Oligopeptides that inhibit angiogenesis and vascular function

Abstract

The present invention provides oligopeptides that inhibit angiogenesis and vascular function. The oligopeptides have a length of 3 to 7 amino acids and have the sequence X2-X3-X4, where X2 is a basic amino acid or an amide amino acid, X3 is a small amino acid, and X4 is a basic amino acid that is charged at neutral pH, or the sequence X1-X2-X3-X4, where X1 is a polar, uncharged amino acid, and X2, X3, and X4 are the X2, X3, and X4 in X2-X3-X4. and X4 in X1-X2-X3-X4, or the sequence X1-X2-X3-X4-X5-X6-X7, wherein X1, X2, X3 and X4 are identical to X1, X2, X3 and X4 in X1-X2-X3-X4, X5 is a small amino acid, X6 is a hydrophobic amino acid and X7 is a hydrophobic amino acid.

Description

Detailed Description of the Invention

[0001] The present invention relates to anti-angiogenic oligopeptides. The present invention further relates to pharmaceutical compositions and uses of the oligopeptides.

[0002] Angiogenesis is the formation of new blood vessels from pre-existing vasculature. Angiogenesis actively occurs during development, determining the growth and differentiation of tissues. In adults, angiogenesis is limited to reproductive events in females and tissue repair resulting from wounds or fractures. Furthermore, the progression of devastating diseases such as cancer, diabetic retinopathy, and rheumatoid arthritis depends on pathological stimulation of angiogenesis. Therefore, molecules capable of inhibiting angiogenesis have great therapeutic potential.

[0003] Several endogenous antiangiogenic factors have been characterized, many of which are molecular fragments derived from specific proteolysis of proteins that are not active in the angiogenic process, such as extracellular matrix and basement membrane proteins, as well as growth factors, cytokines, blood proteins, and hormones.

[0004] Vasoinhibin is an antiangiogenic molecule generated when the hormone prolactin (PRL) loses its fourth alpha-helix after specific proteolytic cleavage by proteases, including cathepsin D, matrix metalloproteinases, and bone morphogenetic protein 1. The remaining fragment, preserving the N-terminal region and the first three helices of PRL, was named vasoinhibin due to its inhibitory effects on angiogenesis and vascular functions, namely vascular permeability and vasodilation. Furthermore, nonvascular effects of vasoinhibin have been reported, including profibrinolytic, inflammatory, anxiolytic, and neuroprotective effects. Vasoinhibin is also known as 16 kDa prolactin and is abbreviated as PRL16K. Furthermore, vasoinhibin blocks various signaling pathways (Ras-Raf-MAPK, Ras-Tiam1-Rac1-Pak1, PI3K-Akt, and PLCγ-IP3-eNOS) induced by proangiogenic factors (VEGF, bFGF, bradykinin, and IL1β). Vasoinhibin blocks angiogenesis by inhibiting endothelial cell proliferation, migration, and survival. Furthermore, vasoinhibin regulates vascular homeostasis by reducing vasodilation and vascular permeability through the reduction of intravascular production of nitric oxide. In animal studies, vasoinhibin induced depression- and anxiety-related behaviors.

[0005] Vasoinhibin has been shown to contribute to the physiological inhibition of angiogenesis in avascular organs and tissues where angiogenesis is highly restricted, such as the retina and cartilage. Furthermore, vasoinhibin action plays a role in the pathogenesis of angiogenesis-dependent diseases such as cancer, rheumatoid arthritis, and diabetic retinopathy, as well as in peripartum cardiomyopathy and preeclampsia.

[0006] The molecular mechanism of vasoinhibin's action is only partially known. It has recently been reported that vasoinhibin binds to the endothelial cell membrane with high affinity and forms a multimeric complex with plasminogen activator inhibitor-1 (PAI-1), urokinase plasminogen activator (uPA), and urokinase receptor (uPAR) on the endothelial cell surface. Vasoinhibin has also been shown to induce endothelial cell apoptosis through specific binding to integrin alpha5beta1.

[0007] Vasoinhibin is not a single molecular species but comprises a family of PRL fragments with different molecular weights determined by the cleavage site of the vasoinhibin-generating protease. These fragments include residues 123, 132, 139, 142, 147, 150, or 159 of mature PRL. Although all of these isoforms inhibit angiogenesis, their relative biological potencies are unknown. Moreno-Carranza, B. et al., Sequence optimization and glycosylation of vasoinhibin: Pitfalls of recombinant production, Protein Expression and Purification. 161 (2019) 49-56, discloses the difficulty of expressing a peptide containing the first 123 amino acids of human prolactin with good antiangiogenic properties in high yields.

[0008] US 7,300,920 B2 discloses an antiangiogenic peptide that is substantially identical to about 10 to about 150 consecutive amino acids selected from the N-terminus of human placental lactogen, human growth hormone, or the growth hormone variant hGH-V. This peptide (i) inhibits the proliferation and organization of capillary endothelial cells; (ii) inhibits angiogenesis in the chick chorioallantoic membrane; and (iii) binds to at least one specific receptor that does not bind to the complete full-length growth hormone, placental lactogen, or growth hormone variant hGH-V.

[0009] Nguyen, N.-Q.-N. et al., "Prolactin / growth hormone-derived antiangiogenic peptides highlight a potential role of tilted peptides in angiogenesis," Proceedings of the National Academy of Sciences. 103 (2006) 14319-14324, demonstrates that tilted peptides exert antiangiogenic activity. Tilted (or obliquely oriented) peptides are short peptides known to destabilize membranes and lipid cores. When helical, they are characterized by an asymmetric distribution of hydrophobic residues along their axis. All of these fragments have been demonstrated to have 14-aa sequences characteristic of tilted peptides. Tilted peptides of human prolactin and human growth hormone induce endothelial cell apoptosis, inhibit endothelial cell proliferation, and inhibit capillary tube formation both in vitro and in vivo.

[0010] US 7,655,626 B2 discloses a composition comprising an isolated anti-angiogenic peptide or a fusion protein comprising a heterologous protein fused to an anti-angiogenic peptide, the peptide having anti-angiogenic activity and consisting of the amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14. Here, X1 is any amino acid residue compatible with helix formation; X2 is an amino acid residue of Leu; X3 is an amino acid residue of Arg or Ser; X4 is an amino acid residue of Ile or Leu; X5 is any amino acid residue compatible with helix formation; X6 is an amino acid residue of Leu or Val; X7 is an amino acid residue of Leu or Ser; X8 is any amino acid residue compatible with helix formation; X9 is any amino acid residue compatible with helix formation; X10 is an amino acid residue of Gln, Glu, or Arg; X11 is an amino acid residue of Ser; X12 is an amino acid residue of Trp; X13 is an amino acid residue of Leu or Asn; and X14 is an amino acid residue of Glu.

[0011] According to Robles, JP et al., Scientific Reports 8 (2018) 17111-17118, vasoinhibin contains a three-helix bundle, and its antiangiogenic domain is located within the first 79 residues. Molecular dynamics simulations (MD) showed that the loss of the fourth α-helix (H4) exposes the hydrophobic core of PRL, causing the three-helix bundle to collapse into the molecule, burying the hydrophobic core. Furthermore, it is speculated that the collapse occurs via the movement of loop 1 (L1) and its interaction with α-helix 1 (H1), generating a new L1 conformation with electrostatic and hydrophobic surfaces distinct from those of PRL. This new L1 conformation may correspond to the bioactive domain. Consistent with this model, a 14-amino acid peptide sequence (residues 45–58) located in the initial portion of L1 of buffalo PRL has been reported to exhibit antiangiogenic effects. This sequence was found to share 35.7% homology with human somatostatin, a known anti-angiogenic factor. The authors found that a recombinant protein containing the first 79 amino acids of human PRL, including H1 and L1, inhibited endothelial cell proliferation and migration and upregulated the vasoinhibin target genes IL1A and ICAM1. This bioactivity was comparable to that of conventional vasoinhibin, which contains 123 residues encompassing H1, L1, H2, L2, and H3 of human PRL. These findings suggested that the tilted peptide, absent from the 79-amino acid vasoinhibin, does not contain the most active biological determinant of vasoinhibin.

