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WO2007013050A1 - Dimeres de peptides derives de l'heparanase et leurs utilisations en tant qu'inhibiteurs de l'heparanase - Google Patents

Dimeres de peptides derives de l'heparanase et leurs utilisations en tant qu'inhibiteurs de l'heparanase Download PDF

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Publication number
WO2007013050A1
WO2007013050A1 PCT/IL2005/000791 IL2005000791W WO2007013050A1 WO 2007013050 A1 WO2007013050 A1 WO 2007013050A1 IL 2005000791 W IL2005000791 W IL 2005000791W WO 2007013050 A1 WO2007013050 A1 WO 2007013050A1
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Prior art keywords
heparanase
peptide
peptide dimer
disorder
catalytic activity
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Inventor
Israel Vlodavsky
Neta Ilan
Flonia Levy-Adam
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Hadasit Medical Research Services and Development Co
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Hadasit Medical Research Services and Development Co
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01166—Heparanase (3.2.1.166)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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.
  • Heparanase is an endo- ⁇ -D-glucuronidase involved in cleavage of heparan sulfate (HS) chains, and hence participates in extracellular matrix (ECM) degradation and remodeling.
  • ECM extracellular matrix
  • 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.
  • 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.
  • 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 lo 9-Ser n ° 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].
  • 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.
  • said two monomers are linked via one of covalent bonds and a chemical linker.
  • said sequence comprises a hep arin-bin ding domain, and is preferably derived from the N' terminus region of heparanase.
  • said sequence comprises amino acid residues selected from the group consisting of: Lys 158 to Asp 171 , as denoted by SEQ ID NO:1, Lys 2 6 2 to Lys 280 , as denoted by SEQ ID NO:2, and Lys 411 to Arg 432 , as denoted by SEQ ID NO:3 of human heparanase, or any functionally equivalent fragment, derivative and variant thereof.
  • said two monomers are linked to each other via one additional cysteine residue at the N'- or C'-terminus.
  • said monomers are linked to each other by disulfide bonds between their C- and/or N'-termini.
  • 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.
  • 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.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising as active ingredient the peptide dimer of the invention, substantially as described above.
  • said composition is for the inhibition of heparanase catalytic activity.
  • said pharmaceutical composition comprising as active ingredient the peptide dimer of the invention substantially as described above, is for medical use.
  • 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.
  • 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.
  • the pharmaceutical composition described herein above further comprises a pharmaceutically acceptable carrier, diluent, excipient and/or additive.
  • 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.
  • 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.
  • dimeric peptide of the invention may be used as an agent for the inhibition of heparanase catalytic activity.
  • 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.
  • 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.
  • said condition is one of angiogenesis, tumor formation, tumor progression, tumor metastasis, malignant proliferative disorder, inflammatory disorder, kidney disorder, and autoimmune disorder.
  • FIG. 1 KKDC peptide dimerization facilitates the inhibition of heparanase enzymatic activity.
  • Heparanase was incubated (2 h, 37 0 C) with 35 S-labeled ECM in the absence ( ⁇ ) or presence of 50 ⁇ g/ml of the KKDC peptide (A), KKDC peptide in a dimer form ( ⁇ ) or its control, scrambled peptide ( ⁇ ). Heparin (15 ⁇ g/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.
  • FIG. 1 KKDC peptide physically interacts with heparin- Sepharose beads, but not when treated with EDT.
  • Figure 3A-3D Binding of the KKDC peptide to cell surface heparan sulfate.
  • CHO Kl Fig.3A-3C
  • HS-deficient CHO 745 Fig. 3D
  • Fig. 3A, Scr control scrambled
  • KKDC-biotin conjugate in the absence
  • Fig. 3B, 3D Fig. 3B, 3D
  • .heparin 50 ⁇ g/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).
  • the inventors show that a dimeric peptide is significantly more potent than the monomer peptide in inhibiting heparanase catalytic activity.
