WO2015087334A1 - Peptides dérivés de vipéristatine et leurs utilisations - Google Patents

Peptides dérivés de vipéristatine et leurs utilisations Download PDF

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WO2015087334A1
WO2015087334A1 PCT/IL2014/051092 IL2014051092W WO2015087334A1 WO 2015087334 A1 WO2015087334 A1 WO 2015087334A1 IL 2014051092 W IL2014051092 W IL 2014051092W WO 2015087334 A1 WO2015087334 A1 WO 2015087334A1
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Prior art keywords
peptide
sequence
seq
peptides
amino acids
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PCT/IL2014/051092
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English (en)
Inventor
Philip Lazarovici
Tatjana MOMIC
Cezary Marcinkiewicz
Jehoshua Katzhendler
Johannes Eble
Hadar ARIEN-ZAKAY
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Yissum Research Develoment Company Of The Hebrew University Of Jerusalem Ltd.
Temple University
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Publication of WO2015087334A1 publication Critical patent/WO2015087334A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • 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 relates to analogs and derivatives of Viperistatin, a snake venom-derived peptide that inhibits the collagen receptor alpha 1 beta 1 ( ⁇ ) integrin. More particularly, the present invention relates to cyclic analogs and derivatives of Viperistatin and their therapeutic uses as anti-angiogenic and anti-adhesive agents.
  • Integrins are a family of various cell surface receptors which play essential roles in cell adhesion and migration. These heterodimeric receptors are composed of non- covalently linked distinct subunits, a and ⁇ , each containing a large extracellular binding domain, a transmembrane region, and a short cytoplasmic domain. Upon ligand binding, integrins cluster and recruit via their cytoplasmic domains cytoskeletal, adaptor and signaling proteins, thus eventually forming the focal adhesions, which not only anchors the cell to the subjacent extracellular matrix (ECM) ligands, but also conveys signals into the cell (Hynes, 1992, Cell 69: 11-25).
  • ECM extracellular matrix
  • ⁇ 5 ⁇ 1 represents the "classical" fibronectin receptor
  • ⁇ 4 ⁇ 1 and ⁇ 4 ⁇ 7 interact with fibronectin and VCAM-1
  • ⁇ 8 ⁇ 1 and ⁇ 9 ⁇ 1 bind tenascin
  • ⁇ and ⁇ 3 are major vitronectin receptors
  • ⁇ 3 is the platelet fibrinogen receptor involved in platelet aggregation
  • integrins ⁇ 3 ⁇ 1, ⁇ 6 ⁇ 1, ⁇ 6 ⁇ 4, and ⁇ 7 ⁇ 1 are laminin receptors.
  • the two major collagen-binding integrins ⁇ and ⁇ 2 ⁇ 1, preferentially bind to collagen type IV and I, respectively (Hynes 1992, supra).
  • ⁇ and ⁇ 2 ⁇ 1 preferentially bind to collagen type IV and I, respectively.
  • a subunits of collagen-binding integrins contain a unique 200 amino acid long A-domain, which is involved in collagen binding (Leitinger & Hogg, 1999, Biochem Soc Trans 27: 826-832).
  • Studies with al/a2 integrin A-domain chimeras confirmed the preference for different collagen types (Abair et al., 2008, Exp Cell Res 314: 3593-3604).
  • integrin receptors are central to the etiology and pathology of many disease states such as cardiovascular, inflammatory and neurodegenerative, and therefore have been actively targeted for drug discovery (Goodman & Picard, 2012, Trends Pharmacol Sci 33: 405- 412). Many approaches have been developed with the aim of identifying integrin inhibitors, ranging from classical small molecules, cyclic peptides and engineered antibodies (Goodman & Picard 2012, supra).
  • Snake venom natural toxins such as disintegrins (Calvete et al., 2005, Toxicon 45: 1063-1074; Marcinkiewicz, 2005, Curr Pharm Des 11 : 815-827) and C-type lectins (Arlinghaus et al., 2012, Toxicon 60: 512-519) are important pharmacological tools since they selectively inhibit integrins.
  • KTS containing disintegrins are very selective for ⁇ integrin, in contrast to RGD (Arg-Gly-Asp) disintegrins which block ⁇ 8 ⁇ 1, ⁇ 5 ⁇ 1, ⁇ , ⁇ 3 and ⁇ 3 ⁇ 4 ⁇ 3.
  • the potency of KTS disintegrin is strictly dependent on KTS motif since mutation of individual amino acids in this motif, decreased potency by 10 to 80 fold (Brown et al., 2009, Biochem J All: 95- 101).
  • KTS motif like RGD
  • Viperistatin and related natural disintegrin peptides have limited value as potential therapeutic molecules for preventing cell adhesion, including potential uses for anti-angiogenesis, due to their relative large size, compared to peptidomimetic drugs, proteolytic degradation upon oral delivery and potential immunogenicity.
  • WO 2002/022571 discloses compounds which are potent and specific inhibitors of ⁇ integrin.
  • the ⁇ integrin-inhibiting compounds of WO 2002/022571 include the peptide Obtustatin as well as fragments, derivatives, homologs and analogs thereof.
  • Viperistatin particularly short analogs and derivatives of Viperistatin with dual antagonistic activity towards both ⁇ and ⁇ 2 ⁇ 1 integrins, with anti-cell adhesion and anti-angiogenic activities.
  • the present invention according to some aspects provides cyclic peptides derived from the disintegrin Viperistatin.
  • the present invention further provides pharmaceutical compositions comprising the peptides and methods for treating diseases involving excessive cell adhesion and/or angiogenesis utilizing the cyclic peptide derivatives.
  • the present invention discloses for the first time analogs and derivatives of Viperistatin which advantageously show dual selectivity and antagonism towards the two major collagen receptors, ⁇ and ⁇ 2 ⁇ 1 integrins. This is in contrast to the parent compound Viperistatin and the structurally-similar Obtustatin, that each has relative selectivity towards only ⁇ integrin receptors.
  • the analogs and derivatives of Viperistatin disclosed herein were found to have lower or intermediate efficacy, meaning lower inhibitory activity, compared to the parent compound.
  • intermediate efficacy is in effect beneficial for therapeutic applications, in that it allows gentle manipulation of the endothelial cell adhesion and angiogenesis.
  • the analogs and derivatives of Viperistatin disclosed herein show increased potency compared to the original compound, meaning that the maximal activity is achieved at lower concentrations.
  • the natural parent compound Viperistatin and its related compound Obtustatin are snake venom toxins with a narrow therapeutic window
  • the peptides disclosed herein were found to have an improved therapeutic index inasmuch as they are non-toxic to endothelial cells and safe upon intravenous injection in mice.
  • the peptides of the present invention have anti-cell adhesion properties.
  • the anti-cell adhesion activity is capable of inhibiting angiogenesis, as exemplified herein below.
  • the peptides of the present invention were found to have antitumor activity, including anti metastatic activity, as exemplified herein below in a mouse model of melanoma.
  • peptides of the present invention are particularly useful for the treatment and/or diagnosis of a variety of medical conditions including, for example, certain types of cancer, retinal disorders involving excessive angiogenesis, e.g. macular degeneration, as well as fibrosis, psoriasis, and other diseases involving ⁇ and ⁇ 2 ⁇ 1 integrins.
  • the peptides of the present invention comprise at least one cyclization.
  • the positions of the cyclization(s) of the peptides of the present invention namely, the residues involved in the cyclization, are different from the positions of the cyclizations in the parent compound Viperistatin or in Obtustatin.
  • the present invention provides a synthetic cyclic peptide of 8-30 amino acids comprising the sequence WKTSXiRTSH (SEQ ID NO: 1), wherein Xi is absent or Cys, the peptide containing said sequence in a cyclic portion thereof.
  • WKTSXiRTSH SEQ ID NO: 1
  • Xi is absent or Cys
  • a peptide of 9-30 amino acids comprising the sequence WKTSRTSHY (SEQ ID NO: 2) in a cyclic portion thereof.
  • a peptide of 11-30 amino acids is provided, comprising the sequence CWKTSRTSHYC (SEQ ID NO: 3) in a cyclic portion thereof.
  • the peptide comprises the sequence CWKTSRTSHYC
  • SEQ ID NO: 3 in a cyclic portion thereof, and is cyclized via a disulfide bridge connecting the side chains of two cysteine residues present in the sequence.
