Oligopeptides that inhibit angiogenesis and vascular function

Abstract

The present invention provides oligopeptides that inhibit angiogenesis and vascular function, the oligopeptides having a length of 3 to 7 amino acids and comprising or consisting of the sequence X2-X3-X4, wherein X2 is a basic or 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, wherein X1 is a polar uncharged amino acid, X2, X3, and X4 are the same as X2, X3, and X4 in X2-X3-X4; or the sequence X1-X2-X3-X4-X5-X6-X7, wherein X1, X2, X3, and X4 are the same as 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

[0001] [Field of Invention]

[0002] This invention relates to anti-angiogenic oligopeptides. The invention also relates to pharmaceutical compositions and uses of these oligopeptides. [Technical Background]

[0004] Angiogenesis is the formation of new blood vessels from a pre-existing vascular system. It occurs actively during development, determining tissue growth and differentiation. In adulthood, angiogenesis is limited to female reproductive events and tissue repair due to wounds or fractures. Furthermore, the progression of high-impact diseases such as cancer, diabetic retinopathy, and rheumatoid arthritis depends on pathological stimuli related to angiogenesis. Therefore, molecules capable of blocking angiogenesis possess enormous therapeutic potential.

[0005] Several endogenous anti-angiogenic factors have been characterized. Many of these are molecular fragments derived from the specific proteolytic breakdown of proteins that are inactive in the angiogenesis process, including extracellular matrix and basement membrane proteins, as well as growth factors, cytokines, circulating proteins, and hormones.

[0006] Angiostatin is an anti-angiogenic molecule produced when the hormone prolactin (PRL) loses its fourth α-helix after proteolytic cleavage by proteases (including cathepsin D, matrix metalloproteinases, and bone morphogenetic protein 1). The first three helices of PRL are named angiostatin because it inhibits angiogenesis and vascular function, namely vascular permeability and vasodilation. In addition, non-angiogenic effects of angiostatin have been reported, such as fibrinolytic, inflammatory, anxiolytic, and neurogenic effects. Angiostatin is also known as 16 kDa prolactin, abbreviated as PRL16K. Furthermore, angiostatin blocks different signaling pathways (Ras-Raf-MAPK, Ras-Tiam1-Rac1-Pak1, PI3K-Akt, and PLCγ-IP3-eNOS) induced by pro-angiogenic factors (VEGF, bFGF, bradykinin, and IL1β). Angiostatin blocks angiogenesis by inhibiting the proliferation, migration, and survival of endothelial cells. Furthermore, angiostatin regulates vascular homeostasis by reducing vasodilation and vascular permeability through decreased nitric oxide production in blood vessels. In animal studies, angiostatin has induced depressive and anxiety-related behaviors.

[0007] It has been shown that angiostatin helps to physiologically inhibit angiogenesis in vascular organs and tissues where angiogenesis is highly restricted, such as the retina and cartilage. Furthermore, angiostatin plays a role in the pathogenesis of angiogenesis-dependent diseases such as cancer, rheumatoid arthritis, diabetic retinopathy, perinatal cardiomyopathy, and preeclampsia.

[0008] The molecular mechanisms of angiostatin action are only partially known. Angiostatin binds to the endothelial cell membrane with high affinity, and recent reports indicate that it forms multimeric complexes with plasminogen activator inhibitor-1 (PAI-1), urokinase plasminogen activator (uPA), and urokinase receptor (uPAR) on the endothelial cell surface. Angiostatin has also been shown to induce endothelial cell apoptosis through its specific binding to integrin α5β1.

[0009] Angiostatin is not a single molecule, but rather a family of PRL fragments with varying molecular weights, determined by the cleavage sites of the proteases that produce it. These fragments include amino acids 1 through 123, 132, 139, 142, 147, 150, or 159 of mature PRL. All of these isotypes inhibit angiogenesis, but their relative biological potency is unknown. Moreno-Carranza, B. et alia, 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 in high yields, which possesses good anti-angiogenic properties.

[0010] According to US 7 300 920 B2, an anti-angiogenic peptide is known to be substantially identical to about 10 to about 150 consecutive amino acids selected from the N-terminus of human placental prolactin, human growth hormone, or growth hormone variant hGH-V, wherein the peptide (i) inhibits capillary endothelial cell proliferation and tissue formation; (ii) inhibits angiogenesis in the chicken chorioallantoic membrane; and (iii) binds to at least one specific receptor for growth hormone, placental prolactin, or growth hormone variant hGH-V that is not bound to the full length of the peptide.

[0011] Nguyen, N.-Q.-N. et alia, “Prolactin / growth hormone-derived dantiangiogenic peptides highlight a potential role of tilted peptides inangiogenesis”, Proceedings of the National Academy of Sciences. 103 (2006)14319-14324. This study demonstrates that tilted peptides exert anti-angiogenic activity. Tilted (or oblique) peptides are short peptides known to disrupt membrane and lipid core stability, characterized by an asymmetric axial distribution of hydrophobic residues when helical. All of these fragments have been shown to possess the 14-aa sequence characteristic of tilted peptides. Tilted peptides of human prolactin and human growth hormone induce endothelial cell apoptosis, inhibit endothelial cell proliferation, and suppress capillary formation in vitro and in vivo.

[0012] US 7,655,626 B2 discloses a composition comprising an isolated anti-angiogenic peptide or a fusion protein comprising a heterologous protein fused to the anti-angiogenic peptide, wherein the peptide has anti-angiogenic activity and comprises the following amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14, wherein X1 is any amino acid residue compatible with forming a helix; X2 is the following amino acid residue: Leu; X3 is the following amino acid residue: Arg, Ser; X4 is the following amino acid residue: Il e, Leu; X5 is any amino acid residue compatible with forming the helix; X6 is the following amino acid residue: Leu, Val; X7 is the following amino acid residue: Leu, Ser; X8 is any amino acid residue compatible with forming the helix; X9 is any amino acid residue compatible with forming the helix; X10 is the following amino acid residue: Gln, Glu, Arg; X11 is the following amino acid residue: Ser; X12 is the following amino acid residue: Trp; X13 is the following amino acid residue: Leu, Asn; X14 is the following amino acid residue: Glu.

[0013] According to Robles, JP et alia, Scientific Reports 8 (2018) 17111-17118, angiostatin contains a triple-helix bundle, and its anti-angiogenic domain is located within the first 79 residues. Molecular dynamics simulations (MD) indicate that the loss of the fourth α-helix (H4) exposes the hydrophobic core of PRL, leading to molecule compression into a triple-helix bundle that hides the hydrophobic core. Further speculation suggests that this compression is generated by the motion of ring 1 (L1) and its interaction with α-helix 1 (H1), resulting in a new L1 conformation with a different electrostatic and hydrophobic surface than PRL, which may correspond to the bioactive domain. Consistent with this model, a 14-amino acid (residues 45–58) peptide sequence located in the early portion of buffalo PRL L1 has been reported to exhibit anti-angiogenic activity. This sequence was revealed by its 35.7% homology with human somatostatin (a known anti-angiogenic factor). The authors discovered that a recombinant protein containing the first 79 amino acids (including H1 and L1) of human PRL inhibits endothelial cell proliferation and migration and upregulates the angiostatin target genes IL1A and ICAM1. This bioactivity is comparable to that of conventional angiostatin, which contains 123 residues including H1, L1, H2, L2, and H3 of human PRL. These findings suggest that the tilted peptide, absent in the 79-amino acid angiostatin, does not contain the most active biological determinants of angiostatin. Summary of the Invention

[0014] The problem this invention aims to solve is to provide an alternative peptide that can exert the function of angiostatin by inhibiting angiogenesis and vascular function. Another object of this invention is to provide uses for recombinant proteins, recombinant nucleic acids, pharmaceutical compositions, pharmaceutical compositions for treating or preventing diseases, and peptides.