  • the inventors prepared homodimeric peptides, and evaluated their capacity to inhibit heparanase catalytic activity.
  • the dimers were composed of two monomeric units of one of the following peptides: Lys 158 to Asp 171 , as denoted by SEQ ID NO:1, Lys 262 to Lys 2 8o, as denoted by SEQ ID NO:2, and Lys 411 to Arg « 2 , as denoted by SEQ ID NO: 3.
  • 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
  • KKDC KKFKNSTYSRSSVDC
  • amino acid locations (Lys 158 to Asp 171 , Lys 262 to Ly s 280 and Lys 411 to Arg 432 ) refer to the amino acid sequence of human heparanase as denoted by GenBank Accession No. AF144325.
  • 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].
  • monomer peptides may be synthesized by chemical procedures, like solid phase peptide synthesis (SPPS), for example.
  • SPPS solid phase peptide synthesis
  • 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).
  • the link may be through covalent bonds or through a linker.
  • 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.
  • 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 Lys 262 -Lys 280 -Cys-linked monomers, each as denoted by SEQ ID NO:8, or the dimer composed of two Lys 411 -Arg 432 - Cys linked monomers, each as denoted by SEQ ID NO: 9.
  • 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.
  • the extra cysteine residue may be added to the N'-terminus of the monomers during synthesis.
  • Monomer linkage 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].
  • 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.
  • 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.
  • 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:
  • 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 ⁇ i ⁇ o. Preparation of PEG-linked peptides has been described previously [Guerra et al. (1998) Pharm. Res. 15:1822-7].
  • PEG polyethyleneglycol
  • amino acid locations (Lys 158 to Asp 171 , Lys 262 to Ly S 28o an( j Lys 411 to Arg 432 ) refer to the amino acid sequence of human heparanase as denoted by GenBank Accession No. AF144325.
  • the dimeric peptide described herein is a potent inhibitor of heparanase catalytic activity.
  • 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 ⁇ -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.
  • 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.
  • 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.
  • fragments 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 Lys 158 to Asp 171 , Ly S 26 2 to Lys 280 and Lys 411 to Arg 432 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.
  • functional is meant having same biological function, for example, being capable of inhibiting heparanase catalytic activity.
  • derivatives and functional derivatives 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%.
  • insertions 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.
  • 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.
  • 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.
  • 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.
  • the peptide may be extended at the N-terminus and/or C-terminus thereof with an N- acetyl group.
  • 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.
  • FGF-2 bFGF
  • ECM extracellular matrix
  • 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].
  • HS heparan sulfate
  • 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.
  • activation signals e.g., thrombin, calcium ionophore, immune complexes, antigens, mitogens
  • 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].
  • heparanase alternative substrates e.g., non-anticoagulant species of low molecular weight heparin
  • EAE experimental autoimmune encephalomyelitis
  • graft rejection e.g., heparanase inhibitors may be applied to inhibit autoimmune and inflammatory diseases
  • 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.
  • compositions are 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.
  • the peptide dimers of the invention are intended for the treatment of said conditions, such as angiogenesis, cancer, inflammation, kidney and autoimmune disorders, as further herein specified.
  • 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.
  • 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.
  • 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.
  • heparanase inhibitors e.g., the peptides described herein, as well as the specific substances and the specific antibody, which will be described hereinafter
  • heparanase catalytic activity such as, but not limited to, cancer, inflammatory disorders, autoimmune diseases or a kidney disorder.
  • compositions of the invention are useful for treating or inhibiting tumors at all stages, namely tumor formation, primary tumors, tumor progression or tumor metastasis.