[0012] The problem to be solved by the present invention is to provide an alternative peptide that can exert the function of vasoinhibin by inhibiting angiogenesis and vascular function. It is a further object of the present invention to provide recombinant proteins, recombinant nucleic acids, pharmaceutical compositions, pharmaceutical compositions for use in the treatment or prevention of disease, and uses of the peptides.

[0013] The problem of the present invention is solved by the features of claims 1, 3, 5, 11, 12, 14, 16 and 18. Embodiments of the invention are the subject of claims 2, 4, 6-10, 13, 15, 17 and 19-22.

[0014] According to a first option of the present invention, there is provided an oligopeptide that inhibits angiogenesis and vascular function. The oligopeptide has a length of 3 to 7 amino acids, A sequence X2-X3-X4, where: X2 is a basic amino acid or an amide amino acid, X3 is a small amino acid, X4 is a basic amino acid that is charged at neutral pH, Array X2-X3-X4, or The sequence X1-X2-X3-X4, wherein: X1 is a polar uncharged amino acid, X2, X3 and X4 are identical to X2, X3 and X4 in X2-X3-X4; Array X1-X2-X3-X4, or The sequence X1-X2-X3-X4-X5-X6-X7, wherein: X1, X2, X3, and X4 are identical to X1, X2, X3, and X4 in X1-X2-X3-X4, X5 is a small amino acid, X6 is a hydrophobic amino acid, X7 is a hydrophobic amino acid, Array X1-X2-X3-X4-X5-X6-X7, It comprises or consists of:

[0015] In one embodiment of this first alternative of the invention, X1 is Thr, Ser, Asn, Glu, Gly or Ala, in particular Thr; X2 is His, Arg, Lys, Gln or Asn, in particular His; X3 is Ala or Gly, in particular Gly; X4 is Arg or Lys, in particular Arg; X5 is Gly, Ser or Ala, in particular Gly; X6 is Phe, Ala, Leu, Ile, Trp or Pro, in particular Phe; X7 is Phe, Ala, Leu, Ile, Trp or Pro, in particular Ile.

[0016] According to a second option of the present invention, there is provided an oligopeptide that inhibits angiogenesis and vascular function. The oligopeptide has a length of 3 to 7 amino acids, The sequence X1-X2-X3, wherein: X1 is an acidic amino acid that is negatively charged at neutral pH, X2 is a polar amino acid, X3 is an amino acid that is positively charged at neutral pH, Array X1-X2-X3, or The sequence X1-X2-X3-X4, wherein: X1, X2, and X3 are identical to X1, X2, and X3 in X1-X2-X3, X4 is a polar aromatic amino acid, Array X1-X2-X3-X4, or The sequence X1-X2-X3-X4-X5-X6-X7, wherein: X1, X2, X3, and X4 are identical to X1, X2, X3, and X4 in X1-X2-X3-X4, X5 is a polar amino acid, X6 is a hydrophobic amino acid, X7 is a hydrophobic amino acid, Array X1-X2-X3-X4-X5-X6-X7, It comprises or consists of:

[0017] In one embodiment of this second alternative of the invention, X1 is Asp or Glu, in particular Glu; X2 is Gln, Asn, Ser or Thr, in particular Gln; X3 is Arg or Lys, in particular Lys; X4 is Tyr, X5 is Gln, Asn, Ser or Thr, in particular Ser; X6 is Phe, Ala, Leu, Ile, Trp or Pro, in particular Phe; X7 is Phe, Ala, Leu, Ile, Trp or Pro, in particular Leu.

[0018] According to a third option of the present invention, there is provided an oligopeptide that inhibits angiogenesis and vascular function, the oligopeptide having a length of 7 amino acids and having the sequence X1-X2-X3-X4-X5-X6-X7, wherein: X1 is the amino acid Thr, Asp or Glu, X2 is the amino acid His or Gln, X3 is the amino acid Gly or Lys, X4 is the amino acid Arg if X3 is Gly, or the amino acid Tyr if X3 is Lys; X5 is the amino acid Gly or Ser, X6 is the amino acid Phe, X7 is the amino acid Ile or Leu.

[0019] In the context of the present disclosure, the terms should be understood as follows: The terms "amino acid" and "amino acid residue" may be used interchangeably and should not be understood as limiting.

[0020] amino acid: Amino acids that make up proteins.

[0021] Amino acid residues: Amino acid residues that make up proteins.

[0022] Amide amino acids: Amino acids with amidated side chains, such as asparagine (Asn) and glutamine (Gln).

[0023] Polar amino acids: Amino acid residues that form hydrogen bonds as donors or acceptors. Among the amino acid residues that make up naturally occurring proteins, there are 10 polar amino acid residues: two that are negatively charged at neutral pH, namely aspartic acid (Asp) and glutamic acid (Glu), three that are positively charged at neutral pH, namely arginine (Arg), lysine (Lys), and histidine (His), and five that are uncharged at neutral pH, namely glutamine (Gln), asparagine (Asn), serine (Ser), threonine (Thr), and tyrosine (Tyr).

[0024] Polar aromatic amino acids: Polar amino acid residues with an aromatic ring, such as tyrosine (Tyr).

[0025] Small amino acids: 100 cubic angstroms (Å), such as alanine (Ala), glycine (Gly), and serine (Ser). 3 ) but is not cysteine ​​(Cys) or any other amino acid with a volume greater than cysteine.

[0026] Hydrophobic amino acids: Amino acid residues that are usually buried inside the protein core, such as phenylalanine (Phe), tryptophan (Trp), isoleucine (Ile), leucine (Leu), methionine (Met), valine (Val), alanine (Ala), and cysteine ​​(Cys). These amino acid residues are non-polar.

[0027] Basic amino acids: Amino acid residues that have a positive charge at neutral pH in their side chains that often form salt bridges, such as arginine (Arg), lysine (Lys), and histidine (His).

[0028] Positively charged amino acids: Basic amino acids.

[0029] Acidic amino acids: Amino acid residues that have a negative charge at neutral pH in their side chains that often form salt bridges, including aspartic acid (Asp) and glutamic acid (Glu).

[0030] Negatively charged amino acids: Acidic amino acids.

[0031] Conservative substitution: Substitution of an amino acid with another amino acid belonging to the same of the above classes of amino acids, i.e., polar amino acids, polar aromatic amino acids, small amino acids, hydrophobic amino acids, basic amino acids, amide amino acids, positively charged amino acids, acidic amino acids, and negatively charged amino acids. Conservative substitution groups include, for example, valine-leucine-isoleucine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0032] Oligopeptide with X% similarity: The percentage of amino acids out of the total number of amino acids in an oligopeptide that are replaced by conservative substitutions. For example, 70% similarity to an oligopeptide having 10 amino acids means that 7 of the 10 amino acids in the oligopeptide are replaced by conservative substitutions.

[0033] peptide A compound consisting of two or more amino acid residues.

[0034] oligopeptides A peptide consisting of fewer than 20 amino acid residues.

[0035] Polypeptides A peptide consisting of at least 20 but not more than 50 amino acid residues.

[0036] protein A peptide consisting of at least 50 amino acid residues.

[0037] It was surprising to find potent antiangiogenic and vascular function inhibitory activity in small oligopeptides such as the oligopeptides of the present invention. The oligopeptides of the present invention consist of only 3 to 7 amino acids. The small size of the oligopeptides provided by the present invention has the advantage of making them easy to produce, purify, handle, and formulate. Despite their small size, the oligopeptides exhibit biological potency that is equal to, better than, or at least similar to that of vasoinhibin in terms of inhibiting angiogenesis and vascular function. The amino acid sequence differs from that of the "tilted" peptide known from Nguyen, N.-Q.-N. et al. This peptide is a novel oligopeptide of the present invention. twist also have a much lower biological potency.

[0038] The ease of production is an important advantage, given the known difficulty in expressing a peptide containing the first 123 amino acids of human prolactin in good yields. As a result, the peptide has good anti-angiogenic properties, a difficulty encountered in producing a variety of other anti-angiogenic proteins derived from prolactin. The small size of the oligopeptides of the present invention is an important advantage for their production, resulting in high yields and stability of the oligopeptides and low production costs.

