Peptides and medical uses thereof

Abstract

To provide strongly demanded synthetic compounds capable of binding to VEGFR-1 and inhibiting the interaction between VEGFR-1 and VEGF-A, PlGF, VEGF-B ligand, and VEGF-A / PlGF heterodimer.SOLUTION: The inventors have found that by introducing a chemical group, in particular, an amino acid characterized by having a side chain having steric hindrance comparable to that of a thiol or thioether group, at the C terminus of a certain peptide, the molecular activity is significantly improved and the modified peptide can inhibit the interaction between PIGF or VEGF and VEGFR1 at a concentration lower than that of the existing peptide. The peptide is unexpectedly effective for treating, preferably by oral administration, pathologies correlated with or in any case caused by VEGFR1 dependent angiogenesis.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to peptides, compositions comprising said peptides, and their use as inhibitors of angiogenesis and / or neovascularization.Furthermore, the present invention relates to the use of said peptides and said compositions for the treatment of pathologies correlated with inappropriate angiogenesis and / or neovascularization.In particular, herein, reference is made to angiogenesis and / or neovascularization correlated with VEGFR1. [Background technology]

[0002] Considering the severity and range of disease conditions for which inhibition of VEGFR-1 activation may be applicable, there is believed to be a strong demand for synthetic compounds capable of binding to VEGFR-1 and disrupting the interaction between VEGF-A, PlGF, VEGF-B ligands and VEGF-A / PlGF heterodimers with VEGFR-1. Indeed, advantageously, synthetic compounds are essentially free of contaminants of biological origin and can also be produced at a significantly lower cost than biological therapeutics of recombinant origin.

[0003] To neutralize the ligand, many therapeutic approaches use monoclonal antibodies, which are molecules characterized by high specificity and affinity. However, synthetic molecules also have the advantage of being easier and cheaper to produce, more stable and easier to deliver.

[0004] In this regard, Ponticelli et al. recently described in 2008 a tetrameric tripeptide selected from a peptide library in which a peptide chain having the formula (R-Glu)-(S-Cys(Bzl))-(S-Cha) is tetramerized on a "core" of three lysines (Tam, JP 1988. Proc. Natl. Acad. Sci. USA 85:5409-5413).

[0005] The structure of this tetrameric peptide is as follows: [ka]

[0006] Scientific evidence reported by Ponticelli et al. indicates that the tetrameric peptide is capable of binding to VEGFR1 and inhibiting the interaction of PlGF, VEGF-A and VEGF-B in vitro with an IC50 of approximately 10 μM. Furthermore, the peptide is unable to bind to VEGFR-2 and does not interfere with its activation by VEGF-A.

[0007] Finally, the peptide 1) exhibits antiangiogenic activity in vitro and blocks the proangiogenic activity of PlGF and VEGF-A; 2) it can replace VEGF-A-sFlt1 binding in the cornea (which is not vascularized under physiological conditions), thus freeing VEGF-A and promoting angiogenesis; 3) when administered intraperitoneally, it reduces tumor growth, angiogenesis, arteriogenesis and metastasis; and 4) When administered intravitreally, it reduces choroidal neovascularization (Cicatiello et al. 2015).

[0008] The anti-angiogenic activity of this peptide is due to both the inhibition of new blood vessel formation and its ability to inhibit the recruitment of inflammatory cells, preferably monocytes-macrophages, at the site of angiogenesis.

[0009] The anti-arteriogenic activity is based on the ability to inhibit the recruitment of smooth muscle cells at sites of angiogenesis.

[0010] Objective of the invention In the present invention, the authors of the present invention have surprisingly found that by inserting at the C-terminus of the peptide an amino acid featuring a side chain with steric hindrance comparable to that of a chemical group, in particular a thiol or thioether group, the activity of the molecule is significantly improved.

[0011] In fact, the above modifications do not impair the selective binding to VEGFR1 and the ability to compete with VEGF-A and / or PlGF in binding to VEGFR1 in a dose-dependent manner.On the contrary, these modifications can inhibit the interaction between PlGF or VEGF and VEGFR1 by 50% (IC50) at a concentration of less than 1000nM.This is a completely unexpected result, considering that the affinity of the peptide reported in Ponticelli et al. to VEGFR1 is 10000nM or more (expressed as IC50).In other words, the peptide of the present invention has an inhibitory ability that is about one order of magnitude greater than that of the peptide reported in Ponticelli et al.

[0012] Moreover, the present authors have surprisingly found that both the peptides described in Ponticelli et al. and the peptides of the present invention show significant ability to inhibit choroidal neovascularization when administered orally or by gavage.Therefore, these molecules are therapeutically effective for treating pathologies that are correlated with or in any case caused by alterations in angiogenesis, preferably VEGFR1-dependent angiogenesis, preferably by oral administration.

[0013] A detailed description of the invention follows, along with non-limiting illustrative examples, with reference to the following figures and definitions. [Brief description of the drawings]

[0014] [Figure 1] Figure 1 shows the inhibitory activity of iVR1 and iVR1-Cys with anti-PlGF monoclonal antibody on PlGF-induced phosphorylation of VEGFR-1. Analysis of VEGFR-1 phosphorylation induced by 20ng / ml PlGF was performed on 293-VEGFR-1 cells by Western blotting. iVR1-Cys and iVR1 were added simultaneously to PlGF at a concentration of 5μM. Human anti-PlGF neutralizing monoclonal antibody was used as an inhibitory control at a concentration of 3.3nM. PBS was used as a negative control. [Diagram 2]It is shown that intravitreally administered iVRI-Cys inhibits laser-induced choroidal neovascularization in a dose-dependent manner. Intravitreous injection of 10 or 50 μg of iVRI-Cys results in a dose-dependent reduction of choroidal neovascularization equal to 48.9% and 75.9% compared to injection of the vehicle (DMSO). The same amount of iVR1 results in CNV inhibition equal to 37.8% and 73.9%. The control peptide (PC) does not show any inhibitory capacity. Quantification of the amount of neovascularization was performed on n=12 and 15 spots for 10 μg and 50 μg of iVR1, n=10 and 8 spots for 10 μg and 50 μg of iVR1-Cys, n=15 spots for PC, and n=14 spots for DMSO. Data are expressed as mean ± SEM compared to control. #p<0.05, *p>0.0002, ¶p<0.02, §p>0.002 vs. PC and DMSO. At the bottom are images depicting CNV. Bars represent 100 μm. [Diagram 3] Figure 1 shows that orally administered iVRI-Cys inhibits laser-induced choroidal neovascularization. Oral administration of iVR1-Cys at 50 mg / Kg twice daily for 7 days reduces choroidal neovascularization by 45.9% compared to vehicle. The same dose of iVR1 results in similar inhibition of CNV (49.7%). Quantification of the amount of neovascularization was performed on n=18 spots for iVRI-Cys, n=20 spots for iVRI, and n=10 spots for vehicle. Data are presented as mean ± SEM compared to control. *p=0.001 and §p=0.007 vs. DMSO. At the bottom are images depicting CNV. Bars represent 100 μm.