  • the peptide comprising SEQ ID NO: 3 is cyclized via a disulfide bridge between residues one and eleven of said sequence.
  • the peptide comprises 15-30 amino acids, for example 15-
  • the peptide is monocyclic, namely, containing a single cyclization.
  • the peptide is bi-cyclic, namely, containing two cyclizations.
  • the peptide is tri-cyclic, namely, containing three cyclizations.
  • the peptide comprises a plurality of cyclizations, wherein each cyclization is independently selected from the group consisting of side-chain to side- chain; end to end; backbone to backbone and backbone to end. In some embodiments, each cyclization is independently selected from the group consisting of side-chain to side- chain and end to end.
  • At least one of said plurality of cyclizations is formed by a disulfide bridge connecting the side chains of two cysteine residues present in the sequence.
  • all of said plurality of cyclizations are formed by disulfide bridges connecting the side chains of two cysteine residues.
  • At least one of said plurality of cyclizations is formed by an amide bond connecting the side chains of two residues in the sequence.
  • all of said plurality of cyclizations are formed by amide bonds connecting the side chains of two residues in the sequence.
  • the peptide further comprises an additional sequence C- terminal to the above-specified sequences, the additional sequence comprising 1-12 contiguous amino acid residues from residues 30-41 of Viperistatin or Obtustatin.
  • the additional sequence comprises 3-6 contiguous amino acid residues derived from residues 30-41 of Viperistatin or Obtustatin.
  • the additional sequence C-terminal to the above-specified sequences comprises the sequence TGKS (SEQ ID NO: 4).
  • the additional sequence is selected from the group consisting of TGKSCG (SEQ ID NO: 5) and TGKSD (SEQ ID NO: 6). Each possibility represents a separate embodiment of the present invention.
  • the additional sequence C-terminal to the above-specified sequences comprises PLY.
  • the additional sequence is selected from the group consisting of PLYPG (SEQ ID NO: 7) and PLYQG (SEQ ID NO: 8).
  • the peptide comprises the sequence CWKTSRTSHYC (SEQ ID NO: 3) and an additional sequence of 3-6 contiguous amino acid residues from residues 30-41 of Viperistatin or Obtustatin C-terminal to CWKTSRTSHYC.
  • the peptide comprises the sequence CWKTSRTSHYC (SEQ ID NO: 3) and further comprises a sequence selected from the group consisting of TGKSCG (SEQ ID NO: 5) and TGKSD (SEQ ID NO: 6) C-terminal to CWKTSRTSHYC.
  • TGKSCG SEQ ID NO: 5
  • TGKSD SEQ ID NO: 6
  • the peptide comprises the sequence CWKTSRTSHYC (SEQ ID NO: 3) and further comprises a sequence selected from the group consisting of PLYPG (SEQ ID NO: 7) and PLYQG (SEQ ID NO: 8) C-terminal to CWKTSRTSHYC.
  • PLYPG SEQ ID NO: 7
  • PLYQG SEQ ID NO: 8
  • the peptide sequence is selected from the group consisting of CCWKTSRTSHYCTGKSCG (SEQ ID NO: 9), CWKTSRTSHYCPLYPG (SEQ ID NO: 10), CWKTSRTSHYCPLYQG (SEQ ID NO: 11), CWKTSRTSHYCTGKSD (SEQ ID NO: 12) and CWKTSCRTSHTGKSD (SEQ ID NO: 13).
  • CCWKTSRTSHYCTGKSCG SEQ ID NO: 9
  • CWKTSRTSHYCPLYPG SEQ ID NO: 10
  • CWKTSRTSHYCPLYQG SEQ ID NO: 11
  • CWKTSRTSHYCTGKSD SEQ ID NO: 12
  • CWKTSCRTSHTGKSD SEQ ID NO: 13
  • the peptide sequence is selected from the group consisting of CCWKTSRTSHYCTGKSCG (SEQ ID NO: 9) and CWKTSRTSHYCPLYPG (SEQ ID NO: 10).
  • the cyclic peptide is selected from the group consisting of compounds (10), (8), (7), (6) and (5):
  • the present invention provides a pharmaceutical composition comprising a peptide of the present invention and optionally a pharmaceutically acceptable excipient.
  • composition of the present invention may be formulated for local or systemic routes of administration.
  • the present invention provides a method for treating a disease or disorder involving excessive cell adhesion and/or angiogenesis in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a peptide of the present invention.
  • the present invention provides a pharmaceutical composition comprising a peptide of the invention, for use in the treatment of a disease or disorder involving excessive angiogenesis.
  • the diseases or disorders amenable to treatment according to the principles of the present invention are those where abnormal, excessive angiogenesis is implicated in their pathogenesis, or facilitates their progression.
  • the disease or disorder is cancer.
  • the disease or disorder is a cancerous solid tumor.
  • Cancer types particularly suitable for treatment by the method of the present invention are those where angiogenesis is known to underlie their progression.
  • the method may be used for the treatment of primary as well as metastatic cancer.
  • the method is particularly suitable for the treatment of vascularized tumors.
  • the disease or disorder is a retinal disorder.
  • proliferative retinopathies may be treated, including retinopathy of prematurity, retrolental fibroplasia, neovascular glaucoma, and age-related macular degeneration (particularly wet age-related macular degeneration (wAMD)). Additional examples include macular edema following retinal vein occlusion (RVO) and diabetic macular edema.
  • RVO retinal vein occlusion
  • diabetic macular edema are a separate embodiment of the invention.
  • non-neoplastic inflammatory conditions may be treated, including, but are not limited to, rheumatoid arthritis, psoriasis, fibrosis, atherosclerosis and thyroid hyperplasias (including Grave's disease).
  • rheumatoid arthritis including, but are not limited to, rheumatoid arthritis, psoriasis, fibrosis, atherosclerosis and thyroid hyperplasias (including Grave's disease).
  • the present invention provides a pharmaceutical composition comprising a peptide of the present invention, preferably attached to a detectable label, for use in the diagnosis of a disease or disorder associated with abnormal expression of ⁇ integrin, ⁇ 2 ⁇ 1 integrin or both.
  • a disease or disorder associated with abnormal expression of ⁇ integrin, ⁇ 2 ⁇ 1 integrin or both.
  • Such disease or disorder may include diseases or disorders associated with up regulation, down regulation, or mutation of ⁇ and/or ⁇ 2 ⁇ 1 integrins.
  • the diseases or disorders to be diagnosed are those associated with upregulation of ⁇ and/or ⁇ 2 ⁇ 1 integrins.
  • the disease or disorder is a disease or disorder involving excessive angiogenesis.
  • the present invention provides a pharmaceutical composition comprising a peptide of the present invention, for use in the imaging of angiogenesis.
  • Figure 2 The inhibitory effect of the peptides on al and a2 overexpressed cell adhesion.
  • Dose response curves of inhibition of al (A-C) (solid symbols) and a2 (D-F) (open symbols) cell adhesion to respective collagens.
  • Viperistatin /Vixapatin (squares) were used as controls.
  • the number of adherent cells (mean ⁇ SD) is derived from three independent experiments.
  • Figure 3 The inhibitory effect of the peptides on binding GST- al and GST- a2 A domains to their respective collagens. Binding of GST- alA domain (A and B) to CB3 (collagen IV fragment); and GST- a2A domain (C and D) to collagen I in the presence of (A and C) 1 ⁇ ; (B and D) 2-1000 ⁇ of the peptides. Solid symbols represent Viperistatin (circles), Rhodocetin (squares), whereas the open symbols represent Compound 6 (circles) and Compound 10 (squares).
  • Figure 4 Effect of the peptides on proliferation of endothelial cells.
  • A HAEC
  • B HUVEC
  • 50 ⁇ g of peptide per sample were used.
  • Obtustatin was used as positive control and proliferation was measured by BrdU assay. /?* ⁇ 0.05 compared to control group.
  • OD optical density.
  • FIG. 5 Inhibitory effect of the peptides on HUVEC migration in the wound healing assay. Representative photos of the wounds at time 0 (left) and 24 hours (right) from wounding in the absence of the peptides (control) (A), in the presence of 5 ⁇ g Obtustatin (B), 50 ⁇ g Compound 6 (C), and 50 ⁇ g Compound 10 (D).