[0015] The problems of the present invention are solved by the features of claims 1, 3, 5, 11, 12, 14, 16 and 18. Embodiments of the present invention are the subject of claims 2, 4, 6 to 10, 13, 15, 17 and 19 to 22.

[0016] According to a first alternative embodiment of the present invention, an oligopeptide that inhibits angiogenesis and vascular function is provided, the oligopeptide being 3 to 7 amino acids in length and comprising or consisting of the following sequences:

[0017] The sequence is X2-X3-X4, where

[0018] X2 is a basic amino acid or an amide amino acid.

[0019] X3 is a small amino acid, and

[0020] X4 is a charged basic amino acid at neutral pH, or

[0021] The sequence is X1-X2-X3-X4, where

[0022] Xl is a polar, uncharged amino acid, and

[0023] X2, X3, and X4 are the same as X2, X3, and X4 in X2-X3-X4, or

[0024] The sequence is X1-X2-X3-X4-X5-X6-X7, where

[0025] X1, X2, X3, and X4 are the same as X1, X2, X3, and X4 in X1-X2-X3-X4.

[0026] X5 is a small amino acid.

[0027] X6 is a hydrophobic amino acid, and

[0028] X7 is a hydrophobic amino acid.

[0029] In the embodiment of the first alternative of the present invention

[0030] Xl is Thr, Ser, Asn, Glu, Gly, or Ala, especially Thr.

[0031] X2 is His, Arg, Lys, Gln, or Asn, especially His.

[0032] X3 is either Ala or Gly, especially Gly.

[0033] X4 is either Arg or Lys, especially Arg.

[0034] X5 is Gly, Ser, or Ala, especially Gly.

[0035] X6 is Phe, Ala, Leu, Ile, Trp, or Pro, especially Phe, and

[0036] X7 is available in Phe, Ala, Leu, Ile, Trp, or Pro, especially Ile.

[0037] According to a second alternative embodiment of the invention, an oligopeptide that inhibits angiogenesis and vascular function is provided, the oligopeptide being 3 to 7 amino acids in length and comprising or consisting of the following sequences:

[0038] The sequence is X1-X2-X3, where

[0039] Xl is an acidic amino acid that carries a negative charge at neutral pH.

[0040] X2 is a polar amino acid, and

[0041] X3 is an amino acid that carries a positive charge at neutral pH, or

[0042] The sequence is X1-X2-X3-X4, where

[0043] X1, X2, and X3 are the same as X1, X2, and X3 in X1-X2-X3, and

[0044] X4 is a polar aromatic amino acid, or

[0045] The sequence is X1-X2-X3-X4-X5-X6-X7, where

[0046] X1, X2, X3, and X4 are the same as X1, X2, X3, and X4 in X1-X2-X3-X4.

[0047] X5 is a polar amino acid.

[0048] X6 is a hydrophobic amino acid, and

[0049] X7 is a hydrophobic amino acid.

[0050] In the second alternative embodiment of the present invention

[0051] Xl is either Asp or Glu, especially Glu.

[0052] X2 is Gln, Asn, Ser, or Thr, especially Gln.

[0053] X3 is either Arg or Lys, especially Lys.

[0054] X4 is Tyr.

[0055] X5 is Gln, Asn, Ser, or Thr, especially Ser.

[0056] X6 is Phe, Ala, Leu, Ile, Trp, or Pro, especially Phe, and

[0057] X7 is available in Phe, Ala, Leu, Ile, Trp, or Pro, especially Leu.

[0058] According to a third alternative of the present invention, an oligopeptide that inhibits angiogenesis and vascular function is provided, the oligopeptide having a length of 7 amino acids and a sequence X1-X2-X3-X4-X5-X6-X7, wherein

[0059] Xl is an amino acid such as Thr, Asp, or Glu.

[0060] X2 is an amino acid, His or Gln.

[0061] X3 is the amino acid Gly or Lys.

[0062] If X3 is Gly and X4 is the amino acid Arg, or if X3 is Lys and X4 is the amino acid Tyr,

[0063] X5 is the amino acid Gly or Ser.

[0064] X6 is the amino acid Phe, and

[0065] X7 is an amino acid, either Ile or Leu.

[0066]

definition

[0067] In the context of this disclosure, the terms should be understood as follows:

[0068] The terms “amino acid” and “amino acid residue” are used interchangeably and should not be construed as restrictive.

[0069] Amino acids: Protein amino acids.

[0070] Amino acid residues: Protein amino acid residues.

[0071] Amide amino acids: Amino acids with amidated side chains, such as asparagine (Asn) and glutamine (Gln).

[0072] Polar amino acids: Amino acid residues that form hydrogen bonds as donors or acceptors. Among the amino acid residues in naturally occurring proteins, there are 10 polar amino acid residues: 2 carry a negative charge at neutral pH, namely aspartic acid (Asp) and glutamic acid (Glu); 3 carry a positive charge at neutral pH, namely arginine (Arg), lysine (Lys), and histidine (His); and 5 are uncharged at neutral pH, namely glutamine (Gln), asparagine (Asn), serine (Ser), threonine (Thr), and tyrosine (Tyr).

[0073] Polar aromatic amino acids: Polar amino acid residues with an aromatic ring, such as tyrosine (Tyr).

[0074] Small amino acids: with a molecular weight of less than 100 cubic angstroms (Å) 3 Amino acid residues of a certain size, such as alanine (Ala), glycine (Gly), and serine (Ser), but not cysteine ​​(Cys), and not other amino acids that are larger than cysteine.

[0075] Hydrophobic amino acids: These are amino acid residues that are typically embedded within 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 nonpolar.

[0076] Basic amino acids: Amino acid residues in the side chain that carry a positive charge at neutral pH, which usually form salt bridges, such as arginine (Arg), lysine (Lys), and histidine (His).

[0077] Positively charged amino acids: basic amino acids.

[0078] Acidic amino acids: Amino acid residues in the side chain that carry a negative charge at neutral pH, which usually form salt bridges and include aspartic acid (Asp) and glutamic acid (Glu).

[0079] Negatively charged amino acids: acidic amino acids.

[0080] Conservative substitution: replacing one amino acid with another amino acid belonging to the same category as those listed above, namely 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.

[0081] X% similarity to an oligopeptide: The percentage of total amino acids in the oligopeptide that have been conservatively substituted. For example, 70% similarity to an oligopeptide with 10 amino acids means that 7 out of the 10 amino acids in the oligopeptide have been conservatively substituted.

[0082] Peptide: A compound consisting of two or more amino acid residues.

[0083] Oligopeptides: Peptides consisting of fewer than 20 amino acid residues.

[0084] Polypeptide: A peptide consisting of at least 20 and less than 50 amino acid residues.

[0085] Protein: A peptide composed of at least 50 amino acid residues. [Detailed Description of the Invention]

[0087] It is surprising to find potent anti-angiogenic and vascular-inhibiting activities in small oligopeptides, as is the case with this invention. The oligopeptides according to the invention consist of only 3 to 7 amino acids. The small size of the oligopeptides provided by this invention offers the advantage of ease of production, purification, processing, and formulation. Despite their small size, these oligopeptides exhibit the same, better, or at least similar biological efficacy to angiostatin in inhibiting angiogenesis and vascular function. The amino acid sequence differs from that of the "tilted" peptides known from Nguyen, N.-Q.-N et al., which possess significantly lower biological efficacy than the oligopeptides according to the invention.

[0088] Ease of production is a significant advantage when considering the difficulty of expressing peptides containing the first 123 amino acids of human prolactin in good yields to impart good anti-angiogenic properties to the peptides and the production of various other anti-angiogenic proteins derived from prolactin. The small size of the oligopeptides of this invention is a major advantage in their production, resulting in high yields, stability, and low production costs.