[0015] definition As used herein, the term "VEGF" refers to vascular endothelial growth factor. In humans, there are five different vascular endothelial growth factors, VEGF-A, VEGF-B, VEGF-C, VEGF-D and PlGF, encoded by five different genes. All are glycosylated dimeric proteins.

[0016] As used herein, the term "VEGF-A" refers to vascular endothelial growth factor-A, also previously known as VPF (vascular permeability factor). VEGF-A is the most potent factor of the VEGF family and plays a crucial role in both physiological and pathological angiogenesis. At least six different isoforms obtained by alternative splicing have been reported in humans. All are capable of interacting with two receptors, termed VEGFR-1 and VEGFR-2.

[0017] As used herein, the term "PlGF" refers to placental growth factor, whose role is restricted to conditions of angiogenesis associated with pathological conditions. Four different isoforms have been described in humans. All are capable of specifically binding to VEGFR-1. VEGF-A and PlGF exhibit potent synergy in pathological conditions, since both interact with VEGFR-1, and when the two respective genes are expressed in the same cell, they can give rise to VEGF-A / PlGF heterodimers that can interact with VEGFR-1 or induce VEGFR-1 / VEGFR-2 heterodimerization.

[0018] As used herein, the term "VEGFR-1" refers to VEGF receptor 1, also known as Flt-1. VEGFR-1 has an intracellular tyrosine kinase domain, while the extracellular portion consists of seven IgG-like domains. VEGF-A, VEGF-B or PlGF lead to dimerization of the receptor, resulting in activation by autophosphorylation of the tyrosine kinase domain. In addition to being expressed in endothelial cells, VEGFR-1 is expressed in many other types of cells, including smooth muscle cells, monocyte-macrophages, fibroblasts and endothelial progenitor cells. It plays a fundamental role in recruiting various types of cells that contribute to angiogenesis. As used herein, the term "soluble VEGFR-1" (sVEGFR-1) refers to the soluble form of VEGF receptor 1, also known as sFlt-1. It consists of the first six IgG-like extracellular domains and the tail of VEGFR-1, and is generated from the VEGFR-1 gene by alternative splicing. It is usually expressed in the same cells as the full-length form of VEGFR-1, but the soluble form is preferentially expressed in the cornea to maintain the cornea in an avascular state. The messenger sequences of full-length and soluble human VEGFR1 are preferably SEQ ID NOs: 1 and 2, respectively, while the protein sequence of full-length human VEGFR1 is SEQ ID NOs: 3 and 4, respectively. Sequences characterized by an identity in the range of 80-99.9% with the sequences described herein must be considered as part of the present specification.

[0019] As used herein, the term "VEGFR-2" refers to VEGF receptor 2, also known as KDR in humans and Flk-1 in mice. VEGFR-2 is specifically bound by VEGF-A and has an intradomain organization and activation mechanism similar to that described for VEGFR-1. Unlike receptor 1, it is constitutively expressed in endothelial cells. It has a fundamental role in stimulating endothelial cell proliferation, migration and differentiation.

[0020] As used herein, the term "angiogenesis" refers to the process of new blood vessel formation from pre-existing blood vessels. As used herein, angiogenesis refers to the process of new blood vessel formation that is associated with various types of pathological conditions, preferably selected from the following: - a neovascular eye disease preferably selected from macular edema, wet forms of age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, central retinal vein occlusion, vitreous hemorrhage and retinal detachment, and combinations thereof, and / or solid tumors and / or tumor metastases, preferably selected from leukemias and lymphomas, preferably acute lymphocytic leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma, Hodgkin's lymphoma, Hodgkin's disease, infant or adult solid tumors, brain tumors, neuroblastoma, retinoblastoma, Wilms' tumor, osteosarcoma and chondrosarcoma, lung tumors, colorectal cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer, urinary tract cancer, bladder cancer, oral cavity tumors, pancreatic tumors, melanomas and tumors of the skin, stomach tumors, brain tumors, thyroid tumors, laryngeal tumors, liver tumors, testicular tumors, and / or - a bone or joint disease, preferably selected from rheumatoid arthritis, synovitis, cartilage and / or bone destruction, osteomyelitis, hypertrophy and / or hyperplasia of synovial tissue, osteophyte formation, neoplasms and / or metastases, and combinations thereof, and / or - a vascular condition, preferably selected from atherosclerosis, hemangioma, hemangioendothelioma, and combinations thereof; and / or - skin diseases, preferably selected from psoriasis, warts, pyogenic granulomas, hair growths, Kaposi's sarcoma, keloids of wounds, allergic edema, neoplasms, and combinations thereof, and / or - angiogenesis observed in adipose tissue pathologies, preferably obesity, and / or diabetes and / or its consequences, preferably retinopathy and / or diabetic foot, and / or Hematopoietic diseases, preferably AIDS and / or Kaposi's sarcoma.

[0021] As used herein, the term "angiogenesis" refers to new blood vessel formation, preferably associated with the formation of new blood vessels in tissues where no blood vessels were previously present and / or an increase in the number of blood vessels in already vascularized tissue. As used herein, angiogenesis is preferably dependent on the activity of VEGFR-1.

[0022] As used herein, the term "vascularization" means angiogenesis, i.e., they are used synonymously.

[0023] As used herein, the term "neovascularization" refers to angiogenesis, preferably angiogenesis that is dependent on the activity of VEGFR-1.