[0039] The oligopeptides of the present invention are soluble in water or a buffer such as Dulbecco's phosphate buffered saline (pH 7). The oligopeptides are soluble in a concentration of approximately 15 mg mL -1 It is soluble at concentrations up to 1000 kJ / kg. Solubility is a distinct advantage over known hydrophobic "gradient" peptides and over the whole vasoinhibin molecule, which exposes hydrophobic patches on its surface that can reduce its solubility and promote its precipitation.

[0040] Chemical modifications of the oligopeptides of the invention can increase their half-life and gastrointestinal resistance, including the incorporation of dextroamino acids or conversion into retro-inverso and cyclic peptides.

[0041] Because the oligopeptides of the present invention preserve the bioactive properties of vasoinhibin, they can be used to design and generate specific antibodies that distinguish between PRL and vasoinhibin, allowing for sensitive and specific quantification of vasoinhibin for its use in clinical trials, diagnostics, and therapy.

[0042] Furthermore, the oligopeptides of the present invention have a direct inhibitory effect on the proliferation and invasion of cancer cells. The oligopeptides of the present invention can inhibit both the proliferation and migration of endothelial cells and the proliferation and migration of cancer cells. This dual effect is advantageous over antiangiogenic drugs used in cancer treatment that only have vasoactive effects.

[0043] Furthermore, the oligopeptides of the present invention can be used in the treatment of angiogenesis-dependent diseases, whether related to reproduction or not. Compared to vasoinhibin, their smaller size, hydrophilicity, and potency allow for the manufacture and formulation of effective drugs containing the oligopeptides, and enhance the stability of the drugs.

[0044] The present invention includes sequences of the oligopeptides according to the invention within recombinant proteins or other structures that can be used as carriers.

[0045] The oligopeptides of the present invention include oligopeptides, particularly agonist oligopeptides. The oligopeptides have the sequence of the oligopeptides defined above or a sequence having at least 70%, particularly at least 80%, particularly at least 85%, particularly at least 90% similarity to the oligopeptides defined above. The oligopeptides may further have a modification at one or both termini of the sequence, or one or more or all amino acids having a D-conformation (D-amino acids) may be replaced by amino acids having an L-conformation (L-amino acids). The modification may be acetylation of the N-terminus and / or amidation of the C-terminus of the oligopeptide, or a covalent bond between the N- and C-terminal amino acids of the oligopeptide, resulting in cyclization of the oligopeptide.

[0046] The oligopeptides of the invention may comprise or consist of the sequence of loop 1 or a sequence which has at least 70%, particularly at least 80%, particularly at least 85%, and particularly at least 90% similarity to loop 1. Loop 1 is loop 1 of PRL, growth hormone or placental lactogen. In particular, the oligopeptides of the invention may consist of or comprise any of the following sequences, or may consist of or comprise a sequence which has at least 70%, particularly at least 80%, particularly at least 85%, and particularly at least 90% similarity to any of the following sequences: SEQ ID NO:1: Thr His Gly Arg Gly Phe Ile (SEQ ID NO: 1), SEQ ID NO:2: Glu Gln Lys Tyr Ser Phe Leu (SEQ ID NO: 2), SEQ ID NO:3: Asp Gln Lys Tyr Ser Phe Leu (SEQ ID NO: 3), SEQ ID NO:4: Thr His Gly Arg (SEQ ID NO: 4), SEQ ID NO:5: Glu Gln Lys Tyr (SEQ ID NO: 5), SEQ ID NO:6: Asp Gln Lys Tyr (SEQ ID NO: 6), SEQ ID NO:7: His Gly Arg, SEQ ID NO:8: Glu Gln Lys, SEQ ID NO:9: Asp Gln Lys.

[0047] The oligopeptide of the present invention may be fused to a carrier protein, which can improve its efficiency, its localization, and / or its half-life.

[0048] The oligopeptides of the present invention, particularly oligopeptides having seven amino acids, may contain approximately 42.86% neutral residues, 28.57% basic or acidic residues, and 28.57% or less hydrophobic residues. Furthermore, the oligopeptides may have a hydrophobicity of +10 Kcal mol according to the experimental scale of Wimley, WC, White, SH, Experimentally determined hydrophobicity scale for proteins at membrane interfaces, Nature Structural Biology. 3 (1996) 842. -1 In particular, the hydrophobicity may be greater than +11.76 to +11.90 Kcal mol -1 Furthermore, the oligopeptides of the present invention may have a particular distribution of hydrophobic residues clustered at the C-terminus.

[0049] The seven amino acid oligopeptides of the invention have a basic amino acid at position X3 or X4, such as Lys or Arg, that is charged at a pH of about 7.4. Additionally, the oligopeptides may contain a basic amino acid at position X2, such as His, that is positively charged at a pH of ≦6, and an acidic amino acid at position X1, such as Asp or Glu, that is negatively charged at neutral pH.

[0050] The present invention further relates to a recombinant protein comprising the sequence of an oligopeptide according to the invention.

[0051] The present invention also relates to a nucleic acid, in particular a recombinant nucleic acid, which consists of or comprises a sequence encoding an oligopeptide according to the invention or a sequence complementary to this sequence. The recombinant nucleic acid may be contained in an expression vector.

[0052] The present invention further relates to a pharmaceutical composition comprising at least one oligopeptide of the present invention, and / or at least one recombinant protein of the present invention, and / or at least one recombinant nucleic acid of the present invention. According to one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is pharmaceutically acceptable for mammalian administration, particularly for humans. The pharmaceutically acceptable carrier may be a physiological salt solution.

[0053] Any physiologically compatible formulation can be used for administering the oligopeptides of the present invention. For example, the formulation may be an aerosol or paste, or may contain lipids. The concentration of the oligopeptide of the present invention in the pharmaceutical composition may vary from about 0.1% w / w to 50% w / w.

[0054] The oligopeptides of the present invention can be administered in a variety of dosage forms. For example, for oral administration, powders, tablets, pills, capsules, or dragees, as well as liquid dosage forms such as suspensions or syrups, may be used. For intraocular or parenteral administration, liquid and sterile forms may be used. For example, to stabilize the pharmaceutical composition, other inactive ingredients such as carriers or excipients, such as glucose, lactose, sucrose, mannitol, starch, cellulose, and one or more derivatives thereof, or pH buffers, may be included in the pharmaceutical compositions of the present invention. The pharmaceutical composition may also contain liposomes, including emulsions, micelles, or liquid crystals. Liposomes may be targeted to a specific target using antibodies or molecules that recognize the target.

[0055] The oligopeptides of the present invention can be administered topically, locally, or systemically by injection, inhalation, suppository, transdermal, and ocular administration, among others. Pharmaceutical compositions containing the oligopeptides of the present invention can be administered, for example, by aerosol for nasal administration. The oligopeptides of the present invention can also be administered through a catheter, thereby enabling their delivery to internal or remote tissues. Pharmaceutical compositions can also include encapsulated oligopeptides for protection and / or controlled long-term release of the oligopeptide. Such pharmaceutical compositions can be implanted near or at a specific target tissue. Suitable formulations for pharmaceutical compositions are reported in various references, such as Shayne Cox, Pharmaceutical Manufacturing Handbook, Wiley Online Books, Canada, 2008. doi:10.1002 / 9780470259818.

[0056] According to a further aspect of the present invention, the pharmaceutical composition of the present invention is for use in the treatment or prevention of an angiogenesis-dependent disease. Any of the oligopeptides of the present invention, any of the recombinant proteins of the present invention, and any of the recombinant nucleic acids of the present invention may be for use in the treatment or prevention of an angiogenesis-dependent disease. The angiogenesis-dependent disease may be cancer, vascular proliferative retinopathy, diabetic retinopathy, or rheumatoid arthritis.