[0089] The oligopeptides of this invention are soluble in water or buffer solutions, such as Dulbecco's phosphate buffer (pH 7). They are soluble at concentrations up to about 15 mg / ml. This solubility is significantly superior to known hydrophobic "tilted" peptides and the entire angiostatin molecule, which exposes hydrophobic plaques on its surface, reducing its solubility and promoting precipitation.

[0090] The chemical modification of the oligopeptides of the present invention can increase their half-life and resistance to the digestive tract. Such modifications include incorporation of dextrorotatory amino acids or conversion into inverse peptides and cyclic peptides.

[0091] Because the oligopeptides of this invention retain the bioactive properties of angiostatin, they can be used to design and generate specific antibodies that distinguish between PRL and angiostatin, allowing for sensitive and specific quantification of angiostatin for use in clinical trials, diagnosis, and treatment.

[0092] 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 superior to anti-angiogenic drugs used to treat cancer that only have vascular effects.

[0093] Furthermore, the oligopeptides of the present invention can be used to treat angiogenesis-dependent diseases, whether related to or unrelated to reproduction. Compared to angiostatin, their smaller size, hydrophilicity, and potency allow for the production and formulation of effective drugs containing oligopeptides and increase drug stability.

[0094] The present invention includes the sequence of an oligopeptide according to the invention within a recombinant protein or another structure that can be used as a carrier.

[0095] The oligopeptides of the present invention include oligopeptides, particularly agonistic oligopeptides, having a sequence of an oligopeptide as defined above or a sequence having at least 70%, particularly at least 80%, particularly at least 85%, particularly at least 90% similarity to an oligopeptide as defined above, and having a modification at one end of its sequence or at both ends of its sequence, or having one, more, or all of its D-conformation amino acids (D-amino acids) substituted by one or more L-conformation amino acids (L-amino acids). The modification may be acetylation of the N-terminus of the oligopeptide and / or amidation of the C-terminus, or covalent bonding between the N-terminal and C-terminal amino acids of the oligopeptide, resulting in cyclization of the oligopeptide.

[0096] The oligopeptides of the present invention may comprise or consist of the following sequences: a sequence of loop 1 or a sequence having at least 70%, particularly at least 80%, particularly at least 85%, particularly at least 90% similarity to loop 1 where loop 1 is PRL, growth hormone, or placental prolactin. Specifically, the oligopeptides of the present invention may consist of or comprise any of the following sequences, or consist of or comprise sequences having at least 70%, particularly at least 80%, particularly at least 85%, particularly at least 90% similarity to any of the following sequences:

[0097] Serial number 1: Thr His Gly Arg Gly Phe Ile (SEQ ID NO: 1)

[0098] Serial No. 2: Glu Gln Lys Tyr Ser Phe Leu (SEQ ID NO: 2)

[0099] Serial No. 3: Asp Gln Lys Tyr Ser Phe Leu (SEQ ID NO: 3)

[0100] Serial number 4: Thr His Gly Arg (SEQ ID NO: 4)

[0101] Serial number 5: Glu Gln Lys Tyr (SEQ ID NO: 5)

[0102] Serial number 6: Asp Gln Lys Tyr (SEQ ID NO: 6)

[0103] Serial Number 7: His Gly Arg

[0104] Serial Number 8: Glu Gln Lys

[0105] Serial Number 9: Asp Gln Lys

[0106] The oligopeptides of this invention can be fused with carrier proteins. The carrier proteins can effectively improve their localization and / or half-life.

[0107] The oligopeptides of the present invention, particularly oligopeptides having 7 amino acids, may contain about 42.86% neutral residues, 28.57% basic or acidic residues, and no more than 28.57% hydrophobic residues. Furthermore, according to the experimental scale described in Wimley, WC, White, SH, Experimentally determined hydrophobicity scale for proteins at membrane interfaces, Nature Structural Biology. 3 (1996) 842, the oligopeptide may have a hydrophobicity exceeding +10 kcal / mol. Specifically, the hydrophobicity can be maintained in the range of +11.76 to +11.90 kcal / mol. Furthermore, the oligopeptides of the present invention may have a specific distribution of hydrophobic residues grouped at the C-terminus.

[0108] The seven-amino acid oligopeptide of the present invention has a basic amino acid that is charged at about pH 7.4, such as Lys or Arg at the X3 or X4 position. Furthermore, the oligopeptide may contain a basic amino acid, such as His, that is positively charged at pH ≤ 6 at the X2 position, and an acidic amino acid, such as Asp or Glu, that is negatively charged at neutral pH at the X1 position.

[0109] The present invention also relates to recombinant proteins comprising oligopeptide sequences according to the present invention.

[0110] The present invention also relates to nucleic acids, particularly recombinant nucleic acids, which consist of or contain the following sequences: sequences encoding oligopeptides according to the invention or sequences complementary to such sequences. The recombinant nucleic acids may be contained in an expression vector.

[0111] The present invention also relates to pharmaceutical compositions comprising at least one oligopeptide according to the invention and / or at least one recombinant protein according to the invention and / or at least one recombinant nucleic acid according to the invention. According to one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable carrier that is pharmaceutically acceptable for administration to mammals, particularly humans. A pharmaceutically acceptable carrier may be a physiological saline solution.

[0112] Any physiologically compatible formulation can be used to administer the oligopeptides according to the invention. For example, the formulation may be an aerosol or paste, or it may contain lipids. The concentration of the oligopeptides of the invention in the pharmaceutical composition may vary between about 0.1% w / w and 50% w / w.

[0113] The oligopeptides of the present invention can be administered in compositions with different dosage forms. For example, for oral administration, powders, tablets, pills, capsules, or sugar-coated pills can be used, as well as liquid dosage forms such as suspensions or syrups. For intraocular or parenteral administration, liquid and sterile forms can be used. The pharmaceutical compositions of the present invention may contain 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, such as for stabilizing pharmaceutical compositions. The pharmaceutical compositions may contain liposomes, including emulsions, micelles, or liquid crystals. The liposomes can be directed to a specific target using antibodies or molecules that recognize the target.

[0114] The oligopeptides of the present invention can be administered locally, regionally, locally, or systemically via injection, inhalation, suppositories, transdermal delivery, and ocular administration. Pharmaceutical compositions containing the oligopeptides of the present invention can be administered, for example, via nasal aerosols. The oligopeptides of the present invention can also be administered via catheters that allow delivery to internal or distal tissues. The pharmaceutical compositions may further include encapsulated oligopeptides for protection and / or control and prolongation of release. Such pharmaceutical compositions can be implanted near or at specific target tissues. Suitable formulations of the pharmaceutical compositions have been reported in various references, such as Shayne Cox, Pharmaceutical Manufacturing Handbook, Wiley Online Books, Canada, 2008. doi:10.1002 / 9780470259818.

[0115] According to another aspect of the invention, the pharmaceutical compositions according to the invention are used to treat or prevent angiogenesis-dependent diseases. Any oligopeptide according to the invention, any recombinant protein according to the invention, and any recombinant nucleic acid according to the invention can be used to treat or prevent angiogenesis-dependent diseases. Angiogenesis-dependent diseases can be cancer, proliferative retinopathy, diabetic retinopathy, or rheumatoid arthritis.

[0116] This invention also relates to the use of the oligopeptides or recombinant proteins according to the invention for antibody production. For this purpose, the oligopeptides may comprise or consist of the following sequences: His Gly Arg, Glu Gln Lys, or Asp Gln Lys, and the recombinant proteins may comprise one of these sequences. The antibodies can be used in in vitro diagnostic methods. These diagnostic methods may relate to the diagnosis of preeclampsia, perinatal cardiomyopathy, fetal growth restriction, conditions associated with abnormal blood pressure, depression, anxiety, or angiogenesis-dependent diseases. Abnormal blood pressure is blood pressure that is lower or higher than normal blood pressure, i.e., hypotension or hypertension. Angiogenesis-dependent diseases are those in which angiogenesis and / or vascular permeability and / or vasodilation are altered, such as rheumatoid arthritis, proliferative retinopathy, diabetic retinopathy, and cancer.