  • 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.
  • 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.
  • malignant proliferative disorder 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.
  • 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.
  • ALL acute lymphocytic leukemia
  • AML acute myelogenous leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • MDS myelodysplastic syndrome
  • mast cell leukemia hairy cell leukemia
  • Hodgkin's disease non-Hodgkin's lymphomas
  • Burkitt's lymphoma multiple myeloma
  • 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 melanom
  • 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.
  • diseases or disorders such as psoriasis, hypertrophic scars, acne and sclerosis/scleroderma
  • other diseases or disorders such as polyps, multiple exostosis, hereditary exostosis, retrolental fibroplasia, hemangioma, and arteriovenous malformation.
  • 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.
  • 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 uveit
  • 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.
  • 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.
  • 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
  • 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).
  • PPN passive Heymann nephritis
  • PAN puromycin aminonucleoside nephrosis
  • compositions of the invention are useful for treatment of or amelioration of any kidney disorder.
  • 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 employeed 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the pharmaceutical composition may further comprise pharmaceutically acceptable additives such as pharmaceutical acceptable carrier, excipient or stabilizer, and optionally other therapeutic constituents.
  • pharmaceutical acceptable carrier, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed.
  • 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.
  • mammalian subjects include monkeys, equines, cattle, canines, felines, rodents such as mice and rats, and pigs.
  • 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.
  • cHRG histidine-rich glycoprotein
  • 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].
  • 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].
  • 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.
  • a sample such as a tumor sample, bodily fluid, or other fluid suspected of having heparanase activity
  • This sample may be pre-treated to remove contaminating or reactive substances such as endogenous biotin.
  • an aliquot or portion of the reaction mixture is removed and placed in a biotin-binding plate.
  • 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.
  • the invention in another embodiment, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • parenteral e.g. intravenous, intraperitoneal or intramuscular injection.
  • 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.
  • the pharmaceutical preparation may be in liquid form, for example, solutions, syrups or suspensions, or in solid form as tablets, capsules and the like.
  • the compositions are conveniently delivered in the form of drops or aerosol sprays.
  • the formulations may be presented in unit dosage form, e.g. in ampoules or in multidose containers with an added preservative.
  • compositions of the invention can also be delivered in a vesicle, for example, in liposomes.
  • 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.
  • an effective amount means an amount necessary to achieve a selected result.
  • 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.
  • the amount must be able to inhibit heparanase catalytic activity.
  • KKFKNSTYSRSSVDC also denoted as KKDC, or SEQ ID NO:4
  • KKDC scrambled analog
  • KDSYTSNCKVKSFSR KDSYTSNCKVKSFSR
  • CHO Kl purchased from American Type Culture Collection, ATCC
  • 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.
  • Peptides 50 ⁇ M were incubated (2 h, 4 0 C) 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.
  • Example 1 - KKDC dimer is a potent heparanase inhibitor
  • ethandithiol 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.
  • 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).
  • EDT-treated KKDC peptide failed to bind heparin (Fig.
  • KKDC- EDT a novel class of heparanase inhibitors to be used in cancer, tumor metastasis and inflammation disorders.
  • 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.
  • 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).
  • Treatment with dimer peptide or scrambled peptide 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.
  • the dimeric peptide, or the scrambled control peptide is dissolved in 10%DMSO/90%H2 ⁇ .