[0057] The present invention further relates to the use of the oligopeptide of the present invention or the recombinant protein of the present invention for the production of antibodies. For this use, the oligopeptide may comprise or consist of the sequence HisGlyArg, GluGlnLys, or the sequence AspGlnLys, and the recombinant protein may comprise one of these sequences. The antibody may be used in a diagnostic method performed in vitro. The diagnostic method may relate to the diagnosis of preeclampsia, peripartum cardiomyopathy, fetal growth restriction, conditions associated with abnormal blood pressure, depressive disorders, anxiety disorders, or angiogenesis-dependent diseases. Abnormal blood pressure is blood pressure that is below or above normal, i.e., hypotension or hypertension. Angiogenesis-dependent diseases are diseases in which angiogenesis and / or vascular permeability and / or vasodilation are altered, such as rheumatoid arthritis, vascular proliferative retinopathy, diabetic retinopathy, and cancer.

[0058] The present invention also relates to pharmaceutical compositions comprising, separately or in combination, one, two or three oligopeptides having the above characteristics. Furthermore, the present invention relates to the recombinant production of precursors of any of the above oligopeptides, and to fusion molecules comprising any of the above sequences.

[0059] The oligopeptides of the present invention can be used as immunizing agents to generate antibodies that recognize whole vasoinhibin but do not recognize PRL.These antibodies allow for the quantification of endogenous levels of vasoinhibin in serum, other biological fluids, and tissues.The quantification of endogenous levels of vasoinhibin is important because it has been shown that vasoinhibin may contribute to the progression of disease states such as preeclampsia, peripartum cardiomyopathy, and diabetic retinopathy.

[0060] The present invention relates to various methods for producing the oligopeptides of the present invention. For example, the oligopeptides can be produced recombinantly from precursors or together with fusion proteins. Furthermore, peptide synthesis, which is the most viable method for producing the oligopeptides of the present invention, can be performed using various known protocols.

[0061] The present invention will now be described by way of examples, in which the oligopeptides of the present invention are presented for illustrative purposes only and should not be construed as limiting the scope of the present invention.

[0062] 1A and 1B show a schematic representation of the position of the seven-amino acid oligopeptide THGRGFI of the present invention in the linear amino acid sequence of vasoinhibin (FIG. 1A) and the chemical structure of this terminally modified oligopeptide at pH 7.4 (FIG. 1B).

[0063] Figures 2A, B show dose-response graphs comparing the biological potency of the 7 amino acid oligopeptide THGRGFI and the 123 amino acid vasoinhibin on growth factor stimulated proliferation of endothelial cells.

[0064] 3A and 3B show the inhibitory effects of the 7-amino acid oligopeptide THGRGFI and vasoinhibin, which has 123 amino acids, on VEGF-stimulated endothelial cell invasion.

[0065] FIG. 4 shows the changes in expression of vasoinhibin target genes, interleukin-1α (IL-1α) and intracellular adhesion molecule 1 (ICAM1), in endothelial cells in response to 100 nM of the 123-amino acid vasoinhibin or the oligopeptide THGRGFI.

[0066] Figure 5A and B show Matrigel TM 1 shows the inhibitory effect of 100 nm of the oligopeptide THGRGFI and vasoinhibin with 123 amino acids on capillary tube formation by endothelial cells cultured on a layer.

[0067] 6A and 6B show the inhibition of vascular permeability by the oligopeptides THGRGFI and vasoinhibin with 123 amino acids for 120 minutes (FIG. 6A) and at 120 minutes (FIG. 6B).

[0068] FIG. 7 shows the inhibition of vascular permeability of endothelial cell monolayers by the oligopeptide THGRGFI and vasoinhibin with 123 amino acids in the absence (control, Ctl) or presence of VEGF alone or in combination with VEGF and oligopeptide or vasoinhibin for 120 minutes.

[0069] FIG. 8 shows the in vivo inhibitory effect of VEGF-induced retinal vascular permeability by the oligopeptide THGRGFI and by vasoinhibin with 123 amino acids.

[0070] 9A and 9B show the effects of the oligopeptide THGRGFI, vasoinhibin with 123 amino acids, and three oligopeptides with scrambled amino acid sequences contained in the oligopeptide THGRGFI on growth factor-stimulated endothelial cell proliferation. Amino acids whose positions remain unchanged are shown in bold.

[0071] Figures 10A and 10B show the location of the oligopeptide THGRGFI and three 7-amino acid oligopeptides with sequences overlapping with this oligopeptide in the linear sequence of vasoinhibin (Figure 10A), as well as the effects of these oligopeptides on endothelial cell proliferation in the presence of VEGF (Figure 10B).

[0072] Figure 11A,B shows the sequences of seven amino acid synthetic oligopeptides (Figure 11A) in which each of the amino acids of the oligopeptide THGRGFI has been replaced with alanine (shown in bold), and the biological efficacy of these oligopeptides on growth factor-stimulated endothelial cell proliferation.

[0073] 12A,B show the sequences of 7, 4 and 3 amino acid oligopeptides of the invention (FIG. 12A) and their biological efficacy on growth stimulated endothelial cell proliferation.

[0074] 1A shows the location of the seven amino acid oligopeptide THGRGFI of the present invention in the linear amino acid sequence of vasoinhibin. The linear diagram of vasoinhibin is shown from its N-terminus (H2N) to its C-terminus (COO). - ) The three major alpha-helices (H1, H2, and H3) and loop 1 (L1) connecting H1 and H2 are shown. The sequence from residues 40 to 65 has been expanded to allow for a better understanding of the sequence. The position of the 7-amino acid oligopeptide THGRGFI is shown in bold and aligned with this sequence.

[0075] 1B shows a diagram of the primary structure of the oligopeptide THGRGFI of the present invention at pH 7.4, the amino and carboxyl termini of which are acetylated and amidated, respectively.

[0076] Example 1. Inhibition of endothelial cell proliferation The inhibitory effect of the 7 amino acid THGRGFI oligopeptide corresponding to SEQ ID NO: 1 on the proliferation of primary cultures of immortalized bovine umbilical vein endothelial cells (BUVEC E6E7) and human umbilical vein endothelial cells (HUVEC) was evaluated and compared with the effect of the conventional 123 amino acid vasoinhibin.

[0077] BUVEC E6E7 and HUVEC cells were plated onto 96-well plates treated for cell culture at approximately 14,000 and 11,000 cells cm, respectively. -2 BUVEC E6E7 cells were maintained in F12K culture medium containing 10% (v / v) fetal bovine serum (FBS), and HUVEC were maintained in F12K culture medium containing 20% ​​FBS and 100 μg heparin ml -1 and endothelial cell growth supplement (ECGS) 25 μg·ml -1 After 24 hours, cells were starved for 16 hours in FBS-reduced medium (0.1% FBS for BUVEC E6E7 and 0.5% FBS for HUVEC) to synchronize them to the G0 phase of the reproductive cycle. After this, HUVECs were supplemented with FBS and heparin. Then, 10 μM of the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU) and 50 ng ml VEGF were added to BUVEC E6E7. -1 or VEGF 25 ng·ml for HUVECs -1 and bFGF 20 ng·ml -1 Cells were treated with vasoinhibin or the oligopeptide TGF-R-GFI at concentrations ranging from 0.001 to 100 nM for 24 hours in the presence of a combination of vasoinhibin and TGF-R-GFI. At the end of the experiment, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in TBS1X, and stained to detect newly synthesized DNA via EdU incorporation using a "click" assay (which involves a copper-catalyzed reaction to covalently attach fluorescent azide to incorporated EdU in DNA). Total DNA was counterstained with Hoechst 33342, and "click"-stained nuclei were quantified and plotted against the total number of Hoechst 33342-stained nuclei.

[0078] The results are shown in Figures 2A and 2B. -1 Regarding the proliferation of immortalized bovine umbilical vein endothelial cells (BUVEC E6E7) stimulated with VEGF (25 ng mL ) (Figure 2A), -1 ) and bFGF (20 ng mL -1 2B shows a dose-response graph comparing the biological potency of the oligopeptide THGRGFI and the 123 amino acid vasoinhibin on primary cultures of human umbilical vein (HUVEC) stimulated with a combination of THGRGFI and vasoinhibin (Figure 2C).

[0079] The proliferation of endothelial cells (BUVEC E6E7 and HUVEC) was inhibited in a dose-response manner by vasoinhibin and the oligopeptide THGRGFI. Both inhibitors had the same effective dose (EC 50 The oligopeptide was active against both types of endothelial cells at concentrations of 1 nM (Figures 2A and 2B), similar to the previously reported potency of vasoinhibin. This result confirms that the seven-amino acid oligopeptide THGRGFI preserves the potency of vasoinhibin for inhibiting endothelial cell proliferation. The oligopeptide exhibits a robust dose-response behavior similar to vasoinhibin.