  • FIG. 6 The inhibitory effect of the peptides on endothelial cells tube formation in Matrigel assay.
  • the tube formation in Matrigel with HAEC (A) and HUVEC (B) was induced by complete EBM-2 medium in the absence (negative control) or in the presence of 100 ⁇ g of each peptide per sample.
  • FIG. 7 The inhibitory effect of the peptides on VEGF induced angiogenesis in Japanese quail CAM model. Representative images of mid-arterial end points of CAMs dissected from embryos appear in the panels. Basal level of angiogenesis (Control); angiogenesis induced by 10 ⁇ g VEGF /embryo (VEGF); the inhibitory effect of 20 ⁇ g Obtustatin/embryo (Obtustatin), 200 ⁇ g peptide/embryo (Compounds 3, 6 and 10) on angiogenesis induced by VEGF. *p ⁇ 0.001 versus Control. **p ⁇ 0.001 versus VEGF. Df, fractal dimension.
  • FIG. 8 The inhibitory effect of the peptides on glioma LN18 cells induced angiogenesis in Japanese quail CAM model.
  • A Representative images of mid-arterial endpoints of CAMs dissected from embryos appear in the panels. Basal level of angiogenesis (Control); angiogenesis induced by LN18 cells (lxl0 7 /embryo) (LN18); the inhibitory effect of 100 ⁇ g peptides (Compounds 6 and 10) on angiogenesis induced by LN18. *p ⁇ 0.001 versus Control. **p ⁇ 0.001 versus LN18.
  • B Representative micrographs of dissected CAMs. Tumors are framed and marked with an arrow.
  • Figure 9 Inhibitory effect of the peptides on bFGF-induced angiogenesis in corneal micropocket assay.
  • the present invention is directed according to some aspects to synthetic cyclic peptides derived from the disintegrin Viperistatin and pharmaceutical compositions comprising the peptides.
  • the peptides and compositions of the present invention are useful for treating diseases involving excessive angiogenesis.
  • the peptides and compositions of the present invention are also useful for analyzing in vitro and in-vivo anti adhesion and anti-angiogenic effects, applicable for anti-angiogenesis and anti-cancer (including anti- metastatic) therapy.
  • the peptides of the present invention can be used as a diagnostic tool for ⁇ and ⁇ 2 ⁇ 1 integrins mediated angiogenesis.
  • the peptides can be utilized in the diagnosis of medical conditions associated with abnormal angiogenesis.
  • the peptides can also be used as imaging agents, for imaging of angiogenesis, e.g., in tumors.
  • the peptides are typically labeled, e.g., with fluorescent, radioactive, near infrared tags.
  • the present invention discloses for the first time non-natural analogs and derivatives of Viperistatin which advantageously show dual selectivity towards the two collagen receptors ⁇ and ⁇ 2 ⁇ 1 integrins with increased potency.
  • both of these integrins are known to be involved in angiogenesis
  • the dual activity of the compounds of the present invention is advantageous in increasing their anti-angiogenesis efficacy.
  • the peptide compounds disclosed herein are characterized by an intermediate efficacy in inhibition of endothelial cell adhesion, such as inhibition of adhesion of al/a2 integrin overexpressor cells to respective collagens.
  • An intermediate efficacy as described herein refers to an efficacy of between about 10-60%, for example between about 20-40%.
  • Vimocin Compound 6
  • Vidapin Compound 10
  • ICso 0.17 nM intermediate efficacy (20 and 40%, respectively) in inhibition of adhesion of al/a2 integrin overexpressor cells to respective collagens
  • Vimocin was more active in inhibition of wound healing (53%) and corneal micropocket (17%) vascularization
  • Vidapin was more potent in inhibition of migration in the Matrigel tube formation assay (90%).
  • synthetic cyclic peptides are provided.
  • peptide indicates a sequence of amino acids linked by peptide bonds.
  • Peptides according to some embodiments of the present invention consist of 8-40, for example, 9-40, 10-30, 11-25 amino acids.
  • amino acid refers to compounds, which have an amino group and a carboxylic acid group, preferably in a 1,2- 1,3-, or 1,4- substitution pattern on a carbon backbone.
  • ot-Amino acids are most preferred, and include the 20 natural amino acids (which are L-amino acids except for glycine) which are found in proteins, the corresponding D-amino acids, the corresponding N-methyl amino acids, side chain modified amino acids, the biosynthetically available amino acids which are not found in proteins (e.g., 4-hydroxy-proline, 5-hydroxy-lysine, citrulline, ornithine, canavanine, djenkolic acid, ⁇ -cyanolanine), and synthetically derived a-amino acids, such as amino- isobutyric acid, norleucine, norvaline, homocysteine and homoserine.
  • ⁇ -Alanine and ⁇ - amino butyric acid are examples of 1,3 and 1,4-amino acids,
  • amino acids used in this invention are those which are available commercially or are available by routine synthetic methods. Certain residues may require special methods for incorporation into the peptide, and either sequential, divergent or convergent synthetic approaches to the peptide sequence are useful in this invention.
  • Natural coded amino acids and their derivatives are represented by one-letter codes or three-letter codes according to IUPAC conventions. When there is no indication, the L isomer was used. The D isomers are indicated by "D" or "(D)" before the residue abbreviation.
  • the peptide is composed of (or consists of) 8-39 amino acids, 8-38 amino acids, 8-37 amino acids, 8-36 amino acids, 8-35 amino acids, 8-34 amino acids, 8-33 amino acids, 8-32 amino acids, 8-31 amino acids, 8-30 amino acids, 8- 29 amino acids, 8-28 amino acids, 8-27 amino acids, 8-26 amino acids, 8-25 amino acids, 8-24 amino acids, 8-23 amino acids, 8-22 amino acids, 8-21 amino acids, 8-20 amino acids, 8-19 amino acids, 8-18 amino acids, 8-17 amino acids, 8-16 amino acids, 8-15 amino acids, 8-14 amino acids, 8-13 amino acids, 8-12 amino acids.
  • Each possibility represents a separate embodiment of the invention.
  • the peptide is composed of (or consists of) 11-39 amino acids, 11-38 amino acids, 11-37 amino acids, 11-36 amino acids, 11-35 amino acids, 11- 34 amino acids, 11-33 amino acids, 11-32 amino acids, 11-31 amino acids, 11-30 amino acids, 11-29 amino acids, 11-28 amino acids, 11-27 amino acids, 11-26 amino acids, 11- 25 amino acids, 11-24 amino acids, 11-23 amino acids, 11-22 amino acids, 11-21 amino acids, 11-20 amino acids, 11-19 amino acids, 11-18 amino acids, 11-17 amino acids, 11- 16 amino acids, 11-15 amino acids, 11-14 amino acids, 11-13 amino acids, 11-12 amino acids.
  • Each possibility represents a separate embodiment of the invention.
  • the peptides of the present invention comprises at least one cyclization, wherein the cyclized sequence, namely, the sequence within a cyclic portion of the peptide, comprises WKTSXiRTSH (SEQ ID NO: 1), wherein Xi is absent or Cys.
  • Xi is absent and the cyclized sequence comprises WKTSRTSH (SEQ ID NO: 26). In other embodiments, Xi is Cys and the cyclized sequence comprises WKTSCRTSH (SEQ ID NO: 27).
  • the peptides comprise a plurality of cyclizations.
  • the term "plurality" indicates at least two.
  • the cyclized sequence comprises WKTSRTSHY (SEQ ID NO: 2).
  • the cyclized sequence comprises CWKTSRTSHYC (SEQ ID NO: 3).
  • the cyclized sequence comprises CWKTSCRTSH (SEQ ID NO: 14).
  • the peptides of the present invention may be monocyclic, bi-cyclic, tri-cyclic or even tetra-cyclic (with four cyclizations). Each possibility represents a separate embodiment of the present invention.
  • each cyclization is independently selected from the group consisting of side -chain to side-chain; end to end; backbone to backbone and backbone to end
  • a peptide of the present invention comprises at least one additional sequence of 1-18 amino acids residues, derived from Viperistatin or Obtustatin.
  • the complete sequences of Viperistatin and Obtustatin are given below.