[0024] As used herein, the term "arteriogenesis" refers to the process of stabilization of new blood vessels by covering them with smooth muscle cells.

[0025] As used herein, "inhibitor" refers to a chemical and / or biological entity that can antagonize the activity of a receptor by binding to the receptor itself and / or its soluble ligand, thus preventing their interaction.

[0026] As used herein, the term "effective dose" refers to a dosing interval during which administration of the active agent according to the present invention can determine the desired biological effect. As is well known to those skilled in the art, the effective dose may vary depending on the health status, physical condition of the individual needing treatment, age, formulation of the active agent, evaluation of the physician taking care of the patient, the ability of the system of a single individual to respond effectively, the degree of the desired response, taxonomic group (e.g., human, non-human primate, primate, etc.), and other relevant factors. It is expected that the effective amount of the active agent described in the present invention will be within an interval wide enough to be determined by routine testing. In general, as reported by Ragan-Shaw et al. (FASEB J. 2008 Mar;22(3):659-61), and therefore also herein, the effective dose administered is preferably in the range of 10-2000 mg / dose, preferably when administered systemically, preferably by the enteral route, more preferably orally, sublingually or rectally. Alternatively, preferably when administered intravitreally, the effective dose administered is in the range of 1-100 mg / dose. Alternatively, the effective dose administered is preferably in the range of 0.16 to 33.3 mg / kg body weight. The treatment regimen provides for single or multiple administrations.

[0027] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE PRESENTINVENTION A first aspect of the present invention relates to a peptide, preferably a multimeric peptide, isolated and characterized by the following general formula (II):

[0028] {{{[Y1-Glu-Cys(Bzl)-Cha]2-Z1}i-Z2}j-Z3}z-Y2-Y3 (Formula II)

[0029] During the ceremony, -Y1 is the amino-terminal function of a peptide (NH2) or at least one chemical group preferably selected in Table I. It is understood that this list also includes chemical groups, preferably amino acids, with steric hindrance and / or chemical properties, in particular the side chains in the case of amino acids, which mimic those of the chemical groups, preferably amino acids, listed in Table I and / or are characterized by a similarity, preferably of at least 70%, which similarity is determined by methods known to those skilled in the art, for example, but not limited to, the methods described in Woong-Hee Shin et al., Molecules 2015, 20, 12841-12862.

[0030] It should be made clear that in this specification the D / L designations suitable for defining the absolute configuration of chiral centers present in the groups herein are interchangeable with the R / S designations according to the rules reported in the literature, as known to the skilled person.

[0031] [Table 1]

[0032] -Glu denotes glutamic acid, preferably in the absolute configuration R (R-Glu) on the Cα of the amino acid.

[0033] -Cys(Bzl) denotes a benzyl cysteine, preferably in the absolute configuration S on the Cα of an amino acid containing a sulfur-linked benzyl group of the amino acid (S-benzyl-cysteine / S-Cys(Bzl) side chain).

[0034] -Cha denotes cyclohexylalanine, preferably cyclohexylalanine in the absolute configuration S on the Cα of the amino acid (S-cyclohexylalanine / S-Cha).

[0035] -Y2 is preferably 1. The tripeptide R-Glu-S-Cys(Bzl)-S-Cha, and an α-amino acid preferably selected from glycine or an α-amino acid characterized by at least one thiol or thioether group, said α-amino acid preferably characterized by at least one thiol or thioether group selected from those shown in Table II and combinations thereof; is selected from.

[0036] This list is also understood to include chemical groups, preferably amino acids, with steric hindrance and / or chemical properties, in particular the side chains in the case of amino acids, which mimic those of the chemical groups, preferably amino acids, listed in Table II and are preferably characterized by at least 70% similarity (this similarity being determined by methods known to the skilled person, such as, but not limited to, the methods described in Woong-Hee Shin et al., Molecules 2015, 20, 12841-12862).

[0037] [Table 2]

[0038] -Y3 is preferably selected from a carboxyl group, a carboxamide group, an N-methyl substituted carboxamide or a disubstituted N,N-dimethyl group, a hydroxyl group and hydrogen.

[0039] -Z1, Z2 and Z3 preferably represent a trifunctional group characterized by the following formula (III): [ka] wherein k is an integer, preferably comprised between 1 and 4, and B is preferably an amino or hydroxyl group. The trifunctional molecule is preferably in the R or S absolute configuration.

[0040] Preferably, Z1, Z2 and Z3 are used to obtain branched structures.In fact, this type of structure is generally used to polymerize peptides according to known methods for this purpose.For example, when B is an amino group, the method described by Tam et al. (Tam JP, 1988, PNAS, 85, 5409-5413) can be used.

[0041] Z1, Z2 and Z3 can be assembled to obtain structures of formula (II) with multiple groups Z1, Z2 and Z3, preferably containing 1, 3 or 7 trifunctional molecules.

[0042] According to a preferred embodiment of the present invention, Z1 and / or Z2 and / or Z3 are preferably linked to each other by an amide bond to form a branched structure, or they may be linked to each other by an ester bond, for example when B is preferably a hydroxyl group.

[0043] -i is preferably 4, 2 or 1.

[0044] -j is preferably 2, 1 or 0.

[0045] -z is preferably 1 or 0.

[0046] According to a preferred embodiment, i=4, j=2 and z=1. According to a further preferred embodiment, i=2, j=1 and z=0.

[0047] According to a further preferred embodiment, i=1, j=z=0.

[0048] When j=0, the Z2 group is omitted, and when z=0, the Z3 group is omitted.

[0049] For purposes of the present invention, a particularly preferred embodiment contemplates i equal to 2, j equal to 1, and Z2 being 0 or omitted (in other words, Z3 not present or absent).

[0050] In a particularly preferred embodiment of the invention, Z1, Z2 and Z3 are R- or S-lysine (k=4), with i preferably being equal to 2.

[0051] A preferred formula for the multimeric peptide of the present invention is represented by the following formula (Figure IIa): [ka]

[0052] According to a particularly preferred embodiment of the invention, the peptide is a tetrameric peptide characterized by formula (IIb). [ka]

[0053] During the ceremony, -Y1 is a hydrogen atom, -Y2 is D-cysteine, -Y3 is an unsubstituted primary amide group; - Z1, Z2 and Z3 are as defined above, -i is equal to 2, -j is equal to 1, and -z is equal to zero or does not exist.