[0080] Example 2. Inhibition of invasive migration of endothelial cells Vasoinhibin can inhibit endothelial cell migration and invasion through mechanisms including inactivation of the Ras-Tiam1-Rac1-Pak1 pathway, inactivation of urokinase-type plasminogen activator (uPA) by increasing the expression of plasminogen activator inhibitor-1 (PAI-1), and inactivation of endothelial nitric oxide synthase (eNOS). To test whether the oligopeptide THGGRGFI of the present invention retains its inhibitory properties against invasive migration of endothelial cells, we used Matrigel as a chemoattractant. TM Migration assays were performed using permeable "transwell" supports with matrix and conditioned medium.

[0081] Area 0.33cm 2and Matrigel on a permeable "Transwell" support in a Transwell chamber with a pore size of 8 μm. TM 100 μl of matrix (380 ng μl -1 ) over a range of 30,000 and 14,000 cells cm for BUVEC E6E7 and HUVEC cells, respectively. -2 Endothelial cells were seeded at a density of 100 μg ml In the upper (luminal) compartment, cells were maintained in starvation medium F12K with 0.1 or 0.5% FBS for BUVEC E6E7 and HUVEC, respectively. Additionally, heparin 100 μg ml was added. -1 HUVEC cells were maintained in the lower abluminal compartment with filtered (0.22 μm) conditioned medium from 3T3-L1 cells (obtained by culturing 3T3-L1 cells in DMEM-10% FBS for 48 h) and VEGF 50 ng ml -1 was used as a chemoattractant. After 24 hours, the medium was removed from both compartments and the luminal cells. The extraluminal cells were fixed with 100% MeOH for 10 minutes, permeabilized with TBS1X-0.5% Triton X-100, and stained with Hoechst 33342. The total number of cells in the extraluminal compartment indicates the invasive activity of endothelial cells.

[0082] The results are shown in Figures 3A and 3B. -1 The inhibitory effect of 100 nM of the oligopeptide THGRGFI on the invasion of immortalized bovine umbilical vein-derived endothelial cells (BUVEC E6E7) (Figure 3A) or primary human umbilical vein-derived cells (HUVEC) (Figure 3B) stimulated with ) was compared with that of 100 nM of the 123 amino acid vasoinhibin ( *** P<0.001).

[0083] Both vasoinhibin and the oligopeptide THGRF1 were formulated with Matrigel TM The peptide significantly inhibited the ability of two types of endothelial cells to invade the endothelial cells and reach the abluminal compartment of the transwell. This result confirms that the peptides from the present invention preserve the properties of vasoinhibin with regard to inhibiting endothelial cell migration.

[0084] Example 3. Induction of expression of vasoinhibin target genes Vasoinhibin induces the expression of various genes through activation of NF-κB, promoting antiangiogenic and inflammatory effects. In particular, interleukin-1 alpha (IL-1α) and intercellular adhesion molecule 1 (ICAM1) are vasoinhibin gene targets in bovine endothelial cells. To evaluate the ability of the oligopeptide THGRGFI to induce IL-1α and ICAM1 expression, BUVEC E6E7 cells were seeded onto 12-well plates containing F12K-10% FBS, grown to 80% confluency, and starved in low-serum (0.1% FBS) culture medium for 24 hours. Next, cells were treated with 100 nM vasoinhibin or the oligopeptide THGRGFI. After 4 hours, RNA was extracted from the cells using Trizol (Invitrogen, Carlsbad, CA) and retrotranscribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). RT-PCR products were quantified in a 10 μl final volume reaction mixture containing template and 0.25 μM of each primer using Maxima SYBRgreen qPCR (Thermo Fisher Scientific). PCR amplification was performed in a CFX96 real-time PCR (BioRad) with denaturation at 95°C for 10 minutes followed by 35 amplification cycles (95°C for 10 seconds, 58°C for 30 seconds, and 72°C for 30 seconds). Primers used were IL-1α forward (5'-TCAAGGAGAATGTGGTGATG-3' = SEQ ID NO: 7) and IL-1α reverse (5'-CTGGAAGCTGTAATGTGCTG-3' = SEQ ID NO: 8); and ICAM1 forward (5'-CGTTAAGCTACACCCACCTT-3' = SEQ ID NO: 9) and ICAM1 reverse (5'-AGGTAAGGGTCTCCATCACA-3' = SEQ ID NO: 10). PCR data were analyzed using a 2-step PCR program. -ΔΔCTThe threshold cycle (CT) was normalized to the constitutive housekeeping gene cyclophilin A (PPIA). The primers for PPIA amplification were PPIA forward (5'-GGTTCCCAGTTTTTCATTTG-3' = SEQ ID NO: 11) and PPIA reverse (5'-ATGGTGATCTTCTTGCTGGT-3' = SEQ ID NO: 12).

[0085] Figure 4 shows the fold change in expression of messenger RNA (mRNA) for the vasoinhibin target genes interleukin-1α (IL-1α) and intercellular adhesion molecule 1 (ICAM1) in endothelial cells derived from bovine umbilical vein (BUVEC E6E7) in response to 100 nM of the 123-amino acid vasoinhibin or the oligopeptide THGRGFI ( *** P<0.001). Relative to untreated controls, vasoinhibin increased IL-1α and ICAM-1 mRNA levels by approximately 20-fold and approximately 12-fold, respectively, whereas the oligopeptide THGRGFI increased IL-1α and ICAM mRNA levels by approximately 30-fold and approximately 13-fold, respectively. These findings indicate that the oligopeptides of the present invention preserve the ability of vasoinhibin to induce IL-1α and ICAM-1 expression.

[0086] Example 4: Inhibition of capillary structure formation Capillary formation is a late stage in the angiogenesis process, involving the migration, interaction, and organization of endothelial cells into tubular capillary structures. Vasoinhibin disrupts this morphogenetic process.

[0087] To investigate whether the oligopeptide THGRGFI shares this property, primary cultures of human umbilical vein-derived endothelial cells (HUVECs) were cultured in 20% FBS, 100 μg·ml heparin, and 100 μg·ml heparin. -1 and endothelial cell growth supplement (ECGS) 25 μg·ml -1Cells from passages 2–4 were rinsed with conditioned PBS 1x, detached from the plate with 0.25% trypsin-EDTA for approximately 3 min, and centrifuged to remove the trypsin. Cells were counted using a hemocytometer and plated in approximately 9.7 μg μl of prepolymerized PBS in a 24-well plate at 37°C for 1 h. -1 Matrigel TM Layer 29,000 cells cm in 300 μl of F12K medium supplemented with 20% FBS and heparin. -2 Cells were seeded at a density of 100 nM. Cells were then treated with 100 nM vasoinhibin or the oligopeptide THGRGFI, and after 6 hours, photomicrographs were taken using an inverted microscope. The photomicrographs are shown in Figure 5A. Images were analyzed and the primary binding area per field was quantified using the software "Angiogenesis Analyzer" [Gilles Carpentier. ImageJ contribution: Angiogenesis Analyzer. ImageJ News, 5 October 2012.] and ImageJ software.

[0088] The results are shown in Figure 5B ( * P<0.001). The capillary structures were formed by the interaction of Matrigel with the angiogenic factors and cells in the culture medium. TM It forms spontaneously upon interaction with the component. Vasoinhibin and the oligopeptide THGRGFI disrupted the capillary structure, confirming that the oligopeptides of the present invention preserve this property of vasoinhibin.

[0089] Example 5. Inhibition of vascular permeability in vitro Vasoinhibin is known to inhibit vascular permeability by directly acting on endothelial cells through the inactivation of endothelial nitric oxide synthase (eNOS) in response to various vasoactive substances. This effect was demonstrated in vitro through confluent monolayers of endothelial cells derived from bovine aorta and umbilical vein, rat retinal capillaries, and mouse brain and retinal endothelial cells. Permeability was tested by measuring the passage of a large protein (radish peroxidase) through the endothelial cell monolayer or by changes in transendothelial resistance (TEER) in the presence of various vascular permeability inducers. Vasoinhibin blocks eNOS activation through signaling pathways including stimulation of protein 2A phosphatase, which dephosphorylates / inactivates eNOS, as well as through the PLC and IP3 systems, as well as by reducing the intracellular calcium concentration required for calcium-calmodulin binding activation of eNOS, and by blocking transient receptor potential (TRP) channels.