  • the additional sequence is C-terminal to the first sequence noted above, namely, to WKTSXiRTSH (or WKTSRTSHY, CWKTSRTSHYC or CWKTSCRTSH, according to some embodiments).
  • the additional sequence is N-terminal to the first sequence.
  • the additional sequence may be attached to the peptide through a linker or spacer.
  • a linker according to some embodiments is a sequence of 1-4 amino acids.
  • the additional sequence is a sequence of 1-12 contiguous amino acids derived from residues 30-41 of Viperistatin or Obtustatin. In some embodiments, the additional sequence is of 3-12, for example, 3-8 or 3-6, contiguous amino acids residues derived from residues 30-41 of Viperistatin or Obtustatin.
  • the additional sequence comprises the sequence TGKS (SEQ ID NO: 4). In some embodiments, the additional sequence is TGKSCG (SEQ ID NO: 5). In additional embodiments, the additional sequence is TGKSD (SEQ ID NO: 6). In yet additional embodiments, the additional sequence is TGKSCDCG (SEQ ID NO: 15).
  • the additional sequence comprises PLY. In some embodiments, the additional sequence is PLYPG (SEQ ID NO: 7). In additional embodiments, the additional sequence is PLYQG (SEQ ID NO: 8).
  • the peptide comprises the sequence CWKTSRTSHYC (SEQ ID NO: 3) and an additional sequence of 1-12, for example 3-8 or 3-6, contiguous amino acid residues derived from residues 30-41 of Viperistatin or Obtustatin, C-terminal to CWKTSRTSHYC.
  • the additional sequence is a sequence of 1-18 contiguous amino acid residues derived from residues 1-18 of Viperistatin or Obtustatin.
  • the additional sequence is TTGPCCRQKKLKPAGTT (SEQ ID NO: 16). In some embodiments, the additional sequence is KTTGPCCRQKKLKPAGTTK (SEQ ID NO: 17).
  • the peptide comprises the sequence WKTSRTSHYC (SEQ ID NO: 1]
  • the peptide sequence is CCWKTSRTSHYCTGKSCG (SEQ ID NO: 9).
  • the peptide sequence is CWKTSRTSHYCPLYPG (SEQ ID NO: 1
  • the peptide sequence is CWKTSRTSHYCPLYQG (SEQ ID NO: 11). In some embodiments, the peptide sequence is CWKTSRTSHYCTGKSD (SEQ ID NO: 12).
  • the peptide sequence is CWKTSCRTSHTGKSD (SEQ ID NO: 13).
  • the peptide sequence is KTTGPCCRQKKLKPAGTTKWKTSRTSHYCTGKSCDCG (SEQ ID NO: 19).
  • Peptides according to the present invention are constructed with one or more cyclizations, including side-chain to side chain (e.g. disulfide bond between two Cysteine residues), backbone to backbone, terminal to terminal as well as backbone to side-chain or any other peptide cyclization.
  • side-chain to side chain e.g. disulfide bond between two Cysteine residues
  • backbone to backbone e.g. terminal to terminal as well as backbone to side-chain or any other peptide cyclization.
  • Solid phase peptide synthesis procedures are well known in the art and further described in "Solid-Phase Synthesis: A Practical Guide", Ed. Steven A. Kates and Fernando Albericio, CRC Press; 1st Edition (2000).
  • a skilled artesian may synthesize any of the peptides of the present invention by using an automated peptide synthesizer using standard chemistry such as, for example, t-Boc or Fmoc chemistry.
  • the methods include exclusive solid phase synthesis, partial solid phase synthesis, fragment condensation, classical solution synthesis.
  • Coupling of the amino acids in solid phase peptide chemistry can be achieved by means of a coupling agent such as but not limited to dicyclohexycarbodiimide (DCC), bis(2-oxo-3 -oxazolidinyl) phosphinic chloride (BOP-C1), benzotriazolyl-N- oxytrisdimethyl-aminophosphonium hexafluoro phosphate (BOP), 1-oxo-l- chlorophospholane (Cpt-Cl), hydroxybenzotriazole (HOBT), or mixtures thereof.
  • a coupling agent such as but not limited to dicyclohexycarbodiimide (DCC), bis(2-oxo-3 -oxazolidinyl) phosphinic chloride (BOP-C1), benzotriazolyl-N- oxytrisdimethyl-aminophosphonium hexafluoro phosphate (BOP), 1-oxo-l- chlor
  • additional coupling reagents including, but not limited to: coupling reagents such as PyBOP (Benzotriazole-l-yl- ⁇ oxy-tris-pyrrolidino-phosphonium hexafluorophosphate), PyBrOP (Bromo-tris-pyrrolidino-phosphonium hexafluoro-iphosphate), HBTU (2-(lH-Benzotriazole-l-yl)-l,l,3,3- tetramethyluronium hexafluoro-phosphate), TBTU (2-(lH-Benzotriazole-l-yl)-l,l,3,3-tetramethyluronium tetrafluoroborate), may be also utilized for synthesizing the peptide compounds of the present invention.
  • coupling reagents such as PyBOP (Benzotriazole-l-yl- ⁇ oxy-tris-pyrrolidino-phosphonium hexafluorophosphate
  • Additional coupling chemistries may be used, such as pre-formed urethane- protected N-carboxy anhydrides (UNCA'S), pre-formed acyl halides most preferably acyl chlorides.
  • UNCA'S pre-formed urethane- protected N-carboxy anhydrides
  • acyl halides most preferably acyl chlorides.
  • Such coupling may take place at room temperature and also at elevated temperatures, in solvents such as toluene, DCM (dichloromethane), DMF (dimethylformamide), DMA (dimethylacetamide), NMP (N-methyl pyrrolidinone), dioxane, tetrahydrofuran, diglyme and 1,3 dichloropropane, or mixtures of the above.
  • solvents such as toluene, DCM (dichloromethane), DMF (dimethylformamide), DMA (dimethylacetamide), NMP (N-methyl pyrrolidinone), dioxane, tetrahydrofuran, diglyme and 1,3 dichloropropane, or mixtures of the above.
  • Synthetic peptides can be purified by preparative high performance liquid chromatography and the composition of which can be confirmed via amino acid sequencing. Some of the peptides of the invention, which include only natural amino acids, may be prepared using recombinant DNA techniques known in the art.
  • Cyclization of peptides has been shown to be a useful approach in developing diagnostically and therapeutically useful peptidic and peptidomimetic agents. Cyclization of peptides reduces the conformational freedom of these flexible, linear molecules, and often results in higher receptor binding affinities by reducing unfavorable entropic effects. Because of the more constrained structural framework, these agents are more selective in their affinity to specific receptor cavities. By the same reasoning, structurally constrained cyclic peptides confer greater stability against the action of proteolytic enzymes (Humphrey, et al., 1997, Chem. Rev., 2243-2266).
  • Methods for cyclization can be classified into cyclization by the formation of an amide bond between the N-terminal and the C-terminal amino acid residues, and cyclizations involving the side chains of individual amino acids.
  • the latter method includes the formation of disulfide bridges between two co-thio amino acid residues (cysteine, homocysteine), the formation of lactam bridges between glutamic/aspartic acid and lysine residues, the formation of lactone or thiolactone bridges between amino acid residues containing carboxyl, hydroxyl or mercapto functional groups, the formation of thioether or ether bridges between the amino acids containing hydroxyl or mercapto functional groups and other special methods.
  • Lambert, et al. reviewed variety of peptide cyclization methodologies (J. Chem. Soc. Perkin Trans., 2001, 1 :471-484).
  • Backbone cyclization is a general method by which conformational constraint is imposed on peptides.
  • backbone cyclization atoms in the peptide backbone (N and/or C) are interconnected covalently to form a ring.
  • Backbone cyclized analogs are peptide analogs cyclized via bridging groups attached to the alpha nitrogens or alpha carbonyl of amino acids.
  • the procedures utilized to construct such peptide analogs from their building units rely on the known principles of peptide synthesis; most conveniently, the procedures can be performed according to the known principles of solid phase peptide synthesis.
  • the protected building unit is coupled to the N-terminus of the peptide chain or to the peptide resin in a similar procedure to the coupling of other amino acids.
  • the protective group is removed from the building unit's functional group and the cyclization is accomplished by coupling the building unit's functional group and a second functional group selected from a second building unit, a side chain of an amino acid residue of the peptide sequence, and an N-terminal amino acid residue.