[0054] For convenience, a particularly preferred embodiment of a peptide characterized by formula IIb is hereinafter referred to as iVR1-Cys.

[0055] The peptides exhibit biological activity, preferably modulating activity, more preferably angiogenesis and / or neovascularization inhibitory activity, which is improved compared to the activity of the peptides described by Ponticelli et al., as reported and explained in the following experimental results (which have the purpose of being non-limiting and illustrative herein). The angiogenesis and / or neovascularization referred to herein is preferably VEGFR1 dependent, as defined above.

[0056] The peptide described in Ponticelli et al. is also a tetrameric peptide characterized by the formula (IIc): [ka]

[0057] During the ceremony, -Y1 is a hydrogen atom, -Y2 is glycine, -Y3 is an unsubstituted primary amide group; - Z1, Z2 and Z3 are as defined above, -i is equal to 2, j is equal to 1, and -z is equal to zero.

[0058] For convenience, a particularly preferred embodiment of a peptide characterized by formula IIc is hereinafter referred to as iVR1.

[0059] The authors of the present invention have surprisingly found that by modifying IVR1, in particular at the terminal carboxyl, preferably by inserting an R-Glu-S-Cys(Bzl)-S-Cha group or an α-amino acid, preferably selected from those shown in Table II and combinations thereof, characterized by at least one thiol or thioether group, the α-amino acid preferably being selected from those shown in Table II and combinations thereof, preferably inhibiting angiogenesis and / or vasculogenesis as defined above, peptides are obtained with improved biological activity, preferably with improved regulatory capacity.

[0060] In fact, as shown and discussed in more detail in the Examples, iVR1-Cys showed the ability to dose-dependently inhibit the interaction of both PlGF and VEGF-A with VEGFR-1, as compared to iVR1. In particular, the concentration (IC50) at which iVR1-Cys can inhibit the interaction of PlGF with VEGFR-1 by 50% is less than 1000 nM, while the IC50 of VEGF-A / VEGFR-1 inhibition is close to or slightly above 1000 nM. Meanwhile, iVR1 can inhibit the interaction of PlGF with VEGFR-1 by 50% (IC50) at a concentration close to 10000 nM. Similarly, the IC50 of VEGF-A / VEGFR-1 inhibition by iVR1 is close to or slightly above 10000 nM.

[0061] Thus, iVR1-Cys exhibits an inhibitory potency that is 10-fold higher than that reported for iVR1.

[0062] Furthermore, the authors showed that in an in vivo assay, iVR1 inhibited choroidal neovascularization by 37.8% and 39.3% compared to vehicle and PC (p<0.05), whereas iVR1-Cys inhibited it by 48.9% and 51.0% compared to vehicle and PC (p<0.02). Thus, iVR1-Cys shows a better inhibitory effect than peptide iVR1, since it further reduces angiogenesis by about 19.3%.

[0063] Finally, when administered orally or by gavage, both peptides tested as examples are able to induce a significant inhibition of angiogenesis compared to vehicle.

[0064] The latter fact is important because, although the ability of iVR1 to inhibit choroidal vascularization and neovascularization by intravitreal injection had already been demonstrated by Ponticelli et al. and Cicatiello et al. (2015), it was never expected that administering the peptide via a different route, in particular by gavage, could maintain or even improve the therapeutic effect, especially in the case of a very complex organ such as the eye and pathologies affecting it that are caused by or in any way correlated with unregulated, preferably increased, angiogenesis / neovascularization. In particular, the neovascular eye diseases mentioned are preferably selected from macular edema, wet forms of age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, central retinal vein occlusion, vitreous hemorrhage and retinal detachment, and combinations thereof.

[0065] In light of this evidence, it is clear that administration of the peptides of the invention via the oral route or by gavage is also therapeutically effective for the treatment of pathologies such as, for example, cancer, which are generally correlated with angiogenesis / angiogenesis. The angiogenesis or vascularization referred to is preferably VEGFR1 dependent.

[0066] According to one embodiment of the present invention, the peptides may be modified to facilitate or improve delivery, preferably by pegylation or by using a container / shuttle / carrier system, preferably a liposome, micelle, capsule, emulsion, matrix, gel, etc.

[0067] A further aspect of the invention relates to a composition comprising a peptide as described in detail and at least one further pharma- ceutically acceptable ingredient.

[0068] The composition preferably comprises at least one peptide characterized by formula IIa, more preferably a peptide characterized by formula IIb, ie, iVR1-Cys.

[0069] As used herein, a pharma- ceutically acceptable ingredient refers to a compound selected from an excipient, a diluent, a carrier, an adjuvant, a preservative, an antibiotic, an anti-inflammatory agent, an oil, a vitamin, an antioxidant, a chelating agent, a solubilizing agent, a viscosity agent, an inert gas, a surfactant, an emulsifier, a buffer substance, an immunosuppressant, an antitumor agent, and combinations thereof.

[0070] For example, according to one embodiment, a composition comprises a peptide of the invention in combination with at least one anti-angiogenic / anti-angiogenic molecule, an antibody that neutralizes the action of PlGF, at least one anti-VEGFR-1, anti-VEGFR-2, anti-VEGFR-3 antibody, at least one anti-VEGF-A, anti-VEGF-B, anti-VEGF-C, anti-VEGF-D, anti-VEGF-E antibody, and combinations thereof.

[0071] A further aspect of the present invention relates to a peptide as defined above, preferably characterized by formula IIa, more preferably by formula IIb, i.e. iVR1-Cys, for use as a medicament.

[0072] A further aspect of the invention relates to a peptide as defined above, preferably characterized by formula IIa, more preferably by formula IIb, i.e. iVR1-Cys, or a composition comprising said peptide, for use in the treatment of a pathological condition associated with or caused by inappropriate angiogenesis / neovascularization, i.e. a condition in which angiogenesis / neovascularization is unregulated, which is preferably increased and therefore needs to be inhibited.

[0073] In addition to being useful in the treatment of this condition, the peptide, preferably characterized by formula IIa, more preferably characterized by formula IIb, i.e., iVR1-Cys, or a composition comprising said peptide, can also be used to follow up further alternative treatments for this condition.