[0090] To assess whether the oligopeptide THGRGFI preserves the inhibitory properties of vasoinhibin on vascular permeability, the passage of Evans Blue-conjugated albumin across endothelial cell (BUVEC E6E7) monolayers was assessed as follows: BUVEC E6E7 cells were plated onto transwell filters (0.4 μm pores) at 10,000 cells cm -2 After 3 days, the monolayers were starved for 48 hours in low serum (0.1% FBS). Subsequently, 100 nM of vasoinhibin or the oligopeptides of the present invention were added to the upper compartment (lumen) of the transwell support and incubated for 1 hour, followed by VEGF 50 ng mL -1The control (Ctl) did not contain VEGF, vasoinhibin, or oligopeptide. After 10 min, the upper (luminal) culture medium was replaced with 300 μl of PBS containing Evans Blue-conjugated albumin, and the medium in the lower abluminal compartment was replaced with 700 μl of PBS. At 10, 20, 30, and 60 min, 50 μl samples of the abluminal compartment were collected and replaced with fresh PBS. The absorbance (620 nm) was measured at all time points using a plate reader iMARK (BioRad). The absorbance values ​​confirmed the passage of Evans Blue-labeled albumin across the endothelial monolayer.

[0091] Figure 6A shows the inhibition of vascular permeability by the 7 amino acid oligopeptide of the invention and the 123 amino acid vasoinhibin over time (Figure 6A) and at 120 minutes (Figure 6B). *** P<0.001). As expected, VEGF stimulated the permeability of the endothelial monolayer, and this effect increased over time (FIG. 6A). Equivalent concentrations of vasoinhibin and the oligopeptide THGRGFI inhibited the VEGF-induced increase in endothelial permeability. This suggests that the peptides of the present invention preserve the ability of vasoinhibin to inhibit vascular permeability.

[0092] Another conventional protocol for assessing vascular permeability is the measurement of transendothelial electrical resistance (TEER), which uses a device that applies an electric current through electrodes on either side of the endothelial cell monolayer. A decrease in electrical resistance indicates a loss of barrier function and, consequently, an increase in permeability. The effect of the oligopeptide THGRGFI on endothelial cell permeability was evaluated by measuring TEER. BUVEC E6E7 were placed in a TEER device at 10,000 cells cm. -2 After 3 days, the monolayers were starved in low-serum medium (0.1% FBS) for 48 hours, after which time 100 nM vasoinhibin or oligopeptides were added to the upper compartment (luminal surface of the monolayer) for 1 hour. At time 0, TEER was recorded and VEGF 50 ng ml -1was added to the luminal side. TEER was measured at 10, 20, 30, 60, 90, and 120 minutes. Determinations were performed using an epithelial volts / ohms (TEER) EVOM2 device (World Precision Instruments, FL, USA) equipped with 4 mm "chopstick" electrodes. Values ​​were normalized to a cell-free device and to an untreated monolayer.

[0093] The results are shown in Figure 7. Vascular permeability was inhibited over time by the 7-amino acid oligopeptide and the 123-amino acid vasoinhibin. Permeability was determined in the absence of VEGF (control, Ctl), or in the presence of VEGF alone, or in the presence of a combination of VEGF and the oligopeptide of the invention or vasoinhibin. The results show the expected decrease in transendothelial resistance by VEGF, indicating that both the oligopeptide THGRGFI and vasoinhibin block VEGF action in a similar manner.

[0094] Example 6. Inhibition of vascular permeability in vivo Diabetic retinopathy and diabetic macular edema are major causes of vision loss in diabetes, and their early signs are retinal vascular permeability deterioration. Because VEGF is the primary factor responsible for these vascular changes, current treatments are based on intravitreal injection of anti-VEGF antibodies that can neutralize VEGF. Vasoinhibin inhibits the increase in retinal vascular permeability in response to intravitreally administered VEGF and also inhibits excessive vascular permeability caused by diabetes in experimental models. These studies have used recombinant vasoinhibin protein and vasoinhibin gene transduction, including recombinant viral vectors.

[0095] To assess whether the oligopeptide THGRGFI preserves the inhibitory properties of vasoinhibin on VEGF-induced vascular permeability in vivo, the effects of intravitreal injection of VEGF alone or in combination with the oligopeptide or vasoinhibin were measured in rats.

[0096] Wistar rats were intravitreally injected with saline (control) or 300 ng of VEGF, either alone, in combination with 20 μM of the oligopeptide of the present invention, or in combination with 20 μM of vasoinhibin. After 24 hours, albumin extravasation into the retina was assessed using the Evans blue method. Briefly, a total dose of 45 mg kg -1 Evans blue dye was intravenously injected into anesthetized rats and allowed to circulate for 2 hours. -1 Animals were perfused with approximately 80 ml of PBS at a flow rate of 100 μl / min. Retinas were dissected, dried, and incubated with 200 μl of formamide (Mallinckrodt Baker, Phillipsburg, NJ) at 72°C for 18 hours, and labeled albumin in retinal extracts was measured.

[0097] The results are shown in Figure 8 ( * P<0.05, ** P<0.02). These results demonstrate that intravitreal administration of the oligopeptides of the present invention, like vasoinhibin, inhibits VEGF-induced increases in retinal vascular permeability. Given the fact that treatment with VEGF-blocking antibodies is effective as a conventional treatment for diabetic retinopathy, diabetic macular edema, and other vascular proliferative retinopathies (juvenile retinopathy and age-related macular degeneration), it is clear that the oligopeptides of the present invention have potential therapeutic value in these diseases.

[0098] Example 7. Structural characterization of oligopeptide THGRGFI To determine whether the antiangiogenic activity of the oligopeptides of the present invention is sequence-specific and not due to their amino acid composition, three scrambled sequences were generated using the amino acids contained in the oligopeptides and tested for their effect on HUVEC cell proliferation at a concentration of 100 nM. The three sequences are GIGHFRT (SEQ ID NO: 13), THIRGGF (SEQ ID NO: 14), and GTRIHFG (SEQ ID NO: 15). They are illustrated in Figure 9A and designated Scr1, Scr2, and Scr3 in Figures 9A and 9B. Amino acids whose positions remain unchanged are shown in bold.

[0099] HUVECs were cultured at approximately 11,000 cells cm -2 Plated onto a 96-well plate at a density of 100 μg ml in 20% FBS and 100 μg ml heparin. -1 and endothelial cell growth supplement (ECGS) 25 μg·ml -1 After 24 hours, cells were maintained in F12K supplemented with 0.5% FBS. After 16 hours of starvation (FBS 0.5%), cells were synchronized in the G0 phase and then supplemented with FBS and heparin. Then, cells were incubated with 10 μM of the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) and 25 ng ml of VEGF. -1 and bFGF 20 ng·ml -1 Cells were treated with TGF-R-GFI, various oligopeptides, and the 123-amino acid vasoinhibin at 100 nM each, in the presence or absence of a combination of TGF-R-GFI and TGF-R-GFI. Finally, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in TBS1X, and newly synthesized DNA was stained using the "click" method, which detects EdU incorporation. Total DNA was stained with Hoechst 33342, and the ratio of "click"-stained nuclei to total nuclei (Hoechst 33342 staining) indicated cell proliferation.

[0100] Figure 9B shows the effect of VEGF (25 ng ml -1 ) and bFGF (20 ng·ml -11 shows the effect of the same concentration (100 nM) of three scrambled oligopeptides, the seven-residue peptide THGRGFI of the present invention, and the 123-amino acid vasoinhibin on the proliferation of HUVECs stimulated with ( *** P<0.001).

[0101] Although the scrambled oligopeptides shared the same amino acid composition as the oligopeptides of the present invention, none of the scrambled oligopeptides inhibited endothelial cell proliferation, indicating that the amino acid sequence is important for the activity of the oligopeptides of the present invention.