  • Backbone cyclization is achieved by covalently connecting at least one amino acid residue in the helix sequence, which was substituted with an ⁇ - ⁇ -functionalized or an Ca-co-functionalized derivative of amino acid residue, with a moiety selected from the group consisting of: another ⁇ - ⁇ -functionalized or an Ca-co-functionalized derivative of amino acid residue, with the side chain of an amino acid in the peptide's sequence, or with one of the peptide terminals. Any covalent bond may be used to connect the anchoring positions of the peptide sequence using backbone cyclization.
  • the peptides of the invention can be used in the form of pharmaceutically acceptable salts.
  • salts refers to both salts of carboxyl groups and to acid addition salts of amino or guanido groups of the peptide molecule.
  • pharmaceutically acceptable means suitable for administration to a subject, e.g., a human.
  • pharmaceutically acceptable can mean approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • Pharmaceutically acceptable salts include those salts formed with free amino groups such as salts derived from non-toxic inorganic or organic acids such as acetic acid, citric acid or oxalic acid and the like, and those salts formed with free carboxyl groups such as salts derived from non-toxic inorganic or organic bases such as sodium, calcium, potassium, ammonium, calcium, ferric or zinc, isopropylamine, triethylamine, procaine, and the like.
  • Analogs and derivatives of the peptides are also within the scope of the present application.
  • “Derivatives” of the peptides of the invention as used herein cover derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C-terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the activity of the peptide, do not confer toxic properties on compositions containing it, and do not adversely affect the immunogenic properties thereof.
  • These derivatives may include, for example, aliphatic esters of the carboxyl groups, amides of the carboxyl groups produced by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues, e.g., N-acetyl, formed by reaction with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups), or O-acyl derivatives of free hydroxyl group (e.g., that of seryl or threonyl residues) formed by reaction with acyl moieties.
  • acyl moieties e.g., alkanoyl or carbocyclic aroyl groups
  • O-acyl derivatives of free hydroxyl group e.g., that of seryl or threonyl residues
  • Analogs of the peptides of the invention as used herein cover compounds which have the amino acid sequence according to the invention except for one or more amino acid changes, typically, conservative amino acid substitutions.
  • the core sequence KTSR should remain unchanged.
  • the two Cys should remain unchanged, or if replaced, should preferably be replaced with two residues capable of forming cyclization.
  • an analog has at least about 75% identity to the sequence of the peptide of the invention, for example at least about 80%, at least about 85%, at least about 90%, at least about 99% identity to the sequence of the peptide of the invention.
  • the term "about”, when referring to a measurable value is meant to encompass variations of +/-10%, more preferably +1-5%, even more preferably +/-1 %, and still more preferably +/-0.1 % from the specified value.
  • Conservative substitutions of amino acids as known to those skilled in the art are within the scope of the present invention.
  • Conservative amino acid substitutions include replacement of one amino acid with another having the same type of functional group or side chain e.g. aliphatic, aromatic, positively charged, negatively charged.
  • One of skill will recognize that individual substitutions, deletions or additions to peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • Analogs according to the present invention may comprise also peptidomimetics.
  • "Peptidomimetic" means that a peptide according to the invention is modified in such a way that it includes at least one non-coded residue or non-peptidic bond. Such modifications include, e.g., alkylation and more specific methylation of one or more residues, insertion of or replacement of natural amino acid by non-natural amino acids, replacement of an amide bond with another covalent bond.
  • a peptidomimetic according to the present invention may optionally comprise at least one bond which is an amide- replacement bond such as urea bond, carbamate bond, sulfonamide bond, hydrazine bond, or any other covalent bond. The design of appropriate analogs may be computer assisted. Analogs are included in the invention as long as they remain pharmaceutically acceptable and their activity is not damaged.
  • the present invention provides pharmaceutical compositions comprising a peptide of the present invention or a salt thereof as the active ingredient.
  • compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers, excipients and auxiliaries, which facilitate processing of the active compounds into preparations which can be used pharmaceutically, as known to a person of skill in the art and detailed, for example, in “Handbook of Pharmaceutical Excipients” ; Pharmaceutical Press, Ed. Raymond C. Rowe et al., 7 th ed., 2012. Proper formulation is dependent upon the route of administration chosen. Techniques for formulation and administration of drugs may be found in “Remington: The Science and Practice of Pharmacy", Pharmaceutical Press, Editor-in-Chief Loyd V Allen, Jr 22 nd ed., 2012.
  • carrier refers to a diluent, adjuvant or vehicle with which the therapeutic compound is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
  • the active ingredients of the present invention are peptides and peptide analogs dictates that the formulation be suitable for delivery of these types of compounds.
  • the pharmaceutical compositions are formulated for parenteral administration.
  • the pharmaceutical compositions may be formulated for injection administration, including but not limited to intravenous, intraarticular, intramuscular, subcutaneous, intradermal or intrathecal. Each possibility represents a separate embodiment of the present invention.
  • the pharmaceutical compositions are formulated for oral administration.
  • enteric-coated preparations or dosage forms, microspheres, liposomes and nanoparticles for oral delivery of peptides and proteins may be used.
  • the pharmaceutical compositions are formulated for ocular delivery.
  • the peptides of the present invention can be supplied in any manner suitable for the provision of the peptide to cells within the tissue of interest.
  • a composition containing the peptides of the present invention can be introduced, for example, into the systemic circulation, which will distribute said peptide to the tissue of interest.
  • a composition can be applied topically (locally) to the tissue of interest (e.g., injected, or pumped as a continuous infusion, or as a bolus within a tissue, applied to all or a portion of the surface of the skin, etc.).
  • the active ingredient may be formulated in aqueous solutions, for example in physiologically compatible buffers including but not limited to Hank's solution, Ringer's solution, or physiological saline buffer.
  • Formulations for injection may be presented in unit dosage forms, for example, in ampoules, or in multi- dose containers with, optionally, an added preservative.
  • the compositions may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.
  • the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredients, to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, for example, a sterile, pyrogen-free, water-based solution, before use.
  • the active ingredient may be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Media such as water, glycols, oils and alcohols can be used in liquid preparations such as suspensions, syrups and solutions.
  • solid carriers such as starches and sugars, lubricants, binders and disintegrating agents can be used.
  • compositions suitable for use in context of the present invention include compositions wherein the active ingredient is contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of a compound effective to prevent, alleviate or ameliorate symptoms of a disease of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
  • Toxicity and therapeutic efficacy of the peptides described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the IC50 (the concentration which provides 50% inhibition) and the LD50 (lethal dose causing death in 50 % of the tested animals) for a subject compound.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.
  • the combination of a compound of the invention with another agent used for treatment of diseases or disorder involving excessive angiogenesis can be used. Such combination can be used simultaneously or sequentially.
  • the composition further comprises at least one more active ingredient.
  • a pharmaceutical composition is provided, consisting of the peptide of the present invention or a salt thereof as the sole an active ingredient.
  • compositions of the present invention are typically employed for the treatment of a mammal, preferably a human.
  • methods for treating diseases or disorders involving excessive angiogenesis in a subject in need thereof comprise administering to the subject a pharmaceutical composition comprising a peptide of the present invention.
  • peptides of the present invention for the preparation of a medicament for the treatment of diseases or disorders involving excessive angiogenesis.
  • Angiogenesis is the physiological process through which new blood vessels form from pre-existing vessels.
  • diseases or disorders involving excessive angiogenesis refers to diseases and disorders where abnormally excessive blood vessel development is observed, and/or where abnormal excessive blood vessel development underlies their pathology.
  • treating refers to reduction, amelioration or even elimination of at least some of the symptoms associated with the relevant disease.
  • the term may also include inhibition of disease progression.
  • the term may include inhibition of tumor growth, reduction in tumor size or even elimination of a tumor, and prevention or reduction of metastasis formation.
  • the term may include, inter alia, inhibition of abnormal formation of blood vessels in the eye.
  • the diseases or disorders amenable to treatment according to the principles of the present invention are those where abnormal, excessive angiogenesis is implicated in their pathogenesis, or facilitates their progression.
  • the disease or disorder is cancer.
  • the disease or disorder is a cancerous solid tumor, namely, a malignancy that forms a discrete tumor mass.