[0074] As already mentioned above, angiogenesis / angiogenesis is preferably dependent on / induced by / regulated by VEGFR1 or the VEGFR1 pathway, as defined above.

[0075] The disease state / condition is preferably selected from the following: - a neovascular eye disease preferably selected from macular edema, wet forms of age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, central retinal vein occlusion, vitreous hemorrhage and retinal detachment, and combinations thereof, and / or solid tumors and / or tumor metastases, which are preferably selected from leukemias and lymphomas, preferably acute lymphocytic leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma, Hodgkin's lymphoma, Hodgkin's disease, infant or adult solid tumors, brain tumors, neuroblastoma, retinoblastoma, Wilms' tumor, osteosarcoma and chondrosarcoma, lung tumors, colorectal cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer, urinary tract cancer, bladder cancer, oral cavity tumors, pancreatic tumors, melanomas and tumors of the skin, stomach tumors, brain tumors, thyroid tumors, laryngeal tumors, liver tumors, testicular tumors, and / or - a bone or joint disease, preferably selected from rheumatoid arthritis, synovitis, cartilage and / or bone destruction, osteomyelitis, hypertrophy and / or hyperplasia of synovial tissue, osteophyte formation, neoplasms and / or metastases, and combinations thereof, and / or - a vascular condition, preferably selected from atherosclerosis, hemangioma, hemangioendothelioma, and combinations thereof; and / or - skin diseases, preferably selected from psoriasis, warts, pyogenic granulomas, hair growths, Kaposi's sarcoma, keloids of wounds, allergic edema, neoplasms, and combinations thereof, and / or - angiogenesis observed in adipose tissue pathologies, preferably obesity, and / or diabetes and / or its consequences, preferably retinopathy and / or diabetic foot, and / or Hematopoietic diseases, preferably AIDS and / or Kaposi's sarcoma.

[0076] For such medical purposes, the peptides and compositions of the present invention can optionally be combined with or used before or after known drugs used to treat such medical conditions.

[0077] Furthermore, the peptides or compositions of the invention may be associated with known surgical, radiotherapeutic or chemotherapeutic treatments used to treat the above mentioned pathologies.

[0078] The peptide of the present invention or a composition comprising said peptide can be formulated to be administered via any route, which is preferably selected from the systemic route, preferably the oral route, gavage, sublingual or rectal route, topical, subcutaneous, intramuscular, intravenous, intraarterial, intraperitoneal, intradermal and intraepidermal routes.

[0079] The peptides or compositions of the invention can be formulated as a solid, e.g., as pills, tablets, granules, soluble granules, pellets, beads, lozenges, etc. Alternatively, the peptides or compositions of the invention can be formulated as a liquid solution or as drops or spray, to be administered, e.g., by injection, inhalation or nebulization.

[0080] The peptide of the invention as described above or a composition comprising this peptide may be administered as a bolus.

[0081] The peptide of the invention as described above or a composition comprising this peptide can be administered by a medical device, for example by a stent, pump or patch.

[0082] Administration may be preferably continuous, by controlled release or constant release, preferably using a device for ocular drug delivery.

[0083] Particularly preferred is the oral route or administration by gavage. Indeed, as described above, the peptides of the present invention (including iVR1) have been shown to be effective in inhibiting angiogenesis / neovascularization even when administered by gavage. They have also been shown to be effective in inhibiting ocular angiogenesis / neovascularization. In other words, it has been surprisingly observed that the peptides of the present invention (including iVR1) inhibit angiogenesis / neovascularization in the eye when administered by gavage. The angiogenesis / neovascularization referred to is preferably VEGFR1 dependent.

[0084] In light of this scientific evidence, a further aspect of the present invention relates to a peptide of the present invention, preferably at least one peptide characterized in Figure IIa, more preferably a peptide characterized in formula IIb, i.e. iVR1-Cys, and / or a peptide characterized in formula IIc, i.e. iVR1, or a composition comprising these peptides administered orally or by gavage, for use in the treatment of a pathology caused by or in any way associated with inappropriate, preferably increased angiogenesis / angiogenesis (preferably VEGFR1 dependent).

[0085] The disease state / condition is preferably selected from the following: - a neovascular eye disease preferably selected from macular edema, wet forms of age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, central retinal vein occlusion, vitreous hemorrhage and retinal detachment, and combinations thereof, and / or solid tumors and / or tumor metastases, preferably selected from leukemias and lymphomas, preferably acute lymphocytic leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma, Hodgkin's lymphoma, Hodgkin's disease, infant or adult solid tumors, brain tumors, neuroblastoma, retinoblastoma, Wilms' tumor, osteosarcoma and chondrosarcoma, lung tumors, colorectal cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer, urinary tract cancer, bladder cancer, oral cavity tumors, pancreatic tumors, melanomas and tumors of the skin, stomach tumors, brain tumors, thyroid tumors, laryngeal tumors, liver tumors, testicular tumors, and / or - a bone or joint disease, preferably selected from rheumatoid arthritis, synovitis, cartilage and / or bone destruction, osteomyelitis, hypertrophy and / or hyperplasia of synovial tissue, osteophyte formation, neoplasms and / or metastases, and combinations thereof, and / or - a vascular condition, preferably selected from atherosclerosis, hemangioma, hemangioendothelioma, and combinations thereof; and / or - skin diseases, preferably selected from psoriasis, warts, pyogenic granulomas, hair growths, Kaposi's sarcoma, keloids of wounds, allergic edema, neoplasms, and combinations thereof, and / or - angiogenesis observed in adipose tissue pathologies, preferably obesity, and / or diabetes and / or its consequences, preferably retinopathy and / or diabetic foot, and / or Hematopoietic diseases, preferably AIDS and / or Kaposi's sarcoma.

[0086] The peptides or compositions of the present invention are administered to any animal in need thereof, preferably an animal in need of inhibiting VEGFR-1 dependent angiogenesis.

[0087] The animal is preferably a mammal, and more preferably it is a human.

[0088] The effective amount of such peptide or composition to be administered is preferably in the following range: - preferably 10-2000 mg / dose when administered systemically, preferably by the systemic enteral route, more preferably orally, sublingually or rectally, or -Preferably 1 to 100 mg per dose when administered intravitreally.