[0102] To determine whether the sequence of the peptide of the present invention is the determining factor for the effect of vasoinhibin on endothelial cell proliferation, or whether this effect is also present in adjacent sequences in the vasoinhibin sequence, the inhibitory effects of 7-amino acid oligopeptides shifted by 2 or 3 residues in the vasoinhibin sequence were evaluated. Figure 10A shows the position of the 7-residue oligopeptide THGRGFI of the present invention in the linear sequence of vasoinhibin. Amino acids and numbers are indicated in the sequence. Three 7-amino acid oligopeptides, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, whose sequences overlap with the peptide of the present invention, are also shown in Figure 10A.

[0103] For analysis, BUVEC E6E7 cells were plated at approximately 14,000 cells cm in 96-well plates with F12K containing 10% (v / v) FBS. -2 After 24 hours, cells were starved (0.1% FBS) for 16 hours to synchronize them in the G0 phase. Then, cells were incubated with 10 μM of the thymidine analogue EdU and 50 ng ml of VEGF. -1 Cells were treated with various oligopeptides in the presence of α-glucan. Finally, cells were fixed, permeabilized, and stained for newly synthesized DNA by EdU incorporation using the "click" assay. Total DNA was stained with Hoechst 33342, and the ratio of "click"-stained nuclei to total nuclei was used as a measure of proliferation.

[0104] Figure 10B shows the effect of VEGF (50 ng ml -1 VEGF (50 ng ml ) alone and in the absence of VEGF and oligopeptide (control, Ctl) on the proliferation of immortalized endothelial cells derived from bovine umbilical vein (BUVEC E6E7). -1 ) in the presence of the shifted 7-residue oligopeptide, the peptide of the present invention, and the same concentration (100 nM) of 123-residue vasoinhibin ( *** P<0.001).

[0105] The oligopeptide GRGFITK (SEQ ID NO: 16), which is shifted by two residues compared to THGRGFI, significantly inhibited VEGF-induced proliferation of endothelial cells. However, this inhibition was significantly lower than that by vasoinhibin and THGRGFI. The other two oligopeptides (GFITKAI (SEQ ID NO: 17) and TKAINSC (SEQ ID NO: 18)) did not show any activity.

[0106] To assess the contribution of each amino acid to the inhibitory potency of the oligopeptide THGRGFI on HUVEC proliferation, an alanine scan was performed using a seven-amino acid peptide to assess the dose-response effect of various alanine substitutions on HUVEC proliferation. Figure 11A shows seven sequences of a seven-amino acid synthetic oligopeptide in which each amino acid was sequentially substituted with alanine. The seven sequences, SEQ ID NOS: 19-25, are shown below the sequence of THGRGFI. The substituted amino acids are shown in bold.

[0107] 20% FBS, 100 μg·ml heparin -1 and ECGS 25 μg·ml -1 Approximately 11,000 HUVEC cells cm were plated onto a 96-well plate in F12K medium containing 1000 mM NaCl. -2 After 24 hours, the cells were starved with 0.5% FBS for approximately 16 hours, and then FBS and heparin were added again, and EdU and VEGF (25 ng ml) were added. -1 and bFGF 20 ng·ml -1Cells were treated with various doses of alanine-substituted oligopeptides for 24 hours in the presence of a combination of VEGF and bFGF. Finally, cells were fixed, permeabilized, and stained to quantify DNA synthesis as a sign of proliferation. Figure 11B shows the biological efficacy of various oligopeptides shown in Figure 11A on the proliferation of HUVEC cells stimulated with a combination of VEGF and bFGF (Figure 11B). *** P<0.001).

[0108] The dose-response effects of most of the oligopeptides with alanine scanning mutations were similar to those of the THGRGFI oligopeptides of the present invention, except for the oligopeptide with a mutation at the histidine residue at X2 (H2A) and the oligopeptide with a mutation at the arginine residue at X4 (R4A). These oligopeptides did not inhibit endothelial proliferation. This indicated that the amino acids at the X2 and X4 positions are important amino acids mediating the inhibitory activity of the oligopeptides of the present invention.

[0109] Since the histidine in X2 (H2) and arginine X4 (R4) appear to be important for the activity of the oligopeptides of the present invention, and since the scrambled peptide Scr2 in Figures 9A and 9B had no effect despite having a histidine in X2 and an arginine in X4, the glycine at position X3 also appears to have a role in biological activity. These results suggest that the peptide THGRGFI can be further compacted. Thus, two peptides of 4 and 3 amino acids, THGR (SEQ ID NO: 4) and HG R were synthesized and tested for their biological efficacy on the proliferation of HUVECs.

[0110] Figure 12A shows the sequences of 7-, 4-, and 3-amino acid oligopeptides of the present invention. Figure 12B shows the sequences of VEGF (25 ng ml -1 ) and bFGF (20 ng·ml -1 12A compares the biological potency of the oligopeptides shown in FIG. 12A on the proliferation of human umbilical vein endothelial cells stimulated with a combination of ).

[0111] Both the tetrapeptide THGR and the tripeptide HGR behaved as vasoinhibins, with dose-response curves very similar to those of the oligopeptide THGRGFI.

[0112] [Use of the invention (industrial applicability)] The oligopeptides of the present invention, and their respective pharmaceutical compositions, that inhibit angiogenesis and vascular function can be used to prevent or treat any condition or disease associated with excessive angiogenesis and vascular permeability. These diseases include, among others, tumor growth, rheumatoid arthritis, atherosclerotic plaque formation, corneal neovascularization, proliferative retinopathies such as diabetic retinopathy and macular degeneration, defective wound repair, glaucoma, psoriasis, chronic varicose ulcers, and reproductive disorders such as follicular cysts. Similarly, the oligopeptides can be used as contraceptives.

[0113] Due to their antiangiogenic properties, the oligopeptides of the present invention can be used to regulate the angiogenic pathological growth of organs and tissues. For example, the oligopeptides of the present invention can be used to inhibit tumor angiogenesis to reduce tumor size and promote tumor regression. The oligopeptides of the present invention can also be used to prevent and avoid metastasis.

[0114] The oligopeptides of the present invention can be used as templates for peptidomimetic protocols to generate peptide or non-peptide analogs or agonists of the oligopeptide's action. Modifications that can be made include mutations or amino acid substitutions with L-amino acids or non-peptide molecules. Additionally, oligopeptides can be further modified by miniaturization techniques or by generating restricted peptides such as cyclic or retro-reverse oligopeptides.

[0115] The oligopeptides of the present invention can be used as templates in peptidomimetic technology to generate peptide or non-peptide antagonists for blocking the effects of endogenous vasoinhibin. Modifications that can be performed include mutation and substitution with L-amino acids or homologous residues with non-peptide structures, miniaturization techniques, or the generation of restricted peptides such as cyclic or retro-inverted peptides. The oligopeptide-based antagonists of the present invention can be used in the treatment of diseases involving elevated endogenous vasoinhibin levels, such as peripartum cardiomyopathy, preeclampsia, conditions associated with abnormal blood pressure, depressive disorders, anxiety disorders, or fetal growth retardation.

[0116] The oligopeptides of the present invention provide insight into the development of methods that allow for the quantification of endogenous levels of vasoinhibin in blood, other body fluids, or tissues. For example, radioimmunoassays or sandwich or multiplex ELISAs can be performed. Any technology in the art for generating diagnostic tools can be used. Currently, the limiting problem in developing this type of assay is the generation of antibodies that recognize vasoinhibin but not PRL. Using the oligopeptides of the present invention, streamlined antibodies that recognize specific domains of vasoinhibin could be generated. Any antibody that recognizes the oligopeptides of the present invention can be used in diagnostic methods and is within the scope of the present invention. Diagnostic methods for specifically detecting vasoinhibin are particularly interesting in the diagnosis of the previously mentioned reproductive disorders (preeclampsia, peripartum cardiomyopathy, fetal growth restriction), conditions associated with abnormal blood pressure, depressive disorders, anxiety disorders, or angiogenesis-dependent diseases.