  • Tumor types particularly suitable for treatment by the method of the present invention are those where angiogenesis is known to underlie their progression.
  • the method may be used for the treatment of primary as well as metastatic tumors.
  • the method is particularly suitable for the treatment of vascularized solid tumors.
  • Cancer types that are amenable to treatment include breast carcinomas, lung carcinomas, gastric carcinomas, esophageal carcinomas, colorectal carcinomas, liver carcinomas, ovarian carcinomas, cervical carcinomas, endometrial carcinoma, endometrial hyperplasia, fibrosarcomas, choriocarcinoma, head and neck cancer, nasopharyngeal carcinoma, laryngeal carcinomas, hepatoblastoma, Kaposi's sarcoma, melanoma, skin carcinomas, hemangioma, cavernous hemangioma, hemangioblastoma, pancreas carcinomas, retinoblastoma, astrocytoma, glioblastoma, Schwannoma, oligodendroglioma, medulloblastoma, neuroblastomas, rhabdomyosarcoma, osteogenic sarcoma, leiomyosarcomas, urinar
  • Lung cancer includes, e.g. small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung.
  • Colorectal cancer includes, e.g. metastatic colorectal cancer.
  • Kidney cancer includes, e.g., metastatic kidney cancer
  • the disease or disorder is a disorder of the eye. In some particular embodiments, the disease or disorder is a retinal disorder.
  • the eye or retinal disorder is selected from the group consisting of a proliferative retinopathy (including retinopathy of prematurity), retrolental fibroplasia, neovascular glaucoma, and age-related macular degeneration (particularly wet age-related macular degeneration).
  • a proliferative retinopathy including retinopathy of prematurity
  • retrolental fibroplasia retrolental fibroplasia
  • neovascular glaucoma neovascular glaucoma
  • age-related macular degeneration particularly wet age-related macular degeneration
  • the eye or retinal disorder is selected from the group consisting of macular edema following retinal vein occlusion (RVO), and diabetic macular edema.
  • RVO retinal vein occlusion
  • diabetic macular edema a separate embodiment of the invention.
  • the disease or disorder is a non-neoplastic inflammatory condition.
  • the non-neoplastic inflammatory condition is selected from the group consisting of rheumatoid arthritis, psoriasis, fibrosis, atherosclerosis and thyroid hyperplasias (including Grave's disease). Each possibility represents a separate embodiment of the invention.
  • the methods of the present invention may be combined with one or more known treatments of the above described disorders/diseases.
  • the present invention further provides methods for diagnosis of diseases or disorders using the peptides of the present invention.
  • the diseases or disorders that can be diagnosed using the peptides of the invention are those associated with abnormal expression of at least one of ⁇ and ⁇ 2 ⁇ 1 integrins, such as up or down regulation, or mutations, of ⁇ and/or ⁇ 2 ⁇ 1 integrins.
  • the diseases or disorders to be diagnosed are those associated with upregulation of ⁇ and/or ⁇ 2 ⁇ 1 integrins, for example diseases associated with abnormal endothelial cell growth, abnormal angiogenesis or abnormal cell adhesion.
  • Non-limiting examples include cancer, particularly solid tumors (e.g., vascularized tumors) and collagen bone and ligament disorders.
  • the present invention further provides methods for imaging angiogenesis in a subject using the peptides of the present invention.
  • a pharmaceutical composition comprising the peptide is used for imaging angiogenesis.
  • the peptides are preferably attached to a detectable label.
  • detectable labels include magnetic, radioactive, fluorescent and near-infrared labels, as known in the art.
  • the peptides can be used for targeting ⁇ and ⁇ 2 ⁇ 1 integrins with MRI, near infrared and fluorescent nanoparticles or carriers.
  • Collagen IV from bovine placenta villi was purchased from Chemicon (Temecula, CA), and collagen I (from rat tail) and Matrigel from BD Biosciences (Bedford, MA).
  • 96-well polystyrene EIA/RIA plates were obtained from Nunc (Roskilde, Denmark), Bovine serum albumin (BSA), Hank's Balanced Salt Solution (HBSS) sulfate, alkaline phosphatase-conjugated anti-rabbit antibody, p-nitrophenyl phosphate, human recombinant basic fibroblast growth factor (bFGF), and vascular endothelial growth factor 165 kDa (VEGF) were purchased from Sigma- Aldrich (St.
  • CMFDA Green 5-Chloromethylfluorescein Diacetate
  • Rabbit polyclonal antibodies against GST were purchased from Molecular Probes (Nijmegen, The Netherlands).
  • BrdU kit was purchased from Roche (Mannheim, Germany).
  • Fertilized Japanese quail eggs (Coturnix coturnix japonica) were purchased from Boyd's Bird Co (Pullman, WA).
  • Lactate Dehydrogenase reagent set was purchased from Pointe Scientific, Inc. (Michigan, USA).
  • Viperistatin was obtained from the venom of Vipera xantina palestinae as previously described (Staniszewska et al., 2009, Cancer Biology & Therapy, 8: 1507-1516). Obtustatin was isolated and purified to homogeneity by two chromatographic steps on HPLC as previously reported (Marcinkiewicz et al., 2003, Cancer Research 63: 2020-2023).
  • Vixapatin was obtained from the venom of Vipera xantina palestinae as previously described (Staniszewska et al 2009, supra). Rhodocetin was purified from C. rhodostoma venom as described (Eble et al., 2001, / Biol Chem 276: 12274-12284).
  • HAECs Human aortic endothelial cells
  • HAVECs Human umbilical vein endothelial cells
  • K562 cells transfected with either al or a2 integrin subunits (Staniszewska et al 2009, supra).
  • LN18 human glioma cells were prepared as previously described (Walsh et al., 2012, Neuro-Oncology 14: 890-901).
  • Mouse brain capillary cells, bEnd.3, were obtained from ATCC (Manassas, VA) and cultured as previously described (Lecht et al., 2010, Mol Cell Biochem 339:201-213).
  • N,N-diisopropylethylamine (DIPEA), 2-(lH- benzotriazole-lyl)-l,l ,3,3-tetramethyluronium hexafluorophosphate (HBTU), 1- hydroxybenzo-triazole (HOBt) were purchased from BioLab Ltd. (Jerusalem, Israel). All coupling reagents, chemicals and solvents were purchased from Sigma-Aldrich (Rehovot, Israel).
  • Synthesized peptides were purified by preparative reverse -phase high-performance liquid chromatography (RP-HPLC) with CI 8 column with an elution gradient of 0-90% acetonitrile with 0.1% trifluoroacetic in water. Peptide purity was verified by Thermo Scientific Dionex UltiMate 3000 analytical HPLC. All peptides showed purity above 95%, based on the chromatographic peak area revealed at 220 nm. The peptides identities were assessed by electrospray ionization mass spectrometry using a ThermoQuest Finnigan LCQ-Duo in the positive ion mode.
  • Fmoc-Lys(Fmoc)-OH was coupled to a glycine already bound to the resin. Following cleavage of Fmoc by 20% piperidine, the peptides were assembled on the two free amino groups of Lys, to form two parallel peptide chains of identical sequence. The pair of cysteines inserted along each chain, were protected by trityl (Trt) and acetamidomethyl (Acm). A one-pot reaction for selective formation of two inter-chain disulfide bonds was carried out by adding 2 ml of 0.1 M in dichloromethane (DCM) for 6h (Bullesbach & Schwabe, 1991, / Biol Chem 266: 10754-10761).
  • DCM dichloromethane
  • This method takes advantage of the difference in the kinetic rates of disulfide bond formation by S-Trt and S-Acm in the presence of iodine, in dichlorometane (DCM).
  • DCM dichlorometane
  • the Cys(Trt) disulfide bond was formed by oxidation in a few seconds
  • formation of the Cys(Acm) disulfide bond occurred after about 2 h.
  • the respective calculated and experimentally found molecular weight values were 3885.36 and 3885.86 for Compound 7 ((C*WKTSRTSHYC**TGKSD,)2K) and 3552.96 and 3553.01 for Compound 8 ((C*WKTSC**RTSHTGKSD)2K).