[0089] Alternatively, the effective dose administered is preferably in the range of 0.16 to 33.3 mg / kg body weight.

[0090] The treatment regimen preferably provides a single dose or multiple doses.

[0091] The sequences of the invention are annotated according to the international standard WIPO ST.25, the description of which is given in the program Patent-In3.5. The description of the sequences is attached hereto.

[0092] As used herein, the sequences identified in Table III and sequences with identities ranging from 80-99.9% are also to be considered as described.

[0093] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] EXAMPLES

[0094] Dose-dependent inhibition of VEGF-A / VEGFR1 and PlGF / VEGFR-1 interactions The assay to test the binding of PlGF or VEGF-A to the VEGFR-1 receptor was based on the ELISA method [Ponticelli et al., JBC. 2008 Dec 5;283(49):34250-9] and was performed using reagents obtained from R&D Systems.

[0095] Human recombinant receptor VEGFR-1, specifically a form consisting of the seven extracellular domains of the receptor fused to the Fc domain of human IgG (R&D Systems, Cat. No. 321-FL), was generated and attached to wells of a 96-well microplate at a concentration of 0.5 μg / ml in PBS pH 7.5 (100 μl / well) for 16 hours at room temperature (RT).

[0096] After blocking nonspecific binding sites in the wells using a buffer consisting of PBS pH 7.5 containing 3% BSA, 5 ng / ml recombinant PlGF of human origin (R&D Systems, Cat. No. 264-PG) or 5 ng / ml recombinant VEGF-A (R&D Systems, Cat. No. 293-VE) in PBET (PBS pH 7.5, 0.1% BSA, 5 mM EDTA, 0.004% Tween) was added to the receptor-attached wells.

[0097] Simultaneously with the ligands, i.e., PlGF or VEGF-A, graded doses of iVR1, iVR1-Cys or a control peptide (PC-[(S-Ser)-(S-Ala)-(S-Cha) tripeptide with a tetrameric structure identical to that of the iVR1 peptide]) were added at concentrations ranging from 780 to 50,000 nM. Binding reactions were carried out at 37 °C for 1 h, followed by 1 h at room temperature.

[0098] At the end of the binding and / or competition steps, anti-human-PlGF biotinylated polyclonal antibody (R&D Systems, Cat. No. BAF264) or anti-human-VEGF-A (R&D Systems, Cat. No. BAF293) was added to the wells at a concentration of 300 ng / ml in PBET. After 1 h of incubation at 37°C followed by 1 h at room temperature, the HRP-conjugated avidin-streptavidin system (Vectastain Elite ABC kit) and substrate for HRP (o-phenylenediamine-Sigma, Cat. No. P1526) were added to the wells. Quantification was performed by measuring absorbance at 490 nM.

[0099] The inhibitory activity of the peptides was expressed as a percentage of residual binding by comparing the data obtained for PlGF or VEGF-A binding to the receptor in the presence of the tetrameric peptide with that in the absence of the tetrameric peptide. iVR1 was a positive control for inhibition of PlGF / VEGFR-1 or VEGF-A / VEGFR-1 interactions.

[0100] The results are shown in Tables IV and V. We demonstrated the ability of iVR1-Cys to inhibit the interaction of both PlGF and VEGF-A with VEGFR-1 in a dose-dependent manner. This shows that.

[0101] The concentration at which iVR1-Cys can inhibit the interaction between PlGF and VEGFR-1 by 50% (IC 50 ) is less than 1000 nM, whereas the IC50 for VEGF-A / VEGFR-1 is close to or slightly above 1000 nM.

[0102] Thus, because iVR1-Cys has approximately 10-fold greater inhibitory potency than iVR1, it is expected that it could be used at a 10-fold lower dose in the same in vitro and in vivo experimental protocols for inhibition of angiogenesis / angiogenesis to achieve the same effects as obtained with iVR1.

[0103] The PC does not block it.

[0104] [Table 4]

[0105] [Table 5]

[0106] The ability of tetrameric peptides having formula (II) but with Y2 different from D-cysteine ​​to inhibit VEGF-A / VEGFR-1 binding was evaluated in the binding assay described above. The IC of peptide Y2 and its respective inhibition of VEGF-A / VEGFR-1 interaction were 50 is shown in Table VI.

[0107] [Table 6]

[0108] Inhibition of PlGF-induced phosphorylation of VEGFR-1 An assay of PlGF-induced phosphorylation of the receptor VEGFR-1 was performed to evaluate the inhibitory potential of peptide iVR1-Cys and to compare its activity with that of iVR1.

[0109] For VEGFR-1 activation, we used a cell line overexpressing the receptor, called 293-VEGFR-1, obtained by stable transfection of HEK-293 cells (Errico, M. et al. 2004 JBC, 279:43929-43939).

[0110] For this purpose, 293-VEGFR-1 cells were cultured until they reached sub-confluence, after which the cells were "starved" by maintaining / incubating them in serum-free medium for at least 16 h.

[0111] At the end of the starvation step, the medium was removed and the cell monolayer was incubated with 100 μM Na 3 VO 4 for 5 min to inhibit the activity of endogenous phosphatases.

[0112] The cells were then stimulated (1) with 20 ng / ml PlGF alone in the medium used for starvation at 37° C. for 10 min, or (2) in the presence of peptide at a concentration of 5 μM.

[0113] Anti-human PlGF neutralizing monoclonal antibody (Thrombogenics) was used as an inhibition control at a concentration of 3.3 nM. PBS was used as a negative control.

[0114] At the end of the incubation, the cells were washed with 100 μM cold Na3VO4 and lysed in a buffer consisting of 20 mM Tris-HCl (pH 8), 5 mM EDTA, 150 mM NaCl, 1% Triton-X100, 10% glycerol, 10 mM zinc acetate, 100 μM Na3VO4 and a mixture of protease inhibitors, and incubated for 1 h at 4 °C with gentle agitation. Finally, the cell lysates were centrifuged at 12000xg for 15 min to remove cell debris. Quantification of the extracts was performed by the Bradford method using Bio-Rad reagents. 100 μg of total protein extract was loaded on SDS-PAGE and reduced to 8.5%, followed by Western blotting, a standard method for analyzing proteins.