[0117] The present invention also relates to a pharmaceutical composition comprising one, two, or three oligopeptides having the above-described characteristics, either isolated or in combination. Furthermore, the present invention relates to the recombinant production of precursors of any of the above-described oligopeptides and fusion molecules comprising any of the above-described sequences.

[0118] The oligopeptides of this invention can be used as immunomodulators to generate antibodies that recognize panangiin but not PRL. These antibodies can quantify the endogenous levels of angiostatin in serum, other bodily fluids, and tissues. Quantification of endogenous levels of angiostatin is important because it has been shown that angiostatin may contribute to the progression of disease states such as preeclampsia, perinatal cardiomyopathy, and diabetic retinopathy.

[0119] This invention relates to different strategies for generating oligopeptides according to the invention. For example, oligopeptides can be generated by precursor recombination or together with a fusion protein. Furthermore, peptide synthesis is the most feasible strategy for generating oligopeptides according to the invention, and can be carried out using various known methods.

[0120] The present invention is described below through examples. In these examples, the oligopeptides of the present invention are for illustrative purposes only and should not be construed as limiting the scope of the invention. Attached Figure Description

[0121] Figure 1A B schematically shows the position of the 7-amino acid oligopeptide THGRGFI according to the invention in the linear amino acid sequence of angiostatin. Figure 1A ) and the chemical structure of the oligopeptide at pH 7.4, wherein the oligopeptide is modified at its terminal ( Figure 1B ).

[0122] Figure 2A Figure B shows a dose-response plot comparing the biological efficacy of the 7-amino acid oligopeptide THGRGFI and the 123-amino acid angiostatin on growth factor-stimulated endothelial cell proliferation.

[0123] Figure 3A B showed the inhibitory effects of the 7-amino acid oligopeptide THGRGFI and the 123-amino acid angiostatin on VEGF-stimulated endothelial cell invasion.

[0124] Figure 4 The expression changes of angiostatin target genes, interleukin-1α (IL-1α), and intracellular adhesion molecule 1 (ICAM1) mRNA in endothelial cells in response to 100 nM of angiostatin or oligopeptide THGRGFI, which has 123 amino acids.

[0125] Figure 5A B shows the effects of 100 nM oligopeptide THGRGFI and angiostatin with 123 amino acids on Matrigel. TM Inhibitory effect on capillary formation of endothelial cells cultured on the lamina.

[0126] Figure 6A B shows within 120 minutes ( Figure 6A ) and at the 120-minute mark ( Figure 6B The inhibitory effects of oligopeptide THGRGFI and angiostatin with 123 amino acids on vascular permeability.

[0127] Figure 7 The study showed that, in the absence of VEGF (control, Ctl) or in the presence of VEGF alone or in combination with oligopeptide or angiostatin, the oligopeptide THGRGFI and angiostatin with 123 amino acids inhibited vascular permeability of monolayer endothelial cells within 120 minutes.

[0128] Figure 8 The study demonstrated the in vivo inhibitory effects of the oligopeptide THGRGFI and angiostatin with 123 amino acids on VEGF-induced retinal vascular permeability.

[0129] Figure 9A Figure B shows the effects of three oligopeptides—THGRGFI, angiostatin with 123 amino acids, and an oligopeptide with a disordered sequence of amino acids contained in THGRGFI—on growth factor-stimulated endothelial cell proliferation. Amino acids whose positions are not altered are shown in bold.

[0130] Figure 10A Figure B shows the positions of the oligopeptide THGRGFI and three other oligopeptides with 7 amino acids overlapping with the linear sequence of angiostatin. Figure 10A And the effects of these oligopeptides on endothelial cell proliferation in the presence of VEGF. Figure 10B ).

[0131] Figure 11AB shows the synthetic oligopeptide sequence of 7 amino acids, in which each amino acid of the oligopeptide THGRGFI is replaced by an alanine, which is indicated in bold. Figure 11A (and the biological efficacy of these oligopeptides on growth factor-stimulated endothelial cell proliferation.)

[0132] Figure 12A B shows the oligopeptide sequences of the 7th, 4th, and 3rd amino acids of the present invention. Figure 12A ) and its biological efficacy on growth factor-stimulated endothelial cell proliferation.

[0133] Figure 1A The position of the 7-amino acid oligopeptide THGRGFI according to the present invention within the linear amino acid sequence of angiostatin is shown. Angiostatin extends from its N-terminus (H2N) to its C-terminus (COO). - A linear plot of the sequence is shown. The three main α-helices (H1, H2, and H3) and loop 1 (L1) connecting H1 and H2 are displayed. The sequence of residues 40–65 is magnified for better understanding. The positions of the 7-amino acid oligopeptide THGRGFI are shown in bold and aligned with the sequence.

[0134] Figure 1B A diagram showing the primary structure of the oligopeptide THGRGFI according to the present invention at pH 7.4 is displayed. The amino and carboxyl terms of the oligopeptide are acetylated and amidated, respectively.

[0135]

Example

[0136] Example 1: Inhibition of endothelial cell proliferation

[0137] The inhibitory effect of the 7-amino acid oligopeptide THGRGFI corresponding to SEQ ID NO: 1 on the proliferation of immortalized bovine umbilical vein endothelial cells (BUVEC E6E7) and human umbilical vein endothelial cells (HUVEC) primary cultures was evaluated and compared with the effect of conventional 123-amino acid angiostatin.

[0138] In 96-well plates that had undergone cell culture treatment, cells were cultured at approximately 14,000 and 11,000 cells / cm², respectively. 2BUVEC E6E7 and HUVEC cells were seeded at appropriate densities. BUVEC E6E7 cells were maintained in F12K medium containing 10% (v / v) fetal bovine serum (FBS), while HUVEC cells were maintained in F12K medium supplemented with 20% FBS, 100 µg / ml heparin, and 25 µg / ml endothelial growth supplement (ECGS). After 24 hours, the cells were starved for 16 hours with a reduced FBS medium (0.1% FBS for BUVEC E6E7 and 0.5% FBS for HUVEC) to synchronize them in the G0 phase of the reproductive cycle. Thereafter, only HUVEC cells were supplemented with FBS and heparin. Cells were then treated with angiostatin or oligopeptide THGRGFI at concentrations ranging from 0.001 to 100 nM for 24 hours in the presence of 10 µM thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) and 50 ng / ml VEGF (for BUVEC E6E7) or a combination of 25 ng / ml VEGF and 20 ng / ml bFGF (for HUVEC). At the end of the experiment, cells were fixed with 4% paraformaldehyde, infiltrated with 0.5% Triton X-100 in TBS1x, and stained using a "click" assay (involving a copper-catalyzed reaction that covalently binds fluorescent azide to DNA-incorporated EDU) to detect newly synthesized DNA. Total DNA was counterstained with Hoechst 33342, and the number of "click"-stained nuclei was quantified and plotted relative to the total number of nuclei stained with Hoechst 33342.

[0139] The result is Figure 2A and 2B The study describes the effects of the oligopeptide THGRGFI and 123-amino acid angiostatin on immortalized bovine umbilical vein endothelial cells (BUVEC E6E7) stimulated with 50 ng / ml VEGF. Figure 2A ) and primary cultures of human umbilical vein endothelial cells (HUVECs) stimulated with a combination of VEGF (25 ng / ml) and bFGF (20 ng / ml) Figure 2B Dose-response plot of the biological efficacy of proliferation.

[0140] Angiostatin and the oligopeptide THGRGFI inhibited the proliferation of endothelial cells (BUVEC E6E7 and HUVEC) in a dose-response manner. Both inhibitors produced approximately 1 nM (ECG) per cell. 50 The same effective dose (≈ 1 nM) is active against both types of endothelial cells. Figure 2A and 2BThis result confirms that the 7-amino acid oligopeptide THGRGFI retains the potency of angiostatin in inhibiting endothelial cell proliferation. The oligopeptide exhibits a similar and robust dose-response behavior to angiostatin.