  • Trt trichloroacetic acid
  • Trt trichloroacetic acid
  • DMT dimethoxytrityl
  • S-CH2COODMT Cys 36
  • Trt and the DMT protecting groups of Lys 10 (Trt) and Cys 34 (S-CH2COODMT), respectively were removed with 3% TCA and the free amine and the carboxylic groups formed were coupled.
  • the next cyclization took place between Cys 29 (CH2CH2N Pht) and Cys 7 (S-CH2COOH).
  • the trityl group was replaced by a carboxymethyl group, as previously described.
  • Cys 6 was transformed into Cys 6 (S-CH2COOH) in the same manner as Cys 34 (Trt), while Acm group was cleaved with 0.1 M iodine in methanol, and the Fmoc protecting group of Boc-Lys ⁇ Fmoc) was cleaved by 1M tetrabutylamonium fluoride (TBAF).
  • TBAF tetrabutylamonium fluoride
  • the free amino and carboxylic groups formed were coupled to yield the cyclization.
  • the respective calculated and experimentally found molecular weight values were 4251.73 and 4251.96 Compound 9 (K*TTGPC*C**RQK***KLKPAGTTK****WKTSRTSHYC**TGKSC***DC****G).
  • Compound 10 The peptide was synthesized as described in General procedure. The two intra-chain disulfide bonds of Compound 10 were prepared in the same manner as Compounds 7 and 8. The respective calculated and experimentally found molecular weight values were 2003.7979 and 2003.7977 for Compound 10 (C*C**WKTSRTSHYC**TGKSC*G).
  • the cells were labeled by incubation with 12.5 ⁇ 5- chloromethylfluorescein diacetate (CMFDA) in HBSS without 1% BSA at 37°C for 30 min. The labeled cells were then centrifuged at 1,000 rpm and washed twice with HBSS containing 1% BSA to remove excess CMFDA. Labeled cells (1 x 10 s cells/well) were added to each well in the absence or presence of inhibitor and incubated at 37°C for 60 min. In the presence of peptide, cells were added to the well after prior incubation with peptide for 30 min at 37°C.
  • CMFDA 5- chloromethylfluorescein diacetate
  • Unbound cells were removed by washing the wells three times with 1% (w/v) BSA in HBSS, and bound cells were lysed by the addition of 0.5% Triton X-100 (diluted in DDW).
  • SPECTRAFluor Plus plate reader Tecan, San Jose, CA
  • Inhibition of GST-aiA/GST-a2A binding to type IV/I collagens by peptides was performed as published previously (Eble & Tuckwell, 2003, Biochem. J. 376: 77-85), with the following modifications: CB3 (collagen IV fragment) or collagen I was immobilized overnight at 4°C on a microtiter plate at 10 ⁇ g/ml in TBS/MgCb (50 mM Tris-HCl, 150 mM NaCl, and 2 mM MgCb, pH 7.4) and 0.1 M acetic acid, respectively.
  • TBS/MgCb 50 mM Tris-HCl, 150 mM NaCl, and 2 mM MgCb, pH 7.4
  • the GST-tagged al A/GST-tagged a2A domain was allowed to bind to type IV collagen/collagen I in the presence or absence of different peptides for 2 h at room temperature.
  • the bound GST- ⁇ /GST- ⁇ 2 ⁇ domain was fixed for 10 min with 2.5% glutaraldehyde in HEPES buffer (50 mM HEPES, 150 mM NaCl, and 2 mM MgCl 2 , and 1 mM MnCh pH 7.4).
  • the bound GST-alA/GST-a2A was quantified with rabbit polyclonal antibodies against GST, followed by alkaline phosphatase-conjugated anti-rabbit antibody, which served as the primary and secondary antibodies, respectively, each diluted in 1% BSA in TBS/MgCb.
  • the conversion of p-nitrophenyl phosphate was measured at 405 nm in an ELISA reader Bio Tek (Bad Friedrichshall, Germany).
  • Nonspecific binding was assessed by binding of GST-alA/GST-a2A to BSA or of ⁇ / ⁇ integrin to collagen IV/collagen I in the presence of 10 mM EDTA.
  • HAEC and HUVEC proliferation assay was performed using (BrdU) kit according to the manufacturer's instructions (Staniszewska et al., 2007, Circ Res 100: 1308-1316).
  • HUVEC migration was measured using a wound healing assay (Lecht et al, 2010, Mol Cell Biochem 339:201-213). In brief, 1 x 10 "6 cells/well were added to a 24-well culture plate. Upon formation of a confluent monolayer, cell migration was initiated by scratching the HUVEC monolayer with a small sterile pipette tip, thus generating a cell-free area (wound) of approximately 1 mm in width. The wounded cells were washed three times with 1% FCS supplemented endothelial cell growth medium 2 and the photos of the wounds at time 0 hours were taken.
  • the culture medium was changed to 2% fetal bovine serum medium as the cell migration stimulator, in the presence or absence of synthetic peptides (50 ⁇ g/ml) or obtustatin (5 ⁇ g/ml) and the cultures were allowed to migrate for 24 hours.
  • another set of photos was taken of the same regions. Images were analyzed using ImageJ software (NIH, Bethesda MD). To assess cell migration at the wound edge and to calculate the area covered by migrating cells, the cell-free areas of the wounds at 24 hours after wounding were subtracted from the area of the wounds at the 0-hour time point and calculated as a percentage of untreated (control) cultures (Lecht et al., 2010, supra).
  • Human aortic endothelial cells tube formation in Matrigel assay was performed using 96-well plate coated with growth factor reduced Matrigel. Briefly, lxlO 4 human aortic endothelial cells (HAEC) per well (in complete EMB-2 media or 2% FBS) were added in the presence or absence of peptides, and the plate was incubated overnight at 37°C in 5% CO2. Images were captured under inverted microscope (Olympus 1X81) with 35x magnification. The images were transferred to ImageJ software and the number of branching points was counted per observation field (Momic et al., 2012, Toxins 4: 862-877).
  • HAEC human aortic endothelial cells
  • Angiogenesis in chorioallantoic membrane (CAM) quail embryonic model Angiogenesis in chorioallantoic membrane (CAM) quail embryonic model.
  • the compounds were applied under sterile conditions to the surface of the CAM and after 24 h the effect on the aortic tree was evaluated.
  • the embryos were divided into experimental groups, each containing at least 10 embryos.
  • the control group received a vehicle (PBS) treatment.
  • PBS vehicle
  • LN18 glioma cells (1 ⁇ 10 7 /50 ⁇ 1) were injected at day 7 under the CAM and tumor induced angiogenesis was measured at day 12.
  • the embryos were fixed with 5 ml of pre- warmed 2% gluteraldehyde, 4% paraformaldehyde in PBS for 48 hours at room temperature.
  • Membranes with and without tumor were dissected and mounted onto the glass slide.
  • the membranes were mounted onto glass slides for evaluation of the fractal dimension (Df).
  • Df fractal dimension
  • Corneal micropocket assay The corneal micropocket assay was performed as previously detailed (Benny et al., 2008, Nat Biotechnol, 26:799-807). In brief, pellets containing 80 ng carrier-free recombinant human bFGF or 160 ng VEGF (R&D Systems, Minneapolis, MN) were implanted into micropockets that were created in the cornea of anesthetized mice. Mice were treated daily by eye drops (8 mg/ml) for 5 days, and the vascular growth area was then measured using a slit lamp.
  • LDH lactate dehydrogenase
  • mice Toxicity to mice. Experiments with animals and animal care were approved by the Committee of Ethics of The Hebrew University and were performed in strict accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
  • the animals were examined for autonomic symptoms by measuring salivation, urinary delivery, pupillary constriction, heart rate, blood pressure, and hair contraction.
  • Neurotoxicity was evaluated by general locomotors activity of the animals in the cage and the ability of the peptide-injected mice to maintain balance and motor coordination by crossing 3 cm, 2 cm and 1 cm width balance beam. Occurrence of either flaccid or spastic paralysis of the legs were also measured.
  • Blood samples were taken from control and peptide-injected mice after 10 hours from injection and submitted from hematocrit and biochemical analysis
  • B16 melanoma cells were incubated with the peptides in PBS for 2 hours (at 37°C) or left untreated (control). Thereafter, male C57BL/6 mice were injected intravenously (iv) with lxlO 5 B 16 melanoma cells in 0.2 ml injection volume (2.5 mg/mL of peptide) (Staniszewska et al 2009, supra). The final concentration of the peptides was 10 mg/kg of mouse. Mice survival was folio wed-up every day for 10 weeks after cells inoculation and death was recorded.