[0115] Normalization was performed by detecting phosphorylated VEGFR-1 using anti-p-VEGFR-1 antibody (R&D Systems, catalog no. AF4170) diluted 1:500, while detecting the nonphosphorylated form of the receptor using anti-VEGFR-1 antibody (Sigma-Aldrich, catalog no. V4262) diluted 1:500.

[0116] As shown in Figure 1, peptide iVR1-Cys had an IC determined in a binding assay. 50 Used at a concentration approximately 5-fold (5000 nM) higher than that of the neutralizing antibody (see Example 1), it potently inhibits receptor phosphorylation, an inhibition similar to that obtained with the neutralizing antibody and clearly greater than that obtained with iVR1 using the same concentration.

[0117] Inhibition of choroidal neovascularization by intravitreal and oral (gavage) administration of iVR1 and iVR1-Cys Experimental models of laser-induced choroidal neovascularization require the injury of Bruch's membrane, which separates the choroid from the retinal pigment epithelium (RPE). The injury is caused by laser-induced burning, which leads to perforation of Bruch's membrane and activates chorioretinal neovascularization. The growth of new blood vessels originates from the choroid and invades the overlying retinal tissue. This mouse model recapitulates the main features of the exudative form of human age-related macular degeneration (AMD) and is, in fact, commonly used as a preclinical model of AMD. It allows the evaluation of the antiangiogenic activity of molecules of interest.

[0118] To be able to visualize the mouse fundus and induce damage with the laser, a Micron IV integrated system was used according to the experimental procedure described below.

[0119] First, dilation of the animal's pupils was induced by applying 0.5% tropicamide eye drops. The animal was then anesthetized by intraperitoneal injection of a solution of ketamine and xylazine (80mg / Kg and 10mg / Kg, respectively). Once sedated, the animal was placed on a stand and a 2.5% aqueous solution of hydroxypropyl methylcellulose was applied to both eyes. By placing the Micron IV camera lens in contact with the solution (a procedure similar to that used in microscopy with immersion objectives), it has the dual function of preventing corneal dehydration and improving visualization of the fundus.

[0120] To induce damage with a laser, a laser pointer is first activated, focused, and the laser beam is directed at the RPE layer. The area where the laser beam is directed must be away from the major blood vessels of the retina to prevent possible bleeding. The efficiency of the burning at the level of Bruch's membrane is confirmed by the formation of bubbles immediately after the laser beam. The conditions for the laser beam were a power of 200 mW for 100 ms.

[0121] From the data present in the literature, well summarized in the article by Lambert et al. (Nature Protocols, 2013, 8:2197), it is known that maximum angiogenesis in this experimental model is obtained 7 days after injury.

[0122] C57Bl6 / J mice were used (n=5 per group). At the end of the laser injury-inducing procedure, intravitreal injections were performed immediately, administering 10 and 50 μg of iVR1-Cys or iVR1 and 50 μg of PC in 1 μL of DMSO using a Hamilton syringe with a 32 g needle. As a control, DMSO was injected alone.

[0123] After 7 days, the animals were sacrificed, the eyes enucleated and fixed in 4% paraformaldehyde. Subsequently, the anterior part of the eye, consisting of the cornea, iris and cornea, was removed under a stereomicroscope. The remaining part, defined as the "eyecup" or posterior part, consisting of the sclera, choroid, RPE and retina, was incubated for 16 h in the presence of 0.7% FITC-Griffonia simplicifolia Isolectin B4 (Vector Laboratories, Burlingame, CA). After a series of washes, the retina was removed and four cuts were made in the RPE / choroid, which allows them to be mounted on slides and observed under a fluorescent microscope. Quantification of choroidal neovascularization is performed on the volume. To evaluate the volume of all spots, a series of images (Z-Satcks, approximately 20-25 images) is acquired. Each image is 1 μm thick and is acquired from the top surface to the deepest focal plane, at the RPE cell level. The volume of fluorescence was measured using the ImageJ program (NIH, Bethesda, MD) by taking the sum of the fluorescent area of ​​all single planes.

[0124] CNV was quantified in n=12 and 15 spots for iVR1 at 10 and 50 μg, n=10 and 8 spots for iVR1-Cys at 10 and 50 μg, n=15 spots for PC, and n=14 spots for DMSO. The results shown in Figure 2 show that both peptides can inhibit angiogenesis in a dose-dependent manner. A higher dose (50 μg) resulted in a potent, significant and comparable angiogenesis inhibition capacity: iVR1-Cys-75.9% and -74.6% (p>0.002) versus vehicle and PC, iVR1-73.9% and -76.5% (p>0.0002) versus vehicle and PC.

[0125] At a dose of 10 μg, iVR1 results in angiogenesis inhibition of 37.8% and 39.3% versus vehicle and PC (p<0.05), whereas iVR1-Cys results in inhibition of 48.9% and 51.0% versus vehicle and PC (p<0.02). Thus, at lower concentrations, peptide iVR1-Cys shows a better inhibitory effect than peptide iVR1, since it reduces angiogenesis by an additional 19.3%. Thus, at the higher doses used, a maximum threshold of the inhibitory capacity of the peptide may have been reached.

[0126] In the oral administration (gavage) experiments, choroidal neovascularization was induced in C57Bl6 / J mice (n=5 animals per group) according to the experimental procedure described above. Administration of peptides iVR1 and iVR1-Cys as well as vehicle was started immediately after injury induction, twice a day for 7 days as defined by the experimental protocol, as soon as the animals recovered from anesthesia. Peptides were administered at 50 mg / Kg, based on data previously obtained for peptide iVR1 administered intraperitoneally (Cicatiello et al. 2015, Oncotarget, 6, 10563-10576).

[0127] To allow for oral administration, the peptide was dissolved in DMSO and then mixed with Nutilis food thickener to give a final mixture consisting of 9 parts Nutilis and 1 part DMSO.

[0128] The substances were prepared in such a concentration that 200 μl of a 9:1 mixture of substance in Nutilis / DMSO was used per administration and administered directly into the stomach of the animals using a needle suitable for gavage with a 20-gauge opening. The control group received 200 μl of the 9:1 Nutilis / DMSO mixture.