[0141] [Example 2: Inhibition of invasive migration of endothelial cells]

[0142] Angiostatin inhibits endothelial cell migration and invasion through mechanisms including inactivation of the Ras-Tiam1-Rac1-Pak1 pathway, inactivation of urokinase-type plasminogen activator (uPA) and endothelial nitric oxide synthase (eNOS) due to increased expression of plasminogen activator inhibitor-1 (PAI-1). To test whether the oligopeptide THGRGFI of the present invention retains its inhibitory properties against endothelial cell invasion and migration, a Matrigel-based... TM The matrix and conditioned medium were used as permeable "transwell" supports for the chemical inducer migration assay.

[0143] Endothelial cells were seeded onto 100 µl of Matrigel on a permeable "transwell" support within the transwell chamber. TM On the matrix (380 ng / µl), the transwell chamber has a diameter of 0.33 cm. 2 With an area of ​​8 µm and a well diameter of 8 µm, the seeding densities for BUVEC E6E7 and HUVEC cells were 30,000 and 14,000 cells / cm², respectively. 2 In the upper (cavity) compartment, cells were maintained in starved medium (F12K) containing 0.1% or 0.5% FBS for BUVEC E6E7 and HUVEC, respectively. HUVEC cells were also maintained with 100 µg / ml heparin. In the lower outer cavity compartment, 3T3-L1 cells (obtained by culturing 3T3-L1 cells in DMEM-10% FBS for 48 hours) and 50 ng / ml VEGF filtered (0.22 µm) conditioned medium were used as chemical inducers. After 24 hours, the medium from both compartments and the cavity cells was removed. The outer cavity cells were fixed with 100% MeOH for 10 minutes, infiltrated with TBS 1x-0.5% Triton X-100, and stained with Hoechst 33342. The total number of cells in the outer cavity compartment indicated the invasive activity of the endothelial cells.

[0144] The results show Figure 3A and 3BIn the study of 100 nM oligopeptide THGRGFI on immortalized endothelial cells (BUVEC E6E7) from bovine umbilical veins stimulated with VEGF (50 ng / ml) in each case. Figure 3A ) or primary cells from the human umbilical vein (HUVEC) Figure 3B The inhibitory effect of ) on invasion was compared with the inhibitory effect of 100 nM of 123 amino acid angiostatin. P < 0.001.

[0145] Both angiostatin and oligopeptide THGRGFI significantly inhibited both types of endothelial cell invasion in Matrigel. TM And the ability to reach the outer compartment of the transwell. This result confirms that the peptide of the present invention retains the angiostatin properties in inhibiting endothelial cell migration.

[0146] [Example 3: Induction of Angiostatin Target Gene Expression]

[0147] Angiostatin induces the expression of multiple genes by activating NF-κB, thereby promoting angiogenesis and inflammation. In particular, interleukin-1α (IL-1α) and intercellular adhesion molecule-1 (ICAM1) are targets of angiostatin genes in bovine endothelial cells. To assess the ability of the oligopeptide THGRGFI to induce IL-1α and ICAM1 expression, BUVEC E6E7 cells were seeded in 12-well plates containing F12K-10% FBS, grown to 80% confluence, and starved for 24 hours in low-serum (0.1% FBS) medium. Cells were then treated with either 100 nM angiostatin or the oligopeptide THGRGFI. After 4 hours, RNA was extracted from the cells using Trizol (Invitrogen, Carlsbad, CA) and reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). RT-PCR products were quantified using a Maxima SYBR green qPCR (ThermoFisher Scientific) in a final volume of 10 µl of reaction mixture containing template and 0.25 µM of each primer. PCR amplification was performed in a CFX96 real-time PCR (BioRad) consisting of denaturation at 95 °C for 10 min, followed by 35 amplification cycles (95 °C for 10 s, 58 °C for 30 s, and 72 °C for 30 s). The primers used were the IL-1α forward primer (5'-TCAAGGAGAA TGTGGTGATG-3' = SEQ ID NO: 7) and the IL-1α reverse primer (5'-CTGGAAGCTG TAATGTGCTG-3' = SEQ ID NO: 8), and the ICAM1 forward primer (5'-CGTTAAGCTACACCCACCTT-3' = SEQ ID NO: 9) and the ICAM1 reverse primer (5'-AGGTAAGGGT CTCCATCACA-3' = SEQ ID NO: 10). Through 2 -ΔΔCT Methods: PCR data were analyzed, and the cycle threshold (CT) was normalized using the constitutive housekeeping gene cyclic protein A (PPIA). Primers used for PPIA amplification were the PPIA forward primer (5'-GGTTCCCAGT TTTTCATTTG-3' = SEQ ID NO: 11) and the PPIA reverse primer (5'-ATGGTGATCT TCTTGCTGGT-3' = SEQ ID NO: 12).

[0148] Figure 4This shows the fold change in the expression of messenger RNA (mRNA) of the angiostatin target genes interleukin-1α (IL-1α) and intracellular adhesion molecule 1 (ICAM1) in endothelial cells (BUVEC E6E7) from bovine umbilical veins in response to 100 nM of the 123 amino acid angiostatin or oligopeptide THGRGFI. (P < 0.001). Compared with the untreated control, angiostatin increased the mRNA levels of IL-1α and ICAM-1 by approximately 20-fold and approximately 12-fold, respectively, while the oligopeptide THGRGFI increased the mRNA levels of IL-1α and ICAM by approximately 30-fold and approximately 13-fold, respectively. These findings indicate that the oligopeptides of the present invention retain the angiostatin-like ability to induce IL-1α and ICAM1 expression.

[0149] [Example 4: Inhibiting the formation of capillary structures]

[0150] Capillary formation is a late step in angiogenesis, involving endothelial cell migration, interaction, and organization into tubular capillaries. Angiostatin disrupts this morphogenesis.

[0151] To investigate whether the oligopeptide THGRGFI possesses this property, primary cultures of human endothelial cells (HUVECs) derived from the umbilical vein were maintained in F12K medium supplemented with 20% FBS, 100 µg / ml heparin, and 25 µg / ml endothelial cell growth supplement (ECGS). Cells from passages 2 to 4 were washed with diluted PBS 1x, dissociated from the plates with 0.25% trypsin-EDTA for approximately 3 minutes, and then centrifuged to remove trypsin. Cells were counted using a hemocytometer at 29,000 cells / cm². 2 The density of approximately 9.7 µg / µl Matrigel prepolymerized at 37°C for 1 hour was inoculated onto 24-well plates. TM The cells were then placed in 300 µl of F12K medium supplemented with 20% FBS and heparin. They were then treated with 100 nM angiostatin or the oligopeptide THGRGFI, and after 6 hours, photomicrographs were obtained using an inverted microscope. The photomicrographs are shown below. Figure 5A As shown. Images were analyzed using the software "Angiogenesis Analyzer" [Gilles Carpentier. ImageJ contribution: Angiogenesis Analyzer. ImageJNews, October 5, 2012] and ImageJ software to quantify the major connectivity regions in each area.

[0152] The results are as follows Figure 5B As shown, P < 0.001. Capillary structure response to angiogenesis factors in culture medium and cell-Matrigel... TM The components interact spontaneously to form the angiogenic structure. The angiogenic inhibitor and oligopeptide THGRGFI disrupt capillary structure, confirming that the oligopeptide of this invention retains this angiogenic inhibitor property.