  • mice showed clinical signs of discomfort or pain, it was euthanized and necropsy was performed.
  • the analysis of survival was done by GraphPad Prism 5, performing both the log-rank (Mantel-Cox) test and the Gehan-Breslow- Wilcoxon test. Experiments were performed under double-blind testing.
  • Fig. 1A shows the sequence and structure of Obtustatin and Viperistatin (SEQ ID NOs: 24 and 25, respectively).
  • Compound 1 Obtustatin analog the sequence of which differs from Compound 3 only by one amino acid at position 24 where Leu 24 ⁇ Arg 24 , had six fold decreased activity.
  • Compound 9 is a modified Viperistatin sequence, where the amino acids at positions 37-40 are eliminated.
  • the peptide is characterized by four intra-strand bonds between Lys 1 and Cys 6 , Lys 10 and Cys 34 , Lys 19 and Cys 36 , and Cys 7 and Cys 29 .
  • Compound 10 includes the original Viperistatin 19-34 sequence and one additional Cys at position 29. Its two intradisulfide bonds involved are between Cys 18 and Cys 34 and Cys 19 and Cys 29 .
  • Viperistatin analogs were used, and potency and efficacy of the analogs were measured in the same range of concentrations, using collagen I-coated plates and Vixapatin, an a2 selective C-type lectin protein (Arlinghaus et al., 2012, Toxicon 60: 512- 519), as positive control.
  • Vixapatin generated a dose-response inhibitory effect on a2 mediated adhesion, with an apparent IC50 of 3 nM and an efficacy of 100%.
  • Viperistatin analogs showed a high potency of inhibition, with an apparent IC50 in the range of 0.12-0.25 nM and efficacy in the range of 10-30% (Fig. 2 D-F, Table 2). These findings indicate dual functional antagonism of synthesized peptides towards both ⁇ and ⁇ 2 ⁇ 1 integrin mediated adhesion.
  • a cell-free assay was performed to assess whether the synthesized peptides directly interact with A domains of al or a2, thus providing a possible mechanism for their inhibitory effect in the cellular adhesion assay.
  • Viperistatin and Rhodocetin another C-type lectin protein selective for ⁇ 2 ⁇ 1 integrin (Eble & Tuckwell 2003, supra), as well as Compounds 3, 5-10 were incubated together with the GST-linked A domains, and allowed to bind to the immobilized collagen I or CB3 (collagen IV fragment). The amount of bound recombinant A domain provided information on the inhibitory potential and biochemical recognition ability of the peptides (Fig. 3).
  • Viperistatin did not inhibit binding of the GST- alA domain to collagen IV in a physiological range of concentrations (Fig. 3A). However, at high concentrations (100-1000 ⁇ ), Compounds 6 and 10 dose-dependently inhibited binding of GST- alA by 50% (Fig. 3B). Without being bound by any theory of a mechanism of action, this finding suggests that Viperistatin affects al integrin outside of the A domain, most probably by conformational change, whereas Compounds 6 and 10 recognized with low potency a motif in the A domain. In the experiments with GST- a2A domain at physiological concentrations, a similar lack of inhibition was observed (Fig. 3C).
  • Rhodocetin inhibited binding of the GST- a2A domain to collagen I in dose-dependent manner (Fig. 3D).
  • Compounds 6 and 10 increased the binding of GST- a2A to collagen I in a dose-dependent manner.
  • Example 3 Viperistatin analogs inhibit proliferation and migration of endothelial cells
  • LN18 glioma cells were injected into the shell-less embryonic CAM system and the effect of Viperistatin analogs on mid-arterial capillary sprouting was measured (Fig. 8A).
  • LN18 glioma tumor induced a significant increase in angiogenesis (Fig. 8B), expressed by an increase in fractal dimension (Df) values from 1.116 to 1.162, 50% greater than that of the control.
  • Treatment with 100 ⁇ g of Compound 6 reduced the LN18 induced-angiogenic effect by 50%, very similar to the effect caused by the same amount of Compound 10 (Fig. 8A,B).
  • Those results characterized Compounds 6 and 10 as anti-angiogenic compounds.
  • mice Male mice were injected intravenously with a dose of 50 mg/kg of Compounds 6 and 10 for 3 consecutive weeks. Acute tolerability was observed. At injection of this high dose of 50 mg/kg body weight, the mice did not suffer from visible weakness and/or exhaustion. No paralysis, altered motor activity, or irregular behavior was observed in mice treated with Compounds 6 and 10, suggesting a lack of neurotoxicity. Cutaneous hematomas around the injection or at distant locations site within 24 hours after injection have not been observed. Furthermore, no mice sudden deaths occurred within 24 hours after the injection or during the 3 weeks of observation. After 10 hours, the blood of mice injected with Compounds 6 and 10 was submitted for hematologic and biochemical analysis.
  • mice injected with Compounds 6 and 10 similarly with the values obtained for control mice. Additional evidence on the lack of hemorrhage or anemic conditions was indicated by the similar hematocrit value in the range of 35-45% and the mean corpuscular hemoglobin of 11.1-12.7 pg/mice, between mice injected with Compounds 6 and 10 compared with control mice.
  • mice C57BL/6 mice were injected intravenously (iv) with B16 melanoma cells (10 s cells/mouse). Cells were incubated with the peptides in PBS for 2 hours (in 37°C) or left untreated (control), and thereafter the cells were injected into the mice. Survival of mice treated with different peptides is shown in Fig. 11. It is evident that Compound 10 (Vidapin) significantly increased the survival of the mice with melanoma from median survival time of 40 days (control) to 73 days. All mice treated with Vidapin did not show any clinical signs at the end point of 73 days.
  • Compound 10 Compound 10

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Abstract

La présente invention concerne des analogues et des dérivés de vipéristatine, un peptide dérivé de venin de serpent qui inhibe les récepteurs de collagène. L'invention concerne en outre des compositions pharmaceutiques comprenant lesdits analogues et dérivés de vipéristatine, et des méthodes pour le traitement et/ou le diagnostic et l'imagerie de maladies ou de pathologies marquées par une angiogenèse excessive à l'aide desdites compositions.
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Publication number Priority date Publication date Assignee Title
WO2018048477A1 (fr) * 2016-09-06 2018-03-15 Debina Diagnostics, Inc. Ingénierie et utilité de particules de nanodiamant fluorescentes (ndp-f) pour le diagnostic et le traitement de caillots sanguins en médecine humaine et vétérinaire
WO2018206535A1 (fr) * 2017-05-08 2018-11-15 Novozymes A/S Domaine de liaison aux glucides et polynucléotides codant pour celui-ci
WO2021112615A1 (fr) * 2019-12-05 2021-06-10 아주대학교산학협력단 Composition contenant un peptide ou un composé peptidique utilisé comme principe actif, et son utilisation médicale
US11085920B2 (en) 2016-09-06 2021-08-10 Debina Diagnostics, Inc. Nanodiamond particles and related devices and methods
WO2022215961A1 (fr) * 2021-04-09 2022-10-13 아주대학교산학협력단 Peptide pour prévenir ou traiter des maladies inflammatoires

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WO2018048477A1 (fr) * 2016-09-06 2018-03-15 Debina Diagnostics, Inc. Ingénierie et utilité de particules de nanodiamant fluorescentes (ndp-f) pour le diagnostic et le traitement de caillots sanguins en médecine humaine et vétérinaire
US11085920B2 (en) 2016-09-06 2021-08-10 Debina Diagnostics, Inc. Nanodiamond particles and related devices and methods
WO2018206535A1 (fr) * 2017-05-08 2018-11-15 Novozymes A/S Domaine de liaison aux glucides et polynucléotides codant pour celui-ci
WO2021112615A1 (fr) * 2019-12-05 2021-06-10 아주대학교산학협력단 Composition contenant un peptide ou un composé peptidique utilisé comme principe actif, et son utilisation médicale
WO2022215961A1 (fr) * 2021-04-09 2022-10-13 아주대학교산학협력단 Peptide pour prévenir ou traiter des maladies inflammatoires

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