[0129] At the end of the experiment, the animals were sacrificed, the eyes enucleated and dissected to isolate the RPE-choroid, and the volume of CNV was determined by immunofluorescence analysis, as described below.

[0130] Quantification of CNV was performed on n=18 spots for iVR1-Cys, n=20 spots for iVR1, and n=10 spots for vehicle.

[0131] The results are shown in Figure 3. Peptide iVR1-Cys was able to induce a significant inhibition of angiogenesis at a level similar to that observed with iVR1 (-49.7%, p=0.001) compared to vehicle (-45.9%, p=0.007). This indicates that.

[0132] Serum protease stability of iVR1-Cys. The stability of peptide iVR1-Cys in 10% serum (fetal calf serum, FCS) in 50 mM phosphate buffer (pH 7.3) for 168 hours was determined as described therein, relying on the RP-HPLC chromatography-based method described by Ponticelli et al. [Ponticelli et al., JBC. 2008 Dec 5;283(49):34250-9].

[0133] Reference curves were made by dissolving the compounds in DMSO at concentrations between 0.1 μmol / L and 1000 μmol / L in order to completely dissolve. The concentration of the molecules remaining in contact with 10% FCS at an initial concentration of 10 μmol / L was then determined by drawing three aliquots at time t = 0, then every hour for the first 12 hours, then at 24, 72, 120 and 168 hours. To isolate any peptides bound to albumin, aliquots were diluted 1:1 with acetic acid 0.1 M, centrifuged to remove precipitated material and analyzed by RP-HPLC under conditions reported in Ponticelli et al. The amount of residual peptide detected in the aliquots was expressed as a percentage of the initial amount and plotted as a function of time. The results are presented in Table VII as the mean ± standard deviation (SD) of triplicate measurements.

[0134] [Table 7]

Claims

1. The following formula (II): {{{[Y1-Glu-Cys(Bzl)-Cha]2-Z1}i-Z2}j-Z3}z-Y2-Y3 (Formula II) During the ceremony, -Y1 is an amino terminal peptide function (NH 2 ) and -Y2 is D-cysteine, L-cysteine, L-homocysteine, D-homocysteine, D-cysteine-S-methyl, L-cysteine-S-methyl, D-cysteine-S-ethyl, L-cysteine-S-ethyl, L-methionine, D-methionine, D-cysteine-S-benzyl, L-cysteine-S-benzyl, L-methionine sulfoxide, D-methionine sulfoxide, L-methionine sulfone, D-methionine sulfone, D-serine, an α-amino acid selected from L-serine, D-serine-O-methyl, L-serine-O-methyl, D-serine-O-ethyl, L-serine-O-ethyl, D-serine-O-benzyl, L-serine-O-benzyl, D-threonine, L-threonine, D-threonine-O-methyl, L-threonine-O-methyl, D-threonine-O-ethyl, L-threonine-O-ethyl, D-threonine-O-benzyl and L-threonine-O-benzyl; -Y3 is selected from a carboxyl group, a carboxamide group, an N-methyl substituted carboxamide or a disubstituted N,N-dimethyl group, a hydroxyl group and hydrogen; Z1, Z2 and Z3 represent a trifunctional molecule; - i is 4, 2 or 1, and / or -j is 2, 1, or 0, and / or -z is 1 or 0, However, when i = 4, j = 2 and z = 1, when i = 2, j = 1 and z = 0, when i = 1, j = z = 0, when j = 0, the Z2 group is omitted, and when z = 0, the Z3 group is omitted. An isolated peptide comprising:

2. The trifunctional molecule has the following formula (III): [Case 1] During the ceremony, k is an integer between 1 and 4, and B is an amino group, S- or R-lysine, or a hydroxyl group; The peptide according to claim 1, characterized in that

3. 3. The peptide according to claim 1 or 2, wherein Z1 and / or Z2 and / or Z3 are linked together by an amide bond or an ester bond to form a branched structure.

4. Formula (IIb): [Case 2] The peptide according to any one of claims 1 to 3, characterized in that

5. A composition comprising at least one peptide according to any one of claims 1 to 4 and at least one further pharma- ceutically acceptable ingredient.

6. A peptide according to any one of claims 1 to 4 or a composition according to claim 5 for use as a medicament.

7. A peptide according to any one of claims 1 to 4 or a composition according to claim 5 for use in the treatment of a pathological condition associated with or caused by unregulated angiogenesis / angiogenesis, angiogenesis / angiogenesis that is VEGFR1 dependent.

8. The pathological condition is - a neovascular eye disease selected from macular edema, wet forms of age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, central retinal vein occlusion, vitreous hemorrhage and retinal detachment, and combinations thereof, and / or solid tumors and / or tumor metastases, said tumors being selected from leukemias and lymphomas, acute lymphocytic leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, multiple myeloma, Hodgkin's lymphoma, Hodgkin's disease, infant or adult solid tumors, brain tumors, neuroblastoma, retinoblastoma, Wilms' tumor, osteosarcoma and chondrosarcoma, lung tumors, colorectal cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer, urinary tract cancer, bladder cancer, oral cavity tumors, pancreatic tumors, melanomas and tumors of the skin, stomach tumors, brain tumors, thyroid tumors, laryngeal tumors, liver tumors, testicular tumors, and / or - a bone or joint disease selected from rheumatoid arthritis, synovitis, cartilage and / or bone destruction, osteomyelitis, hypertrophy and / or hyperplasia of synovial tissue, osteophyte formation, neoplasms and / or metastases, and combinations thereof, and / or - a vascular condition selected from atherosclerosis, hemangioma, hemangioendothelioma, and combinations thereof; and / or - skin diseases selected from psoriasis, warts, pyogenic granulomas, hair growth, Kaposi's sarcoma, keloids of wounds, allergic edema, neoplasms, and combinations thereof; and / or - the angiogenesis observed in adipose tissue pathologies, obesity, and / or - diabetes and / or its consequences, retinopathy and / or diabetic foot, and / or - Hematopoietic disorders, AIDS and / or Kaposi's sarcoma 8. The peptide or composition for use according to claim 7, selected from:

9. A peptide according to any one of claims 1 to 4 or a composition according to claim 5 for use as an inhibitor of VEGFR1, as an inhibitor of its activation.

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