[0153] [Example 5: Inhibition of vascular permeability in vitro]

[0154] Angiostatin is known to inhibit vascular permeability by directly acting on endothelial cells through the inactivation of endothelial nitric oxide synthase (eNOS) in response to different vasoactive substances. This effect has been confirmed in vitro using confluent endothelial monolayers derived from bovine aorta and umbilical vein, rat retinal capillaries, and rat brain and retinal endothelial cells. Permeability was tested by measuring the transport of a large protein (radish peroxidase) across the endothelial monolayer or by changes in transendothelial resistance (TEER) in the presence of different vascular permeability-inducing factors. Angiostatin blocks eNOS activation via a signaling pathway involving stimulation of protein 2A phosphatase, which dephosphorylates / inactivates eNOS by blocking the PLC and IP3 systems and transient receptor potential (TRP) channels that reduce intracellular calcium levels required for eNOS activation by calmodulin binding.

[0155] To assess whether the oligopeptide THGRGFI retains the inhibitory properties of angiostatin on vascular permeability, the transport of Evans blue-linked albumin across the endothelial cell (BUVEC E6E7) monolayer was evaluated as follows:

[0156] BUVEC E6E7 cells were injected at a rate of 10,000 cells / cm³. 2 The sample was seeded at a density on a transwell filter (0.4 µm pores). After 3 days, the monolayer was starved for 48 hours with low serum (0.1 FBS). Subsequently, 100 nM of the angiostatin or oligopeptide of the present invention was added to the upper compartment (luminal portion) of the transwell support, incubated for 1 hour, and then 50 ng / ml of VEGF was added. The control (Ctl) contained no VEGF, angiostatin, or oligopeptide. After 10 minutes, the upper (luminal) medium was replaced with 300 µl of PBS containing Evans blue-linked albumin, and the lower luminal compartment medium was replaced with 700 µl of PBS. At 10, 20, 30, and 60 minutes, 50 µl of sample from the extraluminal compartment was collected and replaced with fresh PBS. Absorbance (620 nm) was measured at all time points using an iMARK (BioRad) plate reader. The absorbance values ​​confirmed that the Evans blue-labeled albumin penetrated the endothelial monolayer.

[0157] Figure 6AThe invention demonstrates the 7-amino acid oligopeptide and 123-amino acid angiostatin throughout time ( Figure 6A ) and in 120 minutes ( Figure 6B Inhibition of vascular permeability ( P < 0.001. As expected, VEGF stimulated the permeability of the endothelial monolayer, and this effect increased over time. Figure 6A Equal concentrations of angiostatin and the oligopeptide THGRGFI inhibited VEGF-induced endothelial permeability increase, indicating that the peptides of the present invention retain the ability of angiostatin to inhibit vascular permeability.

[0158] Another routine protocol for assessing vascular permeability is the measurement of transendothelial resistance (TEER), which uses a device that applies an electric current through electrodes across an endothelial cell monolayer. A decrease in resistance indicates a loss of barrier function, leading to increased permeability. The effect of the oligopeptide THGRGFI on endothelial cell permeability was assessed by measuring TEER. BUVEC E6E7 was used at 10,000 cells / cm². 2 Cells were seeded at a density in a TEER device. After three days, the monolayers were starved in low-serum medium (0.1% FBS) for 48 hours, after which 100 nM angiostatin or oligopeptide was added to the upper compartment (the lumen surface of the monolayer) for 1 hour. At time 0, TEER was recorded, and 50 ng / ml of VEGF was added to the lumen side. TEER was measured at 10, 20, 30, 60, 90, and 120 minutes. Measurements were performed using an Epithelial Volt / Ohm (TEER) EVOM2 device (World Precision Instruments, FL, USA) with 4 mm “chopstick” electrodes. Values ​​were normalized relative to the device with no cells and untreated monolayers.

[0159] Figure 7 The results are shown. Vascular permeability was consistently inhibited by the 7-amino acid oligopeptide and the 123-amino acid angiostatin. Permeability was measured in the absence (control, Ctl) or in the presence of VEGF alone or in combination with the oligopeptide of the present invention or with angiostatin. The results showed the expected reduction in transendothelial resistance by VEGF, and both the oligopeptide THGRGFI and angiostatin blocked the action of VEGF in a similar manner.

[0160] [Example 6: Inhibition of vascular permeability in vivo]

[0161] Diabetic retinopathy and diabetic macular edema are leading causes of vision loss in diabetes, and their early sign is deterioration of retinal vascular permeability. VEGF is a major contributing factor to these vascular changes; therefore, current treatments are based on intravitreal injection of anti-VEGF antibodies to neutralize VEGF. Angiostatin inhibits the increase in retinal vascular permeability in response to intravitreal administration of VEGF, as well as the excessive vascular permeability induced by diabetes in experimental models. Recombinant angiostatin protein and angiostatin gene transduction via recombinant viral vectors have been used in these studies.

[0162] To evaluate whether the oligopeptide THGRGFI retains the inhibitory properties of angiostatin on VEGF-induced vascular permeability in vivo, the effects of intravitreal injection of VEGF alone or in combination with oligopeptide or angiostatin in rats were measured.

[0163] Wistar rats were intravitreally injected with saline (control) or 300 ng VEGF, either alone or in combination with 20 µM of the oligopeptide of the present invention or 20 µM of angiostatin. Twenty-four hours later, albumin infiltration into the retina was assessed using the Evans blue assay. Briefly, anesthetized rats were intravenously injected with 45 mg / kg of Evans blue dye and allowed to circulate for 2 hours. Then, the animals were perfused with approximately 80 ml of PBS at a flow rate of approximately 40 ml / min. The retina was dissected, dried, and incubated with 200 µl of formamide (Mallinckrodt Baker, Phillipsburg, NJ) at 72°C. Labeled albumin in the retinal extract was measured 18 hours later.

[0164] The results are as follows Figure 8 As shown ( P < 0.05 (P < 0.02). The results showed that intravitreal administration of the oligopeptides of the present invention blocked VEGF-induced retinal vascular permeability in a manner similar to angiostatin. Given the effectiveness of treatment with VEGF-blocking antibodies as a routine treatment for diabetic retinopathy, diabetic macular edema, and other proliferative retinopathy (early-onset retinopathy and age-related macular degeneration), it is clear that the oligopeptides of the present invention have potential therapeutic value in these diseases.

[0165] [Example 7: Structural Characterization of Oligopeptide THGRGFI]

[0166] To determine whether the anti-angiogenic activity of the oligopeptides of the present invention is specific to their sequence rather than due to amino acid composition, three disordered sequences were generated from the amino acids contained in the oligopeptides, and their effects on HUVEC cell proliferation at a concentration of 100 nM were tested. These three sequences are GIGHFRT (SEQ ID NO: 13), THIRGGF (SEQ ID NO: 14), and GTRIHFG (SEQ ID NO: 15). Figure 9A As shown in and Figure 9A and 9B The amino acids are designated as Scr1, Scr2, and Scr3. Amino acids whose positions remain unchanged are indicated in bold.

[0167] HUVECs were fed at a density of approximately 11,000 cells / cm². 2 Cells were seeded at a density in 96-well plates and maintained in F12K supplemented with 20% FBS, 100 µg / ml heparin, and 25 µg / ml endothelial growth supplement (ECGS). After 24 hours, cells were synchronized in G0 under starvation conditions (0.5% FBS) for 16 hours, followed by supplementation with FBS and heparin. Cells were then treated for 24 hours with THGRGFI, various oligopeptides, and 123 amino acid angiostatin (100 nM each) in the presence or absence of 10 µM thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) and a combination of 25 ng / ml VEGF and 20 ng / ml bFGF. Finally, cells were fixed with 4% paraformaldehyde, infiltrated in TBS1x with 0.5% Triton X-100, and newly synthesized DNA was stained using a "click" method to detect EdU incorporation. Total DNA was stained with Hoechst 33342, and the percentage of "click" stained cell nuclei relative to total cell nuclei (Hoechst 33342 stained) indicated cell proliferation.