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Abstract

L'invention a pour objet des dimères de peptide, dérivés de la région N' terminale de l'héparanase, lesquels inhibent l'activité catalytique de l'héparanase. L'invention concerne également des compositions pharmaceutiques, leur utilisation et des méthodes qui utilisent lesdits dimères de peptides pour l'inhibition de l'activité de l'héparanase dans des conditions en rapport avec celle-ci.
PCT/IL2005/000791 2005-07-25 2005-07-25 Dimeres de peptides derives de l'heparanase et leurs utilisations en tant qu'inhibiteurs de l'heparanase Ceased WO2007013050A1 (fr)

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WO2007132445A3 (fr) * 2006-05-11 2008-07-24 Hadasit Med Res Service Utilisation d'une forme non catalytique d'héparanase et de peptides de ce composé pour inverser les effets anticoagulants des héparinoïdes
WO2015179635A3 (fr) * 2014-05-21 2016-01-28 President And Fellows Of Harvard College Peptides inhibiteurs de ras et leurs utilisations
WO2017064716A1 (fr) * 2015-10-13 2017-04-20 Rappaport Family Institute For Research Anticorps monoclonaux neutralisant l'héparanase
US10081654B2 (en) 2013-03-13 2018-09-25 President And Fellows Of Harvard College Stapled and stitched polypeptides and uses thereof
US10227390B2 (en) 2013-06-14 2019-03-12 President And Fellows Of Harvard College Stabilized polypeptide insulin receptor modulators
WO2021128255A1 (fr) * 2019-12-27 2021-07-01 深圳市海普瑞药业集团股份有限公司 Charge d'affinité, son procédé de préparation et son utilisation

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A ZETSER ET AL.: "Processing and activation of latent heparanase occurs in lysosomes", JOURNAL OF CELL SCIENCE, vol. 117, 5 January 2004 (2004-01-05), GB CAMBRIDGE UNIVERSITY PRESS, LONDON, pages 2249 - 2258, XP002336686 *
E MCKENZIE ET AL.: "Biochemical characterization of the active heterodimer form of human heparanase (Hpa1) protein expressed in insect cells", BIOCHEMICAL JOURNAL., vol. 373, 2003, GB THE BIOCHEMICAL SOCIETY, LONDON., pages 423 - 435, XP002310698 *
F LEVY-ADAM ET AL.: "Heterodimer formation is essential for heparanase enzymatic activity", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS., vol. 308, 2003, US ACADEMIC PRESS INC. ORLANDO, FL., pages 885 - 891, XP004447196 *
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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007132445A3 (fr) * 2006-05-11 2008-07-24 Hadasit Med Res Service Utilisation d'une forme non catalytique d'héparanase et de peptides de ce composé pour inverser les effets anticoagulants des héparinoïdes
US12258423B2 (en) 2013-03-13 2025-03-25 President And Fellows Of Harvard College Stapled and stitched polypeptides and uses thereof
US11332496B2 (en) 2013-03-13 2022-05-17 President And Fellows Of Harvard College Stapled and stitched polypeptides and uses thereof
US10081654B2 (en) 2013-03-13 2018-09-25 President And Fellows Of Harvard College Stapled and stitched polypeptides and uses thereof
US10227390B2 (en) 2013-06-14 2019-03-12 President And Fellows Of Harvard College Stabilized polypeptide insulin receptor modulators
IL249032B (en) * 2014-05-21 2021-01-31 Harvard College ras inhibitory peptides and their uses
US10533039B2 (en) 2014-05-21 2020-01-14 President And Fellows Of Harvard College Ras inhibitory peptides and uses thereof
IL279547A (en) * 2014-05-21 2021-01-31 Harvard College Peptides inhibit RAS and their uses
US11377476B2 (en) 2014-05-21 2022-07-05 President And Fellows Of Harvard College Ras inhibitory peptides and uses thereof
WO2015179635A3 (fr) * 2014-05-21 2016-01-28 President And Fellows Of Harvard College Peptides inhibiteurs de ras et leurs utilisations
US11161912B2 (en) 2015-10-13 2021-11-02 Technion Research & Development Foundation Limited Heparanase-neutralizing monoclonal antibodies
WO2017064716A1 (fr) * 2015-10-13 2017-04-20 Rappaport Family Institute For Research Anticorps monoclonaux neutralisant l'héparanase
WO2021128255A1 (fr) * 2019-12-27 2021-07-01 深圳市海普瑞药业集团股份有限公司 Charge d'affinité, son procédé de préparation et son utilisation
CN114846137A (zh) * 2019-12-27 2022-08-02 深圳市海普瑞药业集团股份有限公司 一种亲和填料及其制备方法和应用

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