulnerable to proteases in human serum and acts to reduce their affinity for target sites, because only few of the possible conformations may be active. Therefore, it is desirable to optimize the peptide structure, for example by creating different derivatives of the various peptides of the invention.

For every single peptide sequence used by the invention and disclosed herein, this invention includes the corresponding retro-inverso sequence wherein the direction of the peptide chain has been inverted and wherein all the amino acids belong to the D-series.

The involvement of heparanase in tumor angiogenesis has been correlated with the ability to release bFGF (FGF-2) and other growth factors from its storage within the ECM (extracellular matrix). These growth factors provide a mechanism for induction of neovascularization in normal and pathological situations.

Heparanase may thus facilitate not only tumor cell invasion and metastasis but also tumor angiogenesis, both critical steps in tumor progression. Heparanase catalytic activity correlates with the ability of activated cells of the immune system to leave the circulation and elicit both inflammatory and autoimmune responses. Interaction of platelets, granulocytes, T and B lymphocytes, macrophages and mast cells with the subendothelial ECM is associated with degradation of heparan sulfate (HS) by heparanase catalytic activity [Vlodavsky, I. et al. (1992) Invasion & Metastasis 12, 112-127]. The enzyme is released from intracellular compartments (e.g., lysosomes, specific granules) in response to various activation signals (e.g., thrombin, calcium ionophore, immune complexes, antigens, mitogens), suggesting its regulated involvement and presence in inflammatory sites and autoimmune lesions. Heparan sulfate degrading enzymes released by platelets and macrophages are likely to be present in atherosclerotic lesions [Campbell, K. H. et al. (1992) Exp. Cell Res. 200, 156-167]. Treatment of experimental animals with heparanase alternative substrates (e.g., non-anticoagulant species of low molecular weight heparin) markedly reduced the incidence of experimental autoimmune encephalomyelitis (EAE), adjuvant arthritis and graft rejection in experimental animals, indicating that heparanase inhibitors may be applied to inhibit autoimmune and inflammatory diseases [Vlodavsky (1992) ibid.; Lider, O. et al. (1989) J. Clin. Invest. 83:752-756]. The dimeric peptides of the invention may also be presented in the form of compositions. Said compositions are capable of inhibiting heparanase catalytic activity, and may be for medical use.

The preparation of pharmaceutical compositions is well known in the art and has been described in many articles and textbooks, see e.g., Remington's Pharmaceutical Sciences, Gennaro A. R. ed., Mack Publishing Co., Easton, PA, 1990, and especially pp. 1521-1712 therein.

In view of the implications of heparanase activity in various conditions, the peptide dimers of the invention, and compositions comprising the same, are intended for the treatment of said conditions, such as angiogenesis, cancer, inflammation, kidney and autoimmune disorders, as further herein specified.

As used herein in the specification and in the claims section below, the term "treat" or treating and their derivatives includes substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical symptoms of a condition or substantially preventing the appearance of clinical symptoms of a condition.

As used herein in the specification and in the claims section below, the phrase "associated with heparanase catalytic activity" refers to conditions which at least partly depend on the catalytic activity of heparanase. It is understood that the catalytic activity of heparanase under many such conditions may be normal, yet inhibition thereof in such conditions will result in improvement of the affected individual.

It should be further noted that disorders or said conditions may be related to altered function of a HSPG associated biological effector molecule, such as, but not limited to, growth factors, chemokines, cytokines and degradative enzymes. The condition can be, or involve, angiogenesis, tumor cell proliferation, invasion of circulating tumor cells, metastases, inflammatory disorders, autoimmune conditions and/or a kidney disorder. The heparanase inhibitors (e.g., the peptides described herein, as well as the specific substances and the specific antibody, which will be described hereinafter) of the present invention may be used for the treatment of diseases and disorders caused by or associated with heparanase catalytic activity such as, but not limited to, cancer, inflammatory disorders, autoimmune diseases or a kidney disorder.

It is to be therefore understood that the compositions of the invention are useful for treating or inhibiting tumors at all stages, namely tumor formation, primary tumors, tumor progression or tumor metastasis.

Thus, in one embodiment of the present invention, the compositions of the invention can be used for inhibition of angiogenesis, and are thus useful for the treatment of diseases and disorders associated with angiogenesis or neovascularization such as, but not limited to, tumor angiogenesis, opthalmologic disorders such as diabetic retinopathy and macular degeneration, particularly age-related macular degeneration, and reperfusion of gastric ulcer.