[0168] Figure 9B The effects of three out-of-order oligopeptides at the same concentration (100 nM), the seven-residue peptide THGRGFI of this invention, and the 123-amino acid angiostatin on the proliferation of HUVECs stimulated with VEGF (25 ng / ml) and bFGF (20 ng / ml) were shown. P < 0.001.

[0169] Since the disordered oligopeptide has the same amino acid composition as the oligopeptide of the present invention, the disorder-free oligopeptide inhibits the proliferation of endothelial cells. This indicates that the amino acid sequence is important for the activity of the oligopeptide of the present invention.

[0170] To understand whether the sequence of the peptides of the present invention determines the effect of angiostatin on endothelial cell proliferation, or whether such effect still exists in adjacent sequences in the angiostatin sequence, the inhibitory effect of a 7-amino acid oligopeptide that has moved 2 or 3 residues in the angiostatin sequence was evaluated. Figure 10A The position of the 7-residue oligopeptide THGRGFI of the present invention within the linear sequence of angiostatin is shown. Amino acids and their numbers are indicated in the sequence. Three oligopeptides, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, having 7 amino acids overlapping with the peptide of the present invention, are also shown. Figure 10A middle.

[0171] For this assay, BUVEC E6E7 cells were cultured at approximately 14,000 cells / cm³. 2 Cells were seeded at a density of 10% (v / v) FBS in F12K in 96-well plates. After 24 hours, cells were starved with 0.1% FBS for approximately 16 hours for G0 synchronization. Next, cells were treated with different oligopeptides in the presence of 10 µM thymidine analog EdU and 50 ng / ml VEGF. Finally, cells were fixed, permeated, and stained with newly synthesized DNA via "click" analysis, using EdU incorporation. Total DNA was stained with Hoechst 33342; the percentage of nuclei stained by "click" analysis is a measure of proliferation.

[0172] Figure 10B The effects of the same concentration (100 nM) of the translocated 7-residue oligopeptide, the peptide of the present invention, and the 123-residue angiostatin on the proliferation of bovine umbilical vein immortalized endothelial cells (BUVEC E6E7) in the presence of VEGF (50 ng / mL) alone and in the absence of VEGF and oligopeptide (control, Ctl) were shown. P < 0.001.

[0173] The oligopeptide GRGFITK (SEQ ID NO: 16), which is two residues transposed relative to THGRGFI, significantly inhibited VEGF-induced endothelial cell proliferation. However, the inhibition was significantly less than that of angiostatin and THGRGFI. The other two oligopeptides (GFITKAI (SEQ ID NO: 17) and TKAINSC (SEQ ID NO: 18)) did not show any activity.

[0174] To evaluate the contribution of each amino acid to the inhibitory efficacy of the oligopeptide THGRGFI on HUVEC proliferation, alanine was replaced one by one with a peptide containing seven amino acids, and the dose-response effect of various alanine substitutions on HUVEC proliferation was evaluated. Figure 11AThe seven sequences of a synthetic oligopeptide consisting of seven amino acids are shown, wherein each amino acid is successively substituted with alanine. The seven sequences of SEQ ID NO: 19 to SEQ ID NO: 25 are shown below the THGRGFI sequence. The substituted amino acids are indicated in bold.

[0175] Approximately 11,000 HUVEC cells / cm 2 Cells were seeded in F12K medium containing 20% ​​FBS, 100 µg / ml heparin, and 25 µg / ml ECGS in 96-well plates. After 24 hours, the cells were starved with 0.5% FBS for approximately 16 hours, then FBS and heparin were added again, and the cells were treated with different doses of alanine-substituted oligopeptides for 24 hours in the presence of EdU and a combination of 25 ng / ml VEGF and 20 ng / ml bFGF. Finally, the cells were fixed, infiltrated, and stained to quantify DNA synthesis as a marker of proliferation. Figure 11B Showing Figure 11A The biological efficacy of different oligopeptides shown in the study on the proliferation of HUVEC cells stimulated by a combination of VEGF and bFGF. P < 0.001.

[0176] Except for oligopeptides with mutations at the histidine residue (H2A) at X2 and the arginine residue (R4A) at X4, most oligopeptides with alanine substitution mutations exhibit dose-response effects similar to the oligopeptide THGRGFI of this invention. These oligopeptides do not inhibit endothelial proliferation, indicating that the amino acids at X2 and X4 are important amino acids mediating the inhibitory activity of the oligopeptides of this invention.

[0177] Since histidine (H2) of X2 and arginine (R4) of X4 appear to be important for the activity of the oligopeptides of the present invention, and since Figure 9A and 9B The disordered peptide Scr2 had no effect, and although it had histidine at X2 and arginine at X4, glycine at X3 appeared to also play a role in biological activity. These results suggest that the peptide THGRGFI can be further miniaturized. Therefore, two peptides, THGR (SEQ ID NO: 4) and HGR, with 4 and 3 amino acids respectively, were synthesized and their biological efficacy against HUVEC proliferation was tested.

[0178] Figure 12A The oligopeptide sequences of the 7th, 4th, and 3rd amino acids of the present invention are shown. Figure 12B Comparison Figure 12A The shown oligopeptides demonstrate biological efficacy against the proliferation of human umbilical vein endothelial cells stimulated with a combination of VEGF (25 ng / ml) and bFGF (20 ng / ml).

[0179] Both the tetrapeptide THGR and the tripeptide HGR exhibited angiostatin-like behavior in the dose-response curves, which were very similar to those of the oligopeptide THGRGFI.

[0180] [Applications of this invention (industrial applicability)]

[0181] The oligopeptides of the present invention and corresponding pharmaceutical compositions thereof 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 conditions include tumor growth, rheumatoid arthritis, atherosclerotic plaque formation, corneal neovascularization, proliferative retinopathy such as diabetic retinopathy and macular degeneration, wound healing defects, glaucoma, psoriasis, chronic varicose ulcers, follicular cysts, and reproductive disorders. Similarly, the oligopeptides can be used as contraceptives.

[0182] Due to their anti-angiogenic properties, the oligopeptides of the present invention can be used to regulate the vascular-dependent pathological growth of organs and tissues. For example, the oligopeptides of the present invention can be used to inhibit tumor vascularization to reduce their size and promote their regression. Furthermore, the oligopeptides of the present invention can be used to prevent and avoid metastasis.

[0183] The oligopeptides of this invention can be used as templates for peptide analogue schemes to generate peptides or non-peptide analogs or agonists with oligopeptide effects. Possible modifications involve mutations or amino acid substitutions with L-amino acids or non-peptide molecules. Furthermore, the oligopeptides can be further modified by miniaturization techniques or by generating constrained peptides (such as cyclic or inverse oligopeptides).

[0184] The oligopeptides of this invention can be used as templates in peptide mimicry techniques to generate peptide or non-peptide antagonists for blocking the action of endogenous angiostatin. Possible modifications involve mutations and substitutions of homologous residues, L-amino acids, or non-peptide structures, miniaturization techniques, or the generation of constrained peptides (such as cyclic or inverse peptides). Antagonists based on the oligopeptides of this invention can be used to treat diseases involving elevated levels of endogenous angiostatin, such as perinatal cardiomyopathy, preeclampsia, conditions related to abnormal blood pressure, depression, anxiety, or fetal growth retardation.

[0185] The oligopeptides of this invention provide information for generating methods that allow for the quantification of endogenous levels of angiostatin in blood, other bodily fluids, or tissues. For example, radioimmunoassays or sandwich or multi-weight ELISA can be performed. Any technique in the art for generating diagnostic tools can be used. Today, a limiting problem in developing such a detection is generating antibodies that recognize angiostatin but not PRL. Using the oligopeptides of this invention, rationalized antibodies that recognize specific domains of angiostatin can be generated. Any antibody generated that recognizes the oligopeptides of this invention can be used in diagnostic methods and is within the scope of this invention. Diagnostic methods that specifically detect angiostatin are particularly meaningful in diagnosing reproductive disorders (preeclampsia, perinatal cardiomyopathy, fetal growth restriction) mentioned above in the diagnosis of conditions associated with abnormal blood pressure, depression, anxiety, or angiogenesis-dependent diseases.