[0117] The oligopeptides of the present invention can be used for the treatment of cardiovascular disease, ischemic stroke, and thrombosis. Vasoinhibin acts through binding to and antagonizing the effects of plasminogen activator inhibitor 1 (PAI-1), which is associated with thrombosis. Furthermore, angiogenesis inhibition by various agents correlates with an increased risk of thromboembolism. The dual effects of the oligopeptides of the present invention, i.e., antiangiogenic and profibrinolytic effects, help to avoid secondary thrombogenic effects.

[0118] Vasoinhibin has an anti-metastatic effect. The oligopeptides of the present invention can be used to inhibit the invasion of cancer cells in the metastatic process. Furthermore, the oligopeptides can act on cancer cells to directly inhibit their proliferation and migration. Therefore, the anti-tumor effect of the oligopeptides is dual, as it can include the inhibition of tumor angiogenesis and the direct inhibition of tumor cell proliferation and migration.

[0119] The oligopeptides of the present invention can be used as fusion proteins in combination with another protein, such as an antibody, or another anti-angiogenic protein. Furthermore, the oligopeptides can be used as a "linker" between two or more proteins, whether or not related to angiogenesis. Similarly, the sequences or elements of the oligopeptides of the present invention can be converted into non-peptide molecules by peptidomimetic strategies.

[0120] The oligopeptides of the present invention can be used to reduce the formation of tumor metastases.

[0121] The oligopeptides of the invention can be used to stimulate fibrin degradation in thrombotic diseases and hemostatic degeneration and scar formation.

[0122] The oligopeptides of the invention can also be used in veterinary medicine, for example in the treatment of angiogenesis-dependent diseases such as cancer in dogs or cats and other diseases of this type in livestock or domestic animals.

[0123] The features of the present invention can be used individually or in any combination. It should be understood that the embodiments of the present invention are merely illustrative and do not limit the scope of the present invention. [Brief explanation of the drawings]

[0124] [Figure 1A] 1A and 1B show a schematic representation of the position of the seven-amino acid oligopeptide THGRGFI of the present invention in the linear amino acid sequence of vasoinhibin (FIG. 1A) and the chemical structure of this terminally modified oligopeptide at pH 7.4 (FIG. 1B). [Figure 1B] 1A and 1B show a schematic representation of the position of the seven-amino acid oligopeptide THGRGFI of the present invention in the linear amino acid sequence of vasoinhibin (FIG. 1A) and the chemical structure of this terminally modified oligopeptide at pH 7.4 (FIG. 1B). [Figure 2A] Figures 2A, B show dose-response graphs comparing the biological potency of the 7 amino acid oligopeptide THGRGFI and the 123 amino acid vasoinhibin on growth factor stimulated proliferation of endothelial cells. [Figure 2B] Figures 2A, B show dose-response graphs comparing the biological potency of the 7 amino acid oligopeptide THGRGFI and the 123 amino acid vasoinhibin on growth factor stimulated proliferation of endothelial cells. [Figure 3A] 3A and 3B show the inhibitory effects of the 7-amino acid oligopeptide THGRGFI and vasoinhibin, which has 123 amino acids, on VEGF-stimulated endothelial cell invasion. [Figure 3B] 3A and 3B show the inhibitory effects of the 7-amino acid oligopeptide THGRGFI and vasoinhibin, which has 123 amino acids, on VEGF-stimulated endothelial cell invasion. [Figure 4]FIG. 4 shows the changes in expression of vasoinhibin target genes, interleukin-1α (IL-1α) and intracellular adhesion molecule 1 (ICAM1), in endothelial cells in response to 100 nM of the 123-amino acid vasoinhibin or the oligopeptide THGRGFI. [Figure 5A] 5A and 5B show the inhibitory effects of 100 nm of the oligopeptide THGRGFI and vasoinhibin, which has 123 amino acids, on capillary tube formation by endothelial cells cultured on a Matrigel™ layer. [Figure 5B] 5A and 5B show the inhibitory effects of 100 nm of the oligopeptide THGRGFI and vasoinhibin, which has 123 amino acids, on capillary tube formation by endothelial cells cultured on a Matrigel™ layer. [Figure 6A] 6A and 6B show the inhibition of vascular permeability by the oligopeptides THGRGFI and vasoinhibin with 123 amino acids for 120 minutes (FIG. 6A) and at 120 minutes (FIG. 6B). [Figure 6B] 6A and 6B show the inhibition of vascular permeability by the oligopeptides THGRGFI and vasoinhibin with 123 amino acids for 120 minutes (FIG. 6A) and at 120 minutes (FIG. 6B). [Figure 7] FIG. 7 shows the inhibition of vascular permeability of endothelial cell monolayers by the oligopeptide THGRGFI and vasoinhibin with 123 amino acids in the absence (control, Ctl) or presence of VEGF alone or in combination with VEGF and oligopeptide or vasoinhibin for 120 minutes. [Figure 8] FIG. 8 shows the in vivo inhibitory effect of VEGF-induced retinal vascular permeability by the oligopeptide THGRGFI and by vasoinhibin with 123 amino acids. [Figure 9A]9A and 9B show the effects of the oligopeptide THGRGFI, vasoinhibin with 123 amino acids, and three oligopeptides with scrambled amino acid sequences contained in the oligopeptide THGRGFI on growth factor-stimulated endothelial cell proliferation. Amino acids whose positions remain unchanged are shown in bold. [Figure 9B] 9A and 9B show the effects of the oligopeptide THGRGFI, vasoinhibin with 123 amino acids, and three oligopeptides with scrambled amino acid sequences contained in the oligopeptide THGRGFI on growth factor-stimulated endothelial cell proliferation. Amino acids whose positions remain unchanged are shown in bold. [Figure 10A] Figures 10A and 10B show the location of the oligopeptide THGRGFI and three 7-amino acid oligopeptides with sequences overlapping with this oligopeptide in the linear sequence of vasoinhibin (Figure 10A), as well as the effects of these oligopeptides on endothelial cell proliferation in the presence of VEGF (Figure 10B). [Figure 10B] Figures 10A and 10B show the location of the oligopeptide THGRGFI and three 7-amino acid oligopeptides with sequences overlapping with this oligopeptide in the linear sequence of vasoinhibin (Figure 10A), as well as the effects of these oligopeptides on endothelial cell proliferation in the presence of VEGF (Figure 10B). [Figure 11A] Figure 11A,B shows the sequences of seven amino acid synthetic oligopeptides (Figure 11A) in which each of the amino acids of the oligopeptide THGRGFI has been replaced with alanine (shown in bold), and the biological efficacy of these oligopeptides on growth factor-stimulated endothelial cell proliferation. [Figure 11B] Figure 11A,B shows the sequences of seven amino acid synthetic oligopeptides (Figure 11A) in which each of the amino acids of the oligopeptide THGRGFI has been replaced with alanine (shown in bold), and the biological efficacy of these oligopeptides on growth factor-stimulated endothelial cell proliferation. [Figure 12A]12A,B show the sequences of 7, 4 and 3 amino acid oligopeptides of the invention (FIG. 12A) and their biological efficacy on growth stimulated endothelial cell proliferation. [Figure 12B] 12A,B show the sequences of 7, 4 and 3 amino acid oligopeptides of the invention (FIG. 12A) and their biological efficacy on growth stimulated endothelial cell proliferation.

Claims

1. A pharmaceutical composition for use in inhibiting angiogenesis and in treating or preventing diseases associated with angiogenesis, comprising an oligopeptide consisting solely of a sequence selected from the sequences Thr His Gly Arg Gly Phe Ile (SEQ ID NO: 1) and Thr His Gly Arg (SEQ ID NO: 4).

2. The pharmaceutical composition of claim 1 for use in the treatment or prevention of cancer, vascular proliferative retinopathy, diabetic retinopathy, rheumatoid arthritis, atherosclerotic plaque formation, corneal neovascularization, macular degeneration, defect wound repair, glaucoma, psoriasis, chronic varicose ulcers or follicular cysts.

3. The pharmaceutical composition described in claim 1, wherein the oligopeptide is limited to peptides that are acetylated at the N-terminus and / or amidated at the C-terminus, or cyclized via a covalent bond between the N-terminal and C-terminal amino acids, and the modifications have been shown to retain anti-angiogenic activity.

4. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

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