As used herein to describe the present invention, "malignant proliferative disorder", "cancer", "tumor" and "malignancy" all relate equivalently to a hyperplasia of a tissue or organ. If the tissue is a part of the lymphatic or immune systems, malignant cells may include non-solid tumors of circulating cells. Malignancies of other tissues or organs may produce solid tumors. In general, the composition as well as the methods of the present invention may be used in the treatment of non-solid and solid tumors, for example, carcinoma, melanoma, leukemia, and lymphoma. Therefore, according to a preferred embodiment, the dimeric peptides of the invention or a composition comprising the same, can be used for the treatment or inhibition of non-solid cancers, e.g. hematopoietic malignancies such as all types of leukemia, e.g. acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndrome (MDS), mast cell leukemia, hairy cell leukemia, Hodgkin's disease, non-Hodgkin's lymphomas, Burkitt's lymphoma and multiple myeloma, as well as for the treatment or inhibition of solid tumors such as tumors in lip and oral cavity, pharynx, larynx, paranasal sinuses, major salivary glands, thyroid gland, esophagus, stomach, small intestine, colon, colorectum, anal canal, liver, gallbladder, extraliepatic bile ducts, ampulla of vater, exocrine pancreas, lung, pleural mesothelioma, bone, soft tissue sarcoma, carcinoma and malignant melanoma of the skin, breast, vulva, vagina, cervix uteri, corpus uteri, ovary, fallopian tube, gestational trophoblastic tumors, penis, prostate, testis, kidney, renal pelvis, ureter, urinary bladder, urethra, carcinoma of the eyelid, carcinoma of the conjunctiva, malignant melanoma of the conjunctiva, malignant melanoma of the uvea, retinoblastoma, carcinoma of the lacrimal gland, sarcoma of the orbit, brain, spinal cord, vascular system, hemangiosarcoma and Kaposi's sarcoma.

The peptide dimers of the invention or any compositions thereof, may also be useful for inhibiting or treating other cell proliferative diseases or disorders such as psoriasis, hypertrophic scars, acne and sclerosis/scleroderma, and for inhibition or treatment of other diseases or disorders such as polyps, multiple exostosis, hereditary exostosis, retrolental fibroplasia, hemangioma, and arteriovenous malformation.

Therefore, in a further embodiment, the compositions of the invention may be useful for treatment of or amelioration of inflammatory symptoms in any disease, condition or disorder where immune and/or inflammation suppression is beneficial such as, but not limited to, treatment of or amelioration of inflammatory symptoms in the joints, musculoskeletal and connective tissue disorders, or of inflammatory symptoms associated with hypersensitivity, allergic reactions, asthma, atherosclerosis, otitis and other otorhinolaryngological diseases, dermatitis and other skin diseases, posterior and anterior uveitis, conjunctivitis, optic neuritis, scleritis and other immune and/or inflammatory ophthalmic diseases.

In another preferred embodiment, the compositions of the invention are useful for treatment of or amelioration of an autoimmune disease such as, but not limited to, Eaton-Lambert syndrome, Goodpasture's syndrome, Greave's disease, Guillain-Barre syndrome, autoimmune hemolytic anemia (AIHA), hepatitis, insulin-dependent diabetes mellitus (IDDM), systemic lupus erythematosus (SLE), multiple sclerosis (MS), myasthenia gravis, plexus disorders e.g. acute brachial neuritis, polyglandular deficiency syndrome, primary biliary cirrhosis, rheumatoid arthritis, scleroderma, thrombocytopenia, thyroiditis, e.g. Hashimoto's disease, Sjogren's syndrome, allergic purpura, psoriasis, mixed connective tissue disease, polymyositis, dermatomyositis, vasculitis, polyarteritis nodosa, polymyalgia rheumatica, Wegener's granulomatosis, Reiter's syndrome, Behget's syndrome, ankylosing spondylitis, pemphigus, bullous pemphigoid, dermatitis herpetiformis, insulin dependent diabetes, inflammatory bowel disease, ulcerative colitis and Crohn's disease.

The compositions of the invention are also intended for treatment of neurodegenerative diseases, such as Alzheimer's, Huntington's, Parkinson's and prion diseases, as well as various amyloidosis syndromes

Still further, heparanase has been proposed to be involved in the pathogenesis of proteinuria by selectively degrading the negatively charged side chains of heparan sulfate proteoglycans within the glomerular basement membrane. A loss of negatively charged heparan sulfate proteoglycans may result in alteration of the permselective properties of the glomerular basement membrane, loss of glomerular epithelial and endothelial cell anchor points, and liberation of growth factors and potentially leading to different kidney disorders, such as, passive Heymann nephritis (PHN), and puromycin aminonucleoside nephrosis (PAN). As described by Levidiotis, V. et al. [Levidiotis, V. et al. (2004) J. Am. Soc. Nephrol. 15, 68-78], a polyclonal antibody against heparanase, significantly reduced proteinuria without affecting the histologic appearance of glomeruli and the immune mechanisms, which give rise to PHN, and therefore, inhibition of heparanase may be used to reduce proteinuria.

Therefore, in another preferred embodiment, the compositions of the invention are useful for treatment of or amelioration of any kidney disorder.

The composition of the invention may comprise the active substance in free form and be administered directly to the subject to be treated. Alternatively, depending on the size of the active molecule, it may be desirable to conjugate it to a carrier prior to administration. Therapeutic formulations may be administered in any conventional dosage formulation. Formulations typically comprise at least one active ingredient, as denned above, together with one or more acceptable carriers thereof.

Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Formulations include those suitable for oral, rectal, nasal, or parenteral (including subcutaneous, intramuscular, intraperitoneal, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The nature, availability and sources, and the administration of all such compounds including the effective amounts necessary to produce desirable effects in a subject are well known in the art and need not be further described herein.

More specifically, said dimeric peptide, or a composition comprising the same, having heparanase inhibitory activity, may be administered by a route selected from oral, intravenous, parenteral, transdermal, subcutaneous, intravaginal, intranasal, mucosal, sublingual, topical and rectal administration and any combinations thereof.

The pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.

In the case of sterile powders for the preparation of the sterile injectable solutions, the preferred method of preparation are vacuum-drying and freeze drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The therapeutically 'effective amount' for purposes herein is that determined by such considerations as are known in the art. The amount must be sufficient to inhibit one of the processes that enables heparanase to be catalitically active. Said processes may be correct folding of the heparanase molecule, binding of heparanase to its substrate, or formation of the heparanase heterodimer, by binding of the 50 kDa subunit to the 8 kDa subunit of heparanase. Inhibition of any of these processes may thereby be sufficient to inhibit heparanase catalytic activity.

The pharmaceutical composition used by the method of the invention can be prepared in dosage units forms and may be prepared by any of the methods well-known in the art of pharmacy. In addition, the pharmaceutical composition may further comprise pharmaceutically acceptable additives such as pharmaceutical acceptable carrier, excipient or stabilizer, and optionally other therapeutic constituents. Naturally, the acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed.

The magnitude of therapeutic dose of the composition of the invention will of course vary with the group of patients (age, sex, etc.), the nature of the condition to be treated and with the route administration, all of which shall be determined by the attending physician.

Although the method of the invention is particularly intended for the treatment of disorders associated with heparanase catalytic activity in humans, other mammals are included. By way of non-limiting examples, mammalian subjects include monkeys, equines, cattle, canines, felines, rodents such as mice and rats, and pigs.

One example for heparanase catalytic activity assay is an assay developed by Freeman and Parish, wherein the products are separated from the substrate by binding to chicken histidine-rich glycoprotein (cHRG) sepharose [Freeman, C. & Parish, C. R. (1997) Biochem. J. 325; 229-237]. In this method only the lowest molecular weight products that lose the ability to bind to cHRG sepharose are detectable, while other, longer products bind, to the column with the substrate and are therefore excluded. Another example for heparanase assay is based on detection of newly formed reducing ends produced due to cleavage of polysaccharides, such as, heparin or heparan sulfate by heparanase [US 6,190,875].

Still further, there are also some non-radioactive assays available for heparanase. The most used assay for heparanase involves measuring the optical density (at 230 nm) of unsaturated uronic acids formed during degradation of heparin. Another color-based assay for measuring heparanase activity utilizes the ability of heparin to interfere with color development during the interaction of protein with the dye Coomassie brilliant blue [Khan, M. Y. and Newman, S. A. (1991) Anal. Biochem. 196, 373-6]. In yet another assay, disclosed in US 6,656,699, a composition comprising biotin-HS is mixed with a sample (such as a tumor sample, bodily fluid, or other fluid suspected of having heparanase activity), to form a reaction mixture. This sample may be pre-treated to remove contaminating or reactive substances such as endogenous biotin. After incubation, an aliquot or portion of the reaction mixture is removed and placed in a biotin-binding plate. After washing with buffers, a Streptavidin-enzyme conjugate is added to the biotin- binding plate. Reagents for the enzyme are added to form a detectable color product. For example, a decrease in color formation, from a known standard, indicates there was heparanase activity in the sample.

The invention further relates to method for the inhibition of heparanase glycosidase catalytic activity comprising the step of in vivo, ex vivo or in vitro contacting heparanase, under suitable conditions, with an inhibitory effective amount of the substance of the invention, or with a composition comprising the same.

In another embodiment, the invention relates to a method for the inhibition of heparanase glycosidase catalytic activity in a subject in need thereof comprising the step of administering to said subject an inhibitory effective amount of a dimeric peptide, or of a composition comprising the same.

Still further, the invention relates to a method for the inhibition or the treatment of a process or a pathologic disorder associated with heparanase glycosidase catalytic activity comprising the step of administering to a subject in need thereof a therapeutically effective amount of the dimeric peptide of the invention, or of a composition comprising the same.

It should be noted that such methods are applicable for a process associated with heparanase catalytic activity, for example, angiogenesis, tumor formation, tumor progression or tumor metastasis, for a malignant proliferative disorder, such as a solid or non-solid tumor selected from the group consisting of carcinoma, sarcoma, melanoma, leukemia and lymphoma, or for inflammatory disorder, autoimmune disorder and or a kidney disorder.

The pharmaceutical compositions of the invention may be administered by the methods of the invention, systemically, for example by parenteral, e.g. intravenous, intraperitoneal or intramuscular injection. In another example, the pharmaceutical composition can be introduced to a site by any suitable route including intravenous, subcutaneous, transcutaneous, topical, intramuscular, intraarticular, subconjunctival, or mucosal, e.g. oral, intranasal, or intraocular administration. Local administration to the area in need of treatment may be achieved by, for example, local infusion during surgery, topical application, direct injection into the inflamed joint, directly onto the eye, etc.

For oral administration, the pharmaceutical preparation may be in liquid form, for example, solutions, syrups or suspensions, or in solid form as tablets, capsules and the like. For administration by inhalation, the compositions are conveniently delivered in the form of drops or aerosol sprays. For administration by injection, the formulations may be presented in unit dosage form, e.g. in ampoules or in multidose containers with an added preservative.

The compositions of the invention can also be delivered in a vesicle, for example, in liposomes. In another embodiment, the compositions can be delivered in a controlled release system.

The amount of the therapeutic or pharmaceutical composition of the invention which is effective in the treatment of a particular disease, condition or disorder will depend on the nature of the disease, condition or disorder and can be determined by standard clinical techniques. In addition, in vitro assays as well in vivo experiments may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease, condition or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

As used herein, "effective amount" means an amount necessary to achieve a selected result. For example, an effective amount of the composition of the invention useful for inhibition of heparanase activity and thereby for the treatment of said pathology.

The therapeutically 'effective amount' for purposes herein is that determined by such considerations as are known in the art. The amount must be sufficient, e.g., to inhibit the correct folding of the heparanase molecule, or to inhibits its binding to the substrate, or even the formation of the 50/8 kDa heterodimer. Ultimately the amount must be able to inhibit heparanase catalytic activity.

A number of methods of the art of molecular biology are not detailed herein, as they are well known to the person of skill in the art. Such methods include site-directed mutagenesis, PCR cloning, expression of cDNAs, analysis of recombinant proteins or peptides, transformation of bacterial and yeast cells, transfection of mammalian cells, and the like. Textbooks describing such methods are e.g., Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory; ISBN: 0879693096, 1989, Current Protocols in Molecular Biology, by F. M. Ausubel, ISBN: 047150338X, John Wiley & Sons, Inc. 1988, and Short Protocols in Molecular Biology, by F. M. Ausubel et al. (eds.) 3rd ed. John Wiley & Sons; ISBN: 0471137812, 1995. These publications are incorporated herein in their entirety by reference. Furthermore, a number of immunological techniques are not in each instance described herein in detail, as they are well known to the person of skill in the art. See e.g., Current Protocols in Immunology, Coligan et al. (eds), John Wiley & Sons. Inc., New York, NY.