[0186] The oligopeptides of this invention can be used to treat cardiovascular diseases, ischemic stroke, and thrombosis. Angiostatin exerts its effects by binding to plasminogen activator inhibitor 1 (PAI-1) and antagonizing its thrombosis-related effects. Furthermore, the inhibition of angiogenesis by various drugs is associated with an increased risk of thromboembolism. The dual action of the oligopeptides according to this invention, namely anti-angiogenic and fibrinolytic effects, helps to prevent secondary thrombosis.

[0187] Angiostatin has anti-metastatic effects. The oligopeptides of the present invention can be used to block the invasion of cancer cells during metastasis. Furthermore, the oligopeptides can act on cancer cells, directly blocking their proliferation and migration. Therefore, the antitumor effect of the oligopeptides is dual, as it involves both blocking tumor angiogenesis and directly inhibiting tumor cell proliferation and migration.

[0188] The oligopeptides of the present invention can be combined with another protein, such as an antibody or another anti-angiogenic protein, to serve as fusion proteins. Furthermore, they can act as "connectors" between two or more proteins that are related to or unrelated to angiogenesis. Similarly, the sequences or elements of the oligopeptides of the present invention can be converted into non-peptide molecules using a peptide-mimicking strategy.

[0189] The oligopeptides of this invention can be used to reduce the establishment of tumor metastases.

[0190] The oligopeptides of the present invention can be used to stimulate fibrinolysis in thrombotic diseases, hemostatic changes, and scar formation.

[0191] The oligopeptides of the present invention can also be used in veterinary medicine, for example, to treat angiogenesis-dependent diseases, such as canine or feline cancers and other such diseases in farms or livestock.

[0192] The features of this invention can be used alone or in any combination. It should be understood that the embodiments of this invention are merely illustrative and do not constitute a limitation on the scope of this invention. sequence list <110> National Autonomous University of Mexico <120> Oligopeptides that inhibit angiogenesis and vascular function <130> 603958EH <150> PCT / MX / A / 2019 / 013819 <151> 2019-11-20 <160> 25 <170> BiSSAP 1.3.6 <210> 1 <211> 7 <212> PRT <213> Homo sapiens <220> <223> The amino acid composition of human prolactin is 45-51. <400> 1 Thr His Gly Arg Gly Phe Ile 1 5 <210> 2 <211> 7 <212> PRT <213> Homo sapiens <220> <223> The amino acid composition of human growth hormone is 39-45. <400> 2 Glu Gln Lys Tyr Ser Phe Leu 1 5 <210> 3 <211> 7 <212> PRT <213> Homo sapiens <220> <223> The amino acid composition of human placental prolactin is 39-45. <400> 3 Asp Gln Lys Tyr Ser Phe Leu 1 5 <210> 4 <211> 4 <212> PRT <213> Homo sapiens <220> <223> Human prolactin contains 45-48 amino acids. <400> 4 Thr His Gly Arg 1 <210> 5 <211> 4 <212> PRT <213> Homo sapiens <220> <223> Human growth hormone contains 39-42 amino acids. <400> 5 Glu Gln Lys Tyr 1 <210> 6 <211> 4 <212> PRT <213> Homo sapiens <220> <223> The amino acid composition of human placental prolactin is 39-42. <400> 6 Asp Gln Lys Tyr 1 <210> 7 <211> 20 <212> DNA <213> Bovidae (family Bovidae) <220> <223> PCR primers <400> 7 tcaaggagaa tgtggtgatg 20 <210> 8 <211> 20 <212> DNA <213> Bovidae (family Bovidae) <220> <223> PCR primers <400> 8 ctggaagctg taatgtgctg 20 <210> 9 <211> 20 <212> DNA <213> Bovidae (family Bovidae) <220> <223> PCR primers <400> 9 cgttaagcta cacccacctt 20 <210> 10 <211> 20 <212> DNA <213> Bovidae (family Bovidae) <220> <223> PCR primers <400> 10 aggtaagggt ctccatcaca 20 <210> 11 <211> 20 <212> DNA <213> Bovidae (family Bovidae) <220> <223> PCR primers <400> 11 ggttcccagt ttttcatttg 20 <210> 12 <211> 20 <212> DNA <213> Bovidae (family Bovidae) <220> <223> PCR primers <400> 12 atggtgatct tcttgctggt 20 <210> 13 <211> 7 <212> PRT <213> Artificial sequence <220> <223> The disordered sequence of SEQ ID NO 1 <400> 13 Gly Ile Gly His Phe Arg Thr 1 5 <210> 14 <211> 7 <212> PRT <213> Artificial sequence <220> <223> The disordered sequence of SEQ ID NO 1 <400> 14 Thr His Ile Arg Gly Gly Phe 1 5 <210> 15 <211> 7 <212> PRT <213> Artificial sequence <220> <223> The disordered sequence of SEQ ID NO 1 <400> 15 Gly Thr Arg Ile His Phe Gly 1 5 <210> 16 <211> 7 <212> PRT <213> Homo sapiens <220> <223> Human prolactin contains 47-53 amino acids. <400> 16 Gly Arg Gly Phe Ile Thr Lys 1 5 <210> 17 <211> 7 <212> PRT <213> Homo sapiens <220> <223> The amino acid composition of human prolactin is 49-55. <400> 17 Gly Phe Ile Thr Lys Ala Ile 1 5 <210> 18 <211> 7 <212> PRT <213> Homo sapiens <220> <223> The amino acid composition of human prolactin is 52-58. <400> 18 Thr Lys Ala Ile Asn Ser Cys 1 5 <210> 19 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Position 1 of SEQ ID NO 1 is replaced by alanine. <400> 19 Ala His Gly Arg Gly Phe Ile 1 5 <210> 20 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Position 2 of SEQ ID NO 1 is replaced by alanine. <400> 20 Thr Ala Gly Arg Gly Phe Ile 1 5 <210> twenty one <211> 7 <212> PRT <213> Artificial sequence <220> <223> Position 3 of SEQ ID NO 1 is replaced by alanine. <400> twenty one Thr His Ala Arg Gly Phe Ile 1 5 <210> twenty two <211> 7 <212> PRT <213> Artificial sequence <220> <223> Position 4 of SEQ ID NO 1 is replaced by alanine. <400> twenty two Thr His Gly Ala Gly Phe Ile 1 5 <210> twenty three <211> 7 <212> PRT <213> Artificial sequence <220> <223> Position 5 of SEQ ID NO 1 is replaced by alanine. <400> twenty three Thr His Gly Arg Ala Phe Ile 1 5 <210> twenty four <211> 7 <212> PRT <213> Artificial sequence <220> <223> Position 6 of SEQ ID NO 1 is replaced by alanine. <400> twenty four Thr His Gly Arg Gly Ala Ile 1 5 <210> 25 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Position 7 of SEQ ID NO 1 is replaced by alanine. <400> 25 Thr His Gly Arg Gly Phe Ala 1 5

Claims

1. Oligopeptide THGRGFI (SEQ ID NO: 1).

2. The oligopeptide according to claim 1, wherein the N-terminus is acetylated and / or the C-terminus is amidated.

3. The oligopeptide according to claim 1 or 2, wherein it is cyclized by a covalent bond between N-terminal and C-terminal residues.

4. The oligopeptide according to claim 1 or 2, wherein one or more amino acid residues are in the D-configuration.

5. The oligopeptide according to claim 1 or 2, wherein it is fused with a carrier protein.

6. A pharmaceutical composition comprising an oligopeptide according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

7. Use of the oligopeptide THGRGFI (SEQ ID NO: 1) in the preparation of a medicament for reducing retinal vascular permeability associated with diabetic retinopathy.

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