The present invention is defined by the claims, the contents of which are to be read as included within the disclosure of the specification.

Disclosed and described, it is to be understood that this invention is not limited to the particular examples, process steps, and materials disclosed herein as such process steps and materials may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The following Examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.

Examples

Experimental Procedures

- Peptide Synthesis:

The 15-amino acid peptide, KKFKNSTYSRSSVDC (also denoted as KKDC, or SEQ ID NO:4), corresponding to the N-terminal domain of the 50 kDa heparanase subunit, and its scrambled analog, KDSYTSNCKVKSFSR (SEQ ID NO: 10), were prepared on a Wang-resin (Nova Biochem, Laeufelingen, Switzerland) with an automated multiple peptide synthesizer (Abimed, model AMS422, Langenfeld, Germany), using the company's protocol for the Fmoc (N-x-fluorenylmethoxycarbonyl) strategy. For purity determination of products, analytical reverse-phase HPLC was performed by using a prepacked Lichrosp here- 100 RP- 18 column (Merck). The correct amino acid compositions and molecular weights of the crude products (purity >95%) were ascertained by amino acid analysis and by mass spectrometry (VG Tofspec, laser desorption mass spectrometer, Fision Instruments) [Di-Segni et al. (2005) J Pept Sci. 11:45-52, 2005; Pelled et al. (2002) J. Biol. Chem. 277: 1957-1961]. In order to prevent peptide dimerization, ethandithiol (EDT, Sigma, St. Louis) was added immediately following peptide synthesis.

- Heparanase activity assay:

Preparation of ECM-coated 35mm dishes and determination of heparanase activity were performed as described in detail elsewhere [Vlodavsky (1999) ibid.; Levy-Adam (2003) ibid.]. For inhibition studies, heparanase (20 ng) was incubated (2 h, 37⁰C) with ³⁵S-labeled ECM in the presence of the indicated concentration of peptides. The incubation medium containing sulfate labeled degradation fragments was subjected to gel filtration on a Sepharose CL-6B column. Fractions (0.2 ml) were eluted with PBS and their radioactivity counted in a β-scintillation counter. Degradation fragments of HS side chains were eluted at 0.5< K_av<0.8 (peak II, fractions 20-33). Peptide dimerization occurs spontaneously upon oxygenation of the solution. To facilitate dimerization, peptides were dissolved with 10% DMSO in water and incubated for five days at room temperature while stirring to enhance oxygenation required for S-S bond formation between cysteine residues forming a dimer. Control peptide was frozen immediately following dissolving to minimize, but not completely block, dimerization. - Immunocytochemistry:

CHO Kl (purchased from American Type Culture Collection, ATCC) and HS- deficient CHO 745 cells (kindly provided by Dr. J. D. Esko, University of California, San Diego, CA) were incubated with biotin-labeled KKDC or scrambled peptide for 2h. Cells were then washed three times with PBS, fixed with 4% paraformaldehyde and incubated for 45 min with Avidin-FITC conjugate. Cells were then extensively washed with PBS, mounted on slides (Vectashield, Vector, Burlingame, CA) and images were obtained by confocal microscope.

- Heparin /HS binding:

Peptides (50 μM) were incubated (2 h, 4⁰C) with heparin-Sepharose beads in PBS, washed with PBS supplemented with NaCl to a final concentration of 0.35 M, followed by one wash with PBS. Dye-free sample buffer was added and the beads were boiled for 5 min, centrifuged and the supernatants were loaded on Tris-Tricine gel. Subsequently, gels were stained with Commassie Blue to visualize bound peptides.

- General Methods of Molecular Biology:

A number of methods of the molecular biology art are not detailed herein, as they are well known to the person of skill in the art. Such methods include PCR cloning, expression of cDNAs, analysis of recombinant proteins or peptides, transformation of bacterial and yeast cells, transfection of mammalian cells, and the like. Textbooks describing such methods are, e.g., Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, ISBN: 0879693096; F. M. Ausubel (1988) Current Protocols in Molecular Biology, ISBN: 047150338X, John Wiley & Sons, Inc. Furthermore, a number of immunological techniques are not in each instance described herein in detail, as they are well known to the person of skill in the art. See, e.g., Harlow and Lane (1988) Antibodies: a laboratory manual. Cold Spring Harbor Laboratory. Example 1 - KKDC dimer is a potent heparanase inhibitor

Since a cysteine residue was introduced at the peptide C-terminus, dimerization may spontaneously occur and improve the interaction with heparin and HS [Verrecchio et al. (2000) J. Biol. Chem. 275: 7701-7707]. In order to test this possibility, the KKDC and the control, scrambled, peptide were intensively oxygenated to facilitate dimerization and tested for their inhibitory capacity. As shown in Figure 1, no significant inhibition of activity was observed upon incubation of heparanase with the control scrambled peptide (Fig. 1, D). In contrast, the KKDC peptide significantly inhibits heparanase enzymatic activity (Fig. 1, A), in agreement with the inventors' previous finding [Levy- Adam (2005) ibid\. Interestingly, the dimeric form of the KKDC peptide was far more efficient as an inhibitor and resulted in complete loss of heparanase enzymatic activity (Fig. 1, Δ), similar to heparanase inhibition by heparin (Fig. 1, @), a well known and potent heparanase inhibitor [Vlodavsky et al. (1994-95) Invasion Metastasis 14: 290- 302].

Example 2

In order to further examine the importance of dimerization for heparin/HS binding, ethandithiol (EDT) was added to the KKDC peptide preparation immediately following synthesis. This reagent binds covalently to the sulfate group of cysteine, thus preventing di sulfide bridge formation between cysteine residues. As demonstrated in Figure 2, dimerized KKDC peptide physically interacts with heparin-Sepharose beads (Fig. 2, KKDC), while no such binding was noted for the control scrambled peptide (Fig. 2, Scr). In contrast, EDT-treated KKDC peptide failed to bind heparin (Fig. 2, KKDC- EDT), clearly implying that peptide dimerization is required in order to bind heparin with high affinity. These finding supports the notion that heparin/HS biding domains of heparanase are attractive targets for the development of heparanase inhibitors and further suggest that relatively simple modification of the peptide into a dimer form markedly improve its ability to bind heparin and consequently its inhibitory capacity. Thus, the KKDC peptide dimer represents a novel class of heparanase inhibitors to be used in cancer, tumor metastasis and inflammation disorders.

Example 3

The inventors have previously demonstrated that the EZKDC peptide inhibits the interaction of heparanase with heparin-Sepharose beads and, moreover, with cell surface HS [Levy- Adam (2005) ibid], which likely explain its ability to inhibits the enzymatic activity of heparanase. In order to further confirm interaction with the cell surface, the KKDC peptide was labeled with biotin and, following incubation, binding was visualized by Avidin-FITC. As shown in Figure 3B, the KKDC peptide binds and localize to the cell surface of CHO cells, while no such staining was observed with control, scrambled peptide (Fig. 3A). This interaction is mediated by HS since heparin significantly reduced peptide binding (Fig. 3C). Moreover, no significant staining was noted upon incubation of the peptide with CHO-745 cells that devoid of HS (Fig. 3D). This observation supports the previous characterization of the KKDC region as a heparin/HS binding domain of heparanase and clearly indicates that heparanase can interact with extracellular HS in the ECM as well as with cell surface HS, and utilize it as a substrate or as a trafficking route for internalization [Gingis-Velitski, S. et al. (2004) J. Biol. Chem. 279: 44084-44092].

Example 4 - Anti-tumorigenicity of the dimeric peptide

Cells from exponential cultures of human prostate PC3 cells (purchased from ATCC) are detached with trypsin, washed with PBS and brought to a concentration of 3xl0⁷cells/ml. The cell suspension (3xl0⁶/0.1 ml) is inoculated subcutaneously at the right flank of 5-week old female SCID mice (n=5). Xenograft size is determined twice a week by externally measuring tumors in two dimensions using a caliper. Tumor volume (V) is determined by the equation V=L x W² x 0.5, where L is the length and W the width of the xenograft. At the end of the experiment, mice are sacrificed; xenografts are resected, weighted, fixed in formalin and 5 micron sections are analyzed by histology and immunohistologically.

Treatment with dimer peptide or scrambled peptide (as control) is effected twice a week, via i.p. administration of 1 mg of peptide per animal, which is approximately equivalent to 40 mg/kg of peptide. For injection, the dimeric peptide, or the scrambled control peptide, is dissolved in 10%DMSO/90%H2θ.

All animal experiments were approved by the Animal Care Committee of the Technion Institute, Haifa, Israel.

Claims

CLAIMS:

1. A peptide dimer consisting of two identical monomer peptides chemically linked to each other, wherein said monomer peptide has an amino acid sequence derived from the 5OkDa subunit of heparanase.

2. The peptide of claim 1, wherein said two monomers are linked via any one of covalent bonds and a chemical linker.

3. The peptide dimer of any one of claims 1 and 2, wherein said sequence comprises a heparin-binding domain.

4. The peptide dimer of any one of claims 1 to 3, wherein said sequence is derived from the N' terminus region of heparanase.

5. The peptide dimer of any one of claims 1 to 4, wherein said sequence comprises amino acid residues selected from the group consisting of: Lys¹⁵⁸ to Asp¹", _as denoted by SEQ ID NO:1, Lys²⁶² to Lys²⁸°, as denoted by SEQ ID NO:2, and Lys⁴¹¹ to Arg⁴³², as denoted by SEQ ID NO:3 of human heparanase, or any functionally equivalent fragment, derivative and variant thereof.

6. The peptide dimer of any one of claims 1 to 5, wherein said two monomers are linked to each other via one additional cysteine residue at the N'- or C'-terminus.

7. The peptide dimer of claim 6, wherein said monomers are linked to each other by di-sulfi.de bonds between their C- and/or N'-termini.

8. A peptide dimer consisting of two identical monomer peptides chemically linked to each other, wherein said monomer peptides are as denoted by any one of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

9. The peptide dimer of claim 8, wherein said monomer peptides are linked to each other by di-sulfide bonds between their C-termini.

10. The peptide dimer of any one of claims 1 to 9, wherein said peptide is an heparanase inhibitor.

11. A pharmaceutical composition comprising as active ingredient a peptide dimer of any one of claims 1 to 10.

12. The pharmaceutical composition of claim 11, for the inhibition of heparanase catalytic activity.

13. The pharmaceutical composition of any one of claims 11 and 12, for medical use.

14. The pharmaceutical composition of any one of claims 11 to 13, for the treatment of a condition associated with heparanase catalytic activity.

15. The pharmaceutical composition of claim 14, wherein said condition is one of angiogenesis, tumor formation, tumor progression, tumor metastasis, malignant proliferative disorder, inflammatory disorder, kidney disorder, and autoimmune disorder.

16. The pharmaceutical composition of claim 15, wherein said malignant proliferative disorder is any one or solid and non-solid tumor selected from the group consisting of carcinoma, sarcoma, melanoma, leukemia, lymphoma and glioma.

17. The pharmaceutical composition of any one of claims 12 to 16, further comprising a pharmaceutically acceptable carrier, diluent, excipient and/or additive.

18. Use of a peptide dimer as defined in any one of claims 1 to 10, in the preparation of a pharmaceutical composition for the treatment of a condition related to heparanase catalytic activity.

19. The use of claim 18, wherein said condition is one of angiogenesis, tumor formation, tumor progression, tumor metastasis, malignant proliferative disorder, inflammatory disorder, kidney disorder, and autoimmune disorder.

20. Use of a peptide dimer as defined in any one of claims 1 to 10, in the preparation of a composition for the inhibition of heparanase catalytic activity.

21. Use of a dimeric peptide as defined in any one of claims 1 to 10, as an agent for the inhibition of heparanase catalytic activity.

22. A method for the inhibition of heparanase catalytic activity comprising administering a therapeutically effective dosage of a peptide dimer as defined in any one of claims 1 to 10, or a composition comprising the same, as denned in any one of claims 11 to 17, to a subject in need.

23. A method for the treatment of conditions related to heparanase catalytic activity, comprising administering a therapeutically effective dosage of a peptide dimer as defined in any, one of claims 1 to 10, or a composition comprising the same, as defined in any one of claims 11 to 17, to a subject in need.

24. The method of claim 23, wherein said condition is one of angiogenesis, tumor formation, tumor progression, tumor metastasis, malignant proliferative disorder, inflammatory disorder, kidney disorder, and autoimmune